EP4680767A1 - Diagnostic des cancers à partir de l'état de méthylation de petits arn non codants - Google Patents

Diagnostic des cancers à partir de l'état de méthylation de petits arn non codants

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Publication number
EP4680767A1
EP4680767A1 EP24708223.3A EP24708223A EP4680767A1 EP 4680767 A1 EP4680767 A1 EP 4680767A1 EP 24708223 A EP24708223 A EP 24708223A EP 4680767 A1 EP4680767 A1 EP 4680767A1
Authority
EP
European Patent Office
Prior art keywords
sequence
stem
adapter
rna
small non
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24708223.3A
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German (de)
English (en)
Inventor
Rastislav Horos
Alberto Danie MORENO
Carla Bieg Salazar
Tobias SIKOSEK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hummingbird Diagnostics GmbH
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Hummingbird Diagnostics GmbH
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Publication date
Application filed by Hummingbird Diagnostics GmbH filed Critical Hummingbird Diagnostics GmbH
Publication of EP4680767A1 publication Critical patent/EP4680767A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the use of a methylation status of small non-coding RNA to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • the present invention relates to a method for diagnosing cancer in a patient/determining whether a patient suffers from cancer by determining a methylation status of small non-coding RNA in a biological sample.
  • Nucleic acids of interest to be detected in molecular diagnostics include small non-coding RNAs.
  • Small non-coding RNAs particularly with a length of between 18 to 50 nucleotides (nt), are found intracellularly and in extracellular environments, including body fluids such as blood.
  • miRNAs small non-coding RNAs
  • iRNAs microRNAs
  • isomiRs variants having specific terminal sequences
  • mRNAs protein-encoding messenger RNAs
  • RNAs with regulatory functions such as fragments of ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), and others.
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • snoRNA small nucleolar RNA
  • RNAs can be post-transcriptionally edited by chemical modifications such as methylation (m), oxidation, pseudouridylation, etc., which can target a nucleotide base (e.g. methyl-6-adenine, pseudouridine) or a ribose ring (2 ’-ortho-methylation, 2'-O-m).
  • m methylation
  • pseudouridylation oxidation
  • pseudouridylation e.g. methyl-6-adenine, pseudouridine
  • ribose ring e.g. methyl-6-adenine, pseudouridine
  • 2'-O-m ribose ring
  • rRNA is known to contain many pseudouridylation and 2'-O-m sites and these modifications contribute to the folding, stability, and RNase resistance of the ribosome. It has been previously shown that the 2'-O-m sites are modified to various extents (40-100% stoichiometry).
  • DB PCR Dumbbell PCR
  • RT reverse transcription
  • the present inventors have now found that the determination of the methylation status of small non-coding RNA allows to discriminate biological samples of cancer patients from biological samples of non-cancer, i.e. healthy, control subjects. Specifically, the present inventors have now found that the determination of the methylation status of small non-coding RNA extracted from a biological sample such as peripheral blood allows to diagnose cancer such as early-stage cancer in a patient.
  • the present invention relates to the use of a methylation status of small non-coding RNA (in a biological sample) to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • a 3 ’adapter capable of forming a stem-loop structure containing a loop and a double stranded stem is used for determining the methylation status of small non-coding RNA (in a biological sample) to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • the present invention relates to a method for diagnosing cancer in a patient/for determining whether a patient suffers from cancer comprising the step of: determining a methylation status of small non-coding RNA in a biological sample obtained from a patient.
  • the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
  • the term “about” indicates a certain variation from the quantitative value it precedes.
  • the term “about” allows a ⁇ 5% variation from the quantitative value it precedes, unless otherwise indicated or inferred.
  • the use of the term “about” also includes the specific quantitative value itself, unless explicitly stated otherwise. For example, the expression “about 80°C” allows a variation of ⁇ 4°C, thus referring to range from 76°C to 84°C.
  • Nucleic acids of interest to be detected in molecular cancer diagnostics include RNA.
  • target RNA refers to a ribonucleotide sequence that is sought to be detected.
  • the target RNA may be obtained from any source and may comprise any number of different compositional components.
  • the target RNA is isolated from organisms, tissues, cells, or body fluids such as blood.
  • the target RNA encompasses non-coding RNA.
  • the target RNA is small non-coding RNA.
  • the small non-coding RNA has a length of ⁇ 200 ribonucleotides, more particularly a length of between 10 and ⁇ 200 ribonucleotides, even more particularly a length of between 10 and 100 ribonucleotides, and still even more particularly a length of between 18 and 50 ribonucleotides, e.g. a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
  • the small non-coding RNA is a miRNA, a miRNA isoform (an isomiR), a ribosomal RNA fragment (rRNA fragment), a transfer RNA fragment (tRF), or a small nucleolar RNA (snorRNA) fragment.
  • target RNA may refer to the target RNA itself as well as to surrogates thereof, for example, amplification products (e.g. cDNA derived therefrom) and native sequences.
  • the target RNA lacks a poly-A tail.
  • the target RNA described herein may be derived from any number of sources, including without limitation, humans and animals. These sources may include, but are not limited to, whole blood, a tissue biopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen, biowarfare agents, anal secretions, vaginal secretions, perspiration, saliva, or buccal swabs.
  • sources may include, but are not limited to, whole blood, a tissue biopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen, biowarfare agents, anal secretions, vaginal secretions, perspiration, saliva, or buccal swabs.
  • various environmental samples for example, agricultural, water, and soil
  • research samples generally, purified samples generally, culture
  • target RNAs may be isolated from samples using any of a variety of procedures known in the art, for example, the Applied Biosystems ABI Prism® 6100 Nucleic Acid PrepStation (Life Technologies, Foster City, CA) and the ABI Prism® 6700 Automated Nucleic Acid Workstation (Life Technologies, Foster City, CA), Ambion® mirVanaTM RNA isolation kit (Life Technologies, Austin, TX), and the like.
  • small non-coding RNA refers to functional RNA that is not translated into protein. Small non-coding RNA has diverse functionally important roles, which involve, particularly in conjunction with other molecules, gene regulation through RNA interference, RNA modification, or spliceosome involvement.
  • the small non-coding RNA has a length of ⁇ 200 ribonucleotides, more preferably a length of between 10 and ⁇ 200 ribonucleotides, even more preferably a length of between 10 and 100 ribonucleotides, and still even more preferably a length of between 18 and 50 ribonucleotides, e.g.
  • the (above-mentioned) small non-coding RNA is a miRNA, a miRNA isoform (an isomiR), a ribosomal RNA fragment (rRNA fragment), a transfer RNA fragment (tRF), or a small nucleolar RNA (snorRNA) fragment and similar.
  • the rRNA fragment is particularly favoured.
  • RNA refers to a single-stranded RNA.
  • the miRNA has a length of ⁇ 200 ribonucleotides, more preferably a length of between 10 and ⁇ 200 ribonucleotides, even more preferably a length of between 10 and 100 ribonucleotides, and still even more preferably a length of between 18 and 50 ribonucleotides, e.g. a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
  • the miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. miRNAs are non-coding RNAs).
  • the genes encoding miRNAs are longer than the processed mature miRNA molecules.
  • the miRNA is initially transcribed as a longer precursor molecule (>1000 nucleotides long) called a primary miRNA transcript (pri-miRNA).
  • pri-miRNAs have hairpin structures that are processed by the Drosha enzyme (as part of the microprocessor complex).
  • the pri-miRNAs are only 60-100 nucleotides long, and are called precursor miRNAs (pre-miRNAs).
  • pre-miRNAs precursor miRNAs
  • Dicer cuts the miRNA in two, resulting in duplexed miRNA strands.
  • RISC RNA-induced silencing complex
  • the other arm is called the “minor miRNA” or “passenger miRNA”, and is often designated as miR*.
  • miR/miR* name scheme
  • miR-5p/miR-3p the 5’ arm of the miRNA
  • miR-3p the 3’ arm of the miRNA.
  • the present nomenclature is as follows: The prefix “miR” is followed by a dash and a number, the latter often indicating order of naming. For example, hsa- miR-16 was named and likely discovered prior to hsa-miR-342.
  • a capitalized “miR-” refers to the mature forms of the miRNA (e.g. hsa-miR-16-5p and hsa-miR-16-3p), while the uncapitalized “mir-” refers to the pre-miRNA and the pri-miRNA (e.g. hsa-mir-16), and “MIR” refers to the gene that encodes them.
  • AGO Argonaute
  • the complex causes the duplex to unwind, and the passenger RNA strand is discarded, leaving behind a mature RISC carrying the mature, single stranded miRNA.
  • the miRNA remains part of the RISC as it silences the expression of its target genes. While this is the canonical pathway for miRNA biogenesis, a variety of others have been discovered. These include Drosha-independent pathways (such as the mirtron pathway, snoRNA-derived pathway, and shRNA-derived pathway) and Dicer-independent pathways (such as one that relies on AGO for cleavage, and another which is dependent on tRNaseZ).
  • miRBase refers to a well-established repository of validated miRNAs.
  • the miRBase (www.mirbase.org) is a searchable database of published miRNA sequences and annotation. Each entry in the miRBase Sequence database represents a predicted hairpin portion of a miRNA transcript (termed mir in the database), with information on the location and sequence of the mature miRNA sequence (termed miR). Both hairpin and mature sequences are available for searching and browsing, and entries can also be retrieved by name, keyword, references and annotation. All sequence and annotation data are also available for download. In October 2018, miRbase version 22.1 was released. This is the current version.
  • miRNA isoform refers to a miRNA that varies slightly in sequence, which results from variations in the cleavage site during miRNA biogenesis or by processes which affect the mature miRNA after the biogenesis has occurred, such as oligouridylation.
  • imprecise cleavage of Drosha and Dicer or the turnover of miRNAs can result in miRNAs that are heterogeneous in length and/or sequence.
  • IsomiRs can be divided into three main categories: 3' isomiRs (trimmed or addition of one or more nucleotides at the 3' position), 5' isomiRs (trimmed or addition of one or more nucleotides at the 5' position), and polymorphic isomiRs (some nucleotides within the sequence are different from the wild type mature miRNA sequence). It could be envisioned that the increased expression of miRNA variants, or individual isomiRs, lead to the loss or weakening of the function of the corresponding wild-type mature miRNA or result in the regulation of a different transcriptome. Recent studies suggest that isomiRs probably play vital roles in a variety of cancers, tissues, and cell types. The detection of miRNAs as well as isomiRs is, thus, absolutely required to accurately reflect the underlying biological situation and to make the right decisions.
  • ribosomal RNA fragment refers to a fragment derived from a ribosomal RNA (rRNA). Ribosomal RNAs (rRNAs) form a group that includes four (5S, 5.8S, 18S, 28S) rRNAs encoded by the human nuclear genome and two (12S, 16S) by the mitochondrial genome. rRNAs constitute the most abundant RNA type in eukaryotic cells.
  • the rRNA fragment has a length of ⁇ 200 ribonucleotides, more preferably a length of between 10 and ⁇ 200 ribonucleotides, even more preferably a length of between 10 and 100 ribonucleotides, and still even more preferably a length of between 18 and 50 ribonucleotides, e.g.
  • transfer RNA fragment refers to a fragment derived from a transfer RNA.
  • a transfer RNA (tRNA) is a ribonucleic acid which mediates the correct amino acid to the corresponding codon on the mRNA during translation.
  • Transfer RNAs (tRFs) are produced from pre-tRNAs or mature tRNAs. Based on the incision loci, tRFs are classified into several types: tRF-1, tRF-2, tRF-3, tRF-5, and i-tRF. Some tRFs participate in posttranscriptional regulation through microRNA-like actions or by displacing RNA binding proteins and regulating protein translation by promoting ribosome biogenesis or interfering with translation initiation. Other tRFs prevent cell apoptosis by binding to cytochrome c or promoting virus replication.
  • the tRNA fragment has a length of ⁇ 200 ribonucleotides, more preferably a length of between 10 and ⁇ 200 ribonucleotides, even more preferably a length of between 10 and 100 ribonucleotides, and still even more preferably a length of between 18 and 50 ribonucleotides, e.g. a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
  • small nucleolar RNA (snorRNA) fragment refers to a fragment derived from a small nucleolar RNA.
  • Small nucleolar RNA molecules are a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNA, transfer RNA, and small nuclear RNAs.
  • snoRNAs There are two main classes of snoRNA, the C/D box snoRNAs and the H/ACA box snoRNAs.
  • the C/D box snoRNAs are associated with methylation.
  • SnoRNAs are commonly referred to as guide RNAs but should not be confused with the guide RNAs that direct RNA editing in trypanosomes.
  • the snorRNA fragment has a length of ⁇ 200 ribonucleotides, more preferably a length of between 10 and ⁇ 200 ribonucleotides, even more preferably a length of between 10 and 100 ribonucleotides, and still even more preferably a length of between 18 and 50 ribonucleotides, e.g. a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
  • disease refers to an abnormal condition that affects the body of an individual.
  • a disease is often construed as a medical condition associated with specific symptoms and signs.
  • the term “disease” is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one’s perspective on life, and one’s personality.
  • One form of a disease is cancer.
  • the present inventors surprisingly found that the methylation status of small non-coding RNA can be used/allows to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • cancer refers to or describes a physiological condition in a patient that is typically characterized by unregulated cell growth. Specifically, abnormal cells grow uncontrolled anywhere in the body of the patient and form a mass of cancer cells. The abnormal cells are termed cancer cells, malignant cells, or tumor cells.
  • cancer as used herein, also encompasses cancer metastases.
  • the cancer is early-stage cancer such as cancer of stage I. It is more preferred that the cancer is early-stage lung cancer such as lung cancer of stage I.
  • the cancer is lung cancer. It is more preferred that the lung cancer is early stage lung cancer such as lung cancer of stage I.
  • early stage cancer refers to cancer that is early in its growth. It has not grown deeply into nearby tissues. In addition, it has not spread to other parts of the body of a patient. No metastasis has been formed. Specifically, early stage cancer is cancer of stage I.
  • the cancer is lung cancer.
  • lung cancer refers to a disease which consists of uncontrolled cell growth in tissues of the lung. This growth may lead to metastasis, which is the invasion of adjacent tissue and infiltration beyond the lungs.
  • metastasis which is the invasion of adjacent tissue and infiltration beyond the lungs.
  • the vast majority of primary lung cancers are carcinomas of the lung, derived from epithelial cells. Lung cancer is the most common cause of cancer-related death in men and women. The most common symptoms are shortness of breath, coughing (including coughing up blood), and weight loss.
  • Stage / means that the cancer is small. It has not grown deeply into nearby tissues. It hasn’t spread to the lymph nodes or other distant organs. Stage I can also be designated as early lung cancer. Stage I can be divided into IA and IB.
  • Stage IA means the cancer is 3 cm or smaller.
  • Stage IB means the cancer is between 3 cm and 4 cm. It might also be growing into structures such as: the main airway of the lung (main bronchus) or the membrane covering the lung (visceral pleura).
  • Stage II means that the cancer is still small. However, it has grown more deeply into nearby tissues and perhaps the lymph nodes, but not into other parts of the body. Stage II can be divided into stage IIA and IIB. Part of the affected lung might have collapsed.
  • Stage IIA means that the cancer is between 4 cm and 5 cm in size but there are no cancer cells in any lymph nodes.
  • Stage IIB means that the cancer is up to 5 cm in size and there are cancer cells in the lymph nodes close to the affected lung. Alternatively, it is between 5 cm and 7 cm but there are no cancer cells in any lymph nodes. Alternatively, the cancer is not in any lymph nodes but has spread into one or more of the following areas: the chest wall (ribs, muscle or skin), the nerve close to the lung (the phrenic nerve), or the layers that cover the heart (mediastinal pleura and parietal pericardium). Alternatively, the cancer is less than 7 cm but there is more than one tumor in the same lobe of the lung.
  • Stage III can be divided into stage III A, IIIB and IIIC. It is sometimes called locally advanced lung cancer.
  • the cancer is up to 5cm in size and has spread to the lymph nodes in the center of the chest on the same side as the tumor.
  • the cancer it is between 5 cm and 7 cm and there is more than one tumor in the same lobe of the lung.
  • the cancer has spread into one or more of the following areas just outsi de the lung: the chest wall (ribs, muscle or skin), the nerve close to the lung (the phrenic nerve), the layers that cover the heart (mediastinal pleura and parietal pericardium), or lymph nodes in the lung or close to the lung.
  • the cancer is larger than 7 cm.
  • lymph nodes It hasn't spread into lymph nodes but has spread into one or more of the following areas: the muscle under the lung (diaphragm), the center area of the chest (mediastinum), the heart, a main blood vessel, the wind pipe (trachea), the nerve that goes to the voice box (larynx), the food pipe (oesophagus), a spinal bone, or the area where the wind pipe divides (the carina).
  • the cancer is in more than one lobe of the same lung and there might also be cancer cells in lymph nodes close to the affected lung.
  • Stage IIIB can also mean different things.
  • the cancer is less than 5 cm and has spread into lymph nodes in one of these places: the opposite side of the chest from the affected lung, the neck, or above the collarbone.
  • the cancer is between 5 cm to 7 cm and has spread into lymph nodes in the center of the chest.
  • the cancer is any size, has spread into lymph nodes in the center of the chest, and has spread into one or more of the following areas: the chest wall, the muscle under the lung (diaphragm), or the layers that cover the heart (mediastinal pleura and parietal pericardium).
  • the cancer has spread into the lymph nodes in the center of the chest.
  • the lung tumor is more than 7 cm or it has spread into a major structure in your chest such as: the heart, the wind pipe (trachea), the food pipe (oesophagus), or a main blood vessel.
  • Stage IIIC means the cancer is between 5c m and 7 cm in size or has spread into one or more of the following: the nerve close to the lung (phrenic nerve) or the covering of the heart (parietal pericardium) and it has spread into lymph nodes: in the center of the chest on the opposite side from the affected lung or at the top of the lung on the same side or opposite side or above the collar bone.
  • the nerve close to the lung phrenic nerve
  • the covering of the heart parietal pericardium
  • lymph nodes in the center of the chest on the opposite side from the affected lung or at the top of the lung on the same side or opposite side or above the collar bone.
  • lymph nodes in the center of the chest on the opposite side from the affected lung or at the top of the lung on the same side or opposite side or above the collar bone.
  • there is more than one tumor in a different lobe of the same lung there is more than one tumor in a different lobe of the same lung.
  • stage IIIC can mean the cancer is bigger than 7 cm or it has spread into one of the following: the muscle under the lung (the diaphragm), the center of the chest (mediastinum), the heart, a major blood vessel, the wind pipe (trachea), the nerve going to the voice box (the recurrent l aryngeal nerve), the food pipe (oesophagus), a spinal bone, or the area where the windpipe di vides (the carina) and it has spread into lymph nodes: in the center of the chest on the opposite side from the affected lung or at the top of the lung on the same side or opposite side or above the collar bone.
  • Stage IV means that the lung cancer has spread. It can be designated as advanced lung cancer. It is divided into stage IVA and IVB.
  • Stage IVA can mean any of the following: there is cancer in both lungs, the cancer is in the covering of the lung (the pl eura) or the covering of the heart (pericardium), or there is fluid around the lungs or the heart that contains cancer cells. Alternatively, it can mean that there is a single area of cancer that has spread outside the chest to a lymph node or to an organ such as the liver or bone.
  • Stage IVB means that the cancer has spread to several areas in one or more organs.
  • cancer of stage II can also be assigned to the category of “early stage cancer”.
  • “early stage cancer” can encompass both cancer of stage I and II.
  • the early stage cancer is lung cancer. More preferably, the early stage cancer is lung cancer of stage I and/or stage II.
  • diagnosing cancer means determining whether a patient shows signs of or suffers from cancer.
  • the cancer is early-stage cancer such as cancer of stage I. It is more preferred that the cancer is early-stage lung cancer such as lung cancer of stage I.
  • the cancer is lung cancer. It is more preferred that the lung cancer is early stage lung cancer such as lung cancer of stage I and/or stage II.
  • patient refers to any subject for whom it is desired to know whether she or he suffers from cancer.
  • patient refers to a subject suspected to be affected by cancer.
  • the patient may be diagnosed to be affected by cancer, i.e. diseased, or may be diagnosed to be not affected by cancer, i.e. healthy.
  • patient refers to a subject which is affected by cancer, i.e. diseased.
  • the subject may be re-tested for cancer and may be diagnosed as still having cancer or as having no cancer anymore.
  • a patient that is diagnosed as being healthy, i.e. not suffering from cancer may possibly suffer from another disease or condition not tested/known.
  • the patient may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey. Human patients specifically males are particularly preferred.
  • control subject refers to a subject known to be not affected by cancer (negative control), i.e. healthy.
  • control subject which is known to be healthy, i.e. not suffering from cancer, may possibly suffer from another disease or condition not tested/known.
  • the (control) subject may be any mammal, including both a human and another mammal, e.g. an animal such as a rabbit, mouse, rat, or monkey.
  • methylated small non-coding RNA refers to RNA which has been post-transcriptionally edited or modified by methylation.
  • the methylation can occur at a base (e.g. methyl-6-adenine, pseudouridine) and/or ribose ring (2'-ortho-methylated nucleotide (2'-O- m)).
  • the methylation of small non-coding RNA occurring at a base is preferably selected from the group consisting of 6-methyl adenosine (m 6 A), 5-methylcytidine (m 5 C), 5-methyluridine (m 5 U), 3 -methyluridine (m 3 U), 1 -methyladenosine (nkA), and 1 -methyl guanosine (m'G), or is a combination thereof.
  • the 2'-O-methylation of the backbone ribose is the most common and conserved type of small non-coding RNA modification.
  • the methylation of small non-coding RNA occurring at a ribose ring is preferably selected from the group consisting of 3 '-end 2'-O-methyladenosine (Am), 2'-O- methyluridine (Um), 2'-O-methylguanosine (Gm), and 2 '-O-methyl cytidine (Cm), or is a combination thereof.
  • the present inventors surprisingly found that the methylation status of small non-coding RNA can be used/allows to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • the present inventors observed that the methylation status of small non-coding RNA in a biological sample varies between patients suffering from cancer and healthy control subjects. Specifically, they found that the methylation status of small non-coding RNA in a biological sample varies between patients suffering from lung cancer, particularly lung cancer of stage I, and healthy control subjects.
  • a lower methylation status of small non-coding RNA in a biological sample obtained from a patient compared to the methylation status of the small non-coding RNA in a biological sample obtained from healthy control subjects is indicative for lung cancer, particularly lung cancer of stage I.
  • the present inventors have exemplarily analyzed herein the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence of 5’ GCCGCCGGUGAAAUACCACUAC 3’ (SEQ ID NO: 16).
  • This rRNA fragment has two methylations sites, namely G (also designated as Gm4020) and C (also designated as Cm4032). While Gm4020 is a variable methylation site, Cm4032 shows a high degree of non-variable methylation.
  • the methylation status of Gm4020 has been calculated/determined herein. A variation of this methylation status (in a 28S ribosomal RNA (rRNA) fragment population) is indicative for the presence of cancer, specifically lung cancer.
  • methylation status refers to the percentage of small non-coding RNA that is methylated in a biological sample. Specifically, the term “methylation status”, as used herein, refers to the percentage of small non-coding RNA molecules that are methylated in a biological sample.
  • the biological sample may be from a patient to be tested or from one or more control subjects.
  • the methylation status of small non-coding RNA (molecules) ranges between 100% (i.e. fully methylated, or all detected RNA entities are fully methylated) and 0% (i.e. not methylated, or all detected RNA entities are not methylated).
  • the methylation status of a population of small non-coding RNA (molecules) ranges between 100% (i.e. fully methylated, or all detected RNA entities in the population are fully methylated) and 0% (i.e. not methylated, or all detected RNA entities in the population are not methylated).
  • the percentage of small non-coding RNA (molecules) that is (are) methylated in a biological sample is 0, 1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • methylation status of 2’-o-m residues on intact full-length ribosomal RNAs in a biological sample usually varies between 40 and 100%.
  • the present inventors have exemplarily shown herein that the afore-mentioned 28 S ribosomal RNA (rRNA) fragment extracted from peripheral blood of a patient suffering from lung cancer has a methylation status which is lower than the methylation status of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer.
  • a methylation status of the afore-mentioned 28 S ribosomal RNA (rRNA) fragment extracted from peripheral blood of a patient which is lower than the methylation status of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer is indicative for lung cancer in the patient and, thus, allows the diagnosis of lung cancer in said patient.
  • the methylation status of Gm4020 of the afore-mentioned 28 S ribosomal RNA (rRNA) has been calculated/determined herein. Accordingly, the methylation status of Gm4020 of the afore-mentioned 28 S ribosomal RNA (rRNA) extracted from peripheral blood of a patient which is lower than the methylation status of Gm4020 of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer, is indicative for lung cancer in the patient and, thus, allows the diagnosis of lung cancer in said patient.
  • nucleotide refers to an organic molecule consisting of a nucleoside and a phosphate.
  • a nucleotide is composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates.
  • the four nucleobases in DNA are guanine, adenine, cytosine and thymine; in RNA, uracil is used in place of thymine.
  • the nucleotide serves as monomeric unit of nucleic acid polymers, such as deoxyribonucleotide acid (DNA) or ribonucleotide acid (RNA).
  • DNA deoxyribonucleotide acid
  • RNA ribonucleotide acid
  • the nucleotide is a molecular building-block of DNA and RNA.
  • nucleoside refers to a glycosylamine that can be thought of as nucleotide without a phosphate group.
  • a nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or 2'-deoxyribose) whereas a nucleotide is composed of a nucleobase, a five-carbon sugar, and one or more phosphate groups.
  • the anomeric carbon is linked through a glycosidic bond to the N9 of a purine or the N1 of a pyrimindine.
  • nucleotide sequence or “polynucleotide” are interchangeably used herein and refer to single-stranded and double-stranded polymers of nucleotide monomers, including without limitation, 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, or internucleotide analogs, and associated counter ions, e.g., H+, NH4+, trialkylammonium, Mg2+, Na+, and the like.
  • DNA 2'-deoxyribonucleotides
  • RNA ribonucleotides linked by internucleotide phosphodiester bond linkages
  • counter ions e.g., H+, NH4+, trialkylammonium, Mg2+, Na+, and the like.
  • a nucleotide sequence or polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof and may include nucleotide analogs.
  • the nucleotide monomer units may comprise any of the nucleotides described herein, including, but not limited to, nucleotides and/or nucleotide analogs.
  • analog includes synthetic analogs having modified base moieties, modified sugar moieties, and/or modified phosphate ester moieties.
  • Phosphate analogs generally comprise analogs of phosphate wherein the phosphorous atom is in the +5 oxidation state and one or more of the oxygen atoms is replaced with a non-oxygen moiety, e.g. sulfur.
  • Exemplary phosphate analogs include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, boronophosphates, including associated counterions, e.g.
  • Exemplary base analogs include: 2,6-diaminopurine, hypoxanthine, pseudouridine, C-5-propyne, isocytosine, isoguanine, 2-thiopyrimidine.
  • Exemplary sugar analogs include: 2’- or 3’- modifications where the 2’- or 3 ’-position is hydrogen, hydroxy, alkoxy, e.g., methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy, azido, amino or alkylamino, fluoro, chloro, and bromo.
  • modified ribonucleotides are present in/part of the adapters described herein. In one preferred embodiment, the modified ribonucleotides are 2’-o-methyl ribonucleotides.
  • a combination of a 5 ’adapter and 3 ’adapter is used for determining the methylation status of small non-coding RNA (in a biological sample) to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • the term “adapter” refers to a polynucleotide that can be ligated to the 5 ’end of small non-coding RNA (i.e. “5 ’adapter”) or to the 3 ’end of small non-coding RNA (i.e. “3’adapter”).
  • the nucleotides of the 5’adapter and the 3’adapter may be standard or natural (i.e. adenosine, guanosine, cytidine, thymidine, and uridine) as well as nonstandard nucleotides.
  • Non-limiting examples of non-standard nucleotides include inosine, xanthosine, isoguanosine, isocytidine, diaminopyrimidine and deoxyuridine.
  • the adapters may comprise modified or derivatized nucleotides.
  • modifications in the ribose or base moieties include the addition, or removal, of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups and thiol groups.
  • included are 2’-0-methyl and locked nucleic acids (LNA) nucleotides.
  • Suitable examples of derivatized nucleotides include those with covalently attached dyes, such as fluorescent dyes or quenching dyes, or other molecules such as biotin, digoxygenin, or magnetic particles or microspheres.
  • the adapters may also comprise synthetic nucleotide analogs such as morpholinos or peptide nucleic acids (PNA). Phosphodiester bonds or phosphothioate bonds may link the nucleotides or nucleotide analogs of the linkers.
  • the length of the 5’ and 3 ’adapter can vary depending upon, for example, the desired length of the ligation product and the desired features of the adapter.
  • the 5 ’adapter or 3 ’adapter may range from 15 to 60, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, nucleotides in length.
  • the 5’adapter as described herein comprises a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, (random) deoxynucleotides, wherein said 6 to 15 (random) deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA.
  • the 3 ’adapter as described herein comprises a 3 ’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, (random) deoxynucleotides, wherein said 6 to 15 (random) deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA.
  • the 5’adapter and the 3’adapter can be present as linear polynucleotide, e.g. after denaturation/when denatured.
  • the 5’adapter and the 3’adapter is single-stranded.
  • This primary structure may be converted into a secondary structure.
  • the 5’adapter and the 3’adapter is further capable of forming a stem-loop structure.
  • the 5’adapter and the 3’adapter can also have a stem-loop structure, e.g. after re-naturation/when re-natured.
  • stem-loop structure refers to a pattern that can occur in single-stranded RNA.
  • the structure is also known as a “hairpin” or “hairpin loop”. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop.
  • the 5’adapter and the 3’adapter that is capable of forming a stem-loop structure comprises a 5 ’positioned first stem sequence and a 3 ’positioned second stem sequence that are reverse complementary to each other.
  • the first stem sequence and the second stem sequence form the “double-stranded region” or “double-stranded stem” of the stem-loop adaptor.
  • the stem is between 5 and 20, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, nucleotides in length.
  • the stem is between 5 and 10, e.g. 5, 6, 7, 8, 9, or 10, nucleotides in length.
  • a portion of a primer may be encoded in the stem.
  • the stem may be longer.
  • the stem may be shorter.
  • the term “loop” refers to the single-stranded region of the stem-loop structure.
  • the loop is located between the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence.
  • the loop is located between the two reverse complementary strands of the stem and typically the loop comprises single-stranded nucleotides, although other moieties such as modified DNA or RNA molecules are also possible.
  • the loop sequence comprises between 10 and 40 nucleotides, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.
  • the loop sequence comprises between 12 and 20 nucleotides, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.
  • the nucleotides of the loop structure are preferably deoxynucleotides but also ribonucleotides are possible.
  • a portion of a primer may be encoded in the loop.
  • the loop may be longer. In those embodiments in which a primer is not encoded in the loop, the loop may be shorter.
  • the 5 ’adapter as described herein comprises a 5 ’-terminal sequence that is configured such that it forms a single stranded 5’protrusion after formation of the stem-loop structure.
  • the 3 ’adapter as described herein comprises a 3 ’-terminal sequence that is configured such that it forms a single stranded 3 ’protrusion after formation of the stem-loop structure.
  • the adapter e.g. 5’adapter and/or 3 ’adapter, may comprise one or more blocking nucleotides.
  • blocking nucleotide refers to a nucleotide comprising a chemical moiety which prevents or minimizes nucleotide addition by a DNA polymerase. For example, by adding a blocking group to the terminal 3 ’-OH, the nucleotide is no longer able to participate in phosphodiester bond formation catalyzed by the DNA polymerase.
  • Some non-limiting examples include, an alkyl group, non-nucleotide linkers, phosphorothioate, alkane-diol residues, PNA, LNA, nucleotide analogs comprising a 3 ’-amino group in place of the 3 ’-OH group, nucleotide analogs comprising a 5‘-OH group in place of the 5 ’-phosphate group, nucleotide derivatives lacking a 3 ’-OH group, or biotin. These nucleotides are generally not chain extendable. Other examples of non-extendable nucleotides that can be used include nucleotides that have modified ribose moieties.
  • ribonucleotides may serve as non-extendable nucleotides because oligonucleotides terminating in ribonucleotides cannot be extended by certain DNA polymerases.
  • the ribose can be modified to include 3 '-deoxy derivatives including those in which the 3 '-hydroxy is replaced by a functional group other than hydrogen, for example, as an azide group.
  • a non-extendible nucleotide comprises a dideoxynucleotide (ddN), for example but not limited to, a dideoxyadenosine (ddA), a dideoxycytosine (ddC), a dideoxyguanosine (ddG), a dideoxythymidine (ddT), or a dideoxyuridine (ddU).
  • ddN dideoxynucleotide
  • ddA dideoxyadenosine
  • ddC dideoxycytosine
  • ddG dideoxyguanosine
  • ddT dideoxythymidine
  • ddU dideoxyuridine
  • the adapter e.g. 5’adapter and/or 3’adapter, may comprise locked nucleic acids (LNAs).
  • LNAs locked nucleic acids
  • LNAs locked nucleic acids
  • These nucleic acid analogs are also referred to in some circles as “inaccessible ribonucleotides”.
  • LNA nucleotides can be mixed with DNA or RNA residues in the polynucleotide, in effect hybridizing with DNA or RNA according to Watson-Crick base-pairing rules.
  • the inflexible nature of these molecules greatly enhances hybridization stability.
  • polynucleotides containing LNAs offer tremendous discriminatory power, allowing these molecules to distinguish between exact match and mismatched complementary target sequences with very little difficulty.
  • the 5’adapter and/or the 3’adapter comprise(s) locked nucleotides, in particular ribonucleotides.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence of the 5’adapter is (are) LNA enhanced.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence of the 3’adapter is (are) LNA enhanced.
  • the 3’adapter may comprise a 3 ’inverted deoxynucleotide.
  • inverted deoxynucleotide refers to a deoxynucleotide creating a 3 ’-3’ linkage and, thus, prevents undesired nucleotide synthesis from the 3 ’end of the adapter, e.g. during reverse transcriptase (RT) PCR.
  • the 3 ’inverted deoxynucleotide protects the sequence from 3’ exonuclease cleavage.
  • the 3’adapter comprises a 3’inverted deoxynucleotide.
  • the 3’adapter comprises a 3’inverted deoxynucleotide, wherein the deoxynucleotide is inverted dT, dA, dC, or dG.
  • the 5’adapter may further comprise in the loop a base lacking spacer, specifically at the 5’-end of the loop.
  • base lacking spacer refers to a moiety allowing the termination of the reverse transcription in a subsequent step.
  • the reaction terminates at the nucleotide preceding the base lacking spacer in the loop region of the 5’adapter, which prevents the reaction from continuing to the end of the 5’adapter and, thus, generating highly structured cDNAs, which may impair subsequent PCR steps.
  • the base lacking spacer is a 2 ’-dideoxyribose spacer. More particularly, the base lacking spacer is a 1’2’ -dideoxyribose spacer.
  • the 5 ’adapter comprises in the loop a base lacking spacer, preferably at the 5’-end of the loop. In one preferred embodiment, the 5’adapter comprises in the loop a 2’- dideoxyribose spacer, preferably at the 5’-end of the loop.
  • a 5’adaper wherein the 5 ’positioned first stem sequence and/or the 3’positioned second stem sequence of the 5’adapter is (are) LNA enhanced and a 3 ’adapter, wherein the 5 ’positioned first stem sequence and/or the 3’positioned second stem sequence of the 3 ’adapter is (are) LNA enhanced are combined/part of a combination.
  • a 5’adaper wherein the 5 ’positioned first stem sequence and/or the 3’positioned second stem sequence of the 5’adapter is (are) LNA enhanced and a 3 ’adapter, wherein the 5 ’positioned first stem sequence and/or the 3’positioned second stem sequence of the 3 ’adapter is (are) LNA enhanced and wherein the 3 ’adapter comprises a 3 ’inverted deoxynucleotide, e.g. inverted dT, dA, dC, or dG, are combined/part of a combination.
  • the present inventors surprisingly found that the methylation status of small non-coding RNA can be used/allows to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • the present inventors provide a method for diagnosing cancer in a patient or for determining whether a patient suffers from cancer.
  • the methylation status of small non-coding RNA in a biological sample obtained/isolated from a patient is determined.
  • Said method encompasses the step of annealing and ligating adapters to small non-coding RNA present in the biological sample obtained/isolated from a patient.
  • the adapters in particular 5’ and 3 ’adapters, are annealed to the small non-coding RNA.
  • the small non-coding RNA is denatured.
  • the adapters, in particular 5’ and 3 ’adapters are denatured and subsequently renatured.
  • annealing refers to a process of heating and cooling two single-stranded polynucleotides with complementary sequences. Heat breaks all hydrogen bonds and cooling allows new bonds to form between the sequences.
  • the adapters in particular 5’ and 3 ’adapters, attach to the small non-coding RNA, and form their characteristic stem-loop structure.
  • the 5’adapter attaches to the 5 ’end of the small non-coding RNA and the 3 ’adapter attaches to the 3 ’end of the small non-coding RNA.
  • the denaturation/renaturation of the adapters, in particular 5’ and 3 ’adapters preferably takes place separately and in the absence of the small non-coding RNA.
  • the adapters, in particular 5’ and 3 ’adapters are then ligated to the small noncoding RNA, using/with a double stranded RNA ligase, thereby producing a ligation product.
  • the term “ligation product” refers to a (DNA/RNA) hybrid molecule comprising at least one adapter and small non-coding RNA.
  • the ligation product may comprise a 5 ’adapter and small non-coding RNA.
  • the ligation product may comprise a 3 ’adapter and small non-coding RNA.
  • the ligation produced may comprise a 5 ’adapter, a 3 ’adapter and small non-coding RNA.
  • the annealing of the 5 ’adapter with the small non-coding RNA generates a double-stranded (DNA/RNA) hybrid containing a nick of RNA-OH-375’-P-RNA between the 3 ’end of the adapter and the 5 ’end of the small non-coding RNA.
  • This is an efficient substrate for ligation by a double-stranded RNA ligase.
  • the annealing of the 3’adpater with the small non-coding RNA generates a double-stranded (DNA/RNA) hybrid containing a nick of RNA-OH-375’-P-RNA between the 3 ’end of the small non-coding RNA, and the 5 ’end of the adapter.
  • the double stranded RNA ligase is a T4 RNA ligase 2 (Rnl2), a Kodl ligase or RtcB ligase.
  • the double stranded RNA ligase is a T4 RNA ligase 2 (Rnl2).
  • a buffering agent may be used to adjust and maintain the pH at the desired level.
  • suitable buffers include, but are not limited to, MOPS, HEPES, TAPS, Bicine, Tricine, TES, PIPES, MES, sodium acetate and Tris buffer.
  • extension reaction refers to an elongation reaction in which the 3 ’adapter ligated to the 3 ’end of the small non-coding RNA is extended, in particular in 5’ to 3’ direction, to form an “extension reaction product” comprising a strand reverse complementary to the small non-coding RNA.
  • the extension reaction can also be designated as “reverse transcription”.
  • the small non-coding RNA is a miRNA
  • the extension reaction is a reverse transcription reaction comprising a reverse transcriptase, whereby a DNA (in particular cDNA) copy of the ligation product is made.
  • the extension reaction is a reverse transcription reaction comprising a polymerase, such as a reverse transcriptase.
  • reverse transcriptase refers to any enzyme having reverse transcriptase activity.
  • reverse transcriptase refers to an enzyme used to generate DNA (cDNA) from an RNA template in a process termed reverse transcription.
  • the reverse transcriptase has an RNA-dependent DNA polymerase activity. By means of this activity, a hybrid double strand of RNA and DNA is first built up after presentation of a single-stranded RNA by linking complementary paired DNA building blocks (deoxyribonucleotides). Afterwards, its RNA portion is largely degraded by means of an RNase H activity of a special section of the protein.
  • the reverse transcriptase To initiate reverse transcription, the reverse transcriptase requires a primer which serves as a starting point for the reverse transcriptase to synthesize a new strand. This primer is also called RT-primer sequence.
  • the RT-primer depends on the 3’ adapter sequence.
  • the RT-primer is reverse complementary to said sequence.
  • the reverse transcriptase (RT) is Maxima H-RT, Tth polymerase, Protoscript II RT, Luna RT, AMV, or M-MuLV, or any other enzymes derived from these.
  • the reverse transcriptase is Maxima H-RT or Luna RT.
  • the reverse transcription of the ligation product is conducted under limiting conditions (test reaction) as well as under nonlimiting conditions (control reaction).
  • the reverse transcription of the ligation product “under limiting conditions” means performing the reverse transcription (RT) with a desoxynucleosidetriphosphate (dNTP) concentration which is lower than “under non-limiting (normal) conditions”.
  • said reverse transcription of the ligation product is conducted with a desoxynucleosidetriphosphate (dNTP) concentration which is 1/10 or less, e.g. 1/10, 1/11, 1/12, 1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20, 1/21, 1/22, 1/23, 1/24, 1/25, 1/26, 1/27, 1/28, 1/29,
  • dNTP desoxynucleosidetriphosphate
  • said reverse transcription of the ligation product is conducted with a dNTP concentration which is between 1/10 and 1/50, e.g. 1/10, 1/11, 1/12, 1/13, 1/14, 1/15, 1/16, 1/17,
  • said reverse transcription of the ligation product is conducted with between 0.25 mM and 50 mM dNTPs, e.g. with 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mM dNTPs. Still even more preferably, said reverse transcription of the ligation product is conducted with between 0.25 mM and 25 mM dNTPs, e.g. with 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM dNTPs.
  • said reverse transcription of the ligation product is conducted with 1 mM dNTPs.
  • the reverse transcription of the ligation product “under non-limiting (normal) conditions” is preferably conducted with between 2.5 mM and 500 mM dNTPs, more preferably with between 2.5 mM and 250 mM dNTPs, and even more preferably with 10 mM dNTPs.
  • test reaction is specifically performed with 1 mM dNTPs and the reverse transcription reaction under non-limiting (normal) conditions (control reaction) is specifically performed with 10 mM dNTPs.
  • the reverse transcriptase is preferably Maxima RT or Luna RT.
  • methylated nucleotides such as 2'-O-methylated nucleotides induce reverse transcription stops/pauses at low concentrations of desoxynucleosidetriphosphates (dNTPs) (i.e. under limiting conditions), while at high dNTP concentrations (i.e. under nonlimiting conditions) reverse transcriptase can bypass methylated sites such as 2'-O-methylated sites.
  • dNTPs desoxynucleosidetriphosphates
  • reverse transcriptase can bypass methylated sites such as 2'-O-methylated sites.
  • the methylated group such as 2'-O-methyl group acts as a conformational “bump” which hinders the passage of the reverse transcriptase, whose effect is minimized at high dNTP concentrations (i.e. under non-limiting conditions).
  • a difference between the cDNA products produced via reverse transcription under limiting conditions (i.e. at low concentrations of dNTPs) and the cDNA products produced via reverse transcription under non-limiting conditions (i.e. high concentrations of dNTPs) indicates small non-coding RNA methylation in the biological sample.
  • the cDNA product produced with the reverse transcription under limiting conditions i.e. at low dNTP concentrations
  • first cDNA product the cDNA product produced with said reverse transcription under nonlimiting conditions (i.e. at high dNTP concentrations)
  • second cDNA product the cDNA product produced with said reverse transcription under nonlimiting conditions (i.e. at high dNTP concentrations) is designated as second cDNA product.
  • the cDNA product (and consequently the small noncoding RNA based thereon) is further detected.
  • PCR polymerase chain reaction
  • the PCR may be selected from the group consisting of digital PCR, real-time PCR (quantitative PCR or qPCR), preferably Taq-man qPCR, multiplex PCR, nested PCR, high fidelity PR, fast PCR, hot start PCR, and GC-rich PCR.
  • the digital PCR is digital droplet PCR or digital partition PCR.
  • amplicon and amplification product generally refer to the product of an amplification reaction.
  • An amplicon may be double-stranded or single-stranded, and may include the separated component strands obtained by denaturing a double-stranded amplification product.
  • the amplicon of one amplification cycle can serve as a template in a subsequent amplification cycle.
  • amplifying refers to any means by which at least a part of the small non-coding RNA, small non-coding RNA surrogate, or combinations thereof, is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Any of several methods can be used to amplify the target polynucleotide. Any in vitro means for multiplying the copies of a target sequence of nucleic acid can be utilized. These include linear, logarithmic, or other amplification methods.
  • Exemplary methods include polymerase chain reaction (PCR), isothermal procedures (using one or more RNA polymerases, strand displacement, partial destruction of primer molecules, ligase chain reaction (LCR), Q RNA replicase systems, RNA transcription- based systems (e.g., TAS, 3 SR), or rolling circle amplification (RCA).
  • PCR polymerase chain reaction
  • isothermal procedures using one or more RNA polymerases, strand displacement, partial destruction of primer molecules
  • LCR ligase chain reaction
  • Q RNA replicase systems e.g., TAS, 3 SR
  • RCA rolling circle amplification
  • a difference between the cDNA products produced via reverse transcription under limiting conditions (i.e. at low concentrations of dNTPs) and the cDNA products produced via reverse transcription under non-limiting conditions (i.e. high concentrations of dNTPs) indicates small non-coding RNA methylation in the biological sample.
  • the small non-coding RNA is not methylated.
  • a significant difference in this respect preferably means that the experiment is conducted three times and that in all three experiments, a difference could be detected.
  • the difference may reside in different levels of the cDNA products.
  • level refers to an amount (measured for example in grams, mole, or ion counts) or concentration (e.g. absolute or relative concentration, e.g. reads per million (RPM), NGS counts, copies per pl, or cycle thresholds) of the small non-coding RNA or cDNA product derived therefrom.
  • concentration e.g. absolute or relative concentration, e.g. reads per million (RPM), NGS counts, copies per pl, or cycle thresholds
  • level also comprises scaled, normalized, or scaled and normalized amounts or values (e.g. RPM).
  • the level of the small non-coding RNA or cDNA product derived therefrom is determined by sequencing, preferably next generation sequencing (e.g.
  • the PCR is selected from the group consisting of digital PCR, real-time PCR (quantitative PCR or qPCR), preferably TaqMan qPCR, multiplex PCR, nested PCR, high fidelity PR, fast PCR, hot start PCR, and GC-rich PCR.
  • the digital PCR may be digital droplet PCR or digital partition PCR.
  • the difference between the cDNA products produced via reverse transcription is based on a difference in expression levels determined by
  • the reference methylation status is the methylation status of the small non-coding RNA determined empirically by measuring a number of reference biological samples from healthy subjects/ subjects known to not suffer from cancer.
  • methylation status (1- (copies first cDNA product per pl at a dNTP concentration under limiting conditions / copies second cDNA product per pl at a dNTP concentration under non-limiting conditions)) * 100
  • reference methylation status (1- (copies first cDNA product per pl at a dNTP concentration under limiting conditions / copies second cDNA product per pl at a dNTP concentration under non-limiting conditions)) * 100.
  • the dNTP concentration under limiting conditions is 1/10 or less, e.g. 1/10, 1/11, 1/12, 1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20, 1/21, 1/22, 1/23, 1/24, 1/25, 1/26, 1/27, 1/28, 1/29, 1/30, 1/31, 1/32, 1/33, 1/34, 1/35, 1/36, 1/37, 1/38, 1/39, 1/40, 1/41, 1/42, 1/43, 1/44, 1/45, 1/46, 1/47, 1/48, 1/49, 1/50, or less, of the dNTP concentration under non-limiting (normal) conditions.
  • a methylation status which is lower than the reference methylation status indicates that the patient suffers from cancer.
  • Dumbbell PCR refers to an efficient and convenient method to distinctively quantify specific individual small RNA such as miRNA as well as specific individual small RNA variants such as isomiRs.
  • DB-PCR Downlink PCR
  • 5’- and 3’ adapters are specifically hybridized and ligated to the 5’- and 3 ’-ends of target small non-coding RNAs, respectively, by a double stranded RNA ligase, e.g. T4 RNA ligase 2 (Rnl2).
  • the resultant ligation products with ‘dumbbell-like’ structures are subsequently quantified, e.g. by TaqMan RT-PCR.
  • the present inventors found that the use of the proprietary 5’ and 3 ’adapters as described herein as well as high specificity of Rnl2 ligation and TaqMan RT-PCR toward target small non-coding RNAs assured both 5’- and 3 ’-terminal sequences of target small non-coding RNAs with single nucleotide resolution so that Db-PCR specifically detected target small non-coding RNAs but not their corresponding terminal variants.
  • Db-PCR described herein has broad applicability for the quantification of various small RNAs in different cell types. Therefore, Db-PCR provides a much- needed simple method for analyzing RNA terminal heterogeneity.
  • Residues in two or more polynucleotides are said to “correspond” to each other if the residues occupy an analogous position in the polynucleotide structures. It is well known in the art that analogous positions in two or more polynucleotides can be determined by aligning the polynucleotide sequences based on nucleic acid sequence or structural similarities. Such alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, for example, ClustalW or Align using standard settings, preferably for Align EMBOSS: rneedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • sample refers to any sample comprising small non-coding RNA.
  • Said sample is specifically derived from the body of a subject.
  • Said sample specifically comprises small non-coding RNA isolated from organisms, tissues, cells, or body fluids such as blood.
  • the sample is particularly a biological sample.
  • the sample may also be a processed sample which is originated from a biological sample. In other words, the sample may also be a processed sample which has its origin in a biological sample.
  • biological sample refers to any sample having a biological origin and/or comprising biological material.
  • the biological sample may also be a processed sample with biological origin.
  • the biological sample may be a body fluid sample, e.g. a blood sample or urine sample, or a tissue sample, e.g. a tissue biopsy sample.
  • Biological samples may be mixed or pooled, e.g. a sample may be a mixture of a blood sample and a urine sample.
  • body fluid sample refers to any liquid sample comprising small non-coding RNA. Said sample is specifically derived from the body of a patient/subject.
  • Said body fluid sample may be a urine sample, blood sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, gastric juice sample, mucus sample, lymph sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof.
  • body fluid sample also encompasses body fluid fractions, e.g. blood fractions, urine fractions or sputum fractions. Body fluid samples may be mixed or pooled.
  • a body fluid sample may be a mixture of a blood and a urine sample or a mixture of a blood and cerebrospinal fluid sample.
  • blood sample encompasses whole blood or a blood fraction.
  • the blood fraction is selected from the group consisting of a blood cell fraction, plasma, and serum.
  • the blood fraction is selected from the group consisting of a blood cell fraction and plasma or serum.
  • the blood cell fraction encompasses erythrocytes, leukocytes, and/or thrombocytes.
  • the whole blood sample may be collected by means of a blood collection tube. It is, for example, collected in a PAXgene Blood RNA tube, in a Tempus Blood RNA tube, in an EDTA-tube, in a Na-citrate tube, Heparin-tube, or in an ACD-tube (Acid citrate dextrose).
  • a blood collection tube It is, for example, collected in a PAXgene Blood RNA tube, in a Tempus Blood RNA tube, in an EDTA-tube, in a Na-citrate tube, Heparin-tube, or in an ACD-tube (Acid citrate dextrose).
  • the whole blood sample may also be collected by means of a bloodspot technique, e.g. using a Mitra Microsampling Device.
  • This technique requires smaller sample volumes, typically 45-60 pl for humans or less.
  • the whole blood may be extracted from the patient via a finger prick with a needle or lancet.
  • the whole blood sample may have the form of a blood drop.
  • Said blood drop is then placed on an absorbent probe, e.g. a hydrophilic polymeric material such as cellulose, which is capable of absorbing the whole blood.
  • an absorbent probe e.g. a hydrophilic polymeric material such as cellulose, which is capable of absorbing the whole blood.
  • the blood spot is dried in air before transferring or mailing to labs for processing. Because the blood is dried, it is not considered hazardous. Thus, no special precautions need be taken in handling or shipping.
  • the desired components e.g. miRNAs, are extracted from the dried blood spots into a supernatant which is then further analyzed.
  • DB PCR Dumbbell PCR
  • RT reverse transcription
  • the present inventors have now found that the determination of the methylation status of small non-coding RNA allows to discriminate biological samples of cancer patients from biological samples of non-cancer, i.e. healthy, control subjects. Specifically, the present inventors have now found that the determination of the methylation status of small non-coding RNA extracted from a biological sample such as peripheral blood allows to diagnose cancer such as early-stage cancer in a patient.
  • the present invention relates to the (in vitro/ex vivo) use of a methylation status of small non-coding RNA (in a biological sample) to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • the first aspect may alternatively be formulated as follows: (In vitro! Ex vivo) use of a methylation status of a small non-coding RNA population (in a biological sample) to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • a 3 ’adapter capable of forming a stem-loop structure containing a loop and a double stranded stem for determining the methylation status of small non-coding RNA to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • the small non-coding RNA has a length of ⁇ 200 ribonucleotides, more preferably a length of between 10 and ⁇ 200 ribonucleotides, even more preferably a length of between 10 and 100 ribonucleotides, and still even more preferably a length of between 18 and 50 ribonucleotides, e.g.
  • the (above-mentioned) small non-coding RNA is a ribosomal RNA fragment (rRNA fragment), a miRNA, a miRNA isoform (an isomiR), a transfer RNA fragment (tRF), or a small nucleolar RNA (snorRNA) fragment.
  • rRNA fragment is particularly favoured.
  • the 5’adapter comprises in the following order from 5’ to 3’ :
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g. 2, 3, or 4, nucleotides at its 3 ’end are ribonucleotides or modified ribonucleotides and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced.
  • the LNA enhanced sequence comprises between 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 3, locked nucleotides, specifically ribonucleotides.
  • nucleotide sequence of the 5’ adapter comprises deoxynucleotides and ribonucleotides.
  • the 5’adapter may range from 15 to 60, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, nucleotides in length.
  • the 5’adapter may be present as linear polynucleotide, particularly in single- stranded form, e.g. after denaturation/when denatured.
  • the 5’adapter is a polynucleotide that can be attached/ligated to the 5 ’end of small non-coding RNA.
  • the 5’adapter has a stem-loop structure.
  • the attachment/ligation is possible as the 5’adapter comprises between 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to a 5’-terminal sequence of small non-coding RNA.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a 5’positioned first stem sequence and a 3’positioned second stem sequence that are reverse complementary to each other.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence can form the double stranded stem.
  • the double stranded stem may have a length of between 5 and 20, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, nucleotides.
  • each one of the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence has a length of between 5 to 10, e.g. 5, 6, 7, 8, 9, or 10, nucleotides.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence have the same length, e.g. a length of 5, 6, 7, 8, 9, or 10 nucleotides.
  • the 5 ’positioned first stem sequence and/or the 3 ’positioned second stem sequence is (are) LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides. Examples of locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • the 5’positioned first stem sequence is LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides.
  • locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • every, every second, or every third nucleotide may be LNA enhanced in the 5’positioned first stem sequence and/or the 3’positioned second stem sequence.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise (a mixture of) deoxynucleotides and ribonucleotides (e.g. LNA-enhanced) or the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise ribonucleotides.
  • Said ribonucleotides include ribonucleotides which are LNA-enhanced.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a loop sequence which is located between the 5’positioned first stem sequence and the 3’positioned second stem sequence.
  • the loop sequence may comprise between 10 and 40, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, nucleotides.
  • the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, nucleotides, e.g. deoxynucleotides and/or ribonucleotides. More preferably, the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, deoxynucleotides.
  • the nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem comprises deoxynucleotides with the exception of the at least two nucleotides at its 3 ’end which are ribonucleotides or modified ribonucleotides, preferably 2’-o-methyl ribonucleotides, and the locked ribonucleotides.
  • the 5 ’-terminal sequence is configured such that it forms a single stranded 5 ’protrusion after formation of the stem-loop structure.
  • the 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to the 5 ’-terminal sequence of small noncoding RNA encompass G and C but not more than 4, e.g. 1, 2, 3, or 4, in a row.
  • a preferred 5 ’adapter comprises in the following order from 5’ to 3’:
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g. 2, 3, or 4, nucleotides at its 3 ’end are ribonucleotides or modified ribonucleotides and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced.
  • said 6 to 15 deoxynucleotides in (i) which are reverse complementary to a 5’-terminal sequence of small non-coding RNA comprise one or more locked nucleotide- (LNA-) enhanced and/or other modified nucleotide(s).
  • LNA- locked nucleotide-
  • the above described 5 ’adapter comprises in the following order from 5’ to 3’:
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are LNA-enhanced, or
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are otherwise modified (than LNA-enhanced), or
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are LNA-enhanced and otherwise modified, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g. 2, 3, or 4, nucleotides at its 3 ’end are ribonucleotides or modified ribonucleotides.
  • the above described 5 ’adapter comprises in the following order from 5’ to 3’ : (ia) a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are LNA-enhanced, or
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are otherwise modified (than LNA-enhanced), or
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are LNA-enhanced and otherwise modified, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g. 2, 3, or 4, nucleotides at its 3 ’end are ribonucleotides or modified ribonucleotide and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced.
  • the above-mentioned one or more otherwise modified (than LNA-enhanced) nucleotides are preferably 2 ’-ortho-methylated ribonucleotides.
  • the above described 5 ’adapter comprises in the following order from 5’ to 3’ :
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are LNA-enhanced, or
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are replaced by a 2 ’-ortho-methylated ribonucleotide, or
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are LNA-enhanced and replaced by a 2’-ortho-methylated ribonucleotide, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g. 2, 3, or 4, nucleotides at its 3 ’end are ribonucleotides or modified ribonucleotides.
  • the above described 5’adapter comprises in the following order from 5’ to 3’ :
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are LNA-enhanced, or
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are are replaced by a 2’-ortho-methylated ribonucleotide, or
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA and wherein one or more of them are LNA-enhanced and replaced by a 2’-ortho-methylated ribonucleotide, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g. 2, 3, or 4, nucleotides at its 3 ’end are ribonucleotides or modified ribonucleotide and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced.
  • the one or more 2 ’-ortho-methylated ribonucleotides are located at a position (in said nucleotide sequence) corresponding to a position in the non-coding small RNA (molecule) that is presumed to be methylated.
  • the one or more 2 ’-ortho-methylated ribonucleotides are located at a position (in said nucleotide sequence) base-pairing with a position in the noncoding small RNA (molecule) that is presumed to be methylated.
  • the 5’adapter has the following sequence from 5’ to 3’ :
  • every, every second, or every third nucleotide may be LNA enhanced in the underlined portion and/or in the double underlined portion specified above.
  • the 5’adapter has the following sequence from 5’ to 3’ : (6-15x)NCGTGGCGTGGAGTGTGTGCTTTGCCArCrG (SEQ ID NO: 1), wherein “r” stands for ribonucleotide, wherein “(6-15x)N” designates the sequence reverse complementary to a 5 ’terminal sequence of small non-coding RNA, and wherein one or more (e.g. 1, 2, or 3) of the nucleotides in bold letters are LNA enhanced, or is a variant of this sequence.
  • the LNA enhanced nucleotides are ribonucleotides.
  • the 5 ’adapter variant as described above has a sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, and still even more preferably 99%, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the sequence according to SEQ ID NO: 1.
  • Such a 5 ’adapter variant still comprises the at least 2 nucleotides at its 3 ’end which are ribonucleotides or modified ribonucleotides.
  • such a 5’ adapter variant is still LNA enhanced (if this is not an optional feature).
  • such a 5 ’adapter variant is still capable of forming a stem-loop structure containing a loop and a double stranded stem.
  • the skilled person can readily assess whether a 5 ’adapter variant is still capable of forming a stem-loop structure containing a loop and a double stranded stem. For example, the experimental section provides sufficient information in this respect.
  • the 5 ’adapter as described above comprises a base-lacking spacer (e.g. a base-lacking 1’, 2 ’-dideoxyribose spacer) in the loop region.
  • a 5’adapter having a base-lacking spacer in the loop region has preferably the following sequence from 5’ to 3’ : (6- 15x)NCGTGGCG/idSp/TGGAGTGTGTGCTTTGCC ArCrG (SEQ ID NO: 6), wherein “r” stands for ribonucleotide, wherein “(6-15x)N” designates the sequence reverse complementary to a 5 ’terminal sequence of small non-coding RNA, wherein “idSp” stands for base lacking spacer, and wherein one or more nucleotides in the underlined portion and/or one or more nucleotides in the double underlined portion are optionally LNA enhanced, or is a variant of this sequence.
  • the LNA enhanced nucleotides are ribonucleotides.
  • the 5’adapter comprising a base-lacking spacer e.g. a base-lacking 1’, 2’-dideoxyribose spacer
  • a base-lacking spacer e.g. a base-lacking 1’, 2’-dideoxyribose spacer
  • the 5’adapter comprising a base-lacking spacer (e.g. a base-lacking 1’, 2’-dideoxyribose spacer) in the loop region has the following sequence from 5’ to 3’ : (6-15x)NCGTGGCG/idSp/TGGAGTGTGTGCTTTGCCArCrG (SEQ ID NO: 6), wherein “r” stands for ribonucleotide, wherein “(6-15x)N” designates the sequence reverse complementary to a 5 ’terminal sequence of small non-coding RNA, wherein “idSp” stands for base lacking spacer, and wherein one or more (e.g.
  • the 5 ’adapter having a base-lacking spacer has preferably the following sequence from 5’ to 3’ : (6- 15x)NCG/idSp/TGGCGTGGAGTGTGTGCTTTGCC ArCrG (SEQ ID NO: 12), wherein “r” stands for ribonucleotide, wherein “(6-15x)N” designates the sequence reverse complementary to a 5 ’terminal sequence of small non-coding RNA, wherein “idSp” stands for base lacking spacer, and wherein one or more nucleotides in the underlined portion and/or one or more nucleotides in the double underlined portion are optionally LNA enhanced, or is a variant of this sequence.
  • the LNA enhanced nucleotides are ribonucleotides.
  • the 5 ’adapter variant as described above has a sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, and still even more preferably 99%, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the sequence according to SEQ ID NO: 6 or SEQ ID NO: 12.
  • Such a 5’adapter variant still comprises the at least 2 nucleotides at its 3 ’end which are ribonucleotides or modified ribonucleotides.
  • such a 5’ adapter variant is still LNA enhanced (if this is not an optional feature). Furthermore, such a 5’adapter still comprises a base lacking spacer. In addition, such a 5’adapter variant is still capable of forming a stem-loop structure containing a loop and a double stranded stem. The skilled person can readily assess whether a 5’adapter variant is still capable of forming a stem-loop structure containing a loop and a double stranded stem. For example, the experimental section provides sufficient information in this respect.
  • the 5’adapter as described above does not comprise a base-lacking spacer (e.g. a base-lacking 1’, 2’-dideoxyribose spacer) in the loop region.
  • a base-lacking spacer e.g. a base-lacking 1’, 2’-dideoxyribose spacer
  • the 5’adapter having a nucleotide sequence according to SEQ ID NO: 1, 6, or 12 as described above, one or more of the 6 to 15 deoxynucleotides which are reverse complementary to a 5’-terminal sequence of small non-coding RNA are LNA-enhanced and/or replaced by a 2 ’-ortho-methylated ribonucleotide.
  • the 5’adapter as described above can bind to any small non-coding RNA target (specifically to the 5’end of any small non-coding RNA target) just by exchanging the variable protrusions.
  • the 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides in the 5 ’terminal nucleotide sequence of the 5’adapter have only to be selected in a way that they are reverse complementary to the small non-coding RNA target (specifically to the 5’end of the small non-coding RNA target) to be detected.
  • the 5’adapter is especially used jointly with the 3 ’adapter.
  • the 5’ adapter as described above may be present in denatured or renatured form.
  • the present inventors have exemplarily analyzed the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence of 5’ GCCGCCGGUGAAAUACCACUAC 3’ (SEQ ID NO: 16).
  • This rRNA fragment has two methylations sites, namely G (also designated as Gm4020) and C (also designated as Cm4032). While Gm4020 is a variable methylation site, Cm4032 shows a high degree of non-variable methylation.
  • the methylation status of Gm4020 has been calculated/determined herein. A variation of this methylation status is indicative for the presence of cancer, specifically lung cancer.
  • the 5 ’adapter having the following sequence from 5’ to 3’ :
  • CACmCGGCGGCCG/idSp/TGGCGTGGAGTGTGTGCTTTGCCArCrG allows to determine the methylation status of Gm4020 (“G”) of the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence of 5’ GCCGCCGGUGAAAUACCACUAC 3’ (SEQ ID NO: 16).
  • the 3’adapter comprises in the following order from 5’ to 3’ :
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein the 5 ’-terminal nucleotide is phosphorylated and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced, and
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide.
  • the LNA enhanced sequence comprises between 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 3, locked nucleotides, specifically ribonucleotides.
  • nucleotide sequence of the 3’ adapter comprises deoxynucleotides and ribonucleotides.
  • the 3’adapter may range from about 15 to about 60, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, nucleotides in length.
  • the 3’adapter may be present as linear polynucleotide, particularly in single- stranded form, e.g. after denaturation/when denatured.
  • the 3’adapter is a polynucleotide that can be attached/ligated to the 3 ’end of small non-coding RNA.
  • the 3’adapter has a stem-loop structure.
  • the attachment/ligation is possible as the 3’adapter comprises between 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to a 3’-terminal sequence of small non-coding RNA.
  • the small non-coding RNA is preferably a miRNA or isomiR comprised in miRbase version
  • the nucleotide sequence capable of forming a stem-loop structure comprises a 5 ’positioned first stem sequence and a 3 ’positioned second stem sequence that are reverse complementary to each other.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence can form the double stranded stem.
  • the double stranded stem may have a length of between 5 and 20, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, nucleotides.
  • each one of the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence has a length of between 5 to 10, e.g. 5, 6, 7, 8, 9, or 10, nucleotides.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence have the same length, e.g. a length of 5, 6, 7, 8, 9, or 10 nucleotides.
  • the 5 ’positioned first stem sequence and/or the 3 ’positioned second stem sequence is (are) LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides. Examples of locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • the 3 ’positioned second stem sequence is LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides.
  • locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • every, every second, or every third nucleotide may be LNA enhanced in the 5’positioned first stem sequence and/or the 3’positioned second stem sequence.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise (a mixture of) deoxynucleotides and ribonucleotides (e.g. LNA-enhanced) or the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise ribonucleotides.
  • Said ribonucleotides include ribonucleotides which are LNA-enhanced.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a loop sequence which is located between the 5’positioned first stem sequence and the 3’positioned second stem sequence.
  • the loop sequence may comprise between 10 and 40, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, nucleotides.
  • the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, e.g. deoxynucleotides and/or ribonucleotides. More preferably, the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, deoxynucleotides.
  • the 3 ’-terminal sequence is configured such that it forms a single stranded 3 ’protrusion after formation of the stem-loop structure.
  • the 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to the 3 ’-terminal sequence of small noncoding RNA encompass G and C but not more than 4, e.g. 1, 2, 3, or 4, in a row.
  • the inverted deoxynucleotide is inverted dT, dA, dC, or dG.
  • the 3 ’inverted deoxynucleotide creates a 3 ’-3’ linkage and, thus, prevents undesired nucleotide synthesis from the 3 ’end of the adapter, e.g. during RT- PCR.
  • the 3 ’inverted deoxynucleotide protects the sequence from 3’ exonuclease cleavage.
  • a preferred 3 ’adapter comprises in the following order from 5’ to 3’:
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein the 5 ’-terminal nucleotide is phosphorylated and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced, and
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide.
  • said 6 to 15 deoxynucleotides which are reverse complementary to a 5’-terminal sequence of small non-coding RNA comprise one or more locked nucleotide- (LNA-) enhanced and/or other modified nucleotides.
  • LNA- locked nucleotide-
  • the above described 3 ’adapter comprises in the following order from 5’ to 3’:
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein the 5’-terminal nucleotide is phosphorylated
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are LNA-enhanced, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide, or
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are otherwise modified (than LNA-enhanced), and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide, or (iic) a 3’terminal nucleotide sequence comprising 6 to 15, e.g.
  • deoxynucleotides 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are LNA-enhanced and otherwise modified, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide.
  • the above described 3 ’adapter comprises in the following order from 5’ to 3’ :
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein the 5 ’-terminal nucleotide is phosphorylated and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced, and
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are LNA-enhanced, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide, or
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are otherwise modified (than LNA-enhanced), and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide, or
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are LNA-enhanced and otherwise modified, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide.
  • the above-mentioned one or more otherwise modified (than LNA-enhanced) nucleotides are preferably 2 ’-ortho-methylated ribonucleotides.
  • the above described 3 ’adapter comprises in the following order from 5’ to 3’ :
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein the 5’-terminal nucleotide is phosphorylated
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are LNA-enhanced, and wherein the 3 ’terminal deoxynucleotide is an inverted deoxynucleotide, or
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are replaced by a 2’-ortho-methylated ribonucleotide, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide, or
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are LNA-enhanced and replaced by a 2’-ortho-methylated ribonucleotide, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide.
  • the above described 3 ’adapter comprises in the following order from 5’ to 3’ :
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein the 5 ’-terminal nucleotide is phosphorylated and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced, and
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are LNA-enhanced, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide, or
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are replaced by a 2’-ortho-methylated ribonucleotide, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide, or
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein one or more of them are LNA-enhanced and replaced by a 2’-ortho-methylated ribonucleotide, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide.
  • the one or more 2 ’-ortho-methylated ribonucleotides are located at a position (in said nucleotide sequence) corresponding to a position in the non-coding small RNA (molecule) that is presumed to be methylated.
  • the one or more 2 ’-ortho-methylated ribonucleotides are located at a position (in said nucleotide sequence) base-pairing with a position in the noncoding small RNA (molecule) that is presumed to be methylated.
  • the 3 ’adapter has the following sequence from 5’ to 3’: /5Phos/CTC AGTGC AGGGTCCGAGGT ATTCGC ACTGAG/6- 15xW3InvdT/ (SEQ ID NO: 2), wherein “/5Phos/” indicates that the 5’-terminal nucleotide is phosphorylated, wherein one or more nucleotides in the underlined portion and/or one or more nucleotides in the double underlined portion are optionally LNA enhanced, wherein “(6-15x)N” designates the sequence reverse complementary to a 3 ’terminal sequence of small non-coding RNA, and wherein “/3InvdT/” stands for 3 ’inverted deoxynucleotide, or is a variant of this sequence.
  • every, every second, or every third nucleotide may be LNA enhanced in the underlined portion and/or in the double underlined portion specified above.
  • the 3 ’adapter has the following sequence from 5’ to 3’ : /5Phos/CTCAGTGCAGGGTCCGAGGTATTCGCACTGAG(6-15x)N/3InvdT/ (SEQ ID NO: 2), wherein “/5Phos/” indicates that the 5 ’-terminal nucleotide is phosphorylated, wherein one or more (e.g.
  • nucleotides in bold letters are LNA enhanced, wherein “(6-15x)N” designates the sequence reverse complementary to a 3 ’terminal sequence of small non-coding RNA, and wherein “/3InvdT/” stands for 3 ’inverted deoxynucleotide, or is a variant of this sequence.
  • the LNA enhanced nucleotides are ribonucleotides.
  • the 3 ’adapter variant as described above has a sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, still even more preferably 99%, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the sequence according to SEQ ID NO: 2.
  • Such a 3’ adapter variant is still LNA enhanced (if this is not an optional feature). Further, the 5’-terminal nucleotide is still phosphorylated in such a 3 ’adapter variant.
  • the 3 ’terminal deoxynucleotide is still an inverted deoxynucleotide in such a 3 ’adapter variant.
  • such a 3 ’adapter variant is still capable of forming a stemloop structure containing a loop and a double stranded stem.
  • the skilled person can readily assess whether a 3 ’adapter variant is still capable of forming a stem-loop structure containing a loop and a double stranded stem.
  • the experimental section provides sufficient information in this respect.
  • the 3’adapter having a nucleotide sequence according to SEQ ID NO: 2 as described above, one or more of the 6 to 15 deoxynucleotides which are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA are LNA-enhanced and/or replaced by a 2 ’-ortho-methylated ribonucleotide.
  • the 3 ’adapter as described above can bind to any small non-coding RNA target (specifically to the 3 ’end of any small non-coding RNA target) just by exchanging the variable protrusions.
  • the 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides in the 3 ’terminal nucleotide sequence of the 3 ’adapter have only to be selected in a way that they are reverse complementary to the small non-coding RNA target (specifically to the 3 ’end of the small non-coding RNA target) to be detected.
  • the 3 ’adapter is especially used jointly with the 5 ’adapter.
  • the 3’ adapter as described above may be present in denatured or renatured form.
  • methylated small non-coding RNA is RNA which has been post-transcriptionally edited or modified by methylation.
  • the methylation can occur at a base (e.g. methyl-6-adenine, pseudouridine) and/or ribose ring (2'-O-methylated nucleotide (2'-O-m)).
  • the methylation of small non-coding RNA occurring at a base is preferably selected from the group consisting of 6-methyladenosine (m 6 A), 5-methylcytidine (m 5 C), 5-methyluridine (m 5 U), 3 -methyluridine (m 3 U), 1 -methyladenosine (m J A), and 1 -methyl uanosine (nfG), or is a combination thereof.
  • the 2'-O-methylation of the backbone ribose is the most common and conserved type of small non-coding RNA modification.
  • the methylation of small non-coding RNA occurring at a ribose ring is preferably selected from the group consisting of 3 '-end 2'-O-methyladenosine (Am), 2'-O-methyluridine (Um), 2'-O- methylguanosine (Gm), and 2'-O-methylcytidine (Cm), or is a combination thereof.
  • Am 2'-O-methyladenosine
  • Um 2'-O-methyluridine
  • Gm 2'-O- methylguanosine
  • Cm 2'-O-methylcytidine
  • the cancer is preferably early-stage cancer such as cancer of stage I. More preferably, the cancer is early-stage lung cancer such as lung cancer of stage I.
  • the cancer is preferably lung cancer. More preferably, the lung cancer is early stage lung cancer such as lung cancer of stage I and/or stage II.
  • the biological sample may be any sample having a biological origin.
  • the sample may also be a processed biological sample.
  • the biological sample may be a body fluid sample, e.g. a blood sample or urine sample, or a tissue sample, e.g. a tissue (biopsy) sample.
  • Biological samples may be mixed or pooled, e.g. a sample may be a mixture of a blood sample and a urine sample.
  • the body fluid sample may be a urine sample, blood sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, gastric juice sample, mucus sample, lymph sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof.
  • body fluid sample also encompasses body fluid fractions, e.g. blood fractions, urine fractions or sputum fractions. Body fluid samples may be mixed or pooled.
  • a body fluid sample may be a mixture of a blood and a urine sample or a mixture of a blood and cerebrospinal fluid sample.
  • the body fluid sample is a blood sample or the tissue (biopsy) sample is a cancer tissue (biopsy) sample.
  • the blood sample is a whole blood or a blood fraction, preferably blood cells (e.g. erythrocytes, leukocytes, and/or thrombocytes), serum, or plasma.
  • the blood cell fraction encompasses erythrocytes, leukocytes, and/or thrombocytes.
  • the whole blood sample may be collected by means of a blood collection tube.
  • the whole blood sample may also be collected by means of a bloodspot technique, e.g. using a Mitra Microsampling Device. This technique requires smaller sample volumes, typically 45-60 pl for humans or less.
  • the whole blood may be extracted from a subject via a finger prick with a needle or lancet.
  • the whole blood sample may have the form of a blood drop. Said blood drop is then placed on an absorbent probe, e.g.
  • the sample may also be a sample containing total RNA.
  • total RNA includes RNA having a length of ⁇ 200 nucleotides such as a miRNA or a miRNA isoform (an isomiR).
  • the sample used in the first aspect of the present invention contains cellular total RNA.
  • cellular total RNA includes RNA having a length of ⁇ 200 nucleotides such as a miRNA or a miRNA isoform (an isomiR).
  • the cellular total RNA may be obtained from blood cells, e.g. erythrocytes, leukocytes, and/or thrombocytes.
  • the present inventors have exemplarily analyzed the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence of 5’ GCCGCCGGUGAAAUACCACUAC 3’ (SEQ ID NO: 16).
  • This rRNA fragment has two methylations sites, namely G (also designated as Gm4020) and C (also designated as Cm4032). While Gm4020 is a variable methylation site, Cm4032 shows a high degree of non-variable methylation.
  • the methylation status of Gm4020 has been calculated/determined herein. A variation of this methylation status is indicative for the presence of cancer, specifically lung cancer.
  • the 3’adapter having the following sequence from 5’ to 3’ : /5Phos/CTCAGTGCAGGGTCCGAGGTATTCGCACTGAGGTAGTGGTA/3InvdT/ (SEQ ID NO: 15) allows to determine the methylation status of Gm4020 (“G”) of the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence of 5’ GCCGCCGGUGAAAUACCACUAC 3’ (SEQ ID NO: 16).
  • G G
  • rRNA ribosomal RNA
  • the 5’adapter as described above and the 3’adapter as described above can bind/anneal to any small non-coding RNA.
  • the adapters After the ligation of the adapters to the small non-coding RNA, they allow the generation of a cDNA product of said small non-coding RNA via reverse transcription.
  • a small non-coding RNA is methylated, a limited number of cDNA products is produced compared to small non-coding RNA which is not methylated.
  • the adapters allow the determination of methylation of small non-coding RNA and/or the quantification of the methylation status of small non-coding RNA.
  • the combination of said adapters is used for determining the methylation status of small non-coding RNA (in a biological sample) to diagnose cancer in a patient/to determine whether a patient suffers from cancer.
  • CACmCGGCGGCCG/idSp/TGGCGTGGAGTGTGTGCTTTGCCArCrG (SEQ ID NO: 14) in combination with the 3’adapter having the following sequence from 5’ to 3’ : /5Phos/CTCAGTGCAGGGTCCGAGGTATTCGCACTGAGGTAGTGGTA/3InvdT/ (SEQ ID NO: 15) allows to determine the methylation status of Gm4020 (“G”) of the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence of 5’ GCCGCCGGUGAAAUACCACUAC 3’ (SEQ ID NO: 16).
  • G G
  • rRNA ribosomal RNA
  • the present inventors have found that a methylation status of small non-coding RNA which is lower than the reference methylation status of the small non-coding RNA determined empirically by measuring a number of reference biological samples from healthy subjects/subjects known to not suffer from cancer indicates that the patient suffers from cancer.
  • the present inventors have exemplarily shown herein that the 28S ribosomal RNA (rRNA) fragment extracted from peripheral blood of a patient suffering from lung cancer has a methylation status which is lower than the methylation status of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer.
  • a methylation status of the 28 S ribosomal RNA (rRNA) fragment extracted from peripheral blood of a patient which is lower than the methylation status of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer, is indicative for lung cancer in the patient and, thus, allows the diagnosis of lung cancer in said patient.
  • the methylation status of Gm4020 has been calculated/determined herein. Accordingly, the methylation status of Gm4020 of the afore-mentioned 28S ribosomal RNA (rRNA) extracted from peripheral blood of a patient which is lower than the methylation status of Gm4020 of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer, is indicative for lung cancer in the patient and, thus, allows the diagnosis of lung cancer in said patient.
  • rRNA ribosomal RNA
  • the present invention relates to a (an in vitro) method for diagnosing cancer in a patient/for determining whether a patient suffers from cancer comprising the step of: determining a methylation status of small non-coding RNA in a biological sample obtained from a patient.
  • the second aspect may alternatively be formulated as follows: (In vitro) method for diagnosing cancer in a patient/for determining whether a patient suffers from cancer comprising the step of: determining a methylation status of a small non-coding RNA population in a biological sample obtained from a patient.
  • the methylation status indicates whether the patient suffers from cancer or not.
  • the present invention relates to a (an in vitro) method for diagnosing cancer in a patient/for determining whether a patient suffers from cancer comprising the steps of:
  • This comparison allows to determine whether the patient suffers from cancer or not.
  • the reference methylation status is the methylation status of the small non-coding RNA determined empirically by measuring a number of reference biological samples from healthy subjects/subjects known to not suffer from cancer, and/or from subjects known to suffer from cancer.
  • the reference methylation status is the methylation status of the small non-coding RNA determined empirically by measuring a number of reference biological samples from healthy subjects/subjects known to not suffer from cancer and wherein a methylation status which is lower than this reference methylation status indicates that the patient suffers from cancer.
  • the reference methylation status is determined from at least 2, at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000 at least 5000 reference biological samples from healthy subjects/subjects known to not suffer from cancer, and/or the reference methylation is determined from at least 2, at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000 at least 5000 reference biological samples from subjects having cancer/known to have cancer.
  • the reference methylation status is an average reference methylation status. It is determined by measuring reference methylation statuses of control subjects (e.g. healthy subjects or subjects having cancer/known to have cancer) and calculating the “average” value (e.g. mean, median or modal value) thereof. It is preferred that the reference biological samples are from the same source (e.g. blood cells, serum, or plasma) than the biological sample isolated from the patient to be tested. It is further preferred that the reference methylation status is obtained from control subjects (e.g. healthy subjects or subjects having cancer/known to have cancer) of the same gender (e.g. female or male) and/or of a similar age/phase of life (e.g. adults or elderly) than the patient to be tested. In one embodiment, the determination of the methylation status of small non-coding RNA (in the biological sample) comprises the steps of:
  • methylation status (%) (1- (copies first cDNA product under limiting conditions / copies second cDNA product under non-limiting conditions)) * 100.
  • the reference methylation status of the small non-coding RNA is determined.
  • the determination of the reference methylation status of the small non-coding RNA comprises the steps of:
  • reference methylation status (%) (1- (copies first cDNA product under limiting conditions / copies second cDNA product under non-limiting conditions)) * 100.
  • Methylated small non-coding RNA is RNA which has been post-transcriptionally edited or modified by methylation.
  • the methylation can occur at a base (e.g. methyl-6-adenine, pseudouridine) and/or ribose ring (2'-O-methylated nucleotide (2'-O-m)).
  • the methylation of small non-coding RNA occurring at a base is preferably selected from the group consisting of 6-methyladenosine (m 6 A), 5-methylcytidine (m 5 C), 5 -methyluridine (m 5 U), 3 -methyluridine (m 3 U), 1 -methyladenosine (m'A), and 1 -methyl guanosine (m ] G), or is a combination thereof.
  • the 2'-O-methylation of the backbone ribose is the most common and conserved type of small non-coding RNA modification.
  • the methylation of small non-coding RNA occurring at a ribose ring is preferably selected from the group consisting of 3 '-end 2'-O-methyladenosine (Am), 2'-O-methyluridine (Um), 2'-O- methylguanosine (Gm), and 2'-O-methylcytidine (Cm), or is a combination thereof.
  • a ligation product comprising small non-coding RNA to which the 5 ’adapter as defined in the first aspect and the 3 ’adapter as defined in the first aspect are ligated.
  • Said ligation product is preferably produced by
  • the annealing of the adapters, in particular 5’ and 3 ’adapters, to the small non-coding RNA requires that the small non-coding RNA is present in denatured form.
  • the denatured small non-coding RNA is produced by heating the small noncoding RNA at between 65°C and 75°C, e.g. 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C, preferably at 70°C, for between 1 to 3 minutes, e.g. 1, 2, or 3, minutes, preferably for 2 minutes.
  • the denatured small non-coding RNA is produced by heating the small non-coding RNA at 70°C for 2 minutes.
  • the adapters in particular 5’ and 3 ’adapters, a denaturation and a renaturation step is required so that they can from a stem-loop structure which allows annealing to the small noncoding RNA.
  • Annealing is a process of heating and cooling adapters with complementary sequences. Heat breaks all hydrogen bonds and cooling allows new bonds to form between the sequences.
  • the adapters in particular 5’ and 3 ’adapters, attach to the denatured small non-coding RNA and form their characteristic stem-loop structure.
  • the 5 ’adapter attaches to the 5 ’end of the small non-coding RNA and the 3 ’adapter attaches to the 3’end ofthe small non-coding RNA.
  • the adapters in particular s’ and 3 ’adapters, are denatured and renatured together, i.e. in a common reaction vessel. It is further preferred that the denaturation/renaturation of the adapters, in particular 5’ and 3 ’adapters, takes place separately and in the absence of the small non-coding RNA.
  • the renatured 5 ’adapter is produced by denaturing the 5’adapter at between 75°C and 85°C, e.g. 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85°C, preferably at 82°C, for between 1 to 3 minutes, e.g. 1, 2, or 3 minutes, preferably for 2 minutes, and renaturing the 5’adapter by cooling down to 4°C, preferably at a rate of 0. l°C/s.
  • 75°C and 85°C e.g. 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85°C, preferably at 82°C, for between 1 to 3 minutes, e.g. 1, 2, or 3 minutes, preferably for 2 minutes
  • renaturing the 5’adapter by cooling down to 4°C, preferably at a rate of 0. l°C/s.
  • the renatured 5’adapter is produced by denaturing the 5’adapter at 82°C for 2 minutes and by renaturing the 5’adapter by cooling down to 4°C, preferably at a rate of 0.1°C/s.
  • the renatured 3 ’adapter is produced by denaturing the 3’adapter at between 75°C and 85°C, e.g. 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85°C, preferably at 82°C, for between 1 to 3 minutes, e.g. 1, 2, or 3 minutes, preferably for 2 minutes, and renaturing the 3’adapter by cooling down to 4°C, preferably at a rate of 0. l°C/s.
  • the renatured 3’adapter is produced by denaturing the 3’adapter at 82°C for 2 minutes, and by renaturing the 3’adapter by cooling down to 4°C, preferably at a rate of 0.1°C/s.
  • the adapters, in particular 5’ and 3 ’adapters are preferably given to an aqueous buffer, e.g. TNE annealing buffer.
  • the denaturing and renaturing of the adapters, in particular 5’ and 3 ’adapters is preferably carried out in an aqueous buffer, e.g. TNE annealing buffer.
  • composition i.e. the composition comprising denatured small noncoding RNA, the renatured 5’adapter as defined in the first aspect, and the renatured 3’adapter as defined in the first aspect, wherein the 5’adapter and the 3’adapter are annealed to the small noncoding RNA
  • the annealing of the 5’adapter with the small non-coding RNA particularly generates a double-stranded (DNA/RNA) hybrid containing a nick of RNA-OH-375’-P-RNA between the 3’end of the adapter and the 5 ’end of the small non-coding RNA.
  • This is an efficient substrate for ligation by a double stranded RNA ligase.
  • the annealing of the 3’adpater with the small non-coding RNA particularly generates a double-stranded (DNA/RNA) hybrid containing a nick of RNA-OH-375’-P-RNA between the 3’end of the small non-coding RNA and the 5’end of the adapter. This is a substrate for ligation by a double stranded RNA ligase.
  • the ligation, to produce the ligation product provided in step (i) of the method of the second aspect, is usually carried out in a ligation buffer.
  • a ligation buffer comprises polyethylene glycol (PEG), e.g. PEG 8000 (5%), and/or adenosine triphosphate (ATP), e.g. 1 mM ATP.
  • PEG polyethylene glycol
  • ATP adenosine triphosphate
  • the ligation is carried out between 36°C and 38°C, e.g. 36, 37, or 38°C, preferably at 37°C, for between 30 minutes and 1.5 hours, e.g. 30, 35, 40, 45, 50, 55 minutes, 1, 1.25, or 1.5 hour(s), preferably for 1 hour.
  • the ligation is carried out at 37°C for 1 hour.
  • the double stranded RNA ligase can be any ligase capable of ligating double stranded RNA nicks/RNA structures.
  • the double stranded RNA ligase is a T4 RNA ligase 2 (Rnl2), a Kodl ligase or a RtcB ligase.
  • RNA ligase in particular Rnl2.
  • Rnl2 double stranded RNA ligase
  • the adapters, in particular 5’ and 3 ’adapters, described herein provide a dsRNA context with a 6 to 15 nucleotide protrusion that hybridizes to the small non-coding RNA.
  • a 5’ phosphate moiety on the small non-coding RNA is also of advantage for efficient ligation by Rnl2.
  • a ligation product By ligating the adapters, in particular 5’ and 3 ’adapters, to the small non-coding RNA using/with a double stranded RNA ligase, a ligation product is produced.
  • the ligation product can be described as a (DNA/RNA) hybrid molecule comprising at least one adapter and small non-coding RNA.
  • the ligation product may comprise a 5 ’adapter and small non-coding RNA such as miRNA or isomiR.
  • the ligation product may comprise a 3 ’adapter and small non-coding RNA such as miRNA or isomiR.
  • the ligation produced may comprise a 5 ’adapter, a 3 ’adapter and small non-coding RNA such as miRNA or isomiR.
  • Methylated small non-coding RNA such as 2'-O-methylated small non-coding RNA induce reverse transcription stops/pauses at low concentrations of desoxynucleosidetriphosphates (dNTPs) (i.e. under limiting conditions), while at high dNTP concentrations (i.e. under nonlimiting conditions) reverse transcriptase can bypass methylated sites such as 2'-O-methylated sites. It appears that the methylated group such as 2'-O-methyl group acts as a conformational “bump” which hinders the passage of the reverse transcriptase, whose effect is minimized at high dNTP concentrations (i.e. under non-limiting conditions).
  • dNTPs desoxynucleosidetriphosphates
  • step (ii) for determining the small non-coding RNA methylation status/reference methylation status the ligation product is reverse transcribed under limiting conditions, thereby obtaining a first cDNA product.
  • the (same) ligation product is reverse transcribed under non-limiting conditions, thereby obtaining a second cDNA product.
  • the reverse transcription of the ligation product is preferably carried out by
  • said annealing of a primer for reverse transcription (RT- primer) with the ligation product in step (iia) is carried out at between 60°C and 80°C, e.g. 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80°C, preferably at 65°C or 75°C, for between 2 and 7 minutes, e.g. 2, 3, 4, 5, 6, or 7 minutes, preferably 3 or 5 minutes.
  • RT- primer primer for reverse transcription
  • said annealing is carried out at 65°C for between 2 and 7 minutes, e.g. 2, 3, 4, 5, 6, or 7 minutes, preferably 5 minutes.
  • said annealing is carried out at 75°C for between 2 and 7 minutes, e.g. 2, 3, 4, 5, 6, or 7 minutes, preferably 3 minutes.
  • the reverse transcriptase (RT) is a Maxima H-RT, Tth polymerase, Protoscript II RT, Luna RT, AMV, or M-MuLV, or any other enzymes derived from these. More specifically, the reverse transcriptase (RT) is a Maxima H-RT or Luna RT.
  • the 3 ’adapter ligated to the 3 ’end of the small noncoding RNA is extended, in particular in 5’ to 3’ direction, to form a strand reverse complementary to the small non-coding RNA.
  • a cDNA copy of the ligation product is produced in the reverse transcription reaction.
  • the reverse transcriptase (RT) requires a RT primer.
  • the RT-primer is particularly reverse complementary to the nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem of the 3 ’adapter.
  • the RT-primer sequence depends on the 3’ adapter sequence.
  • the RT-primer has the following sequence from 5’ to 3’: CTCAGTGCGAATACCTCGGACCCTGCACTGAGGTAGT (SEQ ID NO: 3) or is a variant of this sequence.
  • the RT-primer is, thus, reverse complementary to at least a part of the 3’adpater sequence as described above.
  • the RT-primer is reverse complementary to nucleotides in the 3’positioned second stem sequence and/or in the loop sequence.
  • the 3 ’adapter sequence is preferably the following from 5’ to 3’: /5Phos/CTC AGTGC AGGGTCCGAGGT ATTCGC ACTGAG/6- 15x)N/3InvdTZ (SEQ ID NO: 2), wherein “/5Phos/” indicates that the 5’-terminal nucleotide is phosphorylated, wherein one or more nucleotides in the underlined portion and/or one or more nucleotides in the double underlined portion are optionally LNA enhanced, wherein (6-15x)N designates the sequence reverse complementary to a 3 ’terminal sequence of a target RNA, and wherein “/3InvdT/” stands for 3 ’inverted deoxynucleotide.
  • the LNA enhanced nucleotides are ribonucleotides.
  • the RT-primer variant has a sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, still even more preferably 99%, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the sequence according to SEQ ID NO: 3.
  • Such a RT-primer variant is still capable of binding the 3 ’adapter sequence and allowing reverse transcription which is performed by a reverse transcriptase (RT), e.g.
  • RT reverse transcriptase
  • said reverse transcribing of the ligation product under limiting conditions means performing the reverse transcription with a desoxynucleosidetriphosphate (dNTP) concentration which is 1/10 or less, e.g. 1/10, 1/11, 1/12, 1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20, 1/21, 1/22, 1/23, 1/24, 1/25, 1/26, 1/27, 1/28, 1/29, 1/30, 1/31, 1/32, 1/33, 1/34, 1/35, 1/36, 1/37, 1/38, 1/39, 1/40, 1/41, 1/42, 1/43, 1/44, 1/45, 1/46, 1/47, 1/48, 1/49, 1/50, or less, of the dNTP concentration under non-limiting conditions.
  • dNTP desoxynucleosidetriphosphate
  • said reverse transcription of the ligation product is conducted with a dNTP concentration which is between 1/10 and 1/50, e.g. 1/10, 1/11, 1/12, 1/13, 1/14, 1/15, 1/16, 1/17,
  • said reverse transcription of the ligation product is conducted with between 0.25 mM and 50 mM dNTPs, e.g. with 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mM dNTPs.
  • said reverse transcription of the ligation product is conducted with between 0.25 mM and 25 mM dNTPs, e.g. with 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM dNTPs.
  • said reverse transcription of the ligation product is conducted with 1 mM dNTPs.
  • the reverse transcription of the ligation product “under non-limiting (normal) conditions” is preferably conducted with between 2.5 mM and 500 mM dNTPs, more preferably with between 2.5 mM and 250 mM dNTPs, and even more preferably with 10 mM dNTPs.
  • test reaction is specifically performed with 1 mM dNTPs and the reverse transcription reaction under non-limiting (normal) conditions (control reaction) is specifically performed with 10 mM dNTPs.
  • the cDNA products derived therefrom have to be amplified in step (iii).
  • the amplification requires a DNA polymerase, e.g. a Taq polymerase.
  • the cDNA is preferably diluted for the PCR, specifically digital PCR, e.g. 1 : 10.
  • the amplification reaction is carried out with a Forward primer having the following sequence from 5’ to 3’: TGGAGTGTGTGCTTTGCCACG (SEQ ID NO: 4) or with a variant of this sequence and a Reverse primer having the following sequence from 5’ to 3’ : GTGCGAATACCTCGGACC (SEQ ID NO: 5) or with a variant of this sequence (see above).
  • Any amplification method may be used.
  • the amplification is carried out using a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the PCR is selected from the group consisting of digital PCR, real-time PCR (quantitative PCR or qPCR), preferably Taq-man qPCR, multiplex PCR, nested PCR, high fidelity PR, fast PCR, hot start PCR, and GC-rich PCR.
  • the digital PCR is preferably a digital droplet PCR or a digital partition PCR.
  • the digital PCR or the TaqMan qPCR is carried out with a Forward primer having the following sequence from 5’ to 3’: TGGAGTGTGTGCTTTGCCACG (SEQ ID NO: 4) or with a variant of this sequence and a Reverse primer having the following sequence from 5’ to 3’ : GTGCGAATACCTCGGACC (SEQ ID NO: 5) or with a variant of this sequence. While the forward primer is derived from the 5 ’adapter, the reverse primer is derived from the 3 ’adapter. These primer designs render the amplification completely dependent on ligation of both the 5’ and 3 ’adapters to exclusively amplify the ligation product.
  • the amplification using a Taq- man qPCR may be carried out as follows: 95°C for 20 seconds, followed by 40 cycles at 95°C for 1 second and 60°C for 20 seconds. The amplification sample is subsequently hold at 4°C.
  • the Forward primer variant as described above has a sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, still even more preferably 99%, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the sequence according to SEQ ID NO: 4.
  • Such a Forward primer variant is still capable of binding the DNA product produced from the 5 ’adapter in the reverse transcription reaction.
  • the Forward primer variant must have at least in part, e.g.
  • the sequences are identical, e.g. over a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or of 21 nucleotides, in the loop region and in the 3 ’positioned second stem sequence of the 5’adapter.
  • the skilled person can readily assess whether a Forward primer variant is still capable binding the DNA product produced from the 5’adapter in the reverse transcription reaction.
  • the experimental section provides sufficient information in this respect.
  • the Reverse primer variant as described above has a sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, still even more preferably 99%, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the sequence according to SEQ ID NO: 5.
  • Such a Reverse primer variant is still capable of binding the 3 ’adapter sequence and allowing cDNA preamplification/amplification.
  • the skilled person can readily assess whether a Reverse primer variant is still capable of binding the 3 ’adapter sequence and allowing cDNA preamplification/amplification.
  • the experimental section provides sufficient information in this respect.
  • the TaqMan qPCR is carried out in the presence of a TaqMan probe.
  • the sequence of the TaqMan probe depends on the sequence of the RNA target.
  • the TaqMan probe is a hydrolysis probe that is designed to increase the specificity of quantitative PCR.
  • the TaqMan probe principle relies on the 5 ’-3’ exonuclease activity of the Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection.
  • the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR.
  • the TaqMan probe significantly increases the specificity of the detection.
  • the TaqMan probe has the following sequence: 16- FAM/TGAGGTAGTGGT ATTTCACCGGCGGCCGT /BHQ-1/ (SEQ ID NO: 7).
  • a difference between the cDNA products produced via reverse transcription under limiting conditions (i.e. at low concentrations of dNTPs) and the cDNA products produced via reverse transcription under non-limiting conditions (i.e. high concentrations of dNTPs) indicates small non-coding RNA methylation in the biological sample.
  • the small non-coding RNA is not methylated.
  • a significant difference in this respect preferably means that the experiment is conducted three times and that in all three experiments, a difference could be detected.
  • the difference may reside in different levels of the cDNA products.
  • the level of the small non-coding RNA or cDNA product derived therefrom is determined by sequencing, preferably next generation sequencing (e.g. ABI SOLID, Illumina Genome Analyzer, Roche 454 GS FL, BGISEQ), nucleic acid hybridization (e.g. microarray or beads), nucleic acid amplification (e.g. polymerase chain reaction (PCR)), polymerase extension, mass spectrometry, flow cytometry (e.g. LUMINEX), or any combination thereof.
  • next generation sequencing e.g. ABI SOLID, Illumina Genome Analyzer, Roche 454 GS FL, BGISEQ
  • nucleic acid hybridization e.g. microarray or beads
  • nucleic acid amplification e.g. polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • polymerase extension e.g. LUMINEX
  • mass spectrometry e.g. LUMINEX
  • the PCR is selected from the group consisting of digital PCR, real-time PCR (quantitative PCR or qPCR), preferably TaqMan qPCR, multiplex PCR, nested PCR, high fidelity PR, fast PCR, hot start PCR, and GC-rich PCR.
  • the digital PCR may be digital droplet PCR or digital partition PCR.
  • the difference between the cDNA products produced via reverse transcription is based on a difference in expression levels determined by
  • step (iv) for determining the small non-coding RNA methylation status/reference methylation status the methylation status/reference methylation status is calculated.
  • the dNTP concentration under limiting conditions is 1/10 or less, e.g.
  • a methylation status which is lower than the reference methylation status indicates that the patient suffers from cancer.
  • the biological sample analysed in the method of the second aspect may be any sample having a biological origin.
  • the sample may also be a processed biological sample.
  • the biological sample may be a body fluid sample, e.g. a blood sample or urine sample, or a tissue sample, e.g. a tissue (biopsy) sample.
  • Biological samples may be mixed or pooled, e.g. a sample may be a mixture of a blood sample and a urine sample.
  • the body fluid sample may be a urine sample, blood sample, sputum sample, breast milk sample, cerebrospinal fluid (CSF) sample, cerumen (earwax) sample, gastric juice sample, mucus sample, lymph sample, endolymph fluid sample, perilymph fluid sample, peritoneal fluid sample, pleural fluid sample, saliva sample, sebum (skin oil) sample, semen sample, sweat sample, tears sample, cheek swab, vaginal secretion sample, liquid biopsy, or vomit sample including components or fractions thereof.
  • body fluid sample also encompasses body fluid fractions, e.g. blood fractions, urine fractions or sputum fractions. Body fluid samples may be mixed or pooled.
  • a body fluid sample may be a mixture of a blood and a urine sample or a mixture of a blood and cerebrospinal fluid sample.
  • the body fluid sample is a blood sample or the tissue (biopsy) sample is a cancer tissue (biopsy) sample.
  • the blood sample is a whole blood or a blood fraction, preferably blood cells (e.g. erythrocytes, leukocytes, and/or thrombocytes), serum, or plasma.
  • the blood cell fraction encompasses erythrocytes, leukocytes, and/or thrombocytes.
  • the whole blood sample may be collected by means of a blood collection tube.
  • the whole blood sample may also be collected by means of a bloodspot technique, e.g. using a Mitra Microsampling Device. This technique requires smaller sample volumes, typically 45-60 pl for humans or less.
  • the whole blood may be extracted from a subject via a finger prick with a needle or lancet.
  • the whole blood sample may have the form of a blood drop. Said blood drop is then placed on an absorbent probe, e.g.
  • the blood spot is dried in air before transferring or mailing to labs for processing. Because the blood is dried, it is not considered hazardous. Thus, no special precautions need be taken in handling or shipping.
  • the desired components e.g. miRNAs, are extracted from the dried blood spots into a supernatant which is then further analyzed.
  • the biological sample may also be a biological sample containing total RNA.
  • total RNA includes RNA having a length of ⁇ 200 nucleotides such as a miRNA or a miRNA isoform (an isomiR).
  • the biological sample analysed in the second aspect of the present invention contains cellular total RNA.
  • cellular total RNA includes RNA having a length of ⁇ 200 nucleotides such as a miRNA or a miRNA isoform (an isomiR).
  • the cellular total RNA may be obtained from blood cells, e.g. erythrocytes, leukocytes, and/or thrombocytes.
  • the cancer is preferably early-stage cancer such as cancer of stage I. More preferably, the cancer is early-stage lung cancer such as lung cancer of stage I.
  • the small non-coding RNA analyzed in the biological sample in the second aspect of the present invention has preferably a length of ⁇ 200 ribonucleotides, more preferably a length of between 10 and ⁇ 200 ribonucleotides, even more preferably a length of between 10 and 100 ribonucleotides, and still even more preferably a length of between 18 and 50 ribonucleotides, e.g. a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
  • the (above-mentioned) small non-coding RNA is a miRNA, a miRNA isoform (an isomiR), a ribosomal RNA fragment (rRNA fragment), a transfer RNA fragment (tRF), or a small nucleolar RNA (snorRNA) fragment.
  • the present inventors have exemplarily analyzed the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence of 5’ GCCGCCGGUGAAAUACCACUAC 3’ (SEQ ID NO: 16).
  • This rRNA fragment has two methylations sites, namely G (also designated as Gm4020) and C (also designated as Cm4032). While Gm4020 is a variable methylation site, Cm4032 shows a high degree of non-variable methylation.
  • the methylation status of Gm4020 has been calculated/determined herein. A variation of this methylation status is indicative for the presence of cancer, specifically lung cancer.
  • the 5’adapter having the following sequence from 5’ to 3’ CACCGGCGGCCG/idSp/TGGCGTGGAGTGTGTGCTTTGCCArCrG (SEQ ID NO: 13), or the 5’adapter having the following sequence from 5’ to 3’ : CACmCGGCGGCCG/idSp/TGGCGTGGAGTGTGTGCTTTGCCArCrG (SEQ ID NO: 14) in combination with the 3’adapter having the following sequence from 5’ to 3’ : /5Phos/CTCAGTGCAGGGTCCGAGGTATTCGCACTGAGGTAGTGGTA/3InvdT/ (SEQ ID NO: 15) allows to determine the methylation status of Gm4020 (“G”) of the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence of 5’ GCCGCCGGUGAAAUACCACUAC 3’ (SEQ ID NO: 16).
  • G G
  • the 28S ribosomal RNA (rRNA) extracted from peripheral blood of a patient suffering from lung cancer has a methylation status which is lower than the methylation status of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer.
  • a methylation status of the 28S ribosomal RNA (rRNA) fragment extracted from peripheral blood of a patient which is lower than the methylation status of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer is indicative for lung cancer in the patient and, thus, allows the diagnosis of lung cancer in said patient.
  • the methylation status of Gm4020 has been calculated/determined herein. Accordingly, the methylation status of Gm4020 of the afore-mentioned 28S ribosomal RNA (rRNA) extracted from peripheral blood of a patient which is lower (about 78.7 %) than the methylation status (about 82.7%) of Gm4020 of the same rRNA fragment extracted from peripheral blood of control subjects being healthy, i.e. not suffering from lung cancer, is indicative for lung cancer in the patient and, thus, allows the diagnosis of lung cancer in said patient.
  • rRNA 28S ribosomal RNA
  • the present invention relates to a (an in vitro) method for diagnosing lung cancer such as early-stage lung cancer in a patient/for determining whether a patient suffers from lung cancer such as early-stage lung cancer comprising the steps of:
  • This comparison allows to determine whether the patient suffers from lung cancer such as lung early-stage lung cancer.
  • the reference methylation status is the methylation status of the small non-coding RNA population determined empirically by measuring a number of reference biological samples from healthy subjects/subjects known to not suffer from lung cancer such as early-stage lung cancer.
  • the methylation status which is lower than this reference methylation status indicates that the patient suffers from lung cancer such as early-stage lung cancer.
  • the present invention relates to a (an in vitro) method for diagnosing lung cancer such as early-stage lung cancer in a patient/for determining whether a patient suffers from lung cancer such as early-stage lung cancer comprising the steps of
  • rRNA ribosomal RNA
  • This comparison allows to determine whether the patient suffers from lung cancer such as lung early-stage lung cancer.
  • the reference methylation status is the methylation status of the 28S ribosomal RNA (rRNA) fragment population determined empirically by measuring a number of reference biological samples from healthy subjects/subjects known to not suffer from lung cancer such as early-stage lung cancer. More particularly, the methylation status which is lower than this reference methylation status indicates that the patient suffers from lung cancer such as early-stage lung cancer.
  • rRNA ribosomal RNA
  • the present invention relates to a (an in vitro) method for diagnosing lung cancer such as early-stage lung cancer in a patient/for determining whether a patient suffers from lung cancer such as early-stage lung cancer comprising the steps of
  • rRNA ribosomal RNA
  • This comparison allows to determine whether the patient suffers from lung cancer such as lung early-stage lung cancer.
  • the reference methylation status is the methylation status of Gm4020 of the 28 S ribosomal RNA (rRNA) fragment population determined empirically by measuring a number of reference biological samples from healthy subjects/subjects known to not suffer from lung cancer such as early-stage lung cancer, specifically early-stage lung cancer of stages I and/or II.
  • rRNA ribosomal RNA
  • the methylation status which is lower (about 78.7 %) than this reference methylation status (about 82.7%) indicates that the patient suffers from lung cancer such as early- stage lung cancer.
  • the 28 S ribosomal RNA (rRNA) fragment having a nucleotide sequence according to SEQ ID NO: 16 is disclosed. Also covered by the present invention, with respect to the first and second aspect, is (the determination of a methylation of) a variant thereof.
  • said variant has
  • nucleotide sequence that is a fragment of the nucleotide sequence according to SEQ ID NO: 16, preferably, a nucleotide sequence that is a fragment which is between 1 and 6, more preferably between 1 and 4, and most preferably between 1 and 2, e.g. 1, 2, 3, 4, 5, or 6, nucleotides shorter than the nucleotide sequence according to SEQ ID NO: 16, or
  • nucleotide sequence that has at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, e.g. at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, sequence identity to the nucleotide sequence according to (i).
  • the methylation sites Gm4020 and/or Cm4032 are excluded from the nucleotide sequence modification. More preferably, the methylation site Gm4020 is excluded from the nucleotide sequence modification.
  • the second aspect relates to a method for diagnosing lung cancer such as early-stage lung cancer in a patient/for determining whether a patient suffers from lung cancer such as early-stage lung cancer.
  • the methylation status of Gm4020 of a 28 S ribosomal RNA (rRNA) fragment population in a blood sample, such as whole blood sample, obtained from a patient is determined.
  • This methylation status is compared to a reference methylation status which is the methylation status of the 28 S ribosomal RNA (rRNA) fragment population determined empirically by measuring a number of reference blood samples, such as whole blood samples, from healthy subjects/ subjects known to not suffer from lung cancer such as early-stage lung cancer.
  • a ligation product comprising the 28S ribosomal RNA (rRNA) fragment of a 28S ribosomal RNA (rRNA) fragment population to which a 5’adapter having a nucleotide sequence according to SEQ ID NO: 13 or having a nucleotide sequence according to SEQ ID NO: 14 and a 3’adapter having a nucleotide sequence according to SEQ ID NO: 15 are ligated.
  • rRNA 28S ribosomal RNA
  • rRNA 28S ribosomal RNA
  • the reverse transcription of the ligation product is carried out under limiting conditions, thereby obtaining a first cDNA product and the reverse transcription of the ligation product is carried out under non-limiting conditions, thereby obtaining a second cDNA product.
  • a RT primer having a nucleotide sequence according to SEQ ID NO: 3 is used.
  • the first cDNA product and the second cDNA product is amplified in a digital PCR (dPCR) reaction using a Forward primer having a nucleotide sequence according to SEQ ID NO: 4 and a Reverse primer having a nucleotide sequence according to SEQ ID NO: 5.
  • dPCR probe TaqMan probe
  • a nucleotide sequence according to SEQ ID NO: 7 is used.
  • the reverse transcription reaction under limiting conditions is ideally performed with 1 mM dNTPs and the reverse transcription reaction under non-limiting (normal) conditions (control reaction) is ideally performed with 10 mM dNTPs.
  • the 28 S ribosomal RNA (rRNA) fragment measured above has a nucleotide sequence according to SEQ ID NO: 16 or is a variant thereof.
  • methylation status (1- (copies first cDNA product under limiting conditions / copies second cDNA product under non-limiting conditions)) * 100.
  • the methylation status and the reference methylation status are compared.
  • a methylation status which is lower than the reference methylation status indicates that the patient suffers from cancer such as early-stage lung cancer.
  • the early stage lung cancer is specifically lung cancer of stage I and/or stage II.
  • the present invention relates to a 5’adapter comprising in the following order from 5’ to 3’ :
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA, and wherein at least one of said 6 to 15 deoxynucleotides which are reverse complementary to the 5 ’-terminal sequence of small non-coding RNA is replaced by a 2’-ortho-methylated ribonucleotide, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2 nucleotides, e.g. 2, 3, or 4, at its 3 ’end are ribonucleotides or modified ribonucleotides and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced.
  • the LNA enhanced sequence comprises between 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 3, locked nucleotides, specifically ribonucleotides.
  • nucleotide sequence of the 5’ adapter comprises deoxynucleotides and ribonucleotides.
  • the 5’adapter may range from 15 to 60, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, nucleotides in length.
  • the 5’adapter may be present as linear polynucleotide, particularly in single-stranded form, e.g. after denaturation/when denatured.
  • the 5’adapter is a polynucleotide that can be attached/ligated to the 5 ’end of small non-coding RNA.
  • the 5’adapter has a stem-loop structure.
  • the attachment/ligation is possible as the 5’adapter comprises between 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to a 5’-terminal sequence of small non-coding RNA.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a 5’positioned first stem sequence and a 3’positioned second stem sequence that are reverse complementary to each other.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence can form the double stranded stem.
  • the double stranded stem may have a length of between 5 and 20, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, nucleotides.
  • each one of the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence has a length of between 5 to 10, e.g. 5, 6, 7, 8, 9, or 10, nucleotides.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence have the same length, e.g. a length of 5, 6, 7, 8, 9, or 10 nucleotides.
  • the 5 ’positioned first stem sequence and/or the 3 ’positioned second stem sequence is (are) LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides. Examples of locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • the 5’positioned first stem sequence is LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides.
  • locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • every, every second, or every third nucleotide may be LNA enhanced in the 5’positioned first stem sequence and/or the 3’positioned second stem sequence.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise (a mixture of) deoxynucleotides and ribonucleotides (e.g. LNA-enhanced) or the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise ribonucleotides.
  • Said ribonucleotides include ribonucleotides which are LNA-enhanced.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a loop sequence which is located between the 5’positioned first stem sequence and the 3’positioned second stem sequence.
  • the loop sequence may comprise between 10 and 40, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, nucleotides.
  • the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, nucleotides, e.g. deoxynucleotides and/or ribonucleotides. More preferably, the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, deoxynucleotides.
  • the nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem comprises deoxynucleotides with the exception of the at least two nucleotides at its 3 ’end which are ribonucleotides or modified ribonucleotides, preferably 2’-o-methyl ribonucleotides, and the locked ribonucleotides.
  • the 5 ’-terminal sequence is configured such that it forms a single stranded 5 ’protrusion after formation of the stem-loop structure.
  • the 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to the 5 ’-terminal sequence of small noncoding RNA encompass G and C but not more than 4, e.g. 1, 2, 3, or 4, in a row.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a 5 ’positioned first stem sequence and a 3 ’positioned second stem sequence that are reverse complementary to each other.
  • the one or more 2’-ortho-methylated ribonucleotide is located at a position (in said nucleotide sequence) corresponding to a position in the non-coding small RNA molecule that is presumed to be methylated.
  • the one or more 2’-ortho- methylated ribonucleotide is located at a position (in said nucleotide sequence) base-pairing with a position in the non-coding small RNA molecule that is presumed to be methylated.
  • a preferred 5’ adapter (with methylation as well as specific for 28S ribosomal RNA (rRNA) fragment) has the following nucleotide sequence: CACmCGGCGGCCG/idSp/TGGCGTGGAGTGTGTGCTTTGCCArCrG (SEQ ID NO: 14).
  • the 5’adapter as described above comprises a base-lacking spacer (e.g. a base-lacking 1’, 2’-dideoxyribose spacer) in the loop region.
  • a base-lacking spacer e.g. a base-lacking 1’, 2’-dideoxyribose spacer
  • the 5’adapter comprises, for example, in the following order from 5’ to 3’ :
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA, and wherein at least one of said 6 to 15 deoxynucleotides which are reverse complementary to the 5 ’-terminal sequence of small non-coding RNA is replaced by a 2’-ortho-methylated ribonucleotide, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2 nucleotides, e.g. 2, 3, or 4, at its 3 ’end are ribonucleotides or modified ribonucleotides, wherein the nucleotide sequence comprises a base-lacking spacer (e.g. a base-lacking 1’, 2’-dideoxyribose spacer), and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced.
  • base-lacking spacer e.g. a base-lacking 1’, 2’-dideoxyribose spacer
  • the present invention relates to a 3 ’adapter comprising in the following order from 5’ to 3’ :
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein the 5 ’-terminal nucleotide is phosphorylated and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced, and (ii) a 3’terminal nucleotide sequence comprising 6 to 15, e.g.
  • deoxynucleotides deoxynucleotides, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein at least one of said 6 to 15 deoxynucleotides which are reverse complementary to the 3’- terminal sequence of small non-coding RNA is replaced by a 2’-ortho-methylated ribonucleotide, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide.
  • the LNA enhanced sequence comprises between 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 3, locked nucleotides, specifically ribonucleotides.
  • nucleotide sequence of the 3’ adapter comprises deoxynucleotides and ribonucleotides.
  • the 3’adapter may range from about 15 to about 60, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, nucleotides in length.
  • the 3’adapter may be present as linear polynucleotide, particularly in single- stranded form, e.g. after denaturation/when denatured.
  • the 3’adapter is a polynucleotide that can be attached/ligated to the 3 ’end of small non-coding RNA.
  • the 3’adapter has a stem-loop structure.
  • the attachment/ligation is possible as the 3’adapter comprises between 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to a 3’-terminal sequence of small non-coding RNA.
  • the small non-coding RNA is preferably a miRNA or isomiR comprised in miRbase version 22.1.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a 5 ’positioned first stem sequence and a 3 ’positioned second stem sequence that are reverse complementary to each other.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence can form the double stranded stem.
  • the double stranded stem may have a length of between 5 and 20, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, nucleotides.
  • each one of the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence has a length of between 5 to 10, e.g. 5, 6, 7, 8, 9, or 10, nucleotides.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence have the same length, e.g. a length of 5, 6, 7, 8, 9, or 10 nucleotides.
  • the 5 ’positioned first stem sequence and/or the 3 ’positioned second stem sequence is (are) LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides. Examples of locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • the 3 ’positioned second stem sequence is LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides.
  • locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • every, every second, or every third nucleotide may be LNA enhanced in the 5’positioned first stem sequence and/or the 3’positioned second stem sequence.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise (a mixture of) deoxynucleotides and ribonucleotides (e.g. LNA-enhanced) or the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise ribonucleotides.
  • Said ribonucleotides include ribonucleotides which are LNA-enhanced.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a loop sequence which is located between the 5’positioned first stem sequence and the 3’positioned second stem sequence.
  • the loop sequence may comprise between 10 and 40, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, nucleotides.
  • the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, e.g. deoxynucleotides and/or ribonucleotides. More preferably, the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, deoxynucleotides.
  • the 3 ’-terminal sequence is configured such that it forms a single stranded 3 ’protrusion after formation of the stem-loop structure.
  • the 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to the 3 ’-terminal sequence of small noncoding RNA encompass G and C but not more than 4, e.g. 1, 2, 3, or 4, in a row.
  • the inverted deoxynucleotide is inverted dT, dA, dC, or dG.
  • the 3’inverted deoxynucleotide creates a 3’-3’ linkage and, thus, prevents undesired nucleotide synthesis from the 3 ’end of the adapter, e.g. during RT- PCR.
  • the 3’inverted deoxynucleotide protects the sequence from 3’ exonuclease cleavage.
  • the one or more 2’-ortho-methylated ribonucleotide is located at a position (in said nucleotide sequence) corresponding to a position in the non-coding small RNA molecule that is presumed to be methylated.
  • the one or more 2’-ortho- methylated ribonucleotide is located at a position (in said nucleotide sequence) base-pairing with a position in the non-coding small RNA molecule that is presumed to be methylated.
  • the present invention relates to a combination of the 5 ’adapter of the third aspect and the 3 ’adapter of the fourth aspect.
  • the present invention relates to a kit comprising the 5’adpater of the third aspect, the 3 ’adapter of the fourth aspect, and/or the combination of the fifth aspect.
  • the 5’adpater of the third aspect, the 3 ’adapter of the fourth aspect, and/or the combination of the fifth aspect can be used for determining the methylation status of small non-coding RNA to diagnose cancer in a patient/to determine whether a patient suffers from cancer (see first aspect of the present invention).
  • the 5’adpater of the third aspect, the 3 ’adapter of the fourth aspect, and/or the combination of the fifth aspect can also be used in a method for diagnosing cancer in a patient/for determining whether a patient suffers from cancer, wherein said method comprises the step of determining a methylation status of small non-coding RNA in a biological sample obtained from a patient (see second aspect of the present invention).
  • the cancer mentioned above is preferably lung cancer such as early stage lung cancer.
  • the early stage lung cancer is specifically lung cancer of stage I and/or stage II.
  • the present invention relates to a 5 ’adapter comprising in the following order from 5’ to 3’ :
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA, and wherein at least one of said 6 to 15 deoxynucleotides which are reverse complementary to the 5 ’-terminal sequence of small non-coding RNA is LNA-enhanced, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2 nucleotides, e.g. 2, 3, or 4, at its 3 ’end are ribonucleotides or modified ribonucleotides and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced.
  • the LNA enhanced sequence comprises between 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 3, locked nucleotides, specifically ribonucleotides.
  • nucleotide sequence of the 5’ adapter comprises deoxynucleotides and ribonucleotides.
  • the 5’adapter may range from 15 to 60, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, nucleotides in length.
  • the 5 ’adapter may be present as linear polynucleotide, particularly in single- stranded form, e.g. after denaturation/when denatured.
  • the 5 ’adapter is a polynucleotide that can be attached/ligated to the 5 ’end of small non-coding RNA.
  • the 5 ’adapter When attached/ligated to the 5 ’end of small non-coding RNA, the 5 ’adapter has a stem-loop structure.
  • the attachment/ligation is possible as the 5’adapter comprises between 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to a 5’-terminal sequence of small non-coding RNA.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a 5’positioned first stem sequence and a 3’positioned second stem sequence that are reverse complementary to each other.
  • the 5’positioned first stem sequence and the 3’positioned second stem sequence can form the double stranded stem.
  • the double stranded stem may have a length of between 5 and 20, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, nucleotides.
  • each one of the 5’positioned first stem sequence and the 3’positioned second stem sequence has a length of between 5 to 10, e.g. 5, 6, 7, 8, 9, or 10, nucleotides.
  • the 5’positioned first stem sequence and the 3’positioned second stem sequence have the same length, e.g. a length of 5, 6, 7, 8, 9, or 10 nucleotides.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence is (are) LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides. Examples of locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • the 5’positioned first stem sequence is LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides.
  • locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • every, every second, or every third nucleotide may be LNA enhanced in the 5’positioned first stem sequence and/or the 3’positioned second stem sequence.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise (a mixture of) deoxynucleotides and ribonucleotides (e.g. LNA-enhanced) or the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise ribonucleotides.
  • Said ribonucleotides include ribonucleotides which are LNA-enhanced.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a loop sequence which is located between the 5’positioned first stem sequence and the 3’positioned second stem sequence.
  • the loop sequence may comprise between 10 and 40, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, nucleotides.
  • the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, nucleotides, e.g. deoxynucleotides and/or ribonucleotides. More preferably, the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, deoxynucleotides.
  • the nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem comprises deoxynucleotides with the exception of the at least two nucleotides at its 3 ’end which are ribonucleotides or modified ribonucleotides, preferably 2’-o-methyl ribonucleotides, and the locked ribonucleotides.
  • the 5 ’-terminal sequence is configured such that it forms a single stranded 5 ’protrusion after formation of the stem-loop structure.
  • the 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to the 5 ’-terminal sequence of small noncoding RNA encompass G and C but not more than 4, e.g. 1, 2, 3, or 4, in a row.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a 5 ’positioned first stem sequence and a 3 ’positioned second stem sequence that are reverse complementary to each other.
  • the at least one 2’-ortho-methylated ribonucleotide is located at a position (in said nucleotide sequence) corresponding to a position in the non-coding small RNA (molecule) that is presumed to be methylated.
  • the at least one 2’-ortho- methylated ribonucleotide is located at a position (in said nucleotide sequence) base-pairing with a position in the non-coding small RNA (molecule) that is presumed to be methylated.
  • the 5’adapter as described above comprises a base-lacking spacer (e.g. a base-lacking 1’, 2’-dideoxyribose spacer) in the loop region.
  • a base-lacking spacer e.g. a base-lacking 1’, 2’-dideoxyribose spacer
  • the 5’adapter comprises, for example, in the following order from 5’ to 3’ :
  • a 5’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 5 ’-terminal sequence of small non-coding RNA, and wherein at least one of said 6 to 15 deoxynucleotides which are reverse complementary to the 5 ’-terminal sequence of small non-coding RNA is LNA-enhanced, and
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2 nucleotides, e.g. 2, 3, or 4, at its 3 ’end are ribonucleotides or modified ribonucleotides, wherein the nucleotide sequence comprises a base-lacking spacer (e.g. a base-lacking 1’, 2’-dideoxyribose spacer), and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced.
  • the present invention relates to a 3 ’adapter comprising in the following order from 5’ to 3’ :
  • nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein the 5 ’-terminal nucleotide is phosphorylated and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced, and
  • a 3’terminal nucleotide sequence comprising 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides, deoxynucleotides, wherein said 6 to 15 deoxynucleotides are reverse complementary to a 3 ’-terminal sequence of small non-coding RNA, wherein at least one of said 6 to 15 deoxynucleotides which are reverse complementary to the 3’- terminal sequence of small non-coding RNA is LNA-enhanced, and wherein the 3’terminal deoxynucleotide is an inverted deoxynucleotide.
  • the LNA enhanced sequence comprises between 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 3, locked nucleotides, specifically ribonucleotides.
  • nucleotide sequence of the 3’ adapter comprises deoxynucleotides and ribonucleotides.
  • the 3’adapter may range from about 15 to about 60, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, nucleotides in length.
  • the 3’adapter may be present as linear polynucleotide, particularly in single- stranded form, e.g. after denaturation/when denatured.
  • the 3’adapter is a polynucleotide that can be attached/ligated to the 3 ’end of small non-coding RNA.
  • the 3’adapter has a stem-loop structure.
  • the attachment/ligation is possible as the 3’adapter comprises between 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to a 3’-terminal sequence of small non-coding RNA.
  • the small non-coding RNA is preferably a miRNA or isomiR comprised in miRbase version 22.1.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a 5 ’positioned first stem sequence and a 3 ’positioned second stem sequence that are reverse complementary to each other.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence can form the double stranded stem.
  • the double stranded stem may have a length of between 5 and 20, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, nucleotides.
  • each one of the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence has a length of between 5 to 10, e.g. 5, 6, 7, 8, 9, or 10, nucleotides.
  • the 5 ’positioned first stem sequence and the 3 ’positioned second stem sequence have the same length, e.g. a length of 5, 6, 7, 8, 9, or 10 nucleotides.
  • the 5 ’positioned first stem sequence and/or the 3 ’positioned second stem sequence is (are) LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides. Examples of locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • the 3 ’positioned second stem sequence is LNA enhanced.
  • the LNA enhanced sequence comprises between 1 to 5, e.g. 1, 2, 3, 4, or 5, more particularly 3, locked nucleotides, specifically ribonucleotides.
  • locked ribonucleotides are LNA-guanine, LNA-adenosine or LNA-cytosine.
  • every, every second, or every third nucleotide may be LNA enhanced in the 5’positioned first stem sequence and/or the 3’positioned second stem sequence.
  • the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise (a mixture of) deoxynucleotides and ribonucleotides (e.g. LNA-enhanced) or the 5’positioned first stem sequence and/or the 3’positioned second stem sequence may comprise ribonucleotides.
  • Said ribonucleotides include ribonucleotides which are LNA-enhanced.
  • the nucleotide sequence capable of forming a stem-loop structure comprises a loop sequence which is located between the 5’positioned first stem sequence and the 3’positioned second stem sequence.
  • the loop sequence may comprise between 10 and 40, e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, nucleotides.
  • the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, e.g. deoxynucleotides and/or ribonucleotides. More preferably, the loop sequence comprises between 12 and 20, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20, deoxynucleotides.
  • the 3 ’-terminal sequence is configured such that it forms a single stranded 3 ’protrusion after formation of the stem-loop structure.
  • the 6 to 15, e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucleotides which are reverse complementary to the 3 ’-terminal sequence of small noncoding RNA encompass G and C but not more than 4, e.g. 1, 2, 3, or 4, in a row.
  • the inverted deoxynucleotide is inverted dT, dA, dC, or dG.
  • the 3 ’inverted deoxynucleotide creates a 3 ’-3’ linkage and, thus, prevents undesired nucleotide synthesis from the 3 ’end of the adapter, e.g. during RT- PCR.
  • the 3 ’inverted deoxynucleotide protects the sequence from 3’ exonuclease cleavage.
  • the one or more 2’-ortho-methylated ribonucleotide is located at a position (in said nucleotide sequence) corresponding to a position in the non-coding small RNA (molecule) that is presumed to be methylated.
  • the one or more 2’-ortho- methylated ribonucleotide is located at a position (in said nucleotide sequence) base-pairing with a position in the non-coding small RNA (molecule) that is presumed to be methylated.
  • the present invention relates to a combination of the 5 ’adapter of the seventh aspect and the 3 ’adapter of the eighth aspect.
  • the present invention relates to a kit comprising the 5’adpater of the seventh aspect, the 3 ’adapter of the eighth aspect, and/or the combination of the ninth aspect.
  • the 5’adpater of the seventh aspect, the 3 ’adapter of the eighth aspect, and/or the combination of the ninth aspect can be used for determining the methylation status of small noncoding RNA to diagnose cancer in a patient/to determine whether a patient suffers from cancer (see first aspect of the present invention).
  • the 5’adpater of the seventh aspect, the 3 ’adapter of the eighth aspect, and/or the combination of the ninth aspect can also be used in a method for diagnosing cancer in a patient/for determining whether a patient suffers from cancer, wherein said method comprises the step of determining a methylation status of small non-coding RNA in a biological sample obtained from a patient (see second aspect of the present invention).
  • the cancer mentioned above is preferably lung cancer such as early stage lung cancer.
  • the early stage lung cancer is specifically lung cancer of stage I and/or stage II.
  • Figure 1 Shows the rRNA fragment of interest (underlined) in the context of the nucleotide sequence of the 28 S rRNA. Two 2-o-m sites are indicated.
  • Figure 2 Shows the assay specificity as measured on synthetic oligos. Unmethylated oligo percentage decreases in steps of 20%, while the Gm oligo percentage increases in the same way.
  • Figure 3 Shows a Gm4020 28 S rRNA methylation assays on clinical lung cancer samples compared to healthy controls. Controls represent non-cancer disease, whereas CaseLC represents a lung cancer group. P value of two-tailed t-test is indicated above.
  • Figure 4 Shows a Gm4020 28 S rRNA methylation assays on clinical early-stage lung cancer samples compared to healthy controls. Controls represent non-cancer disease, whereas stage la represents patients suffering from early-stage lung cancer. P value of two-tailed t-test is indicated above.
  • Figure 5 Shows linear regression illustrating the correlation between G4020 methylation and dNTP ratio.
  • the X-axis represents the percentage of Gm4020, while the Y-axis displays the ratio of ImM to lOmM dNTPs.
  • Serial dilutions of synthetic miLung#l were utilized to generate the data points, with each point representing a 10% increase in methylated G4020.
  • Figure 6 Shows miLung#l 1-D plot acquired from digital PCR (dPCR) analysis for two patients at two different dNTP concentrations (lOmM, ImM) and in duplicates (Rl, R2). Each dot represents an individual partition within the dPCR reaction, with positive partitions indicated in blue and negative partitions in grey. A threshold line is depicted, separating positive from negative partitions based on fluorescence intensity (RFU). A non-template control (NTC) was included.
  • dPCR digital PCR
  • Figure 7 Shows miLung#l Gm4020 2’-o-methylation in %, as measured by mer-idPCR. The p-value of Student’s t-test are indicated above respective group test against control group.
  • the present inventors could show that the determination of the methylation status of this 28 S rRNA fragment allows lung cancer diagnosis, especially at early stages of lung cancer, e.g. lung cancer of stage I and/or stage II. Thereby, lung cancer diagnosis can be improved. Especially, the determination of the methylation status of this 28S rRNA fragment allows to distinguish between patients suffering from lung cancer, specifically early stages of lung cancer, e.g. lung cancer of stage I and/or stage II, and healthy control subjects.
  • DB-PCR adapters are denatured and renatured such that they form required stem loop-like structures.
  • the source RNA is denatured separately, treated with the polynucleotide kinase (to restore the 5’ phosphate), mixed with adapters and used for the ligation by T4 RNA ligase 2.
  • RT primer aligning to the 3’ adapter is used for the cDNA production under limiting conditions.
  • diluted cDNA is used for the digital PCR with a reverse primer aligning to the 5’ end of first strand cDNA and forward primer complementary to the 3’ end of the first strand cDNA.
  • a TaqMan probe is used for the detection of the specific signal in digital PCR. A ratio-based curve indicative of 2-o-m stoichiometry is calculated.
  • RNA denaturation Denature RNA for 2 min at 70°C
  • Methylation status (%) (1- (copies per pl at ImM dNTP/copies per pl at lOmM dNTP))* 100
  • the assay is specific for the Gm and that the dynamic range of such assay is in the 0-88% range.
  • Peripheral blood from 7 lung cancer patients well as from 9 healthy controls was collected in the PAXgene Blood RNA tubes, respectively.
  • the lung cancer group of 7 lung cancer patients covered a broad spectrum of lung cancer types and stages.
  • the tubes were then used for RNA isolation using automated procedure on QIAsymphony liquid handling station.
  • the RNA was used for the assays as described in the protocol details.
  • RNA samples A significant difference (t-test p-value 0.008) in the methylation status between healthy control subject (mean 82.7%) and lung cancer patient (mean 78.7%) RNA samples was determined (see Figure 3). This makes this assay amenable for the detection of (lung) cancer.
  • a significant difference (t-test p-value 0.05) in the methylation status between healthy control subject (mean 82.7%) and early-stage lung cancer patient (mean 79.2%) RNA samples was determined (see Figure 4). Accordingly, this test can also be used for the detection of early-stage (lung) cancer.
  • rRNA ribosomal RNA
  • RT Primer CTCAGTGCGAATACCTCGGACCCTGCACTGAGGTAGT (SEQ ID NO: 3)
  • TaqMan probe FAM/TGAGGTAGTGGTATTTCACCGGCGGCCGT/BHQ-1/ (SEQ ID NO: 7)
  • rRNA 28S ribosomal RNA
  • the method as described herein was applied to a set of clinical samples with the aim to discriminate between the control, non-cancerous patient samples, and samples coming from patients diagnosed with cancer. Specifically, early stages of lung cancer were detected with the method described herein.
  • the patient characteristics i.e. patient demographics and clinical information
  • PAXgene RNA whole blood tubes PreAnalytiX, Hombrechtikon, Switzerland
  • PAXgene blood samples were inverted 10 times, and then frozen at -20°C within a 2-hour timeframe. For extended preservation, the tubes were relocated to a temperature of -80°C.
  • PAXgene tubes were thawed overnight and RNA was extracted using the PAXgene Blood miRNA Kit (Qiagen, Venlo, Netherlands) on the QIAsymphony SP liquid handling station (Qiagen, Venlo,
  • RNA sample typically, a single ligation for RNA sample was performed, followed by duplicated reverse transcription.
  • Adapter and primer sequences (5’ — > 3’ orientation) used in this example.
  • rRNA 28S ribosomal RNA

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Abstract

La présente invention concerne l'utilisation d'un état de méthylation d'un petit ARN non codant pour diagnostiquer un cancer chez un patient/déterminer si un patient souffre d'un cancer. En outre, la présente invention concerne un procédé permettant de diagnostiquer un cancer chez un patient/déterminer si un patient souffre d'un cancer en déterminant un statut de méthylation d'un petit ARN non codant dans un échantillon biologique.
EP24708223.3A 2023-03-15 2024-03-05 Diagnostic des cancers à partir de l'état de méthylation de petits arn non codants Pending EP4680767A1 (fr)

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CN111197070A (zh) * 2018-11-16 2020-05-26 南京迈西可生物科技有限公司 鉴定rna分子中2`-o-甲基化修饰的方法及其应用
EP4251771A1 (fr) * 2020-11-27 2023-10-04 Centre Léon Bérard La 2'o-méthylation des arn ribosomiques comme nouvelle source de biomarqueurs pertinents pour le diagnostic, le pronostic et la thérapie contre les cancers
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