EP4655417A1 - Pcr en haltère pour la détection de petits arn non codants méthylés - Google Patents

Pcr en haltère pour la détection de petits arn non codants méthylés

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Publication number
EP4655417A1
EP4655417A1 EP23805593.3A EP23805593A EP4655417A1 EP 4655417 A1 EP4655417 A1 EP 4655417A1 EP 23805593 A EP23805593 A EP 23805593A EP 4655417 A1 EP4655417 A1 EP 4655417A1
Authority
EP
European Patent Office
Prior art keywords
sequence
adapter
stem
small non
rna
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
EP23805593.3A
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German (de)
English (en)
Inventor
Rastislav Horos
Alberto Daniel MORENO
Bruno STEINKRAUS
Carla BIEG-SALAZAR
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
Original Assignee
Hummingbird Diagnostics GmbH
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Filing date
Publication date
Application filed by Hummingbird Diagnostics GmbH filed Critical Hummingbird Diagnostics GmbH
Publication of EP4655417A1 publication Critical patent/EP4655417A1/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the present invention relates to the use of a combination of a 5’adapter and a 3’adapter for the detection of methylated small non-coding RNA, and/or for the quantification of a methylation status of small non-coding RNA. Further, the present invention relates to a method of detecting methylated small non-coding RNA. Furthermore, the present invention relates to a method of quantifying a methylation status of small non-coding RNA.
  • 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.
  • a subclass of small non-coding RNAs so called microRNAs (miRNAs) or their variants having specific terminal sequences (isomiRs), possess regulatory functions such as gene expression regulation of protein-encoding messenger RNAs (mRNAs).
  • miRNAs microRNAs
  • iRs protein-encoding messenger RNAs
  • mRNAs messenger RNAs
  • rRNA fragments of ribosomal RNA
  • tRNA transfer RNA
  • snoRNA small nucleolar RNA
  • RNAs can be post-transcriptionally edited by chemical modifications such as methylation, oxidation, pseudouridylation, etc., which can target a nucleotide base (e.g. methyl- 6-adenine, pseudouridine) or a ribose ring (2′-O-m).
  • a nucleotide base e.g. methyl- 6-adenine, pseudouridine
  • a ribose ring (2′-O-m
  • 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.
  • RNA is pre-amplified first, followed by the use of a reverse transcription (RT) primer binding downstream of the 2′-O-m site and performing the RT under limiting conditions.
  • RT reverse transcription
  • concentration of deoxynucleotides (dNTPs) in the RT reaction is reduced, which causes pause or stopping of RT at the 2′-O-m site.
  • a control reaction at normal dNTPs concentration is performed, and a difference of the detection levels of the two reactions is indicative of 2′-O-m presence.
  • This approach cannot be used for small non-coding RNA, specifically small non-coding RNA being between 18 to 50 nucleotides long.
  • the NGS method uses as template a full-length RNA (such as ribosomal RNA) that is first fragmented using alkaline hydrolysis and undergoes ligation to an adapter with known sequence. Because 2′-O-m site affects the ligation efficacy on +1 nucleotide position, an under- representation of the reads starting at +1 nucleotide suggests a presence of 2′-O-m site immediately downstream. Yet, this method also requires that the studied RNA is present at full length, or at least in long fragments that allow stochastic fragmentation to shorter fragments and their subsequent ligations.
  • RNA such as ribosomal RNA
  • DB PCR Dumbbell PCR
  • Second layer of specificity is introduced by a TaqMan probe, that can align to a user-defined region of a cDNA sequence.
  • the present inventors have now adapted this method to limiting reverse transcription (RT) conditions and used it for the detection of methylated small non-coding RNA, and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • RT reverse transcription
  • they performed digital PCR to detect the small non-coding RNAs by DB-PCR.
  • the advantage of digital PCR is the direct absolute quantification of the copy numbers of the RNA of interest in the cDNA and, thus, allows linear quantification of methylation stoichiometry.
  • the present invention relates to the use of a combination of (i) a 5’adapter capable of forming a stem-loop structure containing a loop and a double stranded stem, and (ii) a 3’adapter capable of forming a stem-loop structure containing a loop and a double stranded stem for the detection of methylated small non-coding RNA, and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • the present invention relates to a method of detecting methylated small non-coding RNA (in a sample) comprising the steps of: (i) providing 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, (ii) reverse transcribing the ligation product under limiting conditions, thereby obtaining a first cDNA product and reverse transcribing the ligation product under non-limiting conditions, thereby obtaining a second cDNA product, (iii) amplifying the first cDNA product and the second cDNA product, and (iv) determining a difference between the first cDNA product and the second cDNA product, wherein the difference between the first cDNA product and the second cDNA product indicates the presence of methylated small non-coding RNA (in the sample).
  • the present invention relates to a method of quantifying a methylation status of small non-coding RNA (in a sample) comprising the steps of: (i) carrying out the method of the second aspect, and (ii) determining a ratio between the first cDNA product and the second cDNA product.
  • 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.
  • the term “analog”, as used herein, 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. H+, NH4+, Na+.
  • 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.
  • the modified ribonucleotides are 2’-o-methyl ribonucleotides.
  • 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 bodily 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.
  • 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.
  • various environmental samples for example, agricultural, water, and soil
  • purified samples generally, cultured cells and lysed cells may also be used as samples.
  • 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.
  • 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.
  • miRNA the designation “microRNA” is also possible), as used herein, 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.
  • 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). After Drosha processing, the pri-miRNAs are only 60-100 nucleotides long, and are called precursor miRNAs (pre-miRNAs).
  • the pre-miRNA is exported to the cytoplasm, where it encounters the Dicer enzyme.
  • 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*. It was thought that passenger miRNAs were completely degraded, but deep sequencing studies have found that some minor miRNAs persist and in fact have a functional role in gene regulation.
  • miR-5p/miR-3p a miR-5p/miR-3p nomenclature
  • miR-5p the 5’ arm of the miRNA
  • miR-3p the 3’ arm
  • 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 refers to the uncapitalized “mir-” refers to the pre-miRNA and the pri-miRNA (e.g. hsa-mir-16)
  • MIR refers to the gene that encodes them.
  • literature will often refer to the original miR/miR* names.
  • 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.
  • miRNA 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.
  • 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.
  • 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).
  • ribosomal RNA fragment refers to a fragment derived from a ribosomal RNA (rRNA).
  • Ribosomal RNAs 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.
  • 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.
  • 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 combination of a 5’adapter and 3’adapter is used for the detection of methylated small non-coding RNA (in a sample), and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • 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.
  • 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.
  • LNA locked nucleic acids
  • 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. In one preferred embodiment, the stem is between 5 and 10, e.g.5, 6, 7, 8, 9, or 10, nucleotides in length. It should be noted that a portion of a primer may be encoded in the stem. As a general matter, in those embodiments in which a portion of a primer is encoded in the stem, the stem may be longer. In those embodiments in which a portion of a primer is not encoded in the stem, 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. It should be noted that a portion of a primer may be encoded in the loop. As a general matter, in those embodiments in which a primer is 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).
  • the adapter e.g. 5’adapter and/or 3’adapter, may comprise locked nucleic acids (LNAs).
  • LNAs locked nucleic acids
  • LNAs locked nucleic acids
  • ribonucleotides modified nucleotides, specifically ribonucleotides, in which the 2’-O and 4’-C atoms of the ribose are joined through a methylene bridge. This additional bridge limits the flexibility normally associated with the ring, essentially locking the structure into a rigid conformation.
  • 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.
  • 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.
  • a 3’inverted deoxynucleotide e.g. inverted dT, dA, dC, or dG
  • the methods of the present invention are directed to the detection of methylated small non-coding RNA (in a sample), and/or to the quantification of a methylation status of small non-coding RNA (in a sample). These methods require the annealing and the ligation of adapters to the small non-coding RNA (present in a sample).
  • 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 non- coding 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-3’/5’-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-3’/5’-P-RNA between the 3’end of the small non-coding RNA, and the 5’end of the adapter.
  • This is also an efficient substrate for ligation by a double-stranded RNA ligase.
  • any double stranded RNA ligase capable of ligating double stranded RNA nicks/RNA structures may be used for this purpose.
  • the double stranded RNA ligase is a T4 RNA ligase 2 (Rnl2) or a Kod1 ligase.
  • the double stranded RNA ligase is a T4 RNA ligase 2 (Rnl2).
  • the conditions of the ligation reaction are typically adjusted so that the ligase functions near its optimal activity level.
  • 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.
  • a 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).
  • 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 is Maxima H-RT, Tth polymerase, Protoscript II RT, or Luna RT.
  • 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 non-limiting conditions (control reaction).
  • test reaction test reaction
  • control reaction 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, 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.
  • 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, 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, or 1/50, of the dNTP concentration under non-limiting (normal) conditions.
  • 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, 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
  • said reverse transcription of the ligation product is conducted with between 20 ⁇ M and 50 ⁇ M dNTPs, e.g. with 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 ⁇ M dNTPs. Still even more preferably, said reverse transcription of the ligation product is conducted with between 20 ⁇ M and 30 ⁇ M dNTPs, e.g. with 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ⁇ M dNTPs. Most preferably, said reverse transcription of the ligation product is conducted with 25 ⁇ M dNTPs.
  • the reverse transcription of the ligation product “under non-limiting (normal) conditions” is preferably conducted with between 200 ⁇ M and 500 ⁇ M dNTPs, more preferably with between 200 ⁇ M and 300 ⁇ M dNTPs, and even more preferably with 250 ⁇ M dNTPs.
  • the reverse transcription reaction under limiting conditions (test reaction) is specifically performed with 25 ⁇ M dNTPs and the reverse transcription reaction under non-limiting (normal) conditions (control reaction) is specifically performed with 250 ⁇ M dNTPs.
  • the reverse transcriptase is preferably Maxima RT.
  • said reverse transcription of the ligation product is conducted with between 500 ⁇ M and 1200 ⁇ M dNTPs, e.g. with 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 ⁇ M dNTPs. Still even more preferably, said reverse transcription of the ligation product is conducted with between 800 ⁇ M and 1100 ⁇ M dNTPs, e.g. with 800, 850, 900, 950, 1000, 1050, or 1100 ⁇ M dNTPs. Most preferably, said reverse transcription of the ligation product is conducted with 1000 ⁇ M dNTPs.
  • the reverse transcription of the ligation product “under non-limiting (normal) conditions” is, in an alternative, preferably conducted with between 5000 ⁇ M and 12000 ⁇ M dNTPs, more preferably with between 8000 ⁇ M and 11000 ⁇ M dNTPs, and even more preferably with 10000 ⁇ M dNTPs.
  • the reverse transcription reaction under limiting conditions (test reaction) is specifically performed with 1000 ⁇ M dNTPs
  • the reverse transcription reaction under non-limiting (normal) conditions (control reaction) is specifically performed with 10000 ⁇ M dNTPs.
  • the reverse transcriptase is preferably 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 non-limiting 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 at low concentrations of dNTPs (i.e. under limiting conditions) and high concentrations of dNTPs (i.e. under non-limiting conditions) indicates the presence of methylated small non-coding RNA (in a sample).
  • the cDNA product produced with the reverse transcription at low dNTP concentrations is designated as first cDNA product.
  • the cDNA product produced with said reverse transcription at high dNTP concentrations is designated as second cDNA product.
  • methylated small non-coding RNA in a sample
  • a methylation status of mall non-coding RNA in a sample
  • the cDNA product and consequently the small non-coding RNA based thereon
  • the amplification is carried out using a polymerase chain reaction (PCR).
  • 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.
  • RNA transcription- based systems e.g., TAS, 3SR
  • RCA rolling circle amplification
  • a base e.g.
  • the methylation of small non-coding RNA occurring at a base is preferably selected from the group consisting of 6-methyladenosine (m6A), 5-methylcytidine (m5C), 5-methyluridine (m5U), 3-methyluridine (m3U), 1-methyladenosine (m1A), and 1-methylguanosine (m1G), 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.
  • the method of the present invention allows the “detection of methylated small non- coding RNA”.
  • 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.
  • the cDNA product produced with said reverse transcription at low dNTP concentrations (i.e. under limiting conditions) is designated as first cDNA product.
  • the cDNA product produced with said reverse transcription at high dNTP concentrations (i.e. under non-limiting conditions) is designated as second cDNA product.
  • a difference between the cDNA products produced via reverse transcription at low concentrations of dNTPs (i.e. under limiting conditions) and high concentrations of dNTPs (i.e. under non-limiting conditions) indicates the presence of methylated small non-coding RNA (in the sample). If there is no (significant) difference, 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.
  • the term “level”, as used herein, also comprises scaled, normalized, or scaled and normalized amounts or values (e.g. RPM). Particularly, 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.
  • PCR polymerase chain reaction
  • mass spectrometry mass spectrometry
  • flow cytometry 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 (i) cycle thresholds in case of semiquantitative PCR reaction, or (ii) copies per ⁇ l in case of digital PCR such as digital droplet PCR or digital partition PCR.
  • the method of the present invention further allows the “quantification of a methylation status of small non-coding RNA”. In this case, the difference between the first cDNA product and the second cDNA product is quantified (and not only determined). Therefore, a ratio between the first cDNA product and the second cDNA product is determined.
  • methylation ratio copies with a dNTP concentration under limiting conditions / copies with a dNTP concentration under non-limiting conditions, wherein ratio low ⁇ high methylation, and ratio low methylation.
  • dNTP concentration under limiting conditions is 1/10 or less, e.g.
  • methylation ratio copies at 25 ⁇ M dNTP (limiting conditions) / copies at 250 ⁇ M dNTP (non-limiting conditions), wherein ratio low ⁇ high methylation, and ratio high ⁇ low methylation.
  • the ratio between the first cDNA product and the second cDNA product is determined/calculated by determining the level (e.g. the number of copies) of the first cDNA product and the level (e.g. the number of copies) of the second cDNA product, and determining a ratio between the level (e.g. the number of copies) of the first cDNA product and the level (e.g.
  • a ratio of lower than 0.5 indicates a high degree of small non-coding RNA methylation
  • a ratio of higher than or equal to 0.5 indicates a low degree of small non-coding RNA methylation (in the sample).
  • methylation (%) (1- (copies per ⁇ l at a dNTP concentration under limiting conditions / copies per ⁇ l at a dNTP concentration under non-limiting conditions)) * 100.
  • the dNTP concentration under limiting conditions is 1/10 or less, e.g.
  • an exact % methylation (status/degree) can be determined using a calibration curve (as shown in Figure 16).
  • low degree of small non-coding RNA methylation means that between 0% and 50%, e.g.0, 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%, of the small non-coding RNA (molecules) in a sample is (are) methylated.
  • high degree of small non-coding RNA methylation means that between more than 50% and 100%, e.g.
  • RNA methylation may mean no methylation (i.e.
  • methylation of 0% or no small non-coding RNA entities in a sample are methylated).
  • a high degree of small non-coding RNA methylation may mean complete methylation (i.e. methylation of 100% or all small non-coding RNA entities in a sample are methylated).
  • quantifying the methylation status means estimating the ratio of small non-coding RNA (molecules) which are methylated in a sample. Specifically, the status of methylation of small non-coding RNA (molecules) ranges between 0 (i.e. fully methylated, or all detected RNA entities are fully methylated) and 1 (i.e. not methylated, or all detected RNA entities are not methylated).
  • 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::needle, 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 bodily 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.
  • 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 comprises biological material.
  • 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.
  • 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.
  • 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 ⁇ l 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.
  • DB PCR Dumbbell PCR
  • Rnl2 RNA ligase 2
  • Second layer of specificity is introduced by a TaqMan probe, that can align to a user-defined region of a cDNA sequence.
  • the present inventors have now adapted this method to limiting reverse transcription (RT) conditions and used it for the detection of methylated small non-coding RNA (in a sample), and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • RT reverse transcription
  • they performed digital PCR to detect the small non-coding RNAs by DB- PCR.
  • the present invention relates to the use of a combination of (i) a 5’adapter capable of forming a stem-loop structure containing a loop and a double stranded stem, and (ii) a 3’adapter capable of forming a stem-loop structure containing a loop and a double stranded stem for the detection of methylated small non-coding RNA, and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • 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.
  • the 5’adapter comprises in the following order from 5’ to 3’: (i) 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 (ii) a nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g.
  • nucleotides at its 3’end are ribonucleotides or modified ribonucleotides and wherein the nucleotide sequence is optionally locked nucleotide- (LNA-) enhanced.
  • LNA- locked nucleotide-
  • 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.
  • the 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 small non-coding RNA is preferably a miRNA or an isomiR comprised in/part of 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 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.
  • 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 non- coding 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’: (i) 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 (ii) a nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g.
  • nucleotides at its 3’end are ribonucleotides or modified ribonucleotides and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced.
  • 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 nucleotides.
  • the above described 5’adapter comprises in the following order from 5’ to 3’: (i) 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/or otherwise modified, and (ii) a nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g.
  • 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 (ib) 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-en
  • nucleotides at its 3’end are ribonucleotides or modified ribonucleotides.
  • the above described 5’adapter comprises in the following order from 5’ to 3’: (i) 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/or otherwise modified, and (ii) a nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2, e.g.
  • nucleotides at its 3’end are ribonucleotides or modified ribonucleotide and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced.
  • 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 (ib) 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
  • nucleotides at its 3’end are ribonucleotides or modified ribonucleotide and wherein the nucleotide sequence is locked nucleotide- (LNA-) enhanced.
  • LNA- locked nucleotide-
  • 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’: (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 (ib) 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 (ic) a 5’terminal nucleotide sequence comprising 6 to
  • 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 (ib) 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’-
  • 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 non-coding small RNA (molecule) that is presumed to be methylated.
  • 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 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. For example, 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. Specifically, 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).
  • 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/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 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
  • the 5’adapter comprising a base-lacking spacer 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/TGGCGTGGAGTGTGTGCTTTGCCArCrG (SEQ ID NO: 13), 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: 13.
  • Such a 5’adapter variant still comprises the at least 2 nucleotides at its 3’end which are ribonucleotides or modified ribonucleotides. Further, 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. It is preferred that, in the 5’adapter having a nucleotide sequence according to SEQ ID NO: 1, 6, or 13 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.
  • a base-lacking spacer e.g. a base-lacking 1’, 2’-dideoxyribose spacer
  • 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 3’adapter comprises in the following order from 5’ to 3’: (i) a 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.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.
  • the 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 When 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.
  • 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 non- coding 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’: (i) a 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 (ii) 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’: (i) a 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 (ii) 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/or 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’: (i) a 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 (iia) 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 (iib) a 3’terminal nucleotide sequence comprising 6 to 15, e.g.6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, deoxynucle
  • the above described 3’adapter comprises in the following order from 5’ to 3’: (i) a 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 (ii) 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/or 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’: (i) a 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 (iia) 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 (iib) a 3’terminal nucleotide sequence comprising
  • 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’: (i) a 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 (iia) 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 deoxynucleo
  • the above described 3’adapter comprises in the following order from 5’ to 3’: (i) a 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 (iia) 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 (iib) a 3’terminal nucleotide sequence comprising
  • 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 non-coding small RNA (molecule) that is presumed to be methylated.
  • 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 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.1, 2, or 3) of the 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).
  • 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 stem-loop 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.
  • 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.
  • 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. When 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. In this way, 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. Specifically, the combination of said adapters allows for the detection of methylation of small non-coding RNA, and/or for the quantification of a/the methylation status of small non-coding RNA (in a sample).
  • 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 (m6A), 5-methylcytidine (m5C), 5-methyluridine (m5U), 3-methyluridine (m3U), 1-methyladenosine (m1A), and 1-methylguanosine (m1G), 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.
  • the sample is preferably a biological sample.
  • the sample may also be a processed sample which is originated from a biological sample.
  • the sample may also be a processed sample which has its origin in a biological sample.
  • the biological sample may be any sample having a 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.
  • 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.
  • CSF cerebrospinal fluid
  • cerumen earwax
  • gastric juice sample mucus sample
  • lymph sample endolymph fluid sample
  • perilymph fluid sample perilymph fluid sample
  • peritoneal fluid sample pleural fluid sample
  • saliva sample sebum (skin oil) sample
  • 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 biological sample is a blood 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. 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 ⁇ l 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.
  • the 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
  • 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 invention relates to a (an in vitro) method of detecting methylated small non-coding RNA (in a sample) comprising the steps of: (i) providing 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, (ii) reverse transcribing the ligation product under limiting conditions, thereby obtaining a first cDNA product and reverse transcribing the ligation product under non-limiting conditions, thereby obtaining a second cDNA product, (iii) amplifying the first cDNA product and the second cDNA product, and (iv) determining a difference between the first cDNA product and the second cDNA product, wherein the difference between the first cDNA product and the second cDNA product indicates the presence of methylated small non-coding RNA (in the sample).
  • 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 (m6A), 5-methylcytidine (m5C), 5-methyluridine (m5U), 3-methyluridine (m3U), 1-methyladenosine (m1A), and 1-methylguanosine (m1G), 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.
  • the determination of small non-coding RNA methylation requires a ligation product comprising the 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.
  • 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 (i) providing a composition comprising denatured small non-coding 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 non-coding RNA, and (ii) ligating the 5’adapter and the 3’adapter to the small non-coding RNA using/with a double stranded RNA ligase.
  • the denatured small non-coding RNA is produced by heating the small non-coding 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 small non-coding RNA is immediately placed on ice after denaturation.
  • the small non-coding RNA is preferably given to an aqueous solution, e.g. water, or to a buffer solution. It is particularly preferred that the small non-coding RNA is treated after the denaturation with a polynucleotide kinase (to restore or introduce the 5’ phosphate).
  • a denaturation and a renaturation step is required so that they can from a stem-loop structure which allows annealing to the small non- coding RNA. Annealing is a process of heating and cooling adapters with complementary sequences.
  • the 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 of the small non-coding RNA.
  • the adapters, in particular 5’ 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 0.1°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 0.1°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.
  • the above-mentioned composition i.e.
  • the composition comprising denatured small non-coding 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 non-coding RNA, is preferably produced by mixing the denatured small non-coding RNA, the renatured 5’adapter as defined in the first aspect, and the renatured 3’adapter as defined in the first aspect with each other, thereby annealing the 5’adapter and the 3’adapter to the small non-coding 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-3’/5’-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-3’/5’-P-RNA between the 3’end of the small non-coding RNA and the 5’end of the adapter.
  • the 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) or a Kod1 ligase.
  • Rnl2 T4 RNA ligase 2
  • a Kod1 ligase it should be noted that only a perfectly hybridized molecule provides a substrate for the double stranded RNA ligase, in particular Rnl2.
  • the Rnl2 will ligate the molecule with much lower efficiency.
  • 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.
  • 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.
  • 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).
  • 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 (iia) annealing a primer for reverse transcription (RT-primer) with the ligation product, and (iib) reverse transcribing the ligation product by using a reverse transcriptase (RT).
  • RT-primer primer for reverse transcription
  • RT reverse transcriptase
  • 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.
  • 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.
  • said reverse transcribing of the ligation product by using a reverse transcriptase (RT) in step (iib) is carried out at between 40°C and 65°C, e.g.40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65°C, preferably at 50°C, 55°C, 58°C, or 62°C, for between 10 and 40 minutes, 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, 40 minutes, preferably 15 or 30 minutes, and subsequently at between 75°C
  • the reverse transcriptase (RT) is Maxima H-RT or Tth polymerase.
  • said reverse transcribing of the ligation product by using a reverse transcriptase (RT) in step (iib) is carried out at between 40°C and 65°C, e.g.40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65°C, preferably at 50°C, for between 40 and 80 minutes, e.g.
  • the reverse transcriptase (RT) is Luna RT.
  • the reverse transcriptase (RT) can be selected from the group consisting of Maxima H-RT, Tth polymerase, Protoscript II RT, and Luna RT for the above method.
  • 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 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’: CTCAGTGCGAATACCTCGGACCCT (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 RT-primer has the following sequence from 5’ to 3’: CTCAGTGCGAATACCTCGGACCCTGCACTGAGGTAGT (SEQ ID NO: 18) 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 RT-primer has the following sequence from 5’ to 3’: ACCTCGGACCCTGCACTGAGGTAGT (SEQ ID NO: 19) 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/CTCAGTGCAGGGTCCGAGGTATTCGCACTGAG(6-15x)N/3InvdT/ (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, SEQ ID NO: 18, or SEQ ID NO: 19.
  • 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. Maxima H-RT, Tth polymerase, Protoscript II RT, or Luna RT.
  • RT reverse transcriptase
  • the skilled person can readily assess whether a RT primer variant is still capable of binding the 3’adapter sequence and allowing reverse transcription.
  • the experimental section provides sufficient information in this respect.
  • 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.
  • 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, 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, or 1/50, of the dNTP concentration under non-limiting conditions.
  • 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, 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,
  • said reverse transcription of the ligation product is conducted with between 20 ⁇ M and 50 ⁇ M dNTPs, e.g. with 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 ⁇ M dNTPs. Still even more preferably, said reverse transcription of the ligation product is conducted with between 20 ⁇ M and 30 ⁇ M dNTPs, e.g. with 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ⁇ M dNTPs. Most preferably, said reverse transcription of the ligation product is conducted with 25 ⁇ M dNTPs.
  • the reverse transcription of the ligation product “under non-limiting conditions” is preferably conducted with between 200 ⁇ M and 500 ⁇ M dNTPs, more preferably with between 200 ⁇ M and 300 ⁇ M dNTPs, and even more preferably with 250 ⁇ M dNTPs.
  • the reverse transcription reaction under limiting conditions (test reaction) is specifically performed with 25 ⁇ M dNTPs and the reverse transcription reaction under non-limiting conditions (control reaction) is specifically performed with 250 ⁇ M dNTPs.
  • the reverse transcriptase is preferably Maxima RT.
  • said reverse transcription of the ligation product is conducted with between 500 ⁇ M and 1200 ⁇ M dNTPs, e.g. with 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 ⁇ M dNTPs. Still even more preferably, said reverse transcription of the ligation product is conducted with between 800 ⁇ M and 1100 ⁇ M dNTPs, e.g. with 800, 850, 900, 950, 1000, 1050, or 1100 ⁇ M dNTPs. Most preferably, said reverse transcription of the ligation product is conducted with 1000 ⁇ M dNTPs.
  • the reverse transcription of the ligation product “under non-limiting (normal) conditions” is, in an alternative, preferably conducted with between 5000 ⁇ M and 12000 ⁇ M dNTPs, more preferably with between 8000 ⁇ M and 11000 ⁇ M dNTPs, and even more preferably with 10000 ⁇ M dNTPs.
  • the reverse transcription reaction under limiting conditions (test reaction) is specifically performed, in the alternative, with 1000 ⁇ M dNTPs and the reverse transcription reaction under non-limiting (normal) conditions (control reaction) is specifically performed with 10000 ⁇ M dNTPs.
  • the reverse transcriptase is preferably Luna RT.
  • the cDNA products derived therefrom have to be amplified in step (iii) of the method of the second aspect.
  • 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).
  • a Forward primer having the following sequence from 5’ to 3’: TGGAGTGTGTGCTTTGCCACG (SEQ ID NO: 4) or with a variant of this sequence
  • 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).
  • 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. over a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or of 21 nucleotides, the same sequence as the 5’adapter.
  • 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 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: /6-FAM/TGAGGTAGTGGT ATTTCACCGGCGGCCGT /BHQ-1/ (SEQ ID NO: 7).
  • step (iv) of the method of the second aspect a difference between the first cDNA product and the second cDNA product is determined, wherein a difference between the first cDNA product and the second cDNA product indicates the presence of methylated small non- coding RNA (in the sample). If there is no difference, the small non-coding RNA (in the sample) is not methylated.
  • the difference is significant.
  • 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 may be 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 ⁇ l, or cycle thresholds).
  • the difference between the first cDNA product and the second cDNA product is determined by comparing the first cDNA product with the second cDNA product.
  • the difference between the first cDNA product and the second cDNA product is determined/calculated by determining the level, preferably the number of copies, of the first cDNA product and the level, preferably the number of copies, of the second cDNA product, and comparing the level, preferably the number of copies, of the first cDNA product with the level, preferably the number of copies, of the second cDNA product, wherein a lower level, preferably a lower number of copies, of the first cDNA product compared to the level, preferably the number of copies, of the second cDNA product indicates the methylation of the small non-coding RNA (in the sample).
  • the difference is based on a difference in expression values determined by (i) cycle thresholds in case of semiquantitative PCR reaction, (ii) copies per ⁇ l in case digital droplet PCR or digital partition PCR.
  • the small non-coding RNA analyzed (in a 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.
  • 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 invention relates to a method of quantifying a/the methylation status of small non-coding RNA (in a sample) comprising the steps of: (i) carrying out the method of the second aspect, and (ii) determining a ratio between the first cDNA product and the second cDNA product.
  • a/the methylation status of small non-coding RNA is quantified (and not only determined).
  • the dNTP concentration under limiting conditions is 1/10 or less, e.g.
  • methylation ratio copies 25 ⁇ M dNTP (limiting conditions) / copies 250 ⁇ M dNTP (non- limiting conditions), wherein ratio low ⁇ high methylation, and ratio low methylation.
  • methylation ratio copies 1000 ⁇ M dNTP (limiting conditions) / copies 10000 ⁇ M dNTP (non- limiting conditions), wherein ratio low ⁇ high methylation, and ratio low methylation.
  • the ratio between the first cDNA product and the second cDNA product is determined by determining the level (e.g. the number of copies) of the first cDNA product and the level (e.g.
  • a ratio between the level (e.g. the number of copies) of the first cDNA product and the level (e.g. the number of copies) of the second cDNA product wherein a ratio of lower than 0.5 (e.g.0, 0.1, 0.2, 0.3, or 0.4) indicates a high degree of small non-coding RNA methylation, and wherein a ratio of higher or equal to 0.5 (e.g.0.5, 0.6, 0.7, 0.8, 0.9, or 1) indicates a low degree of small non-coding RNA methylation (in the sample).
  • a low degree of small non-coding RNA methylation preferably means that between 0% and 50%, e.g.0, 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%, of the small non-coding RNA (molecules) in a sample are methylated.
  • a high degree of small non-coding RNA methylation preferably means that between more than 50% and 100%, e.g.
  • methylation (%) (1- (copies per ⁇ l at a dNTP concentration under limiting conditions / copies per ⁇ l at a dNTP concentration under non-limiting conditions)) * 100.
  • the dNTP concentration under limiting conditions is 1/10 or less, e.g.
  • an exact % methylation (status/degree) can be determined using a calibration curve (as shown in Figure 16).
  • a low degree of small non-coding RNA methylation may mean no methylation (i.e. methylation of 0% or no small non-coding RNA entities in a sample are methylated).
  • a high degree of small non-coding RNA methylation may mean complete methylation (i.e. methylation of 100% or all small non-coding RNA entities in a sample are methylated).
  • the status of methylation of small non-coding RNA (molecules) ranges between 0 (i.e. fully methylated, or all detected RNA entities are fully methylated) and 1 (i.e. not methylated, or all detected RNA entities are not methylated).
  • the above-mentioned level may be 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 ⁇ l, or cycle thresholds).
  • concentration e.g. absolute or relative concentration, e.g. reads per million (RPM), NGS counts, copies per ⁇ l, or cycle thresholds.
  • the small non-coding RNA analyzed in the third 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.
  • 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 sample is preferably a biological sample.
  • the sample may also be a processed sample which is originated from a biological sample.
  • the sample may also be a processed sample which has its origin in a biological sample.
  • the biological sample may be any sample having a 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.
  • 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.
  • CSF cerebrospinal fluid
  • cerumen earwax
  • gastric juice sample mucus sample
  • lymph sample endolymph fluid sample
  • perilymph fluid sample perilymph fluid sample
  • 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 biological sample is a blood 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. 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 ⁇ l 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.
  • the sample 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
  • 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 methods according to the second and/or third 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.
  • DB-PCR adapters are denaturated and renaturated so that they form the required stem loop-like structures.
  • the source RNA is denaturated separately, treated with the polynucleotide kinase (to restore the 5'phosphate), mixed with adapters and used for the ligation by T4 RNA ligase 2.
  • a RT primer aligning to the 3' adapter is used for the cDNA production under limiting conditions and non-limiting conditions.
  • diluted cDNA is used for 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.
  • the ratio is calculated from: cDNA level under limiting conditions / cDNA level under non-limiting conditions. This ratio is indicative for the methylation status. Subsequently, a ratio-based curve indicative of 2-o-m stoichiometry can be calculated.
  • a combination comprising a 5’adapter having a nucleotide sequence according to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, or SEQ ID NO: 17, and a 3’adapter having a nucleotide sequence according to SEQ ID NO: 12 is used for the detection of methylated small non-coding RNA, and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • a combination comprising a 5’adapter having a nucleotide sequence according to SEQ ID NO: 17, and a 3’adapter having a nucleotide sequence according to SEQ ID NO: 12 is used for the detection of methylated small non-coding RNA, and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • the above combinations can specifically be used to study the methylation profile of the 28S rRNA fragment.
  • the methylation/methylation status of this fragment is obvious from SEQ ID NO: 8 (no methylation), SEQ ID NO: 9 (methylation status: Cm4032), SEQ ID NO: 14 (methylation status: Gm4020), and SEQ ID NO: 15 (methylation status: Gm4020 and Cm4032).
  • SEQ ID NO: 8 no methylation
  • SEQ ID NO: 9 methylation status: Cm4032
  • SEQ ID NO: 14 methylation status: Gm4020
  • SEQ ID NO: 15 methylation status: Gm4020 and Cm4032.
  • the methylation/methylation status of the site Gm4020 of the 28S rRNA fragment which is more variable in comparison to the site Cm4032 of the 28S rRNA fragment, can be detected/quantified with the above combination (see also Figure 8).
  • the method of detecting methylated small non-coding RNA comprises the steps of: (i) providing a ligation product comprising small non-coding RNA to which a 5’adapter having a nucleotide sequence according to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, or SEQ ID NO: 17 and a 3’adapter having a nucleotide sequence according to SEQ ID NO: 12 are ligated, (ii) reverse transcribing the ligation product under limiting conditions, thereby obtaining a first cDNA product and reverse transcribing the ligation product under non-limiting conditions, thereby obtaining a second cDNA product, (iii) amplifying the first cDNA product and the second cDNA product, and (iv) determining a difference between the first cDNA product and the second cDNA product, wherein the difference between the first cDNA product and the second cDNA product and the second cDNA product
  • RT reverse transcriptase
  • Maxima H-RT Tth polymerase, Protoscript II RT, or Luna RT
  • the reverse transcriptase (RT) is Maxima H-RT or Luna RT.
  • primer for reverse transcription RT-primer
  • an RT primer having a nucleotide sequence according to SEQ ID NO: 3, SEQ ID NO: 18, or SEQ ID NO: 19 can be used.
  • the reverse transcription (RT-primer) has a nucleotide sequence according to SEQ ID NO: 18.
  • the reverse transcriptase (RT) is Luna RT and the reverse transcription (RT- primer) has a nucleotide sequence according to SEQ ID NO: 18.
  • the method of detecting methylated small non-coding RNA comprises the steps of: (i) providing a ligation product comprising small non-coding RNA to which a 5’adapter having a nucleotide sequence according to SEQ ID NO: 17 and a 3’adapter having a nucleotide sequence according to SEQ ID NO: 12 are ligated, (ii) reverse transcribing the ligation product under limiting conditions, thereby obtaining a first cDNA product and reverse transcribing the ligation product under non-limiting conditions, thereby obtaining a second cDNA product, (iii) amplifying the first cDNA product and the second cDNA product, and (iv) determining a difference between the first cDNA product and the second cDNA product, wherein the difference between the first cDNA product and the second cDNA product indicates the presence of methylated small non-coding RNA (in
  • the reverse transcription of the ligation product is further carried out by (iia) annealing an RT-primer having a nucleotide sequence according to SEQ ID NO: 18 with the ligation product, and (iib) reverse transcribing the ligation product by using the reverse transcriptase (RT) Luna.
  • the above method can specifically be used to study the methylation profile of the 28S rRNA fragment. The methylation/methylation status of this fragment is obvious from SEQ ID NO: 8 (no methylation), SEQ ID NO: 9 (methylation status: Cm4032), SEQ ID NO: 14 (methylation status: Gm4020), and SEQ ID NO: 15 (methylation status: Gm4020 and Cm4032).
  • the methylation/methylation status of the site Gm4020 of the 28S rRNA fragment which is more variable in comparison to the site Cm4032 of the 28S rRNA fragment, can be detected/quantified in the above method (see also Figure 8).
  • the reverse transcription reaction under limiting conditions (test reaction) is ideally performed with 1000 ⁇ M dNTPs and the reverse transcription reaction under non-limiting (normal) conditions (control reaction) is ideally performed with 10000 ⁇ M dNTPs.
  • the present invention relates to a 5’adapter comprising in the following order from 5’ to 3’: (i) 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 (ii) a nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2 nucleotides, e.g.
  • the 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.
  • 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.
  • 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 non- coding 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.
  • the 5’adapter comprises, for example, in the following order from 5’ to 3’: (i) 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 (ii) a nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2 nucleotides, e.g.
  • 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
  • LNA- locked nucleotide-
  • the present invention relates to a 3’adapter comprising in the following order from 5’ to 3’: (i) a 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.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 replaced by a 2’-ortho- methyl
  • 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.
  • the 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 When 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.
  • 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 non- coding 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 fourth aspect and the 3’adapter of the fifth aspect.
  • the present invention relates to a kit comprising the 5’adpater of the fourth aspect, the 3’adapter of the fifth aspect, and/or the combination of the sixth aspect.
  • the 5’adpater of the fourth aspect, the 3’adapter of the fifth aspect, and/or the combination of the sixth aspect can be used for the detection of methylated small non-coding RNA, and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • the 5’adpater of the fourth aspect, the 3’adapter of the fifth aspect, and/or the combination of the sixth aspect can also be used in a method for detecting methylated small non-coding RNA (in a sample) or in a method for quantifying a methylation status of small non-coding RNA (in a sample).
  • the small non-coding RNA referred to in the fourth or fifth 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.
  • 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.
  • a miRNA 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 invention relates to a 5’adapter comprising in the following order from 5’ to 3’: (i) 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 (ii) a nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2 nucleotides, e.g.
  • the 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.
  • 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.
  • 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 non- coding 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.
  • the 5’adapter comprises, for example, in the following order from 5’ to 3’: (i) 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 (ii) a nucleotide sequence capable of forming a stem-loop structure containing a loop and a double stranded stem, wherein at least 2 nucleotides, e.g.
  • 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
  • LNA- locked nucleotide-
  • the present invention relates to a 3’adapter comprising in the following order from 5’ to 3’: (i) a 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.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
  • 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.
  • the 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 When 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.
  • 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 non- coding 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 eights aspect and the 3’adapter of the ninth aspect.
  • the present invention relates to a kit comprising the 5’adpater of the eighth aspect, the 3’adapter of the ninth aspect, and/or the combination of the tenth aspect.
  • the 5’adpater of the eighth aspect, the 3’adapter of the ninth aspect, and/or the combination of the tenth aspect can be used for the detection of methylated small non-coding RNA, and/or for the quantification of a methylation status of small non-coding RNA (in a sample).
  • the 5’adpater of the eighth aspect, the 3’adapter of the ninth aspect, and/or the combination of the tenth aspect can also be used in a method for detecting methylated small non-coding RNA (in a sample) or in a method for quantifying a methylation status of small non-coding RNA (in a sample).
  • the small non-coding RNA referred to in the eighth or ninth 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.
  • 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.
  • miRNA miRNA
  • rRNA fragment ribosomal RNA fragment
  • tRF transfer RNA fragment
  • snorRNA small nucleolar RNA
  • Figure 1 Schematic hybrid structure of 5’adapter, 3’adapter and the 28S rRNA fragment.
  • the 28S rRNA fragment sequence is underlined, wherein the nucleotide in bold represents a methylated Cytosine (Cm4032) present in the methylated positive control.
  • Figure 4 dNTP gradient for the un-methylated negative control and a methylated positive control as measured on the dPCR.
  • Figure 5 dNTP gradient for un-methylated negative control and methylated positive control. Ratios were calculated using appropriate value divided by 250 ⁇ M value.
  • Figure 6 Linearity of 2-o-m detection. Decreasing percentage of un-methylated negative control results in lower ratio values at 25 ⁇ M dNTPs. Ratios were calculated by dividing 25 ⁇ M value by 250 ⁇ M value.
  • Figure 7 Assay results for 12 clinical samples indicated by ratios of 25 ⁇ M/250 ⁇ M.
  • Figure 8 Schematic hybrid structure of 5’adapter, 3’adapter and the 28S miLung#1 fragment.
  • Figure 10 dNTP gradients of the mGmC miLung#1 synthetic with all three RT primers. This assay was performed with a 5’ adapter containing a spacer (see Figure 8) and Maxima H Minus RT.
  • Figure 11 Detection of 2-o-m using different 5’adapters. Synthetic mGC of miLung#1 was added to a background of unmethylated (GC) fragment. This assay was performed with RT2 primer and Maxima H Minus RT. Ratios were calculated using 25/250 ⁇ M.
  • Figure 12 Detection of 2-o-m using different RT enzymes. Synthetic mGC of miLung#1 was added to a background of unmethylated (GC) fragment.
  • Figure 16 Dilution curve of Gm in miLung#1. dNTP ratios are plotted against % of Gm. Linearity of the detection was calculated at 0.973.
  • Figure 17 Methylation percentage in HCC-827 wildtype and SNORD102 knockout cell lines. Methylation percentages were calculated using the linear regression ( Figure 16).
  • Figure 18 Assay results for 16 clinical samples. Methylation percentages were calculated using the linear regression ( Figure 16).
  • Figure 19 Expression of miLung#1 in copies/ ⁇ l for three PAXgene RNA samples using two different protocols (original and improved). EXAMPLES The examples given below are for illustrative purposes only and do not limit the invention described above in any way.
  • EXAMPLE 1 A) Protocol Details The following process steps were carried out by the present inventors: 1. Adapter preparation Adapter (100 ⁇ M) 5 ⁇ l 10xTNE annealing buffer 10 ⁇ l Nuclease-free water 85 ⁇ l Total 100 ⁇ l - Heat the mixture to 82°C for 2 min - Ramp-down rate of 0.1°C/sec to 4°C - Store at -20°C 2. RNA denaturation - Denature RNA for 2 min at 70°C - Immediately place on ice 3.
  • RNA Volume ( ⁇ l) 96 Denatured RNA 10 - 5' Adapter (5 ⁇ M) 1 110.4 3' Adapter (5 ⁇ M) 1 110.4 PEG8000 (50%) 2 220.8 10x RNA ligation buffer 2 220.8 T4 Rnl2 (10U/ ⁇ l) 1 110.4 ATP 10mM 2 220.8 Nuclease-free water 1 110.4 Total 20 ⁇ l 10 ⁇ L/well - Incubate 1 hour at 37°C with the lid heated to 45°C - Store at -20°C Reverse transcription - Prepare the RT reaction at two different dNTP molarities (250 ⁇ M and 25 ⁇ M): 96 96 Volume ( ⁇ l) 250 ⁇ M 25 ⁇ M Ligated RNA 6 - - dNTPs 0.5 55.2 55.2 RT Primer 1 110.4 110.4 (5 ⁇ M) 1.5 1.5 Total Total 7.5 ⁇ l ⁇ L/well ⁇ L/well - Incubate 5 min at 65°C and cool immediately on
  • FIG. 1 An instance of the 28S fragment DB structure is shown in Figure 1.
  • Dry synthetic RNA was reconstituted to known molarity and a dilution curve with estimated copy numbers was prepared.
  • RT reaction was prepared with standard concentration of dNTPs (1 mM) and PCR performed on digital PCR platform. Determined dPCR copies were then plotted against calculated copies input and a linearity detection threshold (ca 50 copies) was estimated (Figure 2). Linearity of the detection was calculated at 0.996.
  • a similar experiment was performed using PAXgene RNA samples with previously established high concentration of the rRNA fragment, as estimated by NGS measurement. This showed similar threshold of linearity for the digital PCR measurements (at ca 50 copies) as was shown for the synthetic RNA ( Figure 3).
  • a RT under limiting conditions was prepared using a gradient of dNTPs (1mM-5 ⁇ M). Both synthetic fragments were used, that is un-methylated negative control and methylated positive control as RNA templates. It was observed that the detection of negative control RNA was unhindered under conditions down to 50 ⁇ M of dNTPs, while a steady decrease was observed for the methylated RNA (Figure 4). The low starting values at 1mM are likely saturating effects of the RT reaction. Thus, the presence of 2’-o-m hinders the processivity of the RT in the reaction as expected.
  • Adapter preparation Adapter (100 ⁇ M) 5 ⁇ l 10xTNE annealing buffer 10 ⁇ l Nuclease-free water 85 ⁇ l Total 100 ⁇ l - Heat the mixture to 82°C for 2 min. - Ramp-down rate of 0.1°C/sec to 4°C. - Store at -20°C. 2. RNA denaturation - Denature RNA for 2 min at 70°C. - Immediately place on ice. 3.
  • RNA Volume ( ⁇ l) 96 Denatured RNA 8 - 5' Adapter (5 ⁇ M) 0.7 77.28 3' Adapter (5 ⁇ M) 0.7 77.28 PEG8000 (50%) 1.4 154.56 10x RNA ligation buffer 1.4 154.56 T4 Rnl2 (10U/ ⁇ l) 0.7 77.28 ATP 10mM 1.1 121.44 Total 14 ⁇ l 6 ⁇ L/well - Incubate 1 hour at 37°C with the lid heated to 45°C.
  • Dry synthetic RNA (unmethylated miLung#1) was reconstituted to known molarity and a dilution curve with estimated copy numbers was created.
  • RT reaction was prepared with standard concentration of dNTPs (1 mM), Maxima H Minus RT enzyme, and PCR was performed on digital PCR platform (dPCR). Determined dPCR copies were then plotted against calculated copies input and a linearity detection threshold (ca 50 copies) was estimated ( Figure 2). Linearity of the detection was calculated at 0.996.
  • a similar experiment was performed using PAXgene RNA samples with previously established high concentration of the miLung#1 fragment, as estimated by NGS measurement.
  • RT1 SEQ ID NO: 3
  • RT2 SEQ ID NO: 18
  • RT3 SEQ ID NO: 19
  • the LNA adapter showed very low variability between duplicates. However, it had less sensitivity for the methylated G. 3.
  • RT enzyme selection for optimized 2’-o-m detection Because reverse transcriptases can have various processing activities, a head-to-head comparison of three reverse transcriptases were performed: Maxima H Minus RT, Protoscript II RT, and Luna RT. First of all, all three reverse transcriptases worked. However, the results showed that Luna RT exhibits less variability between RT duplicates and, therefore, offers more reliable results (Figure 12).
  • Cell lines SNORD102 is a small nucleolar RNA which directs the site-specific 2'-O-methylation of 28S rRNA residue G4020 (D’Souza et al., 2018).
  • CRISPR/Cas9 was used to generate SNORD102 single-cell-colony knockouts from HCC-827 cells (DSMZ-German Collection of Microorganism and Cell Culture GmbH, no.: ACC 566).
  • sanger data showed crucial deletions of the SNORD102 guide sequence (Table 1), and therefore methylation rates in the rRNA residue G4020 are expected to decrease.
  • HCC-827 InDel Percentage SNORD102 sequence cell line (nt) 1 Wildtype 0 100% GAAGCAATGTGAAAAACACATTT ⁇ CACCGGCTCTGAA SNORD102- 2 -25 100% GAAGC------------------------------------------------------------------------------------------------------------------------------------------------------------------ ⁇ -----GCTCTGAA KO1 SNORD102- 3 -7 100% GAAGCAATGTGAAAAACAC---- ⁇ ---CGGCTCTGAA KO2
  • Table 1 Wildtype and SNORD102-knockout HCC-827 cell lines included for validating our method. Insertion and deletion (InDel) rates are shown for each knockout cell line and their specific location within the SNORD102 gene. SNORD102 guide sequence is underlined.
  • RNA Up to 300 pmol of 5 ⁇ termini 7 6.25 T4 PNK Reaction Buffer (10X) 1 0.8 ATP (10 mM) 1 0.8 T4 PNK (10 units) 0.2 0.16 Nuclease-free Water 0.8 0 Total 10 ⁇ l 8 ⁇ l Incubate 20 min @37°C Denature at 65°C for 10 min Ligation of the adapters to RNA Original Improved Denatured RNA 10 8.01 5’ Adapter-2-o-m (+ spacer) (5 ⁇ M) (SEQ ID NO: 17) 1 0.7 3' Adapter (5 ⁇ M) (SEQ ID NO: 12) 1 0.7 PEG8000 (50%) 2 1.4 10x RNA ligation buffer 2 1.4 T4 Rnl2 (10U/ ⁇ l) 1 0.7 ATP 10mM 2 0 Water 1 1.1 Total 20 ⁇ l 14 ⁇ l Incubate 1 hour at 37°C with the lid heated to 45°C store in -20C 7.2.

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Abstract

La présente invention concerne l'utilisation d'une combinaison d'un adaptateur 5' et d'un adaptateur 3' pour la détection de petits ARN non codants méthylés, et/ou pour la quantification d'un état de méthylation de petits ARN non codants. En outre, la présente invention concerne un procédé de détection de petits ARN non codants méthylés comprenant une transcription inverse sous limitation (faible concentration en dNTP) et des conditions non limitatives. En outre, la présente invention concerne un procédé de quantification d'un état de méthylation d'un petit ARN non codant.
EP23805593.3A 2023-01-25 2023-11-14 Pcr en haltère pour la détection de petits arn non codants méthylés Pending EP4655417A1 (fr)

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EP23153355 2023-01-25
EP23161945 2023-03-15
PCT/EP2023/081720 WO2024156392A1 (fr) 2023-01-25 2023-11-14 Pcr en haltère pour la détection de petits arn non codants méthylés

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