WO2025129663A1 - Procédé de détection d'interaction arn-protéine - Google Patents

Procédé de détection d'interaction arn-protéine Download PDF

Info

Publication number
WO2025129663A1
WO2025129663A1 PCT/CN2023/141158 CN2023141158W WO2025129663A1 WO 2025129663 A1 WO2025129663 A1 WO 2025129663A1 CN 2023141158 W CN2023141158 W CN 2023141158W WO 2025129663 A1 WO2025129663 A1 WO 2025129663A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
nucleic acid
chip
sequence
array
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
PCT/CN2023/141158
Other languages
English (en)
Chinese (zh)
Inventor
吴雪
王欧
周琳
陈奥
吴逸文
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.)
BGI Shenzhen Co Ltd
Original Assignee
BGI Shenzhen Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Priority to PCT/CN2023/141158 priority Critical patent/WO2025129663A1/fr
Publication of WO2025129663A1 publication Critical patent/WO2025129663A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present disclosure relates to the field of bioinformatics, and in particular to a method for constructing an RNA array, a kit for preparing an RNA array, and a method and device for detecting RNA-protein interaction and a kit.
  • RNA RNA binding protein
  • RNA-protein interactions based on high-throughput sequencing chips have been reported: a.
  • the Greenleaf and Lis team used the Illumina second-generation sequencing platform, and connected streptavidin and transcription termination protein Tus to the end of the DNA transcription template as a roadblock, so that RNAP was blocked at the end of the DNA template during transcription, and RNA was tethered to the DNA template through RNAP, thereby constructing a high-throughput RNA array and achieving binding with the target protein.
  • RNA is fixed by the DNA template, and the presence of the DNA template interferes with the subsequent RNA-protein interaction, and the presence of the roadblock protein increases the complexity of the components of the RNA array on the chip surface.
  • the Smith team used nucleic acid in situ synthesis technology, specifically, after the polyethylene glycol chain was seeded on the chip surface, the DNA chain was synthesized at the free end of the polyethylene glycol chain by chemical method, and the synthesized DNA was used as a template for RNA array transcription, and then the DNA template was digested by DNA digestion enzyme to obtain a simple RNA array.
  • the chemical synthesis method used in this method limits the length of RNA to within 33 bases, which is not enough to complete the complete transcription of most functional RNAs. c.
  • RNA arrays by RNA transcription and capture, in which the transcription DNA template and the aptamer-based RNA capture sequence were fixed on different surfaces, and the RNA shed by transcription was captured by the array below through the upper and lower alignment.
  • this RNA transcription capture method can construct RNA up to 600 bases in length, the need for upper and lower alignment results in a larger single site area in the array, resulting in a lower density of the RNA array and a lower throughput.
  • the present disclosure aims to solve one of the technical problems in the related art at least to some extent.
  • the first nucleic acid comprises a first sequence for sequencing and a second sequence for transcription.
  • the second nucleic acid comprises a third sequence for capturing the target RNA strand, wherein the third sequence is at least partially complementary to the target RNA strand.
  • the complementary length between the third sequence and the target RNA chain is at least 20 bp.
  • the third sequence has modifications such as dibenzocyclooctyne (DBCO), cyclooctyne, alkyne or azide for immobilization.
  • DBCO dibenzocyclooctyne
  • cyclooctyne cyclooctyne
  • alkyne alkyne or azide for immobilization.
  • the method further comprises preparing the second nucleic acid fixed to the second chip, specifically comprising: synthesizing the second nucleic acid and optionally amplifying the second nucleic acid; and fixing the second nucleic acid on the second chip.
  • the method further comprises: sequencing the first nucleic acids to determine a first signal based on each of the first nucleic acids, wherein the first signal comprises a sequence and a position of each of the first nucleic acids on the first chip.
  • the method further comprises digesting the first nucleic acid to purify the RNA array.
  • the enzyme used to digest the first nucleic acid is a nuclease, preferably DNase I, DNase II, nuclease S1 and/or nuclease S7, more preferably nuclease S1.
  • the first chip and the second chip are different chips, and the second chip generates the RNA array by diffusion transfer.
  • the target protein carries a fluorescent marker
  • contacting the target protein with the RNA array to obtain RNA-protein interaction data includes: contacting the target protein with the RNA array; after contact, detecting the RNA array to collect a second signal, wherein the second signal is a fluorescent signal of the target protein bound to the RNA array; and obtaining the RNA-protein interaction data based on the second signal.
  • the first chip and the second chip are the same chip.
  • the first nucleic acid comprises a first sequence for sequencing and a second sequence for transcription.
  • the first sequence comprises a linker sequence and an identifier sequence
  • the second sequence comprises a promoter sequence and a DNA sequence encoding a target RNA chain.
  • the complementary length between the third sequence and the target RNA chain is greater than 20 bp, preferably 35 bp.
  • the first nucleic acid is covalently linked to the first chip; and the second nucleic acid is covalently linked to the second chip.
  • the RNA array comprises a plurality of different RNAs, and is used to detect the interaction between the target protein and the plurality of different RNAs to determine the affinity.
  • FIG1 is a schematic diagram of a method for constructing an RNA array according to an embodiment of the present disclosure.
  • FIG. 4 is a schematic diagram of the principle of RNA transcription capture according to an embodiment of the present disclosure.
  • FIG. 5 is a schematic diagram of the fluorescence visualization results of RNA transcription capture according to an embodiment of the present disclosure.
  • FIG. 6 is a schematic diagram of the average fluorescence intensity results of RNA transcription capture according to an embodiment of the present disclosure.
  • FIG. 7 is a schematic diagram showing the principle of RNA crosstalk verification according to an embodiment of the present disclosure.
  • FIG8 is a schematic diagram of the fluorescence visualization results of RNA crosstalk verification according to an embodiment of the present disclosure.
  • FIG. 9 is a schematic diagram of the principle of RNA-protein interaction detection according to an embodiment of the present disclosure.
  • FIG. 10 is a schematic diagram of the fluorescence visualization results of RNA transcription capture according to another embodiment of the present disclosure.
  • FIG. 11 is a schematic diagram of the fluorescence visualization results of RNA-protein interaction detection according to an embodiment of the present disclosure.
  • FIG. 12 is a diagram showing the effect of a morpholino base RNA capture primer according to an embodiment of the present disclosure.
  • RNA-binding proteins are proteins that can coordinate with one or more related RNAs to achieve biological functions. After binding to RNA, they play a role in regulating various biological processes. For example, (1) during transcription, the transcribed RNA can regulate its own transcription by coordinating RNA polymerase (RNAP) II; (2) long noncoding RNAs (lncRNAs) can directly affect the expression of other genes by interacting with proteins, such as XIST lncRNA, which can guide regulatory proteins to chromatin to silence chromosomes; (3) RNA can also affect RNA localization, splicing and other post-transcriptional regulation by interacting with proteins to regulate gene expression.
  • RNA-binding proteins proteins that can coordinate with one or more related RNAs to achieve biological functions. After binding to RNA, they play a role in regulating various biological processes. For example, (1) during transcription, the transcribed RNA can regulate its own transcription by coordinating RNA polymerase (RNAP) II; (2) long noncoding RNAs (lncRNAs) can directly
  • RNA to bind to RBPs lies in the complex structure of RNA and protein itself.
  • RNA In its natural state, RNA generally exists in a single-stranded form. Single-stranded RNA has a wealth of intramolecular interactions, including mismatched base bulges, stem-loops, pseudoknots, G-quadruplexes, divalent cation interactions, and non-classical base pairing.
  • Protein is also a biological macromolecule with a very complex spatial structure. Therefore, due to the complexity of the interaction between RNA and protein, the current methods for analyzing RNA sequences for the interaction between RNA and protein are very limited.
  • the DNA template used as the first nucleic acid in the disclosed embodiment is shown in FIG3 .
  • the DNA template includes a 5'-end circularization linker sequence, a T7 promoter sequence, a GFP adapter sequence, and a 3'-end circularization linker sequence that also serves as a second nucleic acid hybridization sequence.
  • the GFP adapter sequence is used to simulate the nucleic acid encoding the target RNA chain, and in this embodiment is included in the second sequence of the first nucleic acid.
  • the 3'-end circularization linker sequence acts as both a circularization linker sequence and a nucleic acid of the target RNA chain, and therefore can also be included in the second sequence of the first nucleic acid.
  • the DBCO-RNA capture primer (second nucleic acid, SEQ ID NO: 5) was diluted to a concentration of 10 nM-100 nM, and the diluted second nucleic acid was introduced into the chip in (5) and reacted at room temperature overnight to fix the second nucleic acid to the chip through a click chemistry reaction;
  • RNA i.e., the GFP aptamer
  • S1 enzyme Thermo Scientific TM S1 nuclease, catalog number: EN0321
  • RNA transcription capture results are shown in Figures 5 and 6.
  • Channel 1 on the chip is referred to as L1, which is the experimental group and the group for normal transcription capture;
  • L2 is the DNB control group, which is filled with PBS instead of transcription reagent.
  • Figure 5 is a visualization result of the chip photography, in which the L1 group channel shows obvious fluorescent bright spots, indicating that GFP RNA is transcribed on the chip and captured in situ, and no fluorescent signal is shown in the L2 channel.
  • Figure 6 is the average fluorescence intensity in the L1 and L2 channels. The above results show that RNA can be fixed on the chip surface by transcription and in situ capture, proving the feasibility of the RNA array construction method provided by the present disclosure.
  • the embodiment of the present disclosure uses two different nucleic acid sequences encoding different RNAs to prepare a DNA template, and also uses fluorescent probe hybridization to explore the degree of RNA crosstalk.
  • the specific steps are as follows:
  • the DBCO-RNA capture primer (second nucleic acid, SEQ ID NO: 5) was diluted to a concentration of 10 nM-100 nM, and the diluted second nucleic acid was introduced into the chip and reacted at room temperature overnight to fix the second nucleic acid to the chip through a click chemistry reaction;
  • RNA detection primers AF532-P1 primer AF532-CAATTGCGCCACGACGTTTCGTGCT, SEQ ID NO: 8
  • AF647-P2 primer AF647-AGCTTCTGTGGTGGCCCTCTTTTAA, SEQ ID NO: 9
  • AF532 fluorescence and AF647 fluorescence respectively, to the chip, react at 55°C for 2 min and at 30°C for 2 min, so that the primers hybridize with the RNA P1 and P2 transcribed from S1 and S2;
  • the detection primer AF532-P2 (AF532-AGCTTCTGTGGTGGCCCTCTTTTAA, SEQ ID NO: 12) with AF532 fluorescence is introduced into each chip for RNA hybridization detection.
  • the optimized second nucleic acid in this embodiment i.e., the backbone structure of the morpholino base RNA capture primer
  • the morpholino base RNA capture primer can resist digestion by a variety of nucleases and can stably bind to the target RNA single strand in a base pairing manner.
  • the optimization result is shown in FIG12(B).
  • DNase is used to digest DNB
  • the amount of target RNA retained by the morpholino base RNA capture primer is significantly higher than the amount of target RNA that can be retained by the natural base DBCO-RNA capture primer when S1 enzyme is used to digest DNB. This result shows that the construction of high-throughput transcription capture is feasible.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.
  • a feature defined as “first” or “second” may explicitly or implicitly include at least one of the features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • a first feature being “above” or “below” a second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium.
  • a first feature being “above”, “above” or “above” a second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature.
  • a first feature being “below”, “below” or “below” a second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.
  • the terms “one embodiment”, “some embodiments”, “examples”, “specific examples”, or “some examples” and the like mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure.
  • the schematic representations of the above terms do not necessarily refer to the same embodiment or example.
  • the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner.
  • those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de construction d'une matrice d'ARN, un kit de préparation d'une matrice d'ARN, un procédé et un dispositif de détection d'interaction ARN-protéine, et un kit. Le procédé comprend : sur la base d'un premier acide nucléique fixé sur une première puce, la transcription du premier acide nucléique pour obtenir une chaîne d'ARN cible; et sur la base d'un second acide nucléique fixé sur une seconde puce, la capture de la chaîne d'ARN cible à l'aide du second acide nucléique, de façon à obtenir la matrice d'ARN.
PCT/CN2023/141158 2023-12-22 2023-12-22 Procédé de détection d'interaction arn-protéine Pending WO2025129663A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/141158 WO2025129663A1 (fr) 2023-12-22 2023-12-22 Procédé de détection d'interaction arn-protéine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/141158 WO2025129663A1 (fr) 2023-12-22 2023-12-22 Procédé de détection d'interaction arn-protéine

Publications (1)

Publication Number Publication Date
WO2025129663A1 true WO2025129663A1 (fr) 2025-06-26

Family

ID=96136202

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/141158 Pending WO2025129663A1 (fr) 2023-12-22 2023-12-22 Procédé de détection d'interaction arn-protéine

Country Status (1)

Country Link
WO (1) WO2025129663A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008022332A2 (fr) * 2006-08-18 2008-02-21 Board Of Regents, The University Of Texas System Système, procédé et kit pour répliquer une puce à adn
US20140038854A1 (en) * 2011-02-03 2014-02-06 Albert-Ludwigs-Universitaet Freiburg Device and method for the generation of molecular microarrays
US20140235505A1 (en) * 2012-11-06 2014-08-21 Wisconsin Alumni Research Foundation Rna array compositions and methods
CN115244185A (zh) * 2020-01-03 2022-10-25 约翰·霍普金斯大学 使用探针对连接的原位rna分析
CN115698324A (zh) * 2020-02-21 2023-02-03 10X基因组学有限公司 用于整合原位空间测定的方法和组合物
CN116685697A (zh) * 2021-01-08 2023-09-01 安捷伦科技有限公司 使用寡核苷酸微阵列进行空间核酸检测

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008022332A2 (fr) * 2006-08-18 2008-02-21 Board Of Regents, The University Of Texas System Système, procédé et kit pour répliquer une puce à adn
US20140038854A1 (en) * 2011-02-03 2014-02-06 Albert-Ludwigs-Universitaet Freiburg Device and method for the generation of molecular microarrays
US20140235505A1 (en) * 2012-11-06 2014-08-21 Wisconsin Alumni Research Foundation Rna array compositions and methods
CN115244185A (zh) * 2020-01-03 2022-10-25 约翰·霍普金斯大学 使用探针对连接的原位rna分析
CN115698324A (zh) * 2020-02-21 2023-02-03 10X基因组学有限公司 用于整合原位空间测定的方法和组合物
CN116685697A (zh) * 2021-01-08 2023-09-01 安捷伦科技有限公司 使用寡核苷酸微阵列进行空间核酸检测

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PHILLIPS JACK O, BUTT LOUISE E, HENDERSON CHARLOTTE A, DEVONSHIRE MARTIN, HEALY JESS, CONWAY STUART J, LOCKER NICOLAS, PICKFORD AN: "High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD., ENGLAND, vol. 46, no. 14, 21 August 2018 (2018-08-21), England, pages e86 - e86, XP093324801, ISSN: 0305-1048, DOI: 10.1093/nar/gky410 *

Similar Documents

Publication Publication Date Title
WO2021116715A1 (fr) Codage par code-barres spatial
IL293913A (en) Systems and methods for detecting multiple analytes
JP2001013147A (ja) 特定核酸配列の検出方法
CN113005121A (zh) 接头元件、试剂盒及其相关应用
CN116829735A (zh) 检测靶核酸序列的方法
JPH09508268A (ja) 核酸の配列決定
Zhu et al. Cas-Rainbow: Cas12a-driven single-reaction multiplex detection system
WO2003078623A1 (fr) Molecule fonctionnelle et son procede de production
CN107604046A (zh) 用于微量dna超低频突变检测的双分子自校验文库制备及杂交捕获的二代测序方法
US20250369046A1 (en) Aptamer detection techniques
CN109097446A (zh) 一种检测miRNA的方法及试剂盒
CN119899830A (zh) 一种基于链霉亲和素磁珠筛选靶向凝血酶非天然核酸适配体的方法和应用
CN110747514A (zh) 一种高通量单细胞小rna文库构建方法
Zhao et al. Ultra-sensitive detection of microRNAs using an electrochemical biosensor based on a PNA-DNA2 three-way junction nanostructure and dual cascade isothermal amplification
CN109402127A (zh) 一组与结缔组织生长因子特异性结合的高亲和力核酸适配体及其应用
CN121057851A (zh) 筛选核酸适配体的组合元件及其应用，以及核酸适配体高通量筛选方法
WO2025129663A1 (fr) Procédé de détection d'interaction arn-protéine
CN118932023A (zh) 一种多价框架核酸探针及其制备方法和细胞膜蛋白成像分析方法
WO2006095550A1 (fr) Amorce de pcr, methode mettant en application cette amorce et produit amplifie par pcr, dispositif et complexe adn-proteine mettant en application ce produit amplifie par pcr
WO2023092601A1 (fr) Marqueur moléculaire umi et application, adaptateur, réactif de ligature d'adaptateur et son kit, et procédé de construction de banque
CN114317544A (zh) 特异性结合cd133的核酸适配体、其筛选方法及应用
JPH04504059A (ja) 化学発光検出を用いた核酸の配列決定
JP2006153839A (ja) ジップコード方式を用いたｐｎａチップ及びその製作方法
CN112858693A (zh) 一种生物分子检测方法
CN1521272B (zh) 新型配体检测方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23961985

Country of ref document: EP

Kind code of ref document: A1