WO2024067478A1 - 一种测量单分子rna力谱的方法及其应用 - Google Patents

一种测量单分子rna力谱的方法及其应用 Download PDF

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WO2024067478A1
WO2024067478A1 PCT/CN2023/121087 CN2023121087W WO2024067478A1 WO 2024067478 A1 WO2024067478 A1 WO 2024067478A1 CN 2023121087 W CN2023121087 W CN 2023121087W WO 2024067478 A1 WO2024067478 A1 WO 2024067478A1
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handle
rna
chain
primer
molecule
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French (fr)
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史航
康亚峰
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清华大学
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the invention relates to a method for measuring single-molecule RNA force spectrum and application thereof, and belongs to the fields of biophysics and structural biology.
  • RNA molecules have always been an important biological macromolecule that biologists have paid attention to. Early studies believed that RNA was an important molecule that transmitted genetic information between DNA and protein in the central dogma, but it was later discovered that most RNAs are not translated into proteins, but have a variety of important physiological functions, such as genetic coding, gene expression, gene regulation, and enzyme catalysis. From the perspective of molecular structure, these functions can be played by either its primary sequence or its secondary or tertiary structure. However, researchers do not have a good understanding of the complex structure of RNA. Unlike the regular double helix structure of DNA, RNA molecules are generally single-stranded ribonucleic acids that fold into secondary structures or more complex tertiary structures through base pairing.
  • RNA molecules often interact with other molecules (proteins, DNA/RNA, small molecules, etc.), and their conformations often change in real time.
  • the coronavirus genome is a single-stranded positive-sense RNA virus with a pseudoknot structure at the end of the open reading frame 1a, which allows the ribosome to perform a -1 frameshift, thereby continuing to translate the longer polypeptide 1ab on the open reading frame 1b. It is generally believed that this frameshift behavior is related to the special pseudoknot structure of the RNA molecule structure at this location.
  • RNAs with important biomedical value are still unknown, and it is difficult to accurately predict their structures and functions through sequence information.
  • Different methods are needed to analyze the structures and study the functions one by one, such as SHAPE, X-ray, nuclear magnetic resonance and cryo-electron microscopy.
  • SHAPE technology uses electrophilic reagents to react with 2-hydroxyl groups with different activities, and analyzes the structure of RNA molecules through reverse transcription. This method has been successfully used for the structural analysis of many important RNAs. However, this method mainly obtains RNA secondary structures, and the information is relatively simple.
  • X-ray technology and cryo-electron microscopy methods are relatively complex and difficult to be widely used, while nuclear magnetic resonance technology has a large limitation on RNA size and it is difficult to detect larger RNA molecules (>60nt).
  • Optical tweezers technology also known as gradient force optical trap technology, uses focused lasers to exert force on objects. Since the optical tweezers technology was proposed, its application in the biological field has received widespread attention, and Ashkin was awarded the Nobel Prize in Physics in 2018. Compared with other single-molecule force spectroscopy techniques, optical tweezers technology has higher spatial (sub-nanometer), mechanical (sub-piconetonic), and temporal (sub-millisecond) resolution, and its characteristics are consistent with the inherent properties of RNA molecules, such as the length of a single nucleotide (about 0.59nm in length) and mechanics (0-30pN).
  • dual-trap optical tweezers technology has gradually matured, which is different from traditional single-molecule force spectroscopy techniques (single-trap optical tweezers, magnetic tweezers, Unlike atomic force microscopy (AFM), which fixes one end of the molecule to be studied on a glass, substrate or probe surface
  • the dual-trap optical tweezers technique fixes the molecule to be studied between suspended microspheres and uses advanced devices such as piezoelectric platforms and avalanche photodiodes to greatly reduce the impact of the external environment and improve the resolution and stability of the technique.
  • single-molecule force spectroscopy has been widely used in force spectroscopy studies of DNA and proteins, and the technology is relatively mature.
  • due to the instability and complex structure of RNA molecules it is difficult to establish its force spectroscopy experimental method, and there is a lack of research on single-molecule RNA force spectroscopy.
  • the present invention provides a method for measuring single-molecule RNA force spectrum and its application.
  • the present invention provides a method for measuring single-molecule RNA force spectrum, the method comprising:
  • Obtaining a handle chain designing and synthesizing a handle chain primer with a special modification; obtaining a handle chain with a special modification according to the handle chain primer, wherein the handle chain includes a handle chain 1 and a handle chain 2; the handle chain 1 and the handle chain 2 have sticky ends; preferably, the handle chain is a double-stranded DNA with a sticky end; preferably, the sticky end has a length of 30-100 nt, more preferably 30-50 nt;
  • RNA-handle composite chain obtaining a single-molecule RNA, wherein the RNA has a fragment that is complementary to the above-mentioned sticky end; annealing the handle chain with the RNA to obtain a single-molecule RNA-handle composite chain.
  • the method also includes designing and constructing a plasmid; the plasmid contains a template DNA sequence and a handle chain sequence of the RNA to be studied; primers with special modifications are designed according to the plasmid; and the DNA template, handle chain 1 and handle chain 2 of the RNA molecule to be studied are obtained according to the designed plasmid and primers.
  • the method further comprises obtaining single-molecule RNA based on the DNA template of the RNA molecule to be studied.
  • the method further comprises obtaining standard double-stranded DNA; the standard double-stranded DNA is obtained by amplifying using the handle chain primer as an amplification primer; the standard double-stranded DNA molecule is directly used to verify the single-molecule experimental system.
  • the standard double-stranded DNA is obtained by amplifying using the upstream primer of handle chain 1 and the downstream primer of handle chain 2 as amplification primers.
  • the primer and/or handle chain carries one or more special modification molecules
  • the primer and/or handle chain carries 3, 4 or 5 or more special modification molecules.
  • the special modification includes one or a combination of two or more of digoxigenin modification, biotin modification, d-spacer modification, phosphate modification, thiophosphate modification and azide group modification;
  • the specific modification is digoxin.
  • the primer and/or handle strand carries more than 3 digoxigenin modifications
  • the handle chain carries 3-5 digoxigenin modifications.
  • the upstream primer 1F of the handle chain 1 is also modified with digoxigenin, and the downstream primer 1R is modified with d-spacer, and the downstream primer 1R has a sticky end sequence 1 for annealing and connecting with RNA;
  • the upstream primer 2F of the handle chain 2 is modified with phosphate, and the downstream primer 2R is modified with biotin, and the upstream primer 2F has a sticky end sequence 2 for annealing and connecting with RNA.
  • handle chains have special modifications and sticky ends respectively;
  • the handle chains 1 each have a specially modified and sticky end 1 with a length of 30-100 nt;
  • the handle chains 2 each have a special modification and a sticky end 2 with a length of 30-100 nt;
  • the sticky end 1 comprises a fragment of the sequence shown in SEQ ID NO.7;
  • the sticky end 2 includes a fragment of the sequence shown in SEQ ID NO.8.
  • the method comprises obtaining handle chains and/or standard double-stranded DNA of different lengths by changing the spacing distance between the upstream and downstream primers;
  • the length of the handle strand and/or the standard double-stranded DNA can be changed by changing the plasmid size
  • the length of the handle chain and/or the standard double-stranded DNA can be changed by changing the binding position of the primer on the template.
  • the length of the handle chain of the present invention is 1 kb.
  • the primers designed by this method have great scalability and can be used to prepare handle chains of different lengths, which are suitable for realizing force spectrum measurements of molecules of different specifications and expanding their applications.
  • the handle chain is used to connect a single-molecule RNA to a surface; the surface is a surface modified to bind to a special modified molecule of handle chain 1 and/or handle chain 2.
  • the method further comprises designing a primer having a sequence complementary to the sticky end sequence, so as to finally obtain an RNA having a fragment complementary to the sticky end.
  • the handle chain primer includes a primer with a sequence as shown in SEQ ID NO.1-SEQ ID NO.4.
  • the annealing is to anneal after mixing the RNA molecule to be studied with the handle chain 1 and the handle chain 2 in a molar ratio of (0.7-2):1:1;
  • the annealing is performed by mixing the RNA molecule to be studied with handle chain 1 and handle chain 2 in a molar ratio of 1:1:1 and then annealing.
  • the annealing temperature is 50-65°C
  • the annealing temperature is 62°C and/or 52°C.
  • the annealing conditions are: 98° C. for 10 minutes, 62° C. for 1 hour, 52° C. for 1 hour, and 4° C. to terminate the reaction.
  • the annealing buffer is a buffer solution containing formamide and PIPES
  • the annealing buffer contains 60%-80% formamide by volume
  • the annealing buffer is a solution containing: 80% formamide, 400 mM NaCl, 40 mM PIPES and 1 mM EDTA by volume, with a pH of 7.5.
  • the method further comprises performing molecular force spectroscopy detection, and the force spectroscopy detection is performed in a measurement buffer solution containing a deoxygenation system and/or an RNase inhibitor; the deoxygenation system and the RNase inhibitor can keep the RNA molecules in an oxygen-free aqueous solution environment, prevent RNA from being degraded by enzymes, and improve the stability of RNA in the experiment.
  • the deoxygenation system comprises glucose oxidase, catalase and glucose;
  • the deoxygenation system comprises 160 units/ml glucose oxidase, 100 units/ml catalase and 0.8% glucose by mass;
  • the measurement buffer solution is a solution containing NaCl, EDTA + or Mg 2+ ; preferably, the buffer solution is a Hepes or Tris-HCl buffer solution.
  • the force spectrum detection is performed using a microfluidic channel.
  • the sample in the microfluidic channel is: after incubating the single-molecule RNA-handle composite chain or the standard double-stranded DNA single-molecule sample with microsphere 1, a measurement buffer solution is added and placed in channel 1; a measurement buffer solution is added to channel 2; and channel 3 is a microsphere 2 solution.
  • the sample is introduced into the microfluidic channel, and a deoxygenation system and an RNase inhibitor are added to each channel; a force spectrum detection system is formed in the microfluidic channel and molecular force spectrum detection is performed.
  • Microfluidic channels are used to control the separation of samples of different components, avoid interference, and conduct single-molecule force spectrum experiments quickly and efficiently.
  • the force spectrum detection comprises: fixing one or both ends of the RNA-handle composite chain to obtain data; the data is preferably force, time and/or distance;
  • the force spectrum detection comprises: fixing one end of the RNA-handle composite chain and moving the other end in a single direction to obtain data such as force, time and distance;
  • the force spectrum detection includes: fixing the distance or force between the two ends of the RNA-handle composite chain to obtain data such as RNA molecule force or distance, time, etc.;
  • the force spectrum detection includes: fixing both ends of the RNA molecule, and obtaining changes in distance, force parameters, etc. when the RNA molecule interacts with other molecules; the other molecules are preferably small molecules or proteins;
  • dual-trap optical tweezers are used to control the movement and fixation of one end and/or both ends.
  • dual-trap optical tweezers are used to move and fix the microspheres when measuring molecular force spectrum, thereby reducing the contact between the sample and the external physical environment, reducing external environmental interference, and improving the stability of the measurement mechanics and distance.
  • the force spectrum detection and the formation of the force spectrum detection system include: controlling microsphere 1 and microsphere 2 so that a single-molecule RNA composite chain or a standard double-stranded DNA is simultaneously connected to microsphere 1 and microsphere 2 to form a force spectrum detection system.
  • the present invention provides a primer, which consists of a sequence selected from SEQ ID NO.1-SEQ ID NO.4.
  • the primer carries 1 or more than 2 special modification molecules; preferably, the primer carries 3, 4 or more than 5 special modification molecules.
  • the special modification includes one or a combination of two or more of digoxigenin modification, biotin modification, d-spacer modification, phosphate modification, thiophosphate modification and azide group modification; preferably, the special modification is digoxigenin.
  • the primer carries more than 3 digoxigenin modifications
  • the handle chain carries 3-5 digoxigenin modifications.
  • the design of the handle and the sticky end of the present invention enables the single-molecule RNA-handle composite chain obtained by the present invention to withstand a higher force.
  • the higher force is 50-60pN.
  • the present invention provides a sticky end consisting of a sequence selected from SEQ ID NO.7-SEQ ID NO.8.
  • the present invention provides a kit for measuring single-molecule RNA force spectrum, wherein the kit comprises at least one of the primers and/or at least one of the sticky ends.
  • the kit comprises an annealing buffer, and the annealing buffer comprises formamide and PIPES;
  • the annealing buffer contains 60%-80% formamide by volume
  • the annealing buffer is a solution containing: 80% formamide, 400 mM NaCl, 40 mM PIPES and 1 mM EDTA by volume, with a pH of 7.5.
  • the kit comprises a deoxygenation system and/or an RNase inhibitor
  • the deoxygenation system is a buffer solution containing glucose oxidase, catalase and glucose;
  • the deoxygenation system is a buffer solution containing 160 units/mL glucose oxidase, 100 units/mL catalase and 0.8% glucose by mass.
  • This method provides an experimental method for quickly and efficiently measuring single-molecule RNA samples, which can quickly, efficiently and accurately perform force spectroscopy measurement on single RNA molecules. And this method has good repeatability. Specifically, the present invention has the following advantages:
  • the handle chain of the present invention is connected to RNA with a sticky end to form a double strand, so that the connection between the handle and the single-molecule RNA is tighter.
  • the handle of the present invention is a DNA double strand, and the DNA double strand component in the entire single-molecule RNA-handle composite chain is higher, so that the single-molecule RNA-handle composite chain has higher stability and can withstand greater forces, which is suitable for higher forces and more accurate measurements.
  • the present invention is also suitable for RNA structure analysis.
  • the present invention uses a handle chain with special modifications, preferably a handle chain modified with 3 or more special molecules.
  • the handle is more tightly connected to the fixed surface and can withstand a greater force, which is suitable for the measurement of higher forces.
  • the present invention designs a more optimal sticky end length and more efficient annealing conditions, making the annealing connection between the RNA sample and the handle chain more efficient.
  • the present invention designs a measurement system that is more suitable for RNA force spectroscopy measurement, especially by adding a deoxygenation system and/or an RNase inhibitor, so that the measurement environment is more suitable for RNA.
  • Figure 1 is a flow chart of the experiment of this method.
  • FIG2 is a gel electrophoresis diagram of standard double-stranded DNA, handle chain 1, handle chain 2 and RNA molecules.
  • FIG3 is a schematic diagram of a multi-channel microfluidic chip for use in an experiment.
  • FIG4 is a force spectrum diagram of a single molecule of 3 kbp double-stranded DNA.
  • FIG5 is a graph showing the fitting curve of RNA single molecule force spectrum.
  • FIG6 is a schematic diagram of the plasmid design of the standard double-stranded DNA, handle chain 1, handle chain 2 and primers in this method.
  • FIG. 7 is a conditional investigation of annealing solutions with different formamide concentrations.
  • FIG8 shows the use of 1F, 1R, 2F, and 2R to amplify longer handles 3 and 4 by changing the size of the plasmid.
  • the present invention provides a method for measuring single-molecule RNA force spectrum, the method comprising:
  • Obtaining a handle chain designing and synthesizing a handle chain primer with a special modification; obtaining a handle chain with a special modification according to the handle chain primer, wherein the handle chain includes a handle chain 1 and a handle chain 2; the handle chain 1 and the handle chain 2 have sticky ends; preferably, the handle chain is a double-stranded DNA with a sticky end; preferably, the sticky end
  • the terminal length is 30-100 nt;
  • RNA-handle composite chain obtaining a single-molecule RNA, wherein the RNA carries a fragment that is complementary to the above-mentioned sticky end; annealing the handle chain with the RNA to obtain a single-molecule RNA-handle composite chain.
  • the method for measuring single-molecule RNA force spectrum of the present invention comprises:
  • handle chain and other required double-stranded DNA 1) Obtaining the handle chain and other required double-stranded DNA: designing and synthesizing a handle chain primer with a special modification; obtaining a handle chain with a special modification based on the handle chain primer, wherein the handle chain includes handle chain 1 and handle chain 2; the handle chain 1 and handle chain 2 have sticky ends; preferably, the handle chain is a double-stranded DNA with a sticky end; preferably, the sticky end is 30-100 nt in length; obtaining a DNA template of the RNA molecule to be studied.
  • the primers of the handle chain 1 the 5' end of the upstream primer 1F of the handle chain 1 has one or more digoxigenin modifications, the chain of the downstream primer 1R has a d-spacer modification, and the 5' end of the downstream primer 1R has a sticky end sequence 1 to be annealed and connected with the 5' end of the RNA;
  • the primers of the handle chain 2 the 5' end of the upstream primer 2F of the handle chain 2 is modified with Phosphate, the upstream primer 2F chain is modified with Thiophosphate, and the 5' end of the upstream primer 2F is modified with a sticky end sequence 2 to be annealed with the 3' end of the RNA, and the 5' end of the downstream primer 2R of the handle chain 2 is modified with one or more biotins;
  • handle chain 1, handle chain 2 and standard double-stranded DNA were obtained: using the designed plasmid as a template, using the designed primers 1F, 1R, 2F and 2R, a PCR reaction was performed, and the PCR conditions were: 98°C for 1 minute, 98°C for 10 seconds, 58°C for 30 seconds, 72°C for 30 seconds, 60 seconds or 90 seconds, after 35 cycles, the reaction was terminated at 4°C; after the PCR reaction was completed, the products were separated by gel electrophoresis to obtain handle chain 1, handle chain 2 and standard double-stranded DNA respectively;
  • the 5' end and 3' end of the handle chain 1 are respectively modified with digoxigenin and have sticky ends with a sequence length of 30-100 nt;
  • the 5' end and 3' end of the handle chain 2 are respectively biotin-modified and have sticky ends with a sequence length of 30-100 nt;
  • a DNA template of the RNA molecule to be studied is obtained: according to the RNA sequence, an upstream primer 3F and a downstream primer 3R are designed, wherein the 5' end of the upstream primer 3F carries a sequence that is complementary to the sticky end sequence 1 of the handle chain 1, and the 5' end of the downstream primer 3R carries a sequence that is complementary to the sticky end sequence 2 of the handle chain 2; using the designed and constructed plasmid as a template, a PCR reaction is performed using primers 3F and 3R to synthesize a DNA template corresponding to the RNA to be studied;
  • RNA-handle composite chain obtaining a single-molecule RNA, wherein the RNA has a fragment that is complementary to the above-mentioned sticky end; annealing the handle chain with the RNA to obtain a single-molecule RNA-handle composite chain.
  • the RNA molecule to be studied is obtained by using a DNA template corresponding to the RNA to be studied as a substrate and using a T7 kit for transcription to obtain the RNA molecule to be studied; the 5' end and the 3' end of the RNA molecule to be studied have sequences that are complementary to the sticky ends of handle chain 1 and handle chain 2, and the length of the complementary sequence is 30-50nt;
  • the method for obtaining a single-molecule RNA-handle composite chain is as follows: the RNA molecule to be studied is mixed with handle chain 1 and handle chain 2 in a molar ratio of 1:1:1 and then annealed to prepare a single-molecule RNA-handle composite chain, wherein the annealing conditions are: 98°C for 10 minutes, 62°C for 1 hour, 52°C for 1 hour, and 4°C for termination of the reaction; after the reaction is terminated, ethanol is used for precipitation, and 10 ⁇ L of H 2 O is added to dissolve to obtain a single-molecule RNA-handle composite chain;
  • microfluidic channels for molecular force spectroscopy detection introducing samples into microfluidic channels, and adding deoxygenation systems and RNase inhibitors into each channel; forming a force spectroscopy detection system in the microfluidic channels and performing molecular force spectroscopy detection;
  • different samples are introduced into the microfluidic channel: different samples are separated by microfluidic channels, wherein 1 ⁇ L of single-molecule RNA-handle composite chain or standard double-stranded DNA single-molecule sample is incubated with 1 ⁇ L of anti-digoxigenin microsphere 1 at room temperature for 5 minutes, 1 mL of measurement buffer solution is added, and placed in channel 1; measurement buffer solution is added to channel 2; channel 3 is a solution of microsphere 2 modified with streptavidin affinity; deoxygenation system and RNase inhibitor are added to each channel;
  • microspheres 1 and 2 The preparation process of microspheres 1 and 2 is as follows: using 5 ⁇ g/ml EDC and NHS to activate carboxyl-modified microspheres, adding anti-digoxigenin protein to couple with the activated microspheres after 20 minutes to obtain anti-digoxigenin-modified microspheres 1; adding streptomycin to couple after activation to obtain streptomycin-modified microspheres 2;
  • the force spectrum detection system is formed by using optical trap 1 to capture microsphere 1 in channel 1, optical trap 2 to capture microsphere 2 in channel 3, moving optical trap 1 and optical trap 2 to channel 2, and slowly approaching optical trap 1 and optical trap 2, so that the single-molecule RNA complex chain or standard double-stranded DNA sample on microsphere 1 specifically combines with microsphere 2 to form a force spectrum detection system;
  • the force spectrum detection is as follows: fix microball 1 or microball 2, move the other microball in the horizontal direction, and obtain data such as force, time and distance.
  • This embodiment provides a method for quickly and efficiently measuring single-molecule RNA samples.
  • the experimental process of this method is shown in FIG1 .
  • handle chains and other required DNA 1) Obtaining handle chains and other required DNA: designing and constructing plasmids containing the RNA molecule to be studied, handle chain 1 and handle chain 2 DNA sequences; and designing primers with special modifications based on the plasmids; obtaining the DNA template of the RNA molecule to be studied, handle chain 1, handle chain 2 and standard double-stranded DNA based on the designed plasmids and primers; the handle chain 1 and handle chain 2 are double-stranded DNAs with sticky ends;
  • the primers of the handle chain 1 the 5' end of the upstream primer 1F of the handle chain 1 is modified with digoxigenin, the 5' end of the downstream primer 1R is modified with phosphate, and the chain of the downstream primer 1R is modified with d-spacer, and the 5' end of the downstream primer 1R has a sticky end sequence 1 to be annealed and connected with the 5' end of the RNA; the primer sequences are shown in Table 1.
  • the primers of the handle chain 2 the 5' end of the upstream primer 2F of the handle chain 2 is modified with Phosphate, the upstream primer 2F is modified with Thiophosphate, and the 5' end of the upstream primer 2F has a sticky end sequence 2 to be annealed with the 3' end of the RNA, and the 5' end of the downstream primer 2R of the handle chain 2 is modified with biotin; the primer sequences are shown in Table 1. All primers in this experiment were synthesized from GenScript Biotech Co., Ltd. In the present invention, "T” is used to represent uracil "U”.
  • handle chain 1, handle chain 2 and standard double-stranded DNA are obtained: referring to Figure 6, primers 1F, 1R, 2F and 2R are designed and synthesized, and polymerase (F530, Thermo) is used for PCR reaction.
  • primers 1F, 1R, 2F and 2R are designed and synthesized, and polymerase (F530, Thermo) is used for PCR reaction.
  • the PCR conditions are: 98°C for 1 minute, 98°C for 10 seconds, 58°C for 30 seconds, 72°C for 30 seconds, 60 seconds or 90 seconds, and after 35 cycles, the reaction is terminated at 4°C; after the PCR reaction, the products are separated by gel electrophoresis, and the products are recovered using the Tiangen kit (DP209, Tiangen Biochemical Technology (Beijing) Co., Ltd.); handle chain 1, handle chain 2 and standard double-stranded DNA are obtained respectively; the obtained single-molecule sample electrophoresis diagrams of handle chain 1, handle chain 2 and standard double-stranded DNA are shown in Figure 2.
  • the 5' end and 3' end of the handle chain 1 respectively carry 5 digoxigenin modified sticky ends 1 with a sequence length of 36 nt.
  • the sequence of the sticky end 1 is shown in Table 1.
  • the 5' end and 3' end of the handle chain 2 respectively have two biotin modifications and a sticky end 2 with a sequence length of 35 nt.
  • the sequence of the sticky end 2 is shown in Table 1.
  • an upstream primer 3F and a downstream primer 3R are designed, wherein the 5' end of the upstream primer 3F carries a sequence that is complementary to the sticky end sequence 1 of the handle chain 1, and the 5' end of the downstream primer 3R carries a sequence that is complementary to the sticky end sequence 2 of the handle chain 2; the sequences of the primers 3F and 3R are shown in Table 1.
  • Primers 3F and 3R were used to perform PCR reaction to synthesize the DNA template corresponding to the RNA to be studied (the DNA template sequence corresponding to the RNA to be studied is shown in Table 1).
  • RNA-handle composite chain obtaining the RNA molecule to be studied based on the DNA template of the RNA molecule to be studied; the 5' end and 3' end of the RNA molecule to be studied have sequences that are complementary to the sticky ends of handle chain 1 and handle chain 2, and the length of the complementary pairing sequence is 30-100nt; the RNA molecule to be studied is mixed with handle chain 1 and handle chain 2 and then annealed to obtain a single-molecule RNA-handle composite chain.
  • the RNA molecule to be studied is obtained by using a DNA template corresponding to the RNA to be studied as a substrate and using a T7 kit (E2040S, NEB) to transcribe the RNA molecule to be studied; the 5' end and the 3' end of the RNA molecule to be studied have sequences that are complementary to the sticky ends of handle chain 1 and handle chain 2, and the complementary pairing The sequence length is 30-100 nt; the electrophoresis diagram of the RNA molecule sample to be studied is shown in Figure 2.
  • the single-molecule RNA-handle composite chain is obtained: the RNA molecule to be studied is purified and mixed with handle chain 1 and handle chain 2 in a molar ratio of 1:1:1, and then annealed in a solution of 80% formamide (F9037, Sigma), 400mM NaCl, 40mM PIPES, 1mM EDTA, pH 7.5 to prepare a single-molecule RNA-handle composite chain.
  • the annealing conditions are: 98°C for 10 minutes, 62°C for 1 hour, 52°C for 1 hour, and 4°C for termination of the reaction; after the reaction is terminated, ethanol precipitation is used, and 10 ⁇ L H 2 O is added to dissolve to obtain a single-molecule RNA-handle composite chain.
  • the single-molecule RNA composite chain has an affinity molecule end, which can be combined with the corresponding microsphere for large force measurement.
  • microfluidic channels for molecular force spectroscopy detection: using microfluidic channels to separate different samples, introducing different samples into the microfluidic channels, and adding a deoxygenation system and RNase inhibitor (N8080119, Thermo) into each channel; forming a force spectroscopy detection system in the microfluidic channel and performing molecular force spectroscopy detection;
  • RNA-handle composite chain or standard double-stranded DNA single-molecule sample is incubated with 1 ⁇ L of anti-digoxigenin (11222089001, Roche) microsphere 1 at room temperature for 5 minutes, 1 mL of measurement buffer solution is added, and placed in channel 1; channel 2 is added with measurement buffer solution; channel 3 is streptomycin affinity-modified microsphere 2 solution (SVP-08-10, Spherotech); deoxygenation system and RNase inhibitor are added to each channel; the measurement buffer solution used in this method is a buffer solution containing 20 mM Hepes, 100 mM NaCl and 0.2 mM EDTA.
  • the preparation process of the microspheres 1 and 2 is as follows: use 5 ⁇ g/ml EDC and NHS to activate the carboxyl-modified microspheres, add anti-digoxigenin protein to couple with the activated microspheres after 20 minutes to obtain anti-digoxigenin-modified microspheres 1; add streptomycin after activation to obtain streptomycin-modified microspheres 2; the connection method between the nucleic acid macromolecules and between the nucleic acid molecules and the microspheres is shown in Figure 1.
  • the force spectrum detection system is formed: light trap 1 is used to capture microsphere 1 in channel 1, light trap 2 is used to capture microsphere 2 in channel 3, light trap 1 and light trap 2 are moved to channel 2, light trap 1 and light trap 2 are slowly approached, so that the single-molecule RNA complex chain or standard double-stranded DNA sample on microsphere 1 is specifically combined with microsphere 2 to form a force spectrum detection system; this method uses a microfluidic chip, different samples are introduced into the chip, a detection system is quickly formed, and efficient force spectrum detection is performed on the target RNA molecule.
  • the force spectrum detection is as follows: fix microsphere 1 or microsphere 2, move the other microsphere in a single direction, and obtain data such as force, time and distance.
  • This embodiment uses the double-trap optical tweezers produced by Lumicks as an example when measuring single-molecule force spectrum, but is not limited to this device.
  • This example explores the annealing effect of annealing buffer solutions containing different volume fractions of formamide when annealing RNA and handle chains in the process of obtaining single-molecule RNA-handle composite chains.
  • the specific operation is:
  • RNA and handle strands were obtained by the same method as in Example 1;
  • RNA and handle chain were annealed in PIPES buffer solutions containing 30%, 40%, 50%, 60%, 70% and 80% formamide by volume.
  • the annealing conditions of each group were the same except for the concentration of formamide.
  • This example explores the annealing effects of different annealing procedures when annealing RNA and the handle chain in the process of obtaining a single-molecule RNA-handle composite chain.
  • the specific operation is:
  • RNA and handle strands were obtained by the same method as in Example 1;
  • Program 1 98°C for 10 minutes, 62°C for 1 hour, 52°C for 1 hour, and terminate the reaction at 4°C;
  • Procedure 2 95°C for 10 minutes, followed by a gradient temperature decrease at a rate of 1°C/min, and the reaction was terminated at 4°C;
  • RNA-handle complex chains The formation efficiency of RNA-handle complex chains was detected by force spectroscopy.
  • the force spectroscopy test showed that under the reaction conditions of procedure 1, the probability of forming valid data was 10%.
  • the annealing conditions of 62°C for 1 hour and 52°C for 1 hour had the best formation efficiency of RNA-handle complex chains. That is, the most suitable annealing conditions were: 98°C for 10 minutes, 62°C for 1 hour, Incubate at 52°C for 1 hour and terminate the reaction at 4°C.
  • This example provides an experiment of using primers 1F, 1R, 2F and 2R provided in Example 1 to amplify handle chains of different lengths by expanding the plasmid.
  • the PCR conditions were: 98°C for 1 minute, 98°C for 10 seconds, 58°C for 30 seconds, 72°C for 240 seconds, and after 35 cycles, the reaction was terminated at 4°C.
  • the products were separated by gel electrophoresis and recovered using the Tiangen kit (DP209, Tiangen Biochemical Technology (Beijing) Co., Ltd.).
  • Handle chain 1, handle chain 2 and standard double-stranded DNA were obtained respectively.
  • the electrophoresis diagrams of the obtained single-molecule samples of handle chain 1, handle chain 2 and standard double-stranded DNA are shown in Figure 8.
  • handle chains 3 and 4 have molecular weights significantly greater than handle chains 1 and 2. That is, when primers 1F, 1R, 2F and 2R are used to amplify handle chains, handle chains with different lengths can be obtained by changing the plasmid. Handle chains with different lengths are suitable for more types of force spectroscopy experiments and can meet more force spectroscopy measurement needs.
  • This example uses the same method as in Example 1 to measure the force spectra of multiple groups of DNA and RNA molecules.
  • this method uses more stable double-stranded DNA as a handle and can withstand a larger force of about 60pN.
  • the force spectrum curve of the single-molecule RNA composite chain prepared by this method can show the force spectrum changes of the RNA structure under constant speed stretching.
  • the present embodiment provides a kit comprising a sticky end, the sequence of the sticky end being as shown in SEQ ID NO.7 or SEQ ID NO.8, or as shown in the complementary sequence of SEQ ID NO.7 (SEQ ID NO.11: GAATTCGGCTACGTAGCTCAGTTGGTTAGAGCAGCG) or the complementary sequence of SEQ ID NO.8 (SEQ ID NO.12: GTCACAGGTTCGAATCCCGTCGTAGCCACCACTGC).
  • the kit described in this embodiment can be used to construct a RNA with a sticky end such as the structure shown as "sticky end 1-RNA-sticky end 2", and the RNA in the structure can be any single-molecule RNA.
  • the RNA with a sticky end is: GAATTCGGCTACGTAGCTCAGTTGGTTAGAGCAGCG (SEQ ID NO.11)-RNA-GTCACAGGTTCGAATCCCGTCGTAGCCACCACTGC (SEQ ID NO.12).
  • the kit is suitable for measuring the molecular force spectrum of RNA with a length of 40-500 nt.
  • the kit of this embodiment also includes: a solution of 80% by volume formamide, 400 mM NaCl, 40 mM PIPES and 1 mM EDTA at pH 7.5, as well as a deoxygenation system and/or RNase inhibitor.
  • the kit of this embodiment can be applied to the method provided by the present invention to measure force spectroscopy of single-molecule RNA.
  • the method of measuring single-molecule RNA force spectrum of the present invention also has the following advantages:
  • this method simplifies the experimental process of single-molecule force spectroscopy, while improving the detection efficiency and accuracy.
  • This method quickly prepares the molecules required for this method on the constructed plasmid by designing primers with special site modifications: handle chains, standard double-stranded DNA molecules and RNA molecules to be studied, so that they have sticky ends and affinity molecules.
  • the RNA molecules to be studied can efficiently form single-molecule RNA composite chains with affinity molecules with the handle chains through annealing.
  • the single-molecule RNA composite chains are more tightly linked to the microspheres, which can withstand greater forces and are used for the measurement of higher forces.
  • different samples are introduced into the microfluidic chip to quickly form a detection system for efficient force spectroscopy detection of the target RNA molecules.
  • the present invention can be widely used to measure the mechanical and dynamic behavior of nucleic acid molecules, and the interaction between nucleic acid molecules and other molecules such as small molecules, proteins and other nucleic acid-containing complexes.
  • Specific areas of application include basic and applied scientific research with the above needs; high-precision quantitative measurement, comparison and screening of the binding effects of small molecule drugs and various macromolecular drugs with nucleic acids in the process of target nucleic acid drug development; proposal or improvement of design schemes in mRNA drugs or vaccine design; quantitative measurement related to the pathogenic mechanism and treatment scheme of nucleic acid-related patients in medical research.
  • This method can quickly and efficiently construct a single-molecule RNA detection system to be detected, and is suitable for: 1) RNA molecules Force spectroscopy measurements; 2) Single-molecule mechanics experiments measuring higher forces (50-60pN); 3) Studies on the interaction between RNA molecules and other molecules.

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Abstract

本发明提供了一种测量单分子RNA力谱的方法及其应用，所述方法包括以下步骤：1)获得手柄链：设计并合成带有特殊修饰的手柄链引物；根据手柄链引物获得带有特殊修饰的手柄链，所述手柄链包括手柄链1和手柄链2；所述手柄链1和手柄链2带有粘性末端；2)获得单分子RNA-手柄复合链：获得单分子RNA，所述RNA带有与上述粘性末端互补配对的片段；将手柄链与RNA退火，得到单分子RNA-手柄复合链。本方法可以快速高效精准的对单个RNA分子实施力谱测量。本方法制备的单分子RNA复合链可以承受更大的作用力，可以用于较高作用力的测量，也可用于RNA结构分析。

Description

一种测量单分子RNA力谱的方法及其应用 技术领域
本发明涉及一种测量单分子RNA力谱的方法及其应用，属于生物物理，结构生物学领域。
背景技术
RNA分子一直是生物学家关注的重要生物大分子，早期的研究认为RNA在中心法则中作为DNA和蛋白之间传递遗传信息的重要分子，但随后发现，大部分RNA并不会翻译成蛋白质，而是具有多种重要的生理功能，例如：遗传编码、基因表达、基因调控和酶催化功能等。从分子结构角度来讲，在这些功能中起作用的既可以是其一级序列，也可以是二级或三级结构。然而，研究人员对RNA的复杂结构并不十分了解，与DNA规则的双螺旋结构不同，RNA分子一般为单链核糖核酸通过碱基配对折叠成二级结构或更复杂的三级结构，并且，RNA分子常与其他分子(蛋白质、DNA/RNA、小分子等)相互作用，其构象往往会实时变化。例如，冠状病毒基因组为的单股正义RNA病毒，在开放阅读框1a的末端有一个假结结构，可使核糖体执行-1位移码，从而在开放阅读框1b上继续翻译更长的多肽1ab，目前普遍认为该移码行为与该处RNA分子构具有特殊的假结结构有关。
目前，大多数具备重要生物医学价值的RNA结构仍然未知，且很难通过序列信息准确地预测其结构及功能，需要通过不同的方法逐一解析结构和研究功能，例如：SHAPE、X射线、核磁共振和冷冻电子显微镜等技术。SHAPE技术使用亲电试剂与活性不同的2-羟基反应，通过反转录的方法解析RNA分子的结构，该方法已经成功用于多种重要RNA的结构分析，但是，此方法主要获取RNA二级结构，信息较为单一。X射线技术和冷冻电子显微镜方法较为复杂，难以广泛应用，而核磁共振技术对RNA大小具有较大的限制，很难检测较大RNA分子(>60nt)。
光镊技术(Optical Tweezer)也称为梯度力光阱技术，使用聚焦的激光对物体产生力的作用，自光镊技术提出以来，其在生物领域中的应用就受到了广泛的关注，Ashkin也因此在2018年被授予诺贝尔物理学奖。与其他单分子力谱技术相比，光镊技术拥有更高的空间(亚纳米)、力学(亚皮牛顿)、时间(亚毫秒)分辨率，其特点与RNA分子的长度(单核苷酸长度约为0.59nm)、力学(0-30pN)等固有特性一致。随着光镊技术的发展，双光阱光镊技术逐渐成熟，与传统单分子力谱技术(单光阱光镊，磁镊， 原子力显微镜)将待研究分子的一端固定于玻璃、基底或探针表面不同，双光阱光镊技术将待研究分子固定于悬浮微球之间，并且使用压电平台、雪崩光电二极管等先进器件，大大降低外界环境带来的影响，提高技术的分辨率和稳定性。目前，单分子力谱技术已经较为广泛地应用于DNA、蛋白质的力谱研究，技术较为成熟，然而由于RNA分子不稳定和结构较为复杂，其力谱实验方法建立较为困难，缺少单分子RNA力谱的研究。
发明内容
为解决上述技术问题，本发明提供一种测量单分子RNA力谱的方法及其应用。
一方面，本发明提供一种测量单分子RNA力谱的方法，所述方法包括：
1)获得手柄链：设计并合成带有特殊修饰的手柄链引物；根据手柄链引物获得带有特殊修饰的手柄链，所述手柄链包括手柄链1和手柄链2；所述手柄链1和手柄链2带有粘性末端；优选地，所述手柄链为带有粘性末端的双链DNA；优选地，所述粘性末端长度为30-100nt，更优选为30-50nt；
2)获得单分子RNA-手柄复合链：获得单分子RNA，所述RNA带有与上述粘性末端互补配对的片段；将手柄链与RNA退火，得到单分子RNA-手柄复合链。
根据本发明的具体实施方案，所述方法还包括设计并构建质粒；所述质粒包含待研究的RNA的模板DNA序列和手柄链序列；根据质粒设计带有特殊修饰的引物；根据设计的质粒与引物获得待研究RNA分子的DNA模板、手柄链1和手柄链2。
根据本发明的具体实施方案，所述方法还包括根据待研究RNA分子的DNA模板得到单分子RNA。
根据本发明的具体实施方案，所述方法还包括获得标准双链DNA；所述标准双链DNA是以手柄链引物为扩增引物进行扩增得到的；所述标准双链DNA分子直接用于验证单分子实验系统。
优选地，所述标准双链DNA是以手柄链1的上游引物和手柄链2的下游引物为扩增引物进行扩增得到的。
根据本发明的具体实施方案，所述引物和/或手柄链带有1个或2个以上的特殊修饰分子；
优选地，所述引物和/或手柄链带有3个、4个或5个以上的特殊修饰分子。
优选地，所述特殊修饰包括地高辛修饰、生物素修饰、d-spacer修饰、Phosphate修饰、Thiophosphate修饰和叠氮基团修饰中的一种或两种以上的组合；
优选地，所述特殊修饰为地高辛。
优选地，所述引物和/或手柄链带有3个以上的地高辛修饰；
优选地，所述手柄链带有3-5个地高辛修饰。
根据本发明的具体实施方案，其中，所述手柄链1上游引物1F还带有地高辛修饰，且下游引物1R带有d-spacer修饰，下游引物1R带有粘性末端序列1以与RNA退火连接；所述手柄链2的上游引物2F带有Phosphate修饰，下游引物2R带有生物素修饰，且上游引物2F带有粘性末端序列2以与RNA退火连接。
根据本发明的具体实施方案，其中，所述手柄链分别带有特殊修饰和粘性末端；
优选地，所述手柄链1分别带有特殊修饰和长度为30-100nt的粘性末端1；
优选地，所述手柄链2分别带有特殊修饰和长度为30-100nt的粘性末端2；
优选地，所述粘性末端1包括如SEQ ID NO.7所示序列的片段；
优选地，所述粘性末端2包括如SEQ ID NO.8所示序列的片段。
根据本发明的具体实施方案，所述方法包括通过改变上、下游引物的间隔距离得到不同长度的手柄链和/或标准双链DNA；
优选地，可以通过改变质粒大小改变手柄链和/或标准双链DNA的长度；
优选地，可以通过改变引物在模板上的结合位置改变手柄链和/或标准双链DNA的长度。优选地，本发明手柄链的长度为1kb。
本方法设计的引物具有较大的扩展性，可以制备出不同长度的手柄链，适用于实现不同规格的分子的力谱测量，拓展用途。
根据本发明的具体实施方案，其中，所述手柄链用于连接单分子RNA与表面；所述表面为带有修饰以与手柄链1和/或手柄链2的特殊修饰分子结合的表面。
根据本发明的具体实施方案，其中，所述方法还包括设计带有与粘性末端序列互补配对的序列的引物，用以最终得到带有与粘性末端互补配对片段的RNA。
根据本发明的具体实施方案，其中，所述手柄链引物包括如SEQ ID NO.1-SEQ ID NO.4所示序列的引物。
根据本发明的具体实施方案，其中，所述退火是将待研究RNA分子与手柄链1和手柄链2按摩尔比(0.7-2):1:1混合后退火；
优选地，所述退火是将待研究RNA分子与手柄链1和手柄链2按摩尔比1:1:1混合后退火。
根据本发明的具体实施方案，其中，所述退火的温度为50-65℃；
优选地，所述退火的温度为62℃和/或52℃。
进一步优选地，所述退火的条件为：98℃10分钟，62℃1小时，52℃1小时，4℃终止反应。
根据本发明的具体实施方案，其中，所述退火的缓冲液为含有甲酰胺和PIPES的缓冲溶液；
优选地，所述退火的缓冲液中含有体积分数为60％-80％的甲酰胺；
进一步优选地，所述退火的缓冲液为含有：体积分数为80％的甲酰胺、400mM NaCl、40mM PIPES和1mM EDTA的pH 7.5的溶液。
根据本发明的具体实施方案，其中，所述方法还包括进行分子力谱检测，所述力谱检测在含有除氧系统和/或RNase抑制剂的测量缓冲溶液中进行；所述除氧系统和RNase抑制剂可以保持RNA分子处于无氧的水溶液环境中，防止RNA被酶降解，提高RNA在实验中的稳定性。
优选地，所述除氧系统包含葡萄糖氧化酶、过氧化氢酶和葡萄糖；
优选地，所述除氧系统包含160units/ml葡萄糖氧化酶、100units/ml过氧化氢酶和质量分数为0.8％葡萄糖；
优选地，所述测量缓冲溶液为含有NaCl、EDTA+或Mg2+的溶液；优选地，所述缓冲溶液为Hepes或Tris-HCl缓冲溶液。
根据本发明的具体实施方案，其中，所述力谱检测是使用微流控通道进行的。所述微流控通道中的样品为：将单分子RNA-手柄复合链或标准双链DNA单分子样品与微球1孵育后，加入测量缓冲溶液，置于通道1；通道2加入测量缓冲溶液；通道3为微球2溶液。向微流控通道中通入样品，并向每个通道中均加入除氧系统和RNase抑制剂；在微流控通道中形成力谱检测体系并进行分子力谱检测。采用微流控通道用于控制分离不同组分样品，避免干扰，快速高效开展单分子力谱实验。
根据本发明的具体实施方案，其中，所述力谱检测包括：固定RNA-手柄复合链的一端或两端，获取数据；所述数据优选为力、时间和/或距离；
优选地，所述力谱检测包括：固定RNA-手柄复合链一端，在另一端以单一方向移动，获取力、时间和距离等数据；
优选地，所述力谱检测包括：固定RNA-手柄复合链两端的距离或力，获取RNA分子力或距离、时间等数据；
优选地，所述力谱检测包括：固定RNA分子两端，获取RNA分子与其他分子相互作用时距离、力参数等变化；所述其他分子优选为小分子或蛋白质；
优选地，所述力谱检测过程中使用双光阱光镊、磁镊、声镊、原子力显微镜控制其中一端和/或两端的移动和固定。本方法在测量分子力谱时使用双光阱光镊移动和固定微球，减少样品与外界物理环境接触，减少外界环境干扰，提高测量力学和距离的稳定性。
根据本发明的具体实施方案，其中，所述力谱检测所述形成力谱检测体系包括：控制微球1和微球2，使单分子RNA复合链或者标准双链DNA同时连接到微球1和微球2，形成力谱检测体系。
另一方面，本发明提供一种引物，所述引物由选自SEQ ID NO.1-SEQ ID NO.4的序列组成。
根据本发明的具体实施方案，其中，所述引物带有1个或2个以上的特殊修饰分子；优选地，所述引物带有3个、4个或5个以上的特殊修饰分子。
根据本发明的具体实施方案，其中，所述特殊修饰包括地高辛修饰、生物素修饰、d-spacer修饰、Phosphate修饰、Thiophosphate修饰和叠氮基团修饰中的一种或两种以上的组合；优选地，所述特殊修饰为地高辛。
根据本发明的具体实施方案，其中，所述引物带有3个以上的地高辛修饰；
优选地，所述手柄链带有3-5个地高辛修饰。
本发明对手柄和粘性末端的设计，使得本发明所得单分子RNA-手柄复合链可以承受较高的作用力，优选地，所述较高作用力为50-60pN。
另一方面，本发明提供一种粘性末端，其由选自SEQ ID NO.7-SEQ ID NO.8的序列组成。
另一方面，本发明提供一种测量单分子RNA力谱的试剂盒，其中，所述试剂盒包含至少一种所述的引物和/或至少一种所述的粘性末端。
根据本发明的具体实施方案，其中，所述试剂盒包括退火缓冲液，所述退火缓冲液包含甲酰胺和PIPES；
优选地，所述退火缓冲液中含有体积分数为60％-80％的甲酰胺；
进一步优选地，所述退火缓冲液为含有：体积分数为80％的甲酰胺、400mM NaCl、40mM PIPES和1mM EDTA的pH 7.5的溶液。
根据本发明的具体实施方案，其中，所述试剂盒包括除氧系统和/或RNase抑制剂；
优选地，所述除氧系统为含有葡萄糖氧化酶、过氧化氢酶和葡萄糖的缓冲液；
优选地，所述除氧系统为含有160units/mL葡萄糖氧化酶、100units/mL过氧化氢酶和质量分数为0.8％的葡萄糖的缓冲液。
本发明带来的有益效果：
本方法提供了一种快速高效测量单分子RNA样品的实验方法，可以快速高效精准的对单个RNA分子实施力谱测量。并且本方法具有良好的重复性。具体而言，本发明具有以下优势：
1.本发明的手柄链与RNA以粘性末端连接、形成双链，使手柄与单分子RNA的连接更为紧密，本发明的手柄为DNA双链，整个单分子RNA-手柄复合链中DNA双链成分更高，使单分子RNA-手柄复合链具有更高的稳定性，可以承受更大的作用力，适用于较高作用力和更精准的测量。同样的，本发明也适用于RNA结构分析。
2.本发明采用带有特殊修饰的手柄链，优选采用3个及以上的特殊分子修饰手柄链，手柄与固着表面的连接更加紧密，可以承受更大的作用力，适用于较高作用力的测量。
3.本发明设计了更优的粘性末端长度，及更高效的退火条件，使RNA样品与手柄链退火连接更加高效。
4.本发明设计了更适合RNA力谱测量的测量体系，特别是加入了除氧系统和/或RNase抑制剂，使得测量环境更适合RNA。
附图说明
图1为本方法实验流程图。
图2为标准双链DNA、手柄链1、手柄链2和RNA分子凝胶电泳图。
图3为实验用多通道微流控芯片示意图。
图4为3kbp双链DNA单分子力谱图。
图5为RNA单分子力谱拟合曲线图。
图6为本方法标准双链DNA、手柄链1、手柄链2与引物的质粒设计示意图。
图7为不同甲酰胺浓度的退火溶液的条件探究。
图8为改变质粒大小使用1F、1R、2F、2R扩增出较长的手柄3、手柄4。
具体实施方式
本发明提供一种测量单分子RNA力谱的方法，所述方法包括：
1)获得手柄链：设计并合成带有特殊修饰的手柄链引物；根据手柄链引物获得带有特殊修饰的手柄链，所述手柄链包括手柄链1和手柄链2；所述手柄链1和手柄链2带有粘性末端；优选地，所述手柄链为带有粘性末端的双链DNA；优选地，所述粘性 末端长度为30-100nt；
2)获得单分子RNA-手柄复合链：获得单分子RNA，所述RNA带有与上述粘性末端互补配对的片段；将手柄链与RNA退火，得到单分子RNA-手柄复合链。
根据本发明的具体实施方案，本发明所述的测量单分子RNA力谱的方法包括：
1)获得手柄链及其他所需的双链DNA：设计并合成带有特殊修饰的手柄链引物；根据手柄链引物获得带有特殊修饰的手柄链，所述手柄链包括手柄链1和手柄链2；所述手柄链1和手柄链2带有粘性末端；优选地，所述手柄链为带有粘性末端的双链DNA；优选地，所述粘性末端长度为30-100nt；获得待研究RNA分子的DNA模板。
其中，所述手柄链1的引物：手柄链1的上游引物1F的5’端带有1个或2个以上的地高辛修饰，下游引物1R的链中带有d-spacer修饰，下游引物1R的5’端带有粘性末端序列1待与RNA的5’端退火连接；
其中，所述手柄链2的引物：手柄链2的上游引物2F的5’端带有Phosphate修饰，上游引物2F链中带有Thiophosphate修饰，且上游引物2F的5’端带有粘性末端序列2待与RNA的3’端退火连接，手柄链2的下游引物2R的5’端带有1个或2个以上的生物素修饰；
其中，获得手柄链1、手柄链2和标准双链DNA：以设计构建的质粒为模板，利用设计的引物1F、1R、2F和2R，进行PCR反应，PCR条件为：98℃1分钟，98℃10秒，58℃30秒，72℃30秒、60秒或90秒，循环35次后，4℃终止反应；PCR反应结束后凝胶电泳分离产物，分别获得手柄链1、手柄链2和标准双链DNA；
所述手柄链1的5’端和3’端分别带有地高辛修饰和序列长度为30-100nt的粘性末端；
所述手柄链2的5’端和3’端分别带有生物素修饰和序列长度为30-100nt的粘性末端；
其中，获得待研究RNA分子的DNA模板：根据RNA序列，设计上游引物3F和下游引物3R，所述上游引物3F的5’端带有与手柄链1的粘性末端序列1互补配对的序列，下游引物3R的5’端带有与手柄链2的粘性末端序列2互补配对的序列；以设计构建的质粒为模板，利用引物3F和3R进行PCR反应，合成得到待研究RNA对应的DNA模板；
2)获得单分子RNA-手柄复合链：获得单分子RNA，所述RNA带有与上述粘性末端互补配对的片段；将手柄链与RNA退火，得到单分子RNA-手柄复合链。
其中，所述获得待研究RNA分子：以所述待研究RNA对应的DNA模板为底物使用T7试剂盒进行转录，得到待研究RNA分子；所述待研究RNA分子的5’端和3’端具有与手柄链1和手柄链2的粘性末端互补配对的序列，所述互补配对的序列长度为30-50nt；
其中，所述获得单分子RNA-手柄复合链：将待研究RNA分子与手柄链1和手柄链2按摩尔比1:1:1混合后退火，制备单分子RNA-手柄复合链，其中，退火条件为：98℃10分钟，62℃1小时，52℃1小时，4℃终止反应；反应终止后，使用乙醇沉淀，加入10μL H2O溶解即可获得单分子RNA-手柄复合链；
3)使用微流控通道进行分子力谱检测：向微流控通道中通入样品，并向每个通道中均加入除氧系统和RNase抑制剂；在微流控通道中形成力谱检测体系并进行分子力谱检测；
其中，所述向微流控通道中通入不同样品：采用微流控通道分隔不同样品，其中，将1μL单分子RNA-手柄复合链或标准双链DNA单分子样品与1μL抗地高辛微球1室温孵育5分钟，加入1mL测量缓冲溶液，置于通道1；通道2加入测量缓冲溶液；通道3为链霉素亲和修饰的微球2溶液；每个通道中均加入除氧系统和RNase抑制剂；
所述微球1和微球2的制备过程为：使用5μg/ml EDC和NHS活化羧基修饰的微球，20分钟后加入抗地高辛蛋白与活化的微球偶联，获得抗地高辛修饰的微球1；活化后加入链霉素偶联则获得链霉素修饰的微球2；
其中，所述形成力谱检测体系：使用光阱1在通道1捕获微球1，光阱2在通道3捕获微球2，移动光阱1和光阱2至通道2，光阱1和光阱2缓慢靠近，使微球1上的单分子RNA复合链或者标准双链DNA样品特异性的与微球2相结合，形成力谱检测体系；
其中，所述力谱检测：固定微球1或微球2，在水平方向移动另一微球，获取力、时间和距离等数据。
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
实施例中未注明具体条件的实验方法，通常按照常规条件以及手册中所述的条件，或按照制造厂商所建议的条件；所用的通用设备、材料、试剂等，如无特殊说明，均可从商业途径得到，来自于Sigma。
实施例1
本实施例提供快速高效测量单分子RNA样品的方法，本方法的实验流程如图1所示。
所述快速高效测量单分子RNA样品的操作步骤如下：
1)获得手柄链及其他所需DNA：设计并构建含有待研究RNA分子、手柄链1和手柄链2DNA序列的质粒；并根据质粒设计带有特殊修饰的引物；根据设计的质粒与引物获得待研究RNA分子的DNA模板、手柄链1、手柄链2和标准双链DNA；所述手柄链1和手柄链2为带有粘性末端的双链DNA；
其中，所述手柄链1的引物：手柄链1的上游引物1F的5’端带有地高辛修饰，下游引物1R的5’端带有磷酸修饰，且下游引物1R的链中带有d-spacer修饰，下游引物1R的5’端带有粘性末端序列1待与RNA的5’端退火连接；所述引物序列见表1。
其中，所述手柄链2的引物：手柄链2的上游引物2F的5’端带有Phosphate修饰，上游引物2F中带有Thiophosphate修饰，且上游引物2F的5’端带有粘性末端序列2待与RNA的3’端退火连接，手柄链2的下游引物2R的5’端带有生物素修饰；所述引物序列见表1。本实验所有引物合成来自于金斯瑞生物科技股份有限公司。本发明中采用“T”代表尿嘧啶“U”。
表1

其中，获得手柄链1、手柄链2和标准双链DNA：参考图6示意，设计并合成引物1F、1R、2F和2R，使用聚合酶(F530，Thermo)进行PCR反应，PCR条件为：98℃1分钟，98℃10秒，58℃30秒，72℃30秒、60秒或90秒，循环35次后，4℃终止反应；PCR反应结束后凝胶电泳分离产物，使用Tiangen试剂盒(DP209，天根生化科技(北京)有限公司)进行产物回收；分别获得手柄链1、手柄链2和标准双链DNA；得到的手柄链1、手柄链2和标准双链DNA单分子样品电泳图如图2所示。
所述手柄链1的5’端和3’端分别带有5个地高辛修饰和序列长度为36nt的粘性末端1，粘性末端1序列见表1。
所述手柄链2的5’端和3’端分别带有2个生物素修饰和序列长度为35nt的粘性末端2，粘性末端2序列见表1。
其中，获得待研究RNA分子的DNA模板：根据RNA序列，设计上游引物3F和下游引物3R，所述上游引物3F的5’端带有与手柄链1的粘性末端序列1互补配对的序列，下游引物3R的5’端带有与手柄链2的粘性末端序列2互补配对的序列；所述引物3F和3R的序列见表1。
利用引物3F和3R进行PCR反应，合成得到待研究RNA对应的DNA模板(待研究RNA对应的DNA模板序列见表1)。
2)获得单分子RNA-手柄复合链：根据待研究RNA分子的DNA模板得到待研究RNA分子；所述待研究RNA分子的5’端和3’端具有与手柄链1和手柄链2的粘性末端互补配对的序列，所述互补配对的序列长度为30-100nt；将待研究RNA分子与手柄链1和手柄链2混合后退火，得到单分子RNA-手柄复合链。
其中，所述获得待研究RNA分子：以所述待研究RNA对应的DNA模板为底物使用T7试剂盒(E2040S,NEB)进行转录，得到待研究RNA分子；所述待研究RNA分子的5’端和3’端具有与手柄链1和手柄链2的粘性末端互补配对的序列，所述互补配对的 序列长度为30-100nt；得到的待研究RNA分子样品电泳图如图2所示。
其中，所述获得单分子RNA-手柄复合链：将待研究RNA分子与手柄链1和手柄链2各自纯化后按摩尔比1:1:1混合后，在体积分数为80％的甲酰胺(F9037，Sigma)、400mM NaCl、40mM PIPES、1mM EDTA、pH 7.5的溶液中退火反应，制备单分子RNA-手柄复合链。其中，退火条件为：98℃10分钟，62℃1小时，52℃1小时，4℃终止反应；反应终止后，使用乙醇沉淀，加入10μL H2O溶解即可获得单分子RNA-手柄复合链。单分子RNA复合链具有亲和分子末端，可以与对应的微球结合，用于较大力值测定。
3)使用微流控通道进行分子力谱检测：采用微流控通道分隔不同样品，向微流控通道中通入不同样品，并向每个通道中均加入除氧系统和RNase抑制剂(N8080119，Thermo)；在微流控通道中形成力谱检测体系并进行分子力谱检测；
其中，所述向微流控通道中通入不同样品：采用微流控通道分隔不同样品，微流控通道示意图见图3所示。将1μL单分子RNA-手柄复合链或标准双链DNA单分子样品与1μL抗地高辛(11222089001，Roche)微球1室温孵育5分钟，加入1mL测量缓冲溶液，置于通道1；通道2加入测量缓冲溶液；通道3为链霉素亲和修饰的微球2溶液(SVP-08-10,Spherotech)；每个通道中均加入除氧系统和RNase抑制剂；本方法所使用的测量缓冲溶液为含有20mM Hepes、100mM NaCl和0.2mM EDTA的缓冲液。
所述微球1和微球2的制备过程为：使用5μg/ml EDC和NHS活化羧基修饰的微球，20分钟后加入抗地高辛蛋白与活化的微球偶联，获得抗地高辛修饰的微球1；活化后加入链霉素偶联则获得链霉素修饰的微球2；所述核酸大分子之间、核酸分子与微球之间的连接方式见图1所示。
其中，所述形成力谱检测体系：使用光阱1在通道1捕获微球1，光阱2在通道3捕获微球2，移动光阱1和光阱2至通道2，光阱1和光阱2缓慢靠近，使微球1上的单分子RNA复合链或者标准双链DNA样品特异性的与微球2相结合，形成力谱检测体系；本方法使用微流控芯片，向芯片中通入不同样品，快速形成检测体系，对目标RNA分子进行高效的力谱检测。
其中，所述力谱检测：固定微球1或微球2，在单一方向移动另一微球，获取力、时间和距离等数据。
本实施例在测量单分子力谱时使用Lumicks公司生产的双光阱光镊为例，但不限于该设备。
实施例2
本实施例探究获得单分子RNA-手柄复合链过程中，RNA与手柄链退火时含有不同体积分数的甲酰胺的退火缓冲溶液的退火效果。具体操作为：
①采用与实施例1中相同方法获得RNA和手柄链；
②将相同的RNA与手柄链分别在含有体积分数为30％、40％、50％、60％、70％和80％甲酰胺的PIPES缓冲溶液中退火，各组退火条件除甲酰胺的浓度不同外其余退火条件相同；
③将各组获得的单分子RNA-手柄复合链采用与实施例1中相同方法进行后续力谱测量。
退火后的电泳结果如图7所示，仅通过凝胶电泳结果无法判断各组RNA-手柄复合链是否连接成功，需在后续力谱测量中对退火条件的优劣进行判断。经过力谱测量实验，随着甲酰胺浓度增加，形成RNA单分子复合链的几率增加，且在体积分数为80％的甲酰胺溶液中退火后，形成RNA单分子链的效果约为体积分数为50％的甲酰胺溶液中形成RNA单分子链的1.6倍左右。我们判断含有体积分数为80％的甲酰胺的PIPES缓冲溶液是最优的退火条件。即最合适的退火缓冲液为含有：体积分数为80％的甲酰胺、400mM NaCl、40mM PIPES和1mM EDTA的pH 7.5的溶液。
实施例3
本实施例探究获得单分子RNA-手柄复合链过程中，RNA与手柄链退火时不同退火程序的退火效果。具体操作为：
①采用与实施例1中相同方法获得RNA和手柄链；
②将相同的RNA与手柄链分别在相同反应体系、不同反应程序中退火，各组退火条件退火程序不同外其余退火条件相同；其中，所述不同退火反应程序为：
程序1：98℃10分钟，62℃1小时，52℃1小时，4℃终止反应；
程序2：95℃10分钟，之后梯度降温，梯度降温速度为1℃/min进行，4℃终止反应；
③退火结束后，将各组获得的单分子RNA-手柄复合链采用与实施例1中相同方法进行后续力谱测量。
通过力谱检测RNA-手柄复合链的形成效率，力谱检测表明在程序1的反应条件下，形成有效数据的概率为10％。我们判断退火条件为62℃1小时，52℃1小时具有最好的RNA-手柄复合链的形成效率。即最合适的退火条件为：98℃10分钟，62℃1小时， 52℃1小时，4℃终止反应。
实施例4
本实施例提供利用实施例1中提供的引物1F、1R、2F和2R，通过扩大质粒，扩增得到不同长度的手柄链的实验。
PCR条件为：98℃1分钟，98℃10秒，58℃30秒，72℃240秒，循环35次后，4℃终止反应；PCR反应结束后凝胶电泳分离产物，使用Tiangen试剂盒(DP209，天根生化科技(北京)有限公司)进行产物回收；分别获得手柄链1、手柄链2和标准双链DNA；得到的手柄链1、手柄链2和标准双链DNA单分子样品电泳图如图8所示。
由图8可知，手柄链3和手柄链4具有明显大于手柄链1和手柄链2的分子量。即，在使用引物1F、1R、2F和2R扩增得到手柄链时，可以通过改变质粒而得到具有不同的长度的手柄链，不同长度的手柄链适合更多类型的力谱实验，可以满足更多的力谱测量需要。
实施例5
本实施例采用与实施例1中相同方法测量多组DNA、RNA分子的力谱。其中，
①测量3kbp双链DNA单分子力谱图实验结果见图4所示。
②测量RNA单分子力谱实验结果见图5所示。
从图4可以看出，本方法使用更稳定的双链DNA作为手柄，可以承受更大的作用力约60pN的测量
从图5可知，本方法制备的单分子RNA复合链的力谱曲线可以展示出RNA结构在恒定速度拉伸下的力谱变化。
实施例6
本实施例提供一种试剂盒，该试剂盒包含粘性末端，粘性末端的序列如SEQ ID NO.7或SEQ ID NO.8所示，或者如SEQ ID NO.7的互补序列(SEQ ID NO.11：GAATTCGGCTACGTAGCTCAGTTGGTTAGAGCAGCG)或SEQ ID NO.8的互补序列(SEQ ID NO.12：GTCACAGGTTCGAATCCCGTCGTAGCCACCACTGC)所示。
本实施例所述试剂盒可以用于构建结构如：“粘性末端1—RNA—粘性末端2”所示的带有粘性末端的RNA，该结构中的RNA可以为任意单分子RNA。例如，带有粘性末端的RNA为：GAATTCGGCTACGTAGCTCAGTTGGTTAGAGCAGCG(SEQ ID NO.11)—RNA—GTCACAGGTTCGAATCCCGTCGTAGCCACCACTGC(SEQ ID  NO.12)。
优选地，本试剂盒适用于测量长度为40-500nt的RNA的分子力谱。
本实施例的试剂盒还包括：体积分数为80％的甲酰胺、400mM NaCl、40mM PIPES和1mM EDTA的pH 7.5的溶液，以及除氧系统和/或RNase抑制剂。
本实施例的试剂盒可以适用于本发明提供的方法，用以单分子RNA的力谱测量。
上述实施例可由本领域技术人员在不背离被发明原理和宗旨的前提下以不同的方式对其进行局部调整，本发明的保护范围以权利要求书为准且不由上述具体实施所限，在其范围内的各个实现方案均受本发明之约束。
与传统方法相比，本发明测量单分子RNA力谱的方法还具有以下优势：
1.传统方法设计手柄采用DNA和RNA链混合互补配对的方式，由于DNA与RNA的核苷酸组成有不同，所以该方法的手柄力谱数据分析较为困难，缺乏标准数据对比；而本方法中采用双链DNA作用手柄，其力谱数据可根据数学方程计算，具有标准可对照。如图4所示，本方法的双链DNA手柄与理论曲线重合一致，展现本方法制备的手柄具有极高的准确性，可用于后续高精度RNA单分子力谱分析。
2.传统方法缺乏针对RNA分子力谱高精度的测量，本方法填补该研究领域的空白。本方法可快速高效的建立RNA单分子力谱的测量实验体系。
本方法一方面简化单分子力谱的实验流程，同时提高检测效率和精度。本方法通过设计带有特殊位点修饰的引物，在已构建的质粒上快速制备本方法所需的分子：手柄链，标准双链DNA分子和待研究RNA分子，使其具有粘性末端和亲和分子，一方面使得待研究RNA分子可以与手柄链通过退火的方式高效形成带有亲和分子的单分子RNA复合链，同时，使得单分子RNA复合链与微球链接更加紧密，可以承受更大的作用力，用于较高作用力的测量；最后，使用微流控芯片中通入不同样品，快速形成检测体系，对目标RNA分子进行高效的力谱检测。
本发明可以普遍应用于对核酸分子力学及动力学行为进行测量，核酸分子与其他分子如小分子、蛋白质和其他含核酸的复合物之间的相互作用的测量。具体涉及的领域包括有上述需求的基础及应用科研工作；靶标核酸药物研发过程中对小分子药物及各类大分子药物与核酸结合效果的高精度定量测量、比较及筛选；mRNA类药物或疫苗设计中设计方案的提出或改进；在医学研究中与核酸相关病人致病机制及治疗方案相关的定量测量。
本方法可以快速高效构建待检测的单分子RNA检测体系，适用于：1)RNA分子 力谱测量；2)测量较高的单分子力学实验(50-60pN)；3)RNA分子与其他分子相互作用的研究。

Claims (22)

  1. 一种测量单分子RNA力谱的方法，所述方法包括：
    1)获得手柄链：设计并合成带有特殊修饰的手柄链引物；根据手柄链引物获得带有特殊修饰的手柄链，所述手柄链包括手柄链1和手柄链2；所述手柄链1和手柄链2带有粘性末端；
    2)获得单分子RNA-手柄复合链：获得单分子RNA，所述RNA带有与上述粘性末端互补配对的片段；将手柄链与RNA退火，得到单分子RNA-手柄复合链。
  2. 根据权利要求1所述的测量单分子RNA力谱的方法，其中，所述方法还包括获得标准双链DNA；所述标准双链DNA是以手柄链引物为扩增引物进行扩增得到的；
    优选地，所述标准双链DNA是以手柄链1的上游引物和手柄链2的下游引物为扩增引物进行扩增得到的。
  3. 根据权利要求1或2所述的测量单分子RNA力谱的方法，其中，所述引物和/或手柄链带有1个或2个以上的特殊修饰分子；
    优选地，所述引物和/或手柄链带有3个、4个或5个以上的特殊修饰分子。
  4. 根据权利要求1-3任一项所述的测量单分子RNA力谱的方法，其中，所述特殊修饰包括地高辛修饰、生物素修饰、d-spacer修饰、Phosphate修饰、Thiophosphate修饰和叠氮基团修饰中的一种或两种以上的组合。
  5. 根据权利要求1-4任一项所述的测量单分子RNA力谱的方法，其中，所述引物和/或手柄链带有3个以上的地高辛修饰；
    优选地，所述手柄链带有3-5个地高辛修饰。
  6. 根据权利要求1-5任一项所述的测量单分子RNA力谱的方法，其中，所述手柄链分别带有特殊修饰和粘性末端；
    优选地，所述手柄链1分别带有特殊修饰和长度为30-100nt的粘性末端1；
    优选地，所述手柄链2分别带有特殊修饰和长度为30-100nt的粘性末端2；
    优选地，所述粘性末端1包括如SEQ ID NO.7所示序列的片段；
    优选地，所述粘性末端2包括如SEQ ID NO.8所示序列的片段。
  7. 根据权利要求1-6任一项所述的测量单分子RNA力谱的方法，其中，所述方法包括通过改变上、下游引物的间隔距离得到不同长度的手柄链和/或标准双链DNA；
    优选地，可以通过改变质粒大小改变手柄链和/或标准双链DNA的长度；
    优选地，可以通过改变引物在模板上的结合位置改变手柄链和/或标准双链DNA的长度。
  8. 根据权利要求1-7任一项所述的测量单分子RNA力谱的方法，优选地，所述手柄链引物包括如SEQ ID NO.1-SEQ ID NO.4所示序列的引物。
  9. 根据权利要求1-8任一项所述的测量单分子RNA力谱的方法，其中，所述退火是将待研究RNA分子与手柄链1和手柄链2按摩尔比(0.7-2):1:1混合后退火；
    优选地，所述退火是将待研究RNA分子与手柄链1和手柄链2按摩尔比1:1:1混合后退火。
  10. 根据权利要求1-9任一项所述的测量单分子RNA力谱的方法，其中，所述退火的温度为50-65℃；
    优选地，所述退火的温度为62℃和/或52℃；
    进一步优选地，所述退火的条件为：98℃10分钟，62℃1小时，52℃1小时，4℃终止反应。
  11. 根据权利要求1-10任一项所述的测量单分子RNA力谱的方法，其中，所述退火的缓冲液为含有甲酰胺和PIPES的缓冲溶液；
    优选地，所述退火的缓冲液中含有体积分数为60％-80％的甲酰胺；
    进一步优选地，所述退火的缓冲液为含有：体积分数为80％的甲酰胺、400mM NaCl、40mM PIPES和1mM EDTA的pH 7.5的溶液。
  12. 根据权利要求1-11任一项所述的测量单分子RNA力谱的方法，其中，所述粘性末端的长度为30-100nt；
    优选地，粘性末端的长度为30-50nt。
  13. 根据权利要求1-12任一项所述的测量单分子RNA力谱的方法，其中，所述方法还包括进行分子力谱检测，所述力谱检测在含有除氧系统和/或RNase抑制剂的测量缓冲溶液中进行；
    优选地，所述除氧系统包含葡萄糖氧化酶、过氧化氢酶和葡萄糖；
    优选地，所述除氧系统包含160units/mL葡萄糖氧化酶、100units/mL过氧化氢酶和质量分数为0.8％的葡萄糖；
    优选地，所述测量缓冲溶液为含有NaCl、EDTA+或Mg2+的溶液；优选地，所述测量缓冲溶液为Hepes和/或Tris-HCl缓冲溶液。
  14. 根据权利要求13所述的测量单分子RNA力谱的方法，其中，所述力谱检测包 括：固定RNA的一端或两端，获取数据；
    优选地，所述力谱检测过程中使用双光阱光镊、磁镊、声镊、原子力显微镜控制其中一端和/或两端的移动和固定。
  15. 一种引物，所述引物由选自SEQ ID NO.1-SEQ ID NO.4的序列组成。
  16. 根据权利要求15所述的引物，其中，所述引物带有1个或2个以上的特殊修饰分子；优选地，所述引物带有3个、4个或5个以上的特殊修饰分子。
  17. 根据权利要求15或16所述的引物，其中，所述特殊修饰包括地高辛修饰、生物素修饰、d-spacer修饰、Phosphate修饰、Thiophosphate修饰和叠氮基团修饰中的一种或两种以上的组合；
    优选地，所述特殊修饰为地高辛。
  18. 根据权利要求15-17任一项所述的引物，其中，所述引物带有3个以上的地高辛修饰；
    优选地，所述引物带有3-5个地高辛修饰。
  19. 一种粘性末端，其由选自SEQ ID NO.7-SEQ ID NO.8的序列组成。
  20. 一种测量单分子RNA力谱的试剂盒，其中，所述试剂盒包含至少一种如权利要求15-18任一项所述的引物和/或如权利要求19所述的粘性末端。
  21. 根据权利要求20所述的试剂盒，其中，所述试剂盒包括退火缓冲液，所述退火缓冲液包含甲酰胺、PIPES、NaCl和EDTA；
    优选地，所述退火缓冲液中含有体积分数为60％-80％的甲酰胺；
    进一步优选地，所述退火缓冲液为含有：体积分数为80％的甲酰胺、400mM NaCl、40mM PIPES和1mM EDTA的pH 7.5的溶液。
  22. 根据权利要求20或21所述的试剂盒，其中，所述试剂盒包括除氧系统和/或RNase抑制剂。
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