Method for determining dynamic behaviors of different C2 'modified G-quadruplexes based on single-molecule magnetic tweezers

Abstract

The invention discloses a method for determining dynamic behaviors of different C2'modified G-quadruplexes based on single-molecule magnetic tweezers, and belongs to the technical field of biology, the method comprises the following steps: (1) constructing a handle chain 1 and a handle chain 2, the handle chain 1 and the handle chain 2 are respectively modified by different functional groups, and the handle chain 1 and the handle chain 2 have viscous ends; annealing the C2'modified G-quadruplex single chain, the first primer and the second primer, and connecting with the handle chain 1 and the handle chain 2 through ligase to prepare a sample to be detected; and (2) constructing a single-molecule magnetic tweezer system, changing the tensile force applied to the sample to be tested by using a single-molecule magnetic tweezer technology, carrying out a force climbing experiment, collecting real-time data, and analyzing the dynamic behavior of the C2'modified G-quadruplex according to the collected force climbing data through a mathematical model. The method can be used for designing a more stable or controllable G-quadruplex, revealing the influence of modification on a folding path, and providing key data for designing quadruplex-based drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for measuring the dynamic behaviors of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers. Background Art

[0002] The G-quadruplex structure is an unconventional nucleic acid structure in which G-rich sequences can form quadruplex helical structures through Hoogsteen hydrogen bonding. The G-quadruplex is a nucleic acid structure present in human telomeres and oncogene promoter regions. It has attracted widespread attention due to its role in telomere maintenance and gene regulation. In addition, the G-quadruplex can also be used as a supramolecular assembly, a DNA-based nanodevice, a new building block for potential antiviral agents, and a new class of molecular targets for anticancer drugs. G-quadruplexes show significant structural diversity, and current high-resolution structural studies on human telomeric DNA mainly focus on short DNA sequences that can form a single quadruplex. In many cases, these oligonucleotide sequences can adopt and convert between different conformations, making structural analysis complicated.

[0003] Studies have shown that telomeric G-quadruplexes and other RNA quadruplexes tend to form parallel structures, which is related to the strong preference of guanosine ribonucleotides to adopt the trans-anti glycosidic bond conformation. The syn / anti conformational preference between dG and rG has been used to regulate the folding of G-quadruplexes, and different C2′ nucleoside modifications have been proposed based on this strategy. Different C2′ nucleoside-modified G-quadruplexes exhibit different physicochemical properties, thereby realizing biological functions that DNA / RNA do not have. These sugar modifications change the conformation and kinetic behavior of G-quadruplexes, providing ideal physicochemical and biological properties in terms of improving biological stability, binding affinity and tissue specificity, and reducing immune responses. However, to date, the effects of various C2′ modifications on the mechanical stability and folding / unfolding rates of G-quadruplexes have not been studied.

[0004] Single-molecule manipulation technology can apply force to biological macromolecules in a directional manner to directly manipulate biological macromolecules. This type of technology has unique advantages in exploring mechanically induced biological processes. Single-molecule magnetic tweezers technology is one of the single-molecule manipulation technologies. It generally controls the movement of superparamagnetic beads through a magnetic field, and uses this bead to capture single molecules for performance research. For example, the Chinese patent document with publication number CN117191713A discloses a method for determining the thermodynamic properties of hybridization of non-natural nucleic acid chains based on single-molecule magnetic tweezers. The invention constructs a single-molecule hairpin of XNA / DNA hybrid chain, uses the single-molecule XNA hairpin to construct a magnetic tweezers system, and then uses single-molecule magnetic tweezers technology for data collection. Finally, a hidden Markov model is used for fitting. By accurately applying force to the DNA substrate, the energy required for the dissociation of different non-natural nucleic acids and the folding / unfolding rate are quantified. However, the modified G-quadruplex structure is different from the above invention, and the formation rate of the modified G-quadruplex is much slower than that of the fully paired hairpin structure, making it difficult to obtain their folding / unfolding rates through the long-term constant force experiment of the above invention. Summary of the invention

[0005] In order to address the shortcomings of the prior art, the present invention provides a method for measuring the kinetic behavior of G-quadruplexes with different C2′ modifications based on single-molecule magnetic tweezers. This method measures the folding / unfolding rates of G-quadruplexes with different C2′ modifications by directly fitting the collected force climbing data, thereby avoiding the influence of long-term constant force experiments on the structural stability of the G-quadruplex.

[0006] The specific technical solutions adopted are as follows:

[0007] A method for measuring the dynamic behavior of different C2′ modified G-quadruplexes based on single-molecule magnetic tweezers comprises the following steps:

[0008] (1) constructing handle chain 1 and handle chain 2, wherein handle chain 1 and handle chain 2 are modified with different functional groups, respectively, and handle chain 1 and handle chain 2 have sticky ends; annealing the C2′-modified G-quadruplex single strand with the first primer and the second primer, and then connecting them with handle chain 1 and handle chain 2 by ligase to prepare a sample to be tested;

[0009] (2) constructing a single-molecule magnetic tweezers system, using the single-molecule magnetic tweezers technology to change the pulling force applied to the sample to be tested to perform a force ramp experiment and collect real-time data, and using a mathematical model to analyze the kinetic behavior of the C2′-modified G-quadruplex based on the collected force ramp data; the mathematical model is a single exponential model or a Bell model.

[0010] The present invention first constructs a C2′ modified G-quadruplex with a handle chain, and then uses a single-molecule magnetic tweezers technique to connect the handle chain to a magnetic bead and a substrate, respectively. When the external magnetic field pulls the magnetic bead, the C2′ modified G-quadruplex will be stretched or twisted accordingly. By recording the displacement changes of the magnetic beads, combined with the known force-displacement relationship and a specific mathematical model, the mechanical properties of the C2′ modified G-quadruplex, such as tensile strength, unfolding behavior or mechanical properties of structural transformation, are obtained.

[0011] Specifically, the nucleosides in the C2′ modified G-quadruplex are modified with 2′F-ANA, 2′F-RNA or 2′OMe-RNA.

[0012] Optionally, handle chain 1 and handle chain 2 are modified with biotin and thiol-SH, respectively.

[0013] The handle chain 1 and the handle chain 2 are obtained by enzyme digestion, and the protective base during the enzyme digestion is CTGCAGAA. The sequences of the handle chain 1 and the handle chain 2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0014] Optionally, the C2′ modified G-quadruplex single strand and the first primer and the second primer are mixed in equimolar amounts and annealed, the annealing temperature is reduced from 95° C. to 25° C., and the annealing time is 1.5 h.

[0015] Optionally, the annealed product, handle strand 1 and handle strand 2 are ligated by T4 ligase in equimolar amounts, and 1.5 μL of T4 ligase is added to a 100 μL reaction system.

[0016] The single-molecule magnetic tweezers technique can precisely control the force applied to the molecule at the pN level, and can accurately measure the force required for the dissociation of the G-quadruplex. It has the characteristics of high-precision force control. The magnetic tweezers control the movement of the magnetic beads through the magnetic field, and do not directly contact the sample, avoiding interference or damage that may be caused by physical contact. Since the magnetic force does not cause obvious photodamage or other negative effects on the molecular sample, it is suitable for long-term dynamic process observation, and can easily observe the dynamic processes such as molecular folding and unfolding. In addition, the single-molecule magnetic tweezers can analyze a single molecule, avoiding the data confusion caused by sample heterogeneity in group experiments, providing higher resolution, and it can also apply and detect forces from extremely low to high ranges, which is suitable for studying molecular behavior under various mechanical conditions. By accurately changing the force applied to the sample to be tested, the distribution of the force required for the unfolding of different C2′ modified G-quadruplexes and the folding / unfolding rate can be quantified.

[0017] Optionally, in step (2), in the single-molecule flow cell, the handle chain portion in the sample to be tested is connected to the streptavidin magnetic beads and the bottom wall of the single-molecule flow cell, respectively, through the reaction between the sample to be tested, the streptavidin magnetic beads and the modified bottom wall of the single-molecule flow cell, and the single-molecule magnetic tweezers technology is used to change the pulling force applied to the sample to be tested to perform a force climbing experiment.

[0018] During the force ramp experiment, the pulling force was gradually increased from 1pN to 60pN at a rate of 1pN / s. The kinetic behavior included the folding rate and the unfolding rate, and the structure of the C2′-modified G-quadruplex could be determined by the kinetic behavior.

[0019] The single exponential model fitting formula is as follows:

[0020] P f (t) = P st (1-exp(-k f t))

[0021] Among them, P f (t) is the statistically obtained G-quadruplex folding probability, P st is the steady-state folding probability, k f is the folding rate of the G-quadruplex and t is the folding time.

[0022] The Bell model fitting formula is as follows:

[0023]

[0024] in, is the force distribution of G-quadruplex unfolding, r is the loading rate, k B is the Boltzmann constant, T is the absolute temperature, f is the variable force, Δx u is the unfolded transition distance, is the unfolding rate.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The method of the present invention directly fits the collected force ramp data through the Bell model, overcomes the significant impact of long-term constant force experiments on the structural stability of the G-quadruplex, and avoids the possible unfolding, structural transformation or kinetic behavior changes under the action of force, thereby more conveniently and quickly measuring the folding / unfolding rates of G-quadruplexes with different C2′ modifications.

[0027] (2) C2′ modification can significantly affect the stability and folding behavior of G-quadruplexes. The method of the present invention helps to understand the relationship between the structure and function of G-quadruplexes and design more stable or controllable G-quadruplexes for biomedical applications, such as anticancer drug development or gene regulation. The method of the present invention can also reveal the effect of modification on the folding pathway, reveal the mechanism of molecular folding, and provide key data for the design of quadruplex-based drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The sequence of a portion of a G-quadruplex single strand and a schematic diagram of the structure of different C2′ modifications;

[0029] Figure 2 Schematic diagram of the structure of G-quadruplexes with different C2′ modifications;

[0030] Figure 3 Representative stretching curves of G-quadruplexes with different C2′ modifications;

[0031] Figure 4 A representative force-ramp cycling experiment of a DNA G-quadruplex, folding for 60 s at 1 pN;

[0032] Figure 5 The time-evolved folding probability diagram of G-quadruplexes with different C2′ modifications;

[0033] Figure 6 is the distribution diagram of DNA G-quadruplex disintegration force fitted by Bell model;

[0034] Figure 7 is the unfolding rate k of G-quadruplex with different C2′ modifications u And the statistical diagram of the unfolding transition distance Δx. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the examples and drawings. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Contents not described in detail in this specification belong to prior art known to professionals and technicians in the field.

[0036] Example 1

[0037] A method for measuring the dynamic behavior of different C2′ modified G-quadruplexes based on single-molecule magnetic tweezers comprises the following steps:

[0038] (1) Preparation of samples to be tested:

[0039] Handle construction: Using bacteriophage lambda DNA as a template, PCR amplification was performed on two ~500 bp handles (the two handles were modified with thiol-SH and biotin-Biotin (synthesized by Sangon Biotechnology (Shanghai)) at the 5' end. The amplification primers were:

[0040] Handle 1primer forward(SEQ ID NO.3):

[0041] 5'-CTGCAGAACCAACGACATGGCAGGAGGGCGAATG-3';

[0042] Handle 2primer forward(SEQ ID NO.4):

[0043] 5'-CTGCAGAACCAAGTGCATGGTGCTTGAACCCGCCTATG-3';

[0044] Handle 1Biotin(SEQ ID NO.5):

[0045] 5'-CGAAGCAGATCCCACGCAACCAGCTTACGG-3' (5' modification-Biotin);

[0046] Handle 2SH (SEQ ID NO.6):

[0047] 5′-CGACTGAGCTGGCAAGCAACTGACCTG-3′ (5′ modification-SH).

[0048] Reaction system: Ex Taq polymerase 1μL (TAKARA); 10×Taq Buffer 20μL; dNTP mix 16μL; λDNA 7.5μL; Handle 1primer forward, Handle 2primer forward, Handle 1Biotin, Handle 2SH 1μL each; ddH2O 153.5μL.

[0049] Reaction procedure: 95°C for 3 min; 98°C for 10 s, 55°C for 30 s, 72°C for 1 min, 34 cycles; 72°C for 5 min.

[0050] The PCR products were recovered using the Bio-Tech PCR Product Recovery Kit.

[0051] The two handles were then digested with restriction endonuclease BstXI (NEB), with the protective base being CTGCAGAA, exposing the sticky ends. The digestion system was 1.5 μL BstXI endonuclease added to 50 μL, and the sequences of handle chain 1 and handle chain 2 were shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0052] Reaction procedure: 37°C for 3h;

[0053] The products after enzyme digestion were purified by agarose gel electrophoresis at a voltage of 70 V for 60 min.

[0054] Subsequently, C2′ modified G-quadruplex single strands (including DNA G-quadruplex single strands and RNA G-quadruplex single strands), Flank 1, and Flank 2 (both synthesized by Sangon Biotechnology (Shanghai)) were mixed and annealed in a molar ratio of 1:1:1. The mixed system was 50 μL, the concentration was 100 μM, and the buffer was 1×PBS to obtain annealing products.

[0055] Reaction procedure: annealing temperature dropped from 95°C to 25°C, annealing time 1.5h.

[0056] The annealed product was then ligated with handle strand 1 and handle strand 2 in a T4 ligase (NEB) system at a molar ratio of 1:1:1, and 1.5 μL of T4 ligase was added to 100 μL of the system.

[0057] Reaction procedure: 16°C for 16 h;

[0058] Finally, the gel was excised and purified by agarose gel electrophoresis at a voltage of 70 V for 60 min (all gel recovery kits were from Kangwei Century). The single-stranded sequence of the G-quadruplex is: 5'-CTTGTGTACAGACTCGTTTGGGTTAGGGTTAGGGTTAGGGTTTGA AGACAGGTCAGTATCGAC-3' (SEQ ID NO. 7); Figure 1 As shown, the guanine G was modified by 2'F-ANA, 2'F-RNA and 2'OMe-RNA, respectively. Flank1 sequence: 5'-CGAGTCTGTACACAAGGTGC-3' (SEQ ID NO.8); Flank2 sequence: 5'-ATACTGACCTGTCTTC-3' (SEQ ID NO.9). The samples to be tested corresponding to DNA G-quadruplex, RNA G-quadruplex, 2'F-ANA G-quadruplex, 2'F-RNA G-quadruplex and 2'OMe-RNA G-quadruplex were obtained.

[0059] (2) Constructing a single-molecule magnetic tweezers system, using the single-molecule magnetic tweezers technology to change the pulling force applied to the sample to be tested to perform a force ramp experiment and collect real-time data, and using a mathematical model to analyze the dynamic behavior of the C2′-modified G-quadruplex based on the collected force ramp data; the specific steps are as follows:

[0060] (2.1) Connection of single-molecule samples

[0061] First, a single-molecule flow cell was constructed for the experiment: the flow cell consisted of two cover glass slides and a hollow plastic sheet in the middle. Two small holes were opened on the upper glass slide and the cylindrical hole parts obtained by 3D printing were glued with quick-drying glue, which served as the inlet and outlet of the fluid respectively. The volume of the flow cell was about 50 μL. The lower glass slide was ultrasonically treated in deionized water (DI water) for 30 minutes to remove pollutants, and then surface activated by soaking in 10M KOH overnight to enhance the density of surface hydroxyl groups and enhance the binding of APTES (3-aminopropyltriethoxysilane) to the glass slide surface. Then APTES was dissolved in methanol and the solvent was made to be anhydrous to prevent APTES from reacting with water and inactivating it. The glass slide was then soaked in the APTES solution for 25 minutes. After the modification was completed, the glass slide was repeatedly rinsed with methanol to remove the unreacted APTES, and finally dried with nitrogen or in an oven to ensure that there was no residual moisture on the surface.

[0062] The experimental process is as follows: polystyrene balls are mixed with PBS buffer at a volume ratio of 1:1000, added to the flow cell as reference balls, incubated for 20 minutes to fix the reference balls on the surface of the lower glass slide by electrostatic action, and then about 600 μL of 1× PBS buffer is used to wash away the unbound reference balls. Next, the coupling reagent 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) that has been placed at room temperature for 10 minutes is dipped with a pipette tip and dissolved in a mixed solution of 400 μL DMSO and PBS buffer at a volume ratio of 1:1. The mixed solution is allowed to cool to room temperature to avoid a large number of bubbles generated by heat release, and then slowly added to the flow cell and incubated for 25 minutes to react with the amino groups on the surface of the lower glass slide to form an active surface. Finally, the sample prepared in step (1) was added and incubated for 45 minutes, wherein the G-quadruplex sample with a thiol group at the 5' end was covalently bound to the amino surface of the lower glass slide under the action of SMCC, so that the C2' modified G-quadruplex was vertically fixed on the surface of the lower glass slide. 200 μL DNA blocking buffer (including 10 mL 1× PBS buffer, 1% bovine serum albumin and 5 μL β-mercaptoethanol) was added to the flow cell and stored in a 4°C refrigerator overnight.

[0063] (2.2) Magnetic tweezers experiment

[0064] Unfolding force measurement: More than 24 hours in advance, 10 μL of M270 streptavidin magnetic beads (10 mg / mL, Thermo Fisher) were dissolved in 1 mL of blocking buffer (including 10 mL of 1× PBS buffer, 1% bovine serum albumin and 1 μL of Tween) and incubated with continuous rotation at 4°C. Then, taking the constructed C2′-modified G-quadruplex sample as the research object, 200 μL of pretreated M270 streptavidin magnetic beads were first added to the single-molecule flow cell and allowed to stand for more than 15 minutes to ensure that the magnetic beads were fully bound to the sample fixed on the glass substrate, and then 200 μL of experimental buffer (10 mM Tris, 100 Mm KCl) was added to provide the ionic environment required for G-quadruplex folding and to wash the unfixed magnetic beads to prevent blocking the experimental field of view.

[0065] Next, the force ramp program of the single-molecule magnetic tweezers is used to gradually increase the pulling force applied to the sample to be tested. Specifically, the height of the magnet in the magnetic tweezers is controlled to gradually increase the pulling force from 1pN to 60pN at a rate of 1pN / s (the magnet height is about 0.24mm), so that the DNA enters an overstretched state to verify that the collected data is indeed from a single strand of DNA. Figure 2 As shown, during this process, an obvious jump signal can be observed through the real-time curve, indicating that the unwinding (opening) process of the G-quadruplex occurs ( Figure 3 ). The forces required to open different G-quadruplexes were recorded. The experimental results showed that the force required to unfold the 2'F-ANAG-quadruplex sample was the largest, about 58pN; while the force required to unfold the DNA G-quadruplex sample was the lowest, about 22pN ( Figure 3 ).

[0066] Then the tension was controlled to 1pN for 10s to refold the G-quadruplex. The refolding time was set according to different C2′ modified G-quadruplexes. The folding time gradient of 2'F-ANA G-quadruplex was 1s, 10s, 30s, 60s; the folding time gradient of 2'F-RNA G-quadruplex was 10s, 30s, 60s, 120s; the folding time gradient of 2'OMe-RNA G-quadruplex was 30s, 60s, 120s, 300s. The stretching rate was 1pN / s.

[0067] Measurement of folding / unfolding rate: Taking 2'F-ANA G-quadruplex as an example, four folding times were selected as gradients for measurement. Ten force ramp stretching cycles were performed at each time gradient to obtain the probability distribution of G-quadruplex folding / unfolding. The ordinate is the height of the magnetic ball, which represents the length of the tested DNA. The G-quadruplex unfolding signal is manifested as an increase in the ball height, and the G-quadruplex folding signal is manifested as a decrease in the ball height ( Figure 4 ).

[0068] 1) Folding rate quantification

[0069] After obtaining the probability distribution of the folding / unfolding state at different folding times, the single exponential model fitting formula is as follows:

[0070] P f (t) = P st (1-exp(-k f t))

[0071] P f (t) is the statistically obtained G-quadruplex folding probability, P st is the steady-state folding probability, k f is the folding rate of the G-quadruplex, t is the folding time, and P st and k f The two parameters are considered as fitting parameters, and the initial values ​​are not fixed. The fitting results of G-quadruplex folding rates k with different C2′ modifications are: f ( Figure 5 ). The results showed that the folding rate of 2'F-ANA G-quadruplex was the fastest, and the folding rate of 2'OMe-RNAG-quadruplex was the slowest.

[0072] 2) Quantification of unfolding rate

[0073] First, the force climbing data was frequency distributed and then fitted using the Bell model ( Figure 6 )The formula is as follows:

[0074]

[0075] in, is the force distribution of G-quadruplex unfolding, r is the loading rate 1pN / s, k B is the Boltzmann constant, T is the absolute temperature, f is the variable force, Δx u is the unfolded transition distance, is the unfolding rate, k B The value of T is 4.11. and Δx u Both parameters are considered as fitting parameters. The initial value of the fitting is 10 -5 , Δx u The initial value of the fitting is 1.1. The fitting results show the unfolding rate k of G-quadruplex with different C2′ modifications. u and the unfolding transition state distance Δx( Figure 7). The results showed that the unfolding rate of C2′ modified G-quadruplex was several orders of magnitude faster than that of DNA G-quadruplex. This indicates that DNA G-quadruplex is an antiparallel structure, while C2′ modified G-quadruplex, including RNA G-quadruplex-quadruplex, is a parallel structure.

[0076] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers, characterized in that: The following steps are involved: (1) constructing handle chain 1 and handle chain 2, wherein handle chain 1 and handle chain 2 are modified with different functional groups, respectively, and handle chain 1 and handle chain 2 have sticky ends; annealing the C2'-modified G-quadruplex single strand with the first primer and the second primer, and then connecting them with handle chain 1 and handle chain 2 by ligase to prepare a sample to be tested; (2) constructing a single-molecule magnetic tweezers system, using the single-molecule magnetic tweezers technology to change the pulling force applied to the sample to be tested to perform a force ramp experiment and collect real-time data, and using a mathematical model to analyze the kinetic behavior of the C2'-modified G-quadruplex based on the collected force ramp data; the mathematical model is a single exponential model or a Bell model.

2. The method for determining the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers according to claim 1, characterized in that: The nucleosides in the C2' modified G-quadruplex are modified with 2'F-ANA, 2'F-RNA or 2'OMe-RNA.

3. The method for determining the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers according to claim 1, characterized in that: The handle chain 1 and the handle chain 2 are modified with biotin and thiol-SH, respectively.

4. The method for determining the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers according to claim 1, characterized in that: The C2' modified G-quadruplex single strand and the first primer and the second primer are mixed and annealed in equimolar amounts.

5. The method for measuring the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers according to claim 1, characterized in that: The annealed product, handle strand 1 and handle strand 2 were ligated in equimolar amounts by T4 ligase.

6. The method for measuring the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers according to claim 1, characterized in that: In step (2), in the single-molecule flow cell, the handle chain portion in the sample to be tested is connected to the streptavidin magnetic beads and the bottom wall of the single-molecule flow cell, respectively, through the reaction between the sample to be tested, the streptavidin magnetic beads and the modified bottom wall of the single-molecule flow cell, and the force climbing experiment is performed by changing the pulling force applied to the sample to be tested through the single-molecule magnetic tweezers technology.

7. The method for determining the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers according to claim 1, characterized in that: During the force ramp experiment, the pulling force was gradually increased from 1pN to 60pN at a rate of 1pN / s.

8. The method for measuring the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers according to claim 1, characterized in that: The single exponential model fitting formula is as follows: P f (t)=P st (1-exp(-k f t)) Among them, P f (t) is the statistically obtained G-quadruplex folding probability, P st is the steady-state folding probability, k f is the folding rate of the G-quadruplex and t is the folding time.

9. The method for measuring the dynamic behavior of different C2' modified G-quadruplexes based on single-molecule magnetic tweezers according to claim 1, characterized in that: The Bell model fitting formula is as follows: in, is the force distribution of G-quadruplex unfolding, r is the loading rate, k B is the Boltzmann constant, T is the absolute temperature, f is the variable force, Δx u is the unfolded transition distance, is the unfolding rate.

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