A method for determining the rate of toehold-mediated strand displacement reaction based on single-molecule magnetic tweezers

Abstract

The application discloses a method for measuring a toehold-mediated strand displacement reaction rate based on single-molecule magnetic tweezers, and belongs to the technical field of biology. The method comprises the following steps: constructing a single-molecule hairpin DNA, mixing the single-molecule hairpin DNA with a displacement strand, and then performing a strand displacement reaction by using single-molecule magnetic tweezers. The application proves by experiments that the single-molecule magnetic tweezers are a device that can effectively perform a DNA strand displacement reaction, and the visualization of the strand displacement reaction process is realized by tracking a real-time curve. The application provides a method for effectively improving the strand displacement reaction rate without fluorescent labeling by accurately applying force to a DNA substrate.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for determining the rate of Toehold-mediated chain displacement reactions based on single-molecule magnetic tweezers. Background Technology

[0002] DNA strand displacement is a completely thermodynamically driven reaction. The simplest DNA strand displacement process involves three steps: complementary single-strand hybridization (toehold), DNA strand migration via random walk, and the generation of a new hybrid strand. This process is spontaneous, relying solely on the free energy change provided by toehold partial hybridization. The toehold domain plays a crucial role in promoting strand movement, and its length is often used to control the reaction kinetics. Generally, increasing the toehold length accelerates the chain displacement rate because it has a stronger binding energy; that is, a longer toehold thermodynamically increases the energy difference between the product and reactants. For toehold displacement (TD) reactions, it proceeds irreversibly until any reactant is exhausted. Previous experiments by Zhang and Winfree have demonstrated that the rate constant of DNA strand displacement is related to the toehold length; the reaction rate increases with increasing toehold length. When the toehold exceeds 7 nt (nt being the number of bases), the rate constant reaches saturation, and no method has yet been proposed to increase the saturation rate of DNA strand displacement reactions.

[0003] Currently, most chain displacement reactions are performed using fluorescent labeling methods. This method detects collective behavior, but fails to visualize the dissociation and binding of single strands. Furthermore, studies have shown that different fluorescent labels have different effects on the chain displacement reaction rate. Single-molecule manipulation techniques can apply forces to biomolecules directly, offering unique advantages in exploring mechanically induced biological processes. Single-molecule manipulation techniques include single-molecule magnetic tweezers, single-molecule optical tweezers, and atomic force microscopy. These techniques allow for unified measurements of a biomolecule (such as DNA or protein) in three dimensions: time, space, and force. Applying forces to biomolecules can lower the energy barriers of certain biological reactions and control the intrinsic reaction rates of changes in molecular spatial states. Single-molecule manipulation techniques can be used for real-time, precise measurements of DNA under stress. C. Bouchiat et al. determined physical properties such as DNA persistence length under worm-like chain model fitting by stretching single DNA molecules. Woodside et al. measured the equilibrium state of hairpin DNA under stress, obtaining the folding and dissociation rates and energy spectra of hairpin DNA without the need for heating. However, there are currently no reports on single-molecule manipulation techniques for improving the saturation rate of DNA strand displacement reactions. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the rate of Toehold-mediated chain displacement reaction based on single-molecule magnetic tweezers, in order to solve the problems existing in the prior art. By applying force to increase the saturation rate of the chain displacement reaction, a fluorescent label-free method for measuring the chain displacement reaction in real time and with high accuracy is provided.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for determining the rate of Toehold-mediated strand displacement reaction based on single-molecule magnetic tweezers, comprising: constructing single-molecule hairpin DNA, mixing the single-molecule hairpin DNA with the displacement strand, and then performing the strand displacement reaction using single-molecule magnetic tweezers technology.

[0006] Preferably, the method specifically includes the following steps: (1) Using hairpin DNA as a template, amplify the two hybridization handles (Handle 1, Handle 2) of the hairpin DNA, and after enzyme digestion, ligate the hairpin DNA with the two hybridization handles to obtain a single hairpin DNA molecule; The two hybrid handles are respectively modified with thiol and biotin at their 5' ends; (2) The single-molecule hairpin DNA with thiol groups, streptavidin magnetic beads, and aminated glass undergo a covalent reaction to obtain a single-molecule system in which the hairpin DNA is connected to glass and magnetic beads at both ends, respectively. (3) The replacement chain is added to the single-molecule system. The pulling force on the hairpin DNA is controlled based on the single-molecule magnetic tweezers technology. The response signal and reaction time of the single-molecule system that undergoes the chain replacement reaction are tracked by real-time curve, and the reaction time is fitted to obtain the rate of the chain replacement reaction under a specific force.

[0007] Preferably, in step (3), the replacement strand is complementary to the hairpin DNA partial sequence and has a base number of 1-17 bp.

[0008] Preferably, in step (3), the pulling force on the hairpin DNA is controlled based on single-molecule magnetic tweezers technology. The specific method is as follows: first, the hairpin DNA is stretched from 1pN to 65pN, the complementary strand of the hairpin DNA is peeled off, and then the pulling force is adjusted back to 7pN for 3000s. The occurrence of the strand displacement reaction is observed, and the occurrence time is statistically analyzed to fit the strand displacement rate.

[0009] Preferably, the stretching rate is 1 pN / s.

[0010] Preferably, the more base mismatches there are at the junction of the hairpin DNA and the replacement strand, the lower the strand replacement reaction rate.

[0011] Preferably, the more bases in the loop of the hairpin DNA, the higher the chain displacement reaction rate.

[0012] The present invention discloses the following technical effects: This invention proposes a method for determining the rate of DNA strand displacement reactions based on single-molecule magnetic tweezers technology. Experimental verification shows that single-molecule magnetic tweezers are an effective device for performing DNA strand displacement reactions, and the process can be visualized by tracking real-time curves. This method requires no fluorescent labeling and allows for precise application of force to the DNA substrate. Furthermore, by applying force to hairpin DNA, the strand displacement reaction rate can be effectively increased, enabling precise determination of the reaction rate after the toehold exceeds 7 nt (nt is the number of bases). Therefore, this invention provides a method to improve the saturation rate of DNA strand displacement reactions, offering a scientific basis for the current real-time and accurate measurement of hairpin DNA strand displacement reaction rates. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is a schematic diagram of a DNA strand displacement reaction; Figure 2 A schematic diagram of the connection of a single-molecule magnetic tweezers system; Figure 3 The hairpin DNA stretching state under single-molecule magnetic tweezers; Figure 4 To observe the chain displacement reaction by tracking real-time curves; Figure 5 A schematic diagram illustrating how a DNA hairpin opens under the action of the replacement strand; Figure 6 To obtain the reaction rate by curve fitting of the reaction time; Figure 7 The effect of Toehold length on the rate of DNA strand displacement reaction; Figure 8 To analyze the relationship between the formation of secondary structures by the substitution chain and the reaction rate curve using NUPACK calculations; Figure 9 Compare the rates of substitutional chains that do not form secondary structures with those that do form secondary structures; Figure 10 To set the positions for base mismatches; Figure 11 The effect of base mispairing on the chain substitution reaction rate; Figure 12 The effect of the number of loop bases in DNA hairpin substrates on the rate of strand substitution reactions; Figure 13 Schematic diagrams of the structures of non-natural nucleic acids FANA and TNA; Figure 14 The effect of substitutional chains after replacement with non-natural nucleic acids FANA and TNA on the chain substitution reaction rate. Detailed Implementation

[0015] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0016] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0017] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0018] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0019] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0020] See the diagram illustrating the principle of DNA strand displacement reaction. Figure 1 This invention focuses on single-molecule hairpin DNA and describes a method for real-time and precise measurement of the saturation rate of DNA strand displacement reaction based on single-molecule magnetic tweezers technology. Specific embodiments are described below for further explanation.

[0021] Example 1 1. Sample preparation (1) Construction of single-molecule hairpin DNA Using phage lambda DNA as a template, PCR amplification was performed on two ~500bp handles (each handle having a thiol-SH group modified at the 5' end and a biotin group modified at the 5' end, respectively). The amplification primers were: Handle 1 primer (SEQ ID NO: 1): 5'-ATCACCAACGACATGGCAGGAGGGCGAATG-3'; Handle 2 primer (SEQ ID NO: 2): 5'-ATCACCAAGTGCATGGTGCTTGAACCCGCCTATG-3'; Handle 1 Biotin (SEQ ID NO: 3): 5'-CGAAGCAGATCCCACGCAACCAGCTTACGG-3' (5' modified-Biotin); Handle 2 SH (SEQ ID NO: 4): 5'-CGACTGAGCTGGCAAGCAACTGACCTG-3' (5' modified-SH); Reaction system: 2 μL Ex Taq polymerase; 40 μL 10×Taq Buffer; 32 μL dNTP mix; 15 μL λDNA; 2 μL each of Handle 1 primer, Handle 2 primer, Handle 1 Biotin, and Handle 2 SH; 307 μL ddH2O.

[0022] Reaction program: 95℃ for 3 min; 98℃ for 10 s, 55℃ for 30 s, 72℃ for 1 min, cycled 34 times; 72℃ for 5 min.

[0023] The two handles were then digested with the restriction endonuclease BstXI to expose the sticky ends. Hairpin DNA, Flank 1, and Flank 2 (synthesized by Sangon Biotech (Shanghai)) were then mixed in a 1:1:1 molar ratio, annealed, and ligated with Handle 1 and Handle 2 using the T4 ligase (TAKARA) system. Finally, the DNA was purified by agarose gel electrophoresis (see...). Figure 2 Among them, the DNA hairpin sequence (SEQ ID NO: 5): 5'-CCACATACATATGGAGACGTAGGGTATTGAATGAGGGTCTCGTTCCCTCATTCAATACCCTACGAGCTAGCTTT-3'; the Flank1 sequence (SEQ ID NO: 6): 5'-CGAGTCTGTACACAAGGTGC-3'; and the Flank2 sequence (SEQ ID NO: 7): 5'-ATACTGACCTGTCTTC-3'.

[0024] (2) Linkage of single-molecule samples Thiol-containing DNA (100 μL, 1-2 ng / μL) can covalently react with the lower glass surface, which is aminolated by γ-aminopropyltriethoxysilane (APTES), in the presence of the cross-linking agent N-maleimidemethyl (SMCC). We constructed a single-molecule flow cell (approximately 60 μL) by adhesively bonding the upper and lower glass surfaces together; the upper glass surface serves as a fixation element and does not require APTES modification. The SMCC was dissolved in a mixture of 200 μL each of DMSO and PBS, with a trace amount of SMCC added using a 1000 μL pipette tip. Because the DNA has a biotinylated end, it can specifically bind to streptavidin-coated magnetic beads, forming a single-molecule system with the glass plate at the bottom and the magnetic beads at the top. The sample is attached to the lower surface within the single-molecule flow cell.

[0025] 2. Magnetic tweezers experiment Rate measurement of chain displacement reaction with different toehold lengths.

[0026] At least one day in advance, dissolve 1 μL of M270 streptavidin magnetic beads (10 mg / mL, Thermo Fisher Scientific) in 1 mL of buffer (10 mL 1×PBS, 0.1 g bovine serum albumin, 0.01 g Tween) and incubate continuously by rotation. Using the single-molecule hairpin DNA constructed above as the research object, first add 200 μL of the aforementioned M270 streptavidin magnetic beads to the single-molecule flow cell and wait for ten minutes to allow the magnetic beads to fully ligate with the DNA attached to the glass surface substrate. Next, add 400 µL of 10 nM DNA replacement strands with toeholds of 1, 3, 5, 7, 9, 11, 13, 15, and 17 bases respectively to the single-molecule flow cell (see...). Figure 3 The Force ramp program was set up to control the pulling force on the hairpin DNA by gradually increasing the pulling force from 1 pN to 65 pN at a rate of 1 pN / s by controlling the height of the magnets in the single-molecule magnetic tweezers, thus overstretching the DNA (to strip the complementary strand of the short DNA). The pulling force was then reduced to 7 pN and held for 3000 s (see...). Figure 4 During this period, a clear jump signal can be observed by tracking the real-time curve, indicating that the DNA hairpin has opened (see...). Figure 5 In the second cycle, the tension is gradually increased from 1 pN to 65 pN to ensure complete stripping of the replacement strand from the hairpin DNA substrate, thus allowing for the next strand replacement reaction and completing the cycle. The reaction times are recorded and fitted (see [link to relevant documentation]). Figure 6 ), which can yield the rate of chain displacement reaction under specific forces.

[0027] (4) Analyze the factors affecting the rate of substitution chain reaction. 1) The effect of substitutional chain secondary structure on reaction rate The results showed that the chain displacement reaction rate exhibited a low-speed, high-speed, and medium-speed range (see...). Figure 7 When the toehold length increases to 13, 15, and 17 nt, the substitution chain itself forms a secondary structure, and the energy decreases accordingly. This is consistent with the transition of the rate curve from a high-speed region to a medium-speed region, indicating that the chain substitution reaction rate follows a low-speed, high-speed, and medium-speed range, which is related to the formation of a secondary structure by the substitution chain itself (see...). Figure 8 ); In addition, four substitutional chains that do not form secondary structures were synthesized through sequence design, with toehold lengths of 9, 11, 13, and 15 nt, respectively. Their chain substitution reaction rates were calculated.

[0028] like Figure 9 As shown, the results indicate that when the added substitution chain does not form a secondary structure, the chain reaction rate does not decrease with the increase of the toehold length.

[0029] 2) Effect of base mispairing at the substitutional chain and hairpin junction on the reaction rate One and two base mismatches were respectively placed at the hairpin binding site of the 11nt toehold DNA replacement strand (see...). Figure 10 ), and the timing of the chain permutation.

[0030] like Figure 11 As shown, the chain substitution reaction rate decreases significantly with the increase in the number of mismatched bases.

[0031] 3) Effect of the number of loop bases in DNA hairpin substrate on the rate of strand substitution reaction The number of bases in the loop region of the DNA hairpin substrate was changed to 3nt, 7nt, and 11nt respectively, and the timing of strand substitution was recorded.

[0032] like Figure 12 As shown, the more loop bases a DNA hairpin substrate has, the faster the strand displacement reaction rate.

[0033] 4) Effect of substitutional chain sequence on reaction rate Prepare substitution strands with a toehold length of 7 bases, and replace the toehold positions with non-natural nucleic acids FANA and TNA, respectively. The synthesis method is referenced in (Wang Y, Ngor AK, Nikoomanzar A, et al. Evolution of a general RNA-cleaving FANA enzyme[J]. Nature communications, 2018, 9(1): 1-10.) (Structures are shown in [link to structural diagram]). Figure 13 ).

[0034] like Figure 14 As shown, compared to DNA replacement strands, the strand replacement reaction rate decreased after FANA replacement, and the decrease was more pronounced for TNA.

[0035] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for determining the rate of Toehold-mediated chain substitution reaction based on single-molecule magnetic tweezers, characterized in that, The method specifically includes the following steps: (1) Using phage lambda DNA as a template, amplify the two hybrid handles of hairpin DNA, digest with enzymes, mix and anneal the hairpin DNA, Flank 1 and Flank 2, and ligate with Handle 1 and Handle 2 to obtain single-molecule hairpin DNA; The two hybrid handles are respectively modified with thiol and biotin at their 5' ends; (2) The single-molecule hairpin DNA with thiol groups, streptavidin magnetic beads, and aminated glass undergo a covalent reaction to obtain a single-molecule system in which the hairpin DNA is connected to glass and magnetic beads at both ends, respectively. (3) The replacement chain is added to the single-molecule system, and the pulling force on the hairpin DNA is controlled based on the single-molecule magnetic tweezers technology. The response signal and reaction time of the single-molecule system that undergoes the chain replacement reaction are tracked by real-time curve, and the reaction time is fitted to obtain the rate of the chain replacement reaction under a specific force. The Handle 1 primer is: 5'-ATCACCAACGACATGGCAGGAGGGCGAATG-3'; The Handle 2 primer is: 5'-ATCACCAAGTGCATGGTGCTTGAACCCGCCTATG-3'; The DNA hairpin sequence is: 5'-CCACATACATATGGAGACGTAGGGTATTGAATGAGGGTCTCGTTCCCTCATTCAATACCCTACGAGCTAGCTTT-3'; the Flank1 sequence is: 5'-CGAGTCTGTACACAAGGTGC-3'; the Flank2 sequence is: 5'-ATACTGACCTGTCTTC-3'; In step (3), the replacement strand is complementary to the hairpin DNA partial sequence and has a base number of 1-17 bp; the strand replacement reaction rate is in the low-speed, high-speed, and medium-speed range; through the design of the replacement strand sequence, a replacement strand that does not form a secondary structure itself is synthesized. In step (3), the pulling force on the hairpin DNA is controlled based on single-molecule magnetic tweezers technology. The specific method is as follows: first, the hairpin DNA is stretched from 1pN to 65pN, the complementary strand of the hairpin DNA is peeled off, and then the pulling force is adjusted back to 7pN and held for 3000s. The occurrence of the strand replacement reaction is observed, and the occurrence time is statistically analyzed to fit the strand replacement rate. The more base mismatches at the junction of the hairpin DNA and the replacement strand, the lower the strand replacement reaction rate. The more bases in the loop of the hairpin DNA, the higher the chain displacement reaction rate.

2. The method as described in claim 1, characterized in that, The stretching rate is 1 pN / s.

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