Method for accelerating DNA strand displacement reaction by using n-butyl alcohol

Abstract

The invention discloses a method for accelerating DNA strand displacement reaction by using n-butyl alcohol, and belongs to the technical field of nucleic acid reaction. The method comprises the following steps: providing a water-phase DNA solution containing an input chain and double-chain compound; under proper conditions, the aqueous-phase DNA solution is in full contact with n-butyl alcohol, so that an input chain and a bottom chain of the double-chain compound are subjected to complementary pairing, a top chain is released, and the DNA chain replacement reaction is completed. Instantaneous phase separation and dehydration concentration of a water phase / an organic phase are carried out on a system by utilizing n-butyl alcohol, and the effective concentration and the molecular collision frequency of DNA molecules are instantaneously improved, so that the reaction rate of DNA strand displacement is remarkably accelerated. The method can provide a new strategy for DNA calculation and acceleration of a nucleic acid molecule logic circuit.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nucleic acid reactions, and particularly relates to a method for accelerating DNA strand displacement reaction by using n-butanol. BACKGROUND

[0002] DNA strand displacement reaction (SDR) is a molecular reaction process based on the principle of base complementary pairing, and is widely used in fields such as DNA computing, molecular logic circuit, signal amplification and biosensing. The reaction is usually started by a short single-stranded region (called “toehold domain”), the input strand is first combined with the toehold domain, and then gradually displaces the original complementary strand through a branch migration process, so as to realize the displacement of the strand. Since the DNA strand displacement reaction has high programmability and specificity, it is widely used in various molecular system designs.

[0003] However, the DNA strand displacement reaction in the prior art generally has a slow rate, especially in systems with a short toehold domain (for example, only 1 nucleotide, 1nt), the reaction has a high starting energy barrier, the initial combination between the input strand and the double-stranded complex is weak, and the branch migration process is inefficient, thereby significantly reducing the overall reaction rate. Although some studies have tried to accelerate the strand displacement process by extending the toehold domain length, changing the reaction ionic strength or introducing auxiliary molecules, these methods often affect the reaction specificity or system stability, and the acceleration effect on short toehold domain systems is still limited.

[0004] Short toehold domain systems are of great significance in DNA reaction network design, and can be used to improve reaction selectivity, reduce cross-reactions and simplify system structure. However, the slow reaction problem seriously restricts the application of related systems in high-throughput DNA computing, rapid signal response and real-time biological detection.

[0005] Therefore, it is urgent to develop a new method that can efficiently start and complete the DNA strand displacement reaction under the condition of a short toehold domain, so as to significantly improve the reaction rate and efficiency while maintaining the specificity and simplicity of the system. SUMMARY

[0006] Therefore, the primary purpose of the present application is to provide a method for accelerating DNA strand displacement reaction by using n-butanol, which introduces n-butanol into the aqueous DNA solution containing the input strand and the double-stranded complex, so that the system undergoes transient phase separation and dehydration concentration of the aqueous / organic phase, transiently increases the effective concentration and collision frequency of DNA molecules, and thus significantly accelerates the reaction rate of DNA strand displacement.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: One aspect of the present application discloses a method for accelerating DNA strand displacement reaction by using n-butanol, comprising the following steps: Providing an aqueous DNA solution containing input strand and double-stranded complex; Adding n-butanol to the aqueous DNA solution, and making the n-butanol fully contact with the aqueous DNA solution; Adjusting the type of anions in the aqueous DNA solution and controlling the volume ratio of n-butanol to the aqueous DNA solution to make the reaction system produce a controllable degree of transient dehydration effect, so as to adjust the effective concentration of DNA molecules and the collision frequency, and make the input strand and the bottom strand of the double-stranded complex to be complementary paired and release the top strand, and complete the DNA strand displacement reaction.

[0008] Another aspect of the present application discloses the application of the method described in the present application in the acceleration of DNA computing, molecular diagnosis, dynamic DNA circuit or nucleic acid molecule logic circuit.

[0009] The beneficial effects of the present application are as follows: The present application introduces n-butanol into the aqueous DNA solution containing input strand and double-stranded complex, makes the reaction system fully contact with n-butanol, the reaction system undergoes transient aqueous / organic phase separation and is accompanied by dehydration and concentration, significantly improves the effective concentration and collision frequency of DNA molecules, and thus promotes the invasion and displacement reaction of the input strand to the bottom strand, and realizes the acceleration of DNA strand displacement reaction.

[0010] The method for accelerating DNA strand displacement reaction provided by the present application can increase the rate of DNA strand displacement reaction by more than 100 times, and shorten the reaction time from several hours to about 1 minute. Especially for the short-toe region (1 nt) system which is traditionally considered to be difficult to react, the present application also realizes efficient DNA strand displacement reaction, significantly improves the reaction rate and accurately controls the kinetic behavior.

[0011] In general, the acceleration method provided by the present application has the following significant advantages: (1) Significantly accelerating the reaction rate: under the condition of not changing the temperature and ionic strength of the system, the rate of DNA strand displacement is increased by 100 to 1000 times.

[0012] (2) Breaking the 1 nt system kinetic bottleneck: for the first time, detectable and high-yield strand displacement reaction is realized in the 1 nt toe region system.

[0013] (3) Programmable reaction kinetics: the reaction process is adjusted by the number of n-butanol treatment cycles, volume and centrifugation conditions, and the rate is predictably controlled.

[0014] (4) Mild conditions and simple operation: no enzyme or complex device is needed.

[0015] (5) Wide applicability: This method is applicable to catalytic hairpin assembly (CHA), chain hybridization reaction (HCR), chain displacement reaction (SDR) signal amplification, DNA logic circuits and molecular diagnostic systems, providing a new strategy for constructing rapid, stable and controllable DNA reaction networks. Attached Figure Description

[0016] Figure 1 A real-time DNA computing system based on dehydration-driven chain displacement reaction was demonstrated. Figure 1 Figure A illustrates the diffusion (initial) and compartmentalization (intermediate) states that the molecular complex undergoes during dehydration, promoting the reaction between active molecules (final state). Figure 1 B in the figure shows that n-butanol accelerates DNA circuit equilibrium by n times compared to the natural state.

[0017] Figure 2 This diagram illustrates the DNA strand displacement reaction accelerated by n-butanol. Figure 2 In diagram A, n-butanol is used to accelerate DNA computation. The input strand (X) m,8 ) and partial double-stranded substrate complex (C m,8 The combination of ) through the scaffolding structural domain Triggered branch migration leads to the replacement and release of the output chain (Out); the reporter complex R8 undergoes a stoichiometric reaction with the output chain Out, resulting in enhanced fluorescence. Figure 2 B represents toe X 4,8 Speed ​​of computation by introducing n-butanol and DNA under natural conditions; Figure 2 C represents the toe X 3,8 Speed ​​of computation by introducing n-butanol and DNA under natural conditions; Figure 2 D represents the toe X 2,8 Speed ​​of computation by introducing n-butanol and DNA under natural conditions; Figure 2 E in the middle represents the toe X 1,8 The calculation rates of n-butanol and DNA under natural conditions are introduced. The gray dashed line indicates the time required to reach reaction equilibrium under natural conditions. All experiments were performed at a strand concentration of 50 nM in TE buffer (pH 8.0) containing 12.5 mM Mg(NO3)2. b V represents the volume of n-butanol. TE This indicates the volume of the aqueous DNA solution, representing the natural reaction at room temperature without any treatment.

[0018] Figure 3 The effect of different anions on the acceleration of DNA computation by n-butanol. Figure 3 A in the middle is NO3 - The effect of n-butanol on accelerating DNA computation; Figure 3 B is Cl- The effect of n-butanol on accelerating DNA computation; Figure 3 C is Ac - The effect of n-butanol on accelerating DNA computation; Figure 3 D in the middle is SO4 2- The effect of n-butanol on accelerating DNA computation.

[0019] Figure 4 Experimental results for constructing AND logic gates using n-butanol-accelerated DNA strand substitution method. Figure 4 A in the diagram is an AND logic gate; Figure 4 In the equation B, the reaction kinetics are under natural conditions at room temperature. Figure 4 C represents the output of n-butanol:water at a volume ratio of 5:1 and after 10 hours of reaction under natural conditions. The numbers in the loop represent the calculation time for DNA calculations performed under n-butanol dehydration. Figure 4 In the figure, D represents the fluorescence value of the product after dehydration of different volumes of n-butanol. Figure 4 E in the table represents the truth table, with gray dashed lines marking the threshold of 0.4 (true if the value is above 0.4, otherwise false).

[0020] Figure 5 Experimental results for constructing OR logic gates using n-butanol-accelerated DNA strand substitution method. Figure 5 A in the diagram is a schematic of an OR logic gate. Figure 5 Figure B illustrates the reaction kinetics under natural, room-temperature conditions. Figure 5 C represents the output of n-butanol:water at a volume ratio of 4:1 and after 10 hours of reaction under natural conditions. The numbers in the loop represent the computation time for DNA calculations performed under n-butanol dehydration. Figure 5 In the figure, D represents the fluorescence value of the product after dehydration of different volumes of n-butanol. Figure 5 E in the table represents the truth table, with gray dashed lines marking the threshold of 0.4 (true if the value is above 0.4, otherwise false).

[0021] Figure 6 Experimental results for constructing AND-OR gates using n-butanol-accelerated DNA strand substitution method. Figure 6 A in the diagram is a schematic of a two-level cascaded AND-OR logic gate. Figure 6 In the equation B, the reaction kinetics are under natural conditions at room temperature. Figure 6 C represents the output of n-butanol:water at a volume ratio of 5:1 and after 10 hours of reaction under natural conditions. The numbers in the loop represent the computation time for DNA calculations performed under n-butanol dehydration. Figure 6 In the figure, D represents the fluorescence value of the product after dehydration of different volumes of n-butanol. Figure 6 E in the table represents the truth table, with gray dashed lines marking the threshold of 0.4 (true if the value is above 0.4, otherwise false).

[0022] Figure 7Experimental results for constructing an OR-AND-OR gate using the n-butanol-accelerated DNA strand substitution method. Figure 7 A in the diagram is a schematic of a two-level cascaded AND-OR logic gate. Figure 7 In the equation B, the reaction kinetics are under natural conditions at room temperature. Figure 7 C represents the output of n-butanol:water at a volume ratio of 5:1 and after 10 hours of reaction under natural conditions. The numbers in the loop represent the computation time for DNA calculations performed under n-butanol dehydration. Figure 7 Fluorescence values ​​of products after dehydration of n-butanol of different volumes in D. Figure 7 The gray dashed line in the E truth table marks the threshold of 0.4 (above 0.4 is true, otherwise it is false). Detailed Implementation

[0023] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0024] The first aspect of this application discloses a method for accelerating DNA strand replacement reaction using n-butanol. Specifically, the input strand and the double-stranded complex are prepared as an aqueous DNA solution. Under suitable conditions, n-butanol is brought into full contact with the aqueous DNA solution. Due to the instantaneous dehydration effect of n-butanol, the hybridization reaction between DNA strands is rapidly driven, and the input strand and the bottom strand (double-stranded complex) quickly pair up, thereby releasing the top strand of the double-stranded complex and accelerating the DNA strand replacement reaction.

[0025] As used in this application, the term "introducing strand," also known as the invading strand, is a single-stranded DNA molecule used to initiate a DNA replacement reaction. It binds to the complementary region of the bottom strand via its terminal toehold domain (also known as the toehold region), and then gradually replaces the top strand through a branching migration process, ultimately releasing the top strand.

[0026] As used in this application, the term "top chain" refers to the chain located above the initial double-stranded complex, which typically has a longer sequence and contains branching migration regions. It initially binds complementary to the bottom chain portion, but upon intrusion of the input chain, the top chain is completely displaced and released, and it typically acts as the output chain to trigger downstream reactions.

[0027] As used in this application, the term "bottom chain" refers to a short sequence located below the initial double-stranded complex that includes a support region complementary to the input chain. The bottom chain acts as a "scaffold" and binds to the top chain. The input chain recognizes and binds to the bottom chain through the support region, thereby initiating a replacement process. After the replacement is completed, the bottom chain and the input chain pair up to form a new double-stranded structure.

[0028] As used in this application, the term "double-stranded complex" refers to double-stranded DNA formed by the complementary pairing of two single strands of DNA. In the initial state, it is formed by the complementary pairing of the top and bottom strands; in the intermediate state, it is formed by the complementary pairing of the input and bottom strands.

[0029] As used in this application, the term "reporter complex" also refers to a double-stranded DNA composed of two single-stranded DNA molecules that pair complementary bases. However, one single-stranded DNA molecule is labeled with a quencher, and the other is labeled with a fluorescent label, so that when the input or output strand replaces its components, the fluorescence signal is amplified. It is primarily used to output a signal, allowing visualization of the reaction process or result by observing changes in the fluorescence signal. Specifically, in the specific example of this application, to observe the occurrence of the strand replacement reaction, a reporter complex is introduced to collect the fluorescence signal, thus monitoring the DNA strand replacement reaction.

[0030] In this application, the sequence involved in the DNA strand substitution reaction is not particularly limited. The design is based on the premise of achieving the DNA strand substitution reaction and is tailored to specific needs. Those skilled in the art possess the ability to do so, typically using Watson... The Crick base pairing principle is used in the design. As an example, the DNA sequence is designed using a three-letter coding system (A, T, C), which eliminates the co-occurrence of G bases and C bases, thereby reducing the complementarity of ssDNA itself.

[0031] In some specific examples of this application, the input chain (X) m,8 It is a single-stranded DNA, composed of three structural domains: Tm, Pi, and T8. Tm is the toe domain, which consists of 1-4 nucleotides and provides the initial invasion site in the strand displacement reaction. Pi and T8 are binding domains, which are usually 6-12 bases in length and are used to pair with complementary bases of the double-stranded DNA complex, thereby driving the strand displacement reaction.

[0032] Double-chain complex (C m,8 It consists of a top chain and a bottom chain. The top chain contains four functional domains: Pi, T8, Sr, and T, which are responsible for binding to the input chain and the reporter complex; the bottom chain contains... , and Three complementary domains are perfectly paired with the Tm, Pi, and T8 of the input chain, respectively. This design allows the input chain to successfully invade the bottom chain and release the top chain under n-butanol-assisted conditions.

[0033] The reporter complex (R8), used for signal output, also consists of a top chain and a bottom chain. Its top chain contains Sr and T, and its terminal is modified with a fluorescent group. The fluorescent group can be of types well-known in the art, such as FAM, Cy5, etc., but is not limited to these. The bottom chain contains... , and The end of the fluorophore is modified with a quenching group compatible with the aforementioned fluorescent group, such as BHQ-1. The specific quenching group can be appropriately selected based on its type; those skilled in the art possess this capability, and therefore, no specific limitation is made here. Initially, the fluorescent group and the quenching group are spatially close, and the fluorescence signal is quenched. When the strand displacement reaction occurs, the fluorescence signal is activated, and the DNA strand displacement reaction is monitored by detecting the fluorescence signal.

[0034] In the examples of this application, all DNA strands employ a "long recognition domain + short toe domain" structural design: the term "short toe domain" refers to a short nucleotide sequence (typically 3-8 nucleotides) at the end of the DNA strand. Its primary function is initial recognition and binding. At the start of the reaction, the toe domain of one DNA strand binds briefly and reversibly to a complementary toe domain on another strand through base pairing, like "hooking up." The term "long recognition domain" is a longer nucleotide sequence adjacent to the toe domain. Once successfully "hooked" through the toe domain, the long recognition domain binds stably and persistently to a complementary region on the target strand, ultimately completing strand replacement through branching migration. To avoid the formation of unnecessary secondary structures and non-specific interactions, the DNA sequences in this application are designed using a three-letter coding system (A, T, C), excluding the co-occurrence of G and C bases. The sequences are further limited to no more than four consecutive A or T bases and no more than three consecutive C bases to reduce the synthesis error rate. In the overall design, the C content is strictly controlled between 30% and 70% to ensure the stability of base pairing and the efficiency of chain substitution reaction.

[0035] In this application, the aqueous DNA solution refers to an aqueous solution formed by dissolving dry DNA sequence powder in a buffer solution. The buffer solution is a TE buffer, which contains divalent magnesium ions and anionic electrolytes. As an example, the TE buffer (pH 8.0) contains 12.5 mM of divalent soluble magnesium salt. The specific concentration can be adjusted as needed; in the example of this application, the strand concentration in the aqueous DNA solution is 50 nM.

[0036] In this application, the sufficient contact in the DNA strand displacement reaction can be achieved using any mixing method well-known in the art, ensuring thorough mixing and contact between the aqueous DNA solution and n-butanol. In some specific examples of this application, the sufficient contact can be achieved using a vortex mixing method, which specifically includes the following steps: (i) Add n-butanol to the aqueous DNA solution, vortex to mix, centrifuge, and add buffer solution; (ii) Repeat step (i) once, and finally vortex mix and centrifuge, then discard the n-butanol.

[0037] In this application, the supplemented buffer solution is dehydrated at a ratio of 19.6% per volume of n-butanol.

[0038] In this application, reaction control at different rate levels can be achieved by adjusting the amount of n-butanol, the number of treatments, or the centrifugation conditions, demonstrating excellent programmability.

[0039] As an example, the vortexing time can be 20-40 seconds, and the centrifugation speed can be 3000-5000 rpm, which can be adjusted according to the specific needs, without any particular limitation.

[0040] In some specific examples, the volume of n-butanol added is 1 to 8 times the volume of the aqueous DNA solution, for example, any value among 1, 2, 3, 4, 5, 6, 7, and 8 times, or any range between the two.

[0041] In this application, suitable conditions include the DNA concentration in the aqueous solution, the ratio of n-butanol to the aqueous phase, the type of anionic electrolyte, and the reaction temperature. These conditions can be determined experimentally by those skilled in the art, and therefore are not particularly limited. Regarding the temperature of the DNA strand displacement reaction, in this application, the DNA strand displacement reaction can be carried out at room temperature, which is between 20-30°C, with 25°C being the preferred temperature, without the need for enzyme catalysis or other complex equipment.

[0042] In a preferred embodiment of this application, the aqueous DNA solution further includes at least one anionic electrolyte. The type of anion has a significant impact on the reaction rate. Regulation of anion hydration capacity following the Hofmeister sequence can further finely control the phase separation intensity and reaction kinetics, achieving ion-specific regulation. Those skilled in the art can select the appropriate anion as needed. In some specific examples, the anion in the anionic electrolyte is NO3. - Cl - Ac - SO4 2- It may be at least one of the following, but is not limited to this.

[0043] Based on the acceleration of DNA strand displacement reactions by n-butanol, the method of this application can be applied to the acceleration of DNA computing, molecular diagnostics, dynamic DNA circuits, or nucleic acid molecular logic circuits, thereby providing a new strategy for constructing fast, stable, and controllable DNA reaction networks.

[0044] Furthermore, in this application, there are no particular limitations on the preparation of the double-chain complex and / or reporter complex; they can be carried out with reference to conventional processes known in the art, without any particular limitation. As an example, the preparation steps of the double-chain complex and reporter complex described in this application are as follows: The corresponding top and bottom chains were mixed and annealed to obtain a double-chain complex and a reporter complex, respectively. All complexes were annealed at a concentration of 4 μM. The report complex, as well as the transport and switching modules (if applicable in DNA computation), are annealed with a 20% (mole percentage) excess of the bottom and top strands; all other complexes are annealed with a 1:1 molar ratio of top to bottom strands. The annealing reaction buffer is a 1×TE buffer (pH 8.0) containing 12.5 mM of divalent soluble magnesium salt; wherein the divalent soluble magnesium salt may be magnesium nitrate, magnesium acetate, magnesium chloride or magnesium sulfate.

[0045] The mixture was placed in a thermal circulator, heated to 95°C and held for 5 minutes, then slowly cooled to 30°C at a rate of 0.1°C / 8s and held at 4°C. The resulting hybrid molecules are stored at 4°C for future use.

[0046] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0048] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0049] The DNA sequences in the following examples were purchased from Shanghai Sangon Biotech. The DNA sequences were purified by polyacrylamide gel electrophoresis (PAGE). The DNA single strands modified with FAM fluorescent groups or BHQ quenching groups were purified by high performance liquid chromatography (HPLC).

[0050] All DNA concentrations were determined using a UV spectrophotometer. A fluorescence spectrophotometer was used to scan within the excitation wavelength range of 488 nm and the emission wavelength range of 500–600 nm. All data were normalized from the raw fluorescence levels to the relative concentration of the output signal. Each set of parallel experiments was performed under the same circuitry and different input conditions, and the output data were normalized during data analysis.

[0051] For n-butanol-accelerated DNA strand displacement reactions, the output was read using the reporter complex R8 to monitor the DNA strand displacement reaction. The maximum fluorescence value was determined as the original fluorescence, and subsequent output fluorescence intensities were normalized to relative concentrations. Parallel experiments were performed on the same circuit with various inputs, and the outputs were normalized together for data analysis. The obtained fluorescence signals were converted to the corresponding concentration of the unquenched fluorescent chain using a standard curve for each reporter. To construct the standard curve, solutions containing reporter molecules labeled with predetermined concentrations of fluorophores were tested. Independently repeated experiments were performed to minimize operational fluctuations. Linear fitting results were used for signal conversion. For the readout of the logic results, the fluorescence intensity was normalized. The minimum level (output = 0) was determined by the minimum value of all data at t = 0; the maximum level (output = 1) was determined by the maximum value of the parallel experiments. 0.4 × 10⁻⁶ was used as the threshold in this paper. Signals below 0.4 × 10⁻⁶ were considered 0, and signals above 0.4 × 10⁻⁶ were considered 1.

[0052] Example 1: Transiently driven DNA strand displacement reaction by n-butanol This embodiment discloses the steps of the n-butanol-driven DNA strand displacement reaction. For details, please refer to [link to relevant documentation]. Figure 1 and Figure 2 A.

[0053] 1.1 n-Butanol accelerates DNA strand replacement (1) Design the required DNA sequence. For example... Figure 2 As shown in Figure A, design an input strand DNA molecule (X). m,8 ) and double-stranded DNA (C m,8 The top and bottom chains of the input chain contain a toe domain (Tm) of 1-4 nucleotides in length, which serves as the entry site for the chain substitution reaction. Figure 2 (A)

[0054] (2) Preparation of double-stranded complex / reporter complex The top and bottom strands designed in step (1) are mixed and annealed to obtain double-stranded DNA complexes (C1, C2, C3, C4, C5, C6, C7, C8, C9, C1, C2m,8 ) and reporter complex (R8); All complexes were annealed at a concentration of 4 μM. The reported complex was annealed with a 20% (molar percentage) excess of the bottom chain and top chain; all other complexes were annealed with a top chain to bottom chain ratio of 1:1. The annealing reaction buffer was 1×TE buffer (pH 8.0) containing 12.5 mM Mg(NO3)2; The mixture was placed in a thermal circulator, heated to 95°C and held for 5 minutes, then slowly cooled to 30°C at a rate of 0.1°C / 8s and held at 4°C. The resulting hybrid molecules are stored at 4°C for future use.

[0055] (3) Prepare aqueous DNA solution The input chain and the double-stranded complex were mixed at a volume ratio of 1:1 and dissolved in 1×TE buffer (containing 12.5 mM Mg(NO3)2, pH 8.0), wherein the input chain X m,8 The concentration was 50 nM, the reporter complex R8 concentration was 50 nM, and the double-stranded complex C m,8 The concentration is 50 nM.

[0056] (4) Chain displacement reaction At 25°C, different volumes of n-butanol were added to the aqueous DNA solution, vortexed for 30 seconds to mix, and centrifuged at 4000 rpm for 30 seconds. Buffer was then added to make up to the same volume as the removed aqueous phase. The reporter complex was then added, and n-butanol was added in the correct proportion. The above steps were repeated, and finally, the mixture was vortexed and centrifuged to remove the n-butanol.

[0057] 1.2 n-Butanol accelerates DNA strand displacement reaction in the short toe region 1.2.1 DNA Molecular Sequence Design Table 1 DNA molecular sequence

[0058] Note: X in Table 1 1,8 -X 4,8 In the sequence, bold indicates the length of the toe domain, C 1,8 -C 4,8 Bold text in the sequence indicates regions complementary to the toe domain.

[0059] 1.2.2 n-Butanol accelerates DNA strand displacement reactions The input chain is X in Table 1 4,8 The double-chain complex is composed of C m,8 -Q and C 4,8 The report complex was obtained from R8-P and R8-Q. Following the method in Example 1, experimental groups (0-8) with different volume ratios of n-butanol Vb and aqueous DNA solution were set up to perform the DNA strand displacement accelerated reaction, and the results are as follows. Figure 2 As shown in B.

[0060] The results showed that in the control group (i.e., Vb:V) TE In the case of 0), the chain substitution reaction, under natural conditions, depends on diffusion and thermodynamic equilibrium, typically requiring more than 20 hours to reach complete equilibrium. However, in the instantaneous n-butanol-driven method provided in this application, the input chain (X) is... 4,8 ) and double-chain complex (C 4,8 After mixing, a certain volume of n-butanol is added, followed by rapid vortexing and centrifugation. TE buffer is then added to restore the aqueous phase. Subsequently, the reporter complex is added, and n-butanol is added again, repeating the above steps. This treatment method can achieve instantaneous dehydration and concentration of the system within minutes, allowing the input chain to come into close proximity with the base chain of the double-stranded complex and pair rapidly, thereby triggering the chain displacement reaction.

[0061] In the input chain (X) 4,8 Under the condition of n-butanol, the reaction body driven by n-butanol can reach the maximum kinetic equilibrium in less than 1 minute. Compared with the reaction time of more than 20 hours under conventional conditions, its apparent reaction rate is increased by about 1200 times. This result shows that the method of this application can realize DNA strand replacement reaction in a very short time.

[0062] The n-butanol-assisted DNA computation mechanism proposed in this application has significant advantages in accelerating the reaction.

[0063] 1.2.3 Butanol accelerates DNA strand displacement reaction in toe regions of different lengths By designing input chains with toe domains of different lengths (1-nt, 2-nt, and 3-nt) (see Table 1 for details, X...), 1,8 X 2,8 X 3,8 The reaction was carried out with the corresponding double-stranded complex in a DNA strand displacement reaction as described in Example 1 to investigate the accelerating effect of the n-butanol transient-driven method on shorter, thermodynamically less favorable scaffold strand displacement reactions at room temperature. Results are as follows... Figure 2 As shown in CE.

[0064] Experimental results show that the average reaction rate of the 3-nt toe domain was significantly increased after transient n-butanol-driven treatment, achieving an acceleration effect of three orders of magnitude. Furthermore, the apparent reaction rate increased further with increasing n-butanol and water phase ratio. In contrast, the reactions in the 1-nt and 2-nt toe domains were more thermodynamically constrained, requiring more n-butanol to drive them to near equilibrium. This indicates that the transient n-butanol-driven method can also iteratively push the DNA strand replacement reaction in the short toe domain towards thermodynamic stability, especially under less favorable thermodynamic conditions, where its acceleration and promoting effects are more significant—an effect unattainable by other methods. This further verifies the universality and effectiveness of the method presented in this application.

[0065] And according to Figure 2 The results further show that, without the addition of n-butanol, the DNA strand displacement reaction depends entirely on diffusion and thermodynamic equilibrium, and its rate is significantly slower than the instantaneously driven n-butanol-based reaction. Even in the input strand (X... m,8 In the presence of [a specific substance], the reaction typically requires more than 20 hours to approach equilibrium; and for reaction systems where the toe region is only 1-nt or 2-nt, the yield is significantly lower, and equilibrium is difficult to reach even after a long reaction period. Figure 2 B, Figure 2 C in the middle Figure 2 China D and Figure 3 (E). This further illustrates that conventional diffusion conditions at room temperature are insufficient to drive DNA strand replacement reactions on short scaffolds in a short time, thus highlighting the significant advantages of the n-butanol-driven instantaneous method proposed in this application in accelerating the reaction rate and increasing the apparent reaction rate, up to approximately 1200 times.

[0066] Example 2: Ion-specifically regulated n-butanol accelerates DNA strand displacement reaction This embodiment further investigates a method for regulating the DNA strand displacement reaction (SDR) with n-butanol using anion-specific hydration kinetics. By selecting different anion environments, precise control of the SDR reaction rate and kinetics is achieved. In the experiment, nitrate (NO3) was selected. - ), chloride (Cl) - Acetate (Ac) - ) and sulfates (SO4) 2- As an anion source, it is used to regulate DNA strand displacement reactions. Anion-specific interactions are quantified using radial charge density parameters, based on literature reports (GREGORY KP, WANLESS EJ, WEBBER GB, et al. The electrostaticorigins of specific ion effects: quantifying the Hofmeister series for anions[J]. Chemical Science, 2021, 12(45): 15007-15): •NO3 - : = -5.78 •Cl - : = -6.25 •Ac - : = -7.12 •SO4 2- : = -8.19 This series of anions exhibits a distinct Hofmeister effect hierarchy, with its enthalpy of hydration ( H hyd The following are respectively: NO3 - ( H hyd = -312kJ·mol -1 )>Cl - ( H hyd = -367kJ·mol -1 )>Ac - ( H hyd = -425kJ·mol -1 SO4 2- ( H hyd = -1035kJ·mol -1 ).

[0067] The anions further influence the kinetics of DNA strand displacement reactions by regulating the mobility of free water molecules and the stability of the hydration shell in the solution. Strongly hydrophilic anions (such as SO42-) 2- ): Forms a highly ordered hydrated shell, restricts the movement of water molecules, hinders the acceleration of chain displacement reaction rate, and relatively delays the reaction.

[0068] Unstable anions in the hydrated shell (such as NO3-) - Rapid exchange of water molecules promotes solvent penetration and cellular phase separation, enhancing the molecular crowding effect and thus significantly accelerating the chain displacement reaction.

[0069] This mechanism not only regulates the reaction rate but also maintains the hydrogen bond network and structural stability of DNA molecules during strand replacement.

[0070] For specific experimental procedures, please refer to Example 1. The input chain is X. 3,8 By placing the same DNA double-stranded complex in buffers containing different anions (i.e., replacing the divalent magnesium salt in the buffer in Example 1 with magnesium chloride, magnesium acetate and magnesium sulfate respectively), different volumes of n-butanol were added at room temperature to carry out chain displacement reactions.

[0071] Statistical results show that, based on nitrate (NO3) - Acetate (Ac) - ) and chloride (Cl - The system can achieve an equivalent yield of approximately 80% when the dehydration ratio is greater than or equal to 3:1.Figure 4 ), and rich in sulfate (SO4) 2- The buffer solution requires a higher dehydration ratio (4:1) to achieve a conversion rate of approximately 80%. The ionized anion NO3- - The introduction of [a specific substance] significantly accelerated the reaction rate, increasing it by approximately 1200 times compared to the reaction under natural conditions. In contrast, using the more hydrophilic anionic SO42- [another substance]... 2- Replace NO3 - At this point, a higher proportion of n-butanol is required to achieve a comparable rate increase, indicating that anion-specific hydration and electrostatic interactions play a key regulatory role in the molecular crowding effect.

[0072] Therefore, it is evident that n-butanol accelerates the chain substitution reaction, significantly shortening the reaction time (from hours to seconds) with an overall acceleration of approximately 1200 times. Furthermore, it maintains controllable adjustment of the reaction rate under different anionic conditions, thereby enabling rapid, flexible, and efficient DNA computational operations. This result fully validates the significant technical advantages of the acceleration method provided in this application in DNA logic circuits and molecular computation.

[0073] Application Example 1: AND gate based on DNA strand substitution This application example provides an AND gate constructed based on a DNA strand substitution reaction, specifically referring to the reaction system in Example 1. The logic circuit includes input strands denoted as X1 and X2, a switching module, a transmission module, a threshold module, and a reporter module modified with fluorescent and quenching groups. Figure 4 ).

[0074] When X1=1 and X2=1, input strands X1 and X2 coexist in the reaction system and bind to the corresponding domains of the switching module to form a complex. This complex releases the top strand via a DNA strand displacement reaction, which further binds to the recognition region of the transport module, driving the signal downstream. Under the regulation of the threshold module, the signal eventually hybridizes with the recognition sequence of the reporter probe, triggering a conformational change in the probe, causing the fluorophore to separate from the quencher group, thereby generating a significant fluorescent signal in the system (output=1).

[0075] When there is no input (X1=0, X2=0) or only a single input (X1=1, X2=0 or X1=0, X2=1), the reaction system cannot form a complete complex, the chain substitution process cannot be effectively initiated, and therefore the reporting probe remains in a quenched state, generating a fluorescence signal less than the threshold (output=0). Thus, this application example 1 implements the logic characteristics of an AND gate.

[0076] The above-mentioned AND gate system was constructed under transient dehydration conditions with different volumes of n-butanol introduced at room temperature (specific reaction system as described in Example 1). Experimental results showed that, at room temperature and without the introduction of n-butanol (in its natural state), even with X1=1 and X2=1, a complete chain substitution reaction required at least 6 hours (approximately 360 minutes) to produce a correct signal output. However, under the transient dehydration conditions mediated by n-butanol, the same input conditions triggered a complete chain substitution reaction within approximately 1 minute, resulting in rapid dissociation of the report probe and the generation of a significant fluorescence signal. Figure 4 (B)

[0077] Therefore, the method described in this embodiment, while performing AND gate logic operations, reduces the response time from hours to seconds. The minimum time required for the output to reach the threshold under all input combinations is 6 hours, resulting in an acceleration rate of over 360 times. Figure 5 (CE). This result demonstrates that this method significantly improves the operational efficiency and signal response speed of DNA logic circuits, providing strong technical support for rapid and controllable molecular computing.

[0078] Application Example 2: OR gate based on DNA strand substitution This application example provides an OR gate constructed based on a DNA strand substitution reaction. The logic circuit includes input strands X1 and X2, a switching module, a transmission module, and a reporter module modified with fluorescent and quenching groups. (See attached diagram.) Figure 5 .

[0079] When X1=1 and X2=0, the input chain X1 can bind to the corresponding structural domain of the switching module and trigger a chain substitution reaction. The released top chain further binds to the transmission module, driving the signal downstream. This signal hybridizes with the recognition region of the reporter probe, causing a conformational change in the probe, separating the fluorophore from the quencher group, thereby generating a significant fluorescent signal in the system (output=1).

[0080] When X1=0 and X2=1, the input chain X2 independently triggers the same reaction path as above, and can also generate a fluorescence signal (output=1).

[0081] When X1=1 and X2=1, both input chains exist simultaneously, which can trigger chain displacement reactions and enhance signal output. A significant fluorescence signal was also detected in the system (output=1).

[0082] Only when X1=0 and X2=0 is there no input chain in the system capable of triggering the reaction, the chain substitution reaction cannot be initiated, the reporting probe remains quenched, and therefore no fluorescence signal is generated (output=0). Figure 6 ).

[0083] Therefore, this application example implements the logical characteristics of the OR gate: that is, the output is 0 except when there is no input, and 1 in all other cases.

[0084] The aforementioned OR gate system was constructed under transient dehydration conditions with different volumes of n-butanol at room temperature (specific reaction systems are described in Example 1). The results showed that, under natural conditions, even with X1=1 or X2=1, a complete chain substitution reaction required at least 7 hours (420 minutes) to achieve stable signal output. However, under n-butanol-mediated transient dehydration conditions, the same input conditions triggered the chain substitution reaction within approximately 1 minute, rapidly activating the reporter probe and generating a significant fluorescence signal.

[0085] Therefore, the method described in this application example reduces the response time from hours to seconds while implementing OR gate logic operations. The minimum time required for the output to reach above the threshold under all input combinations is 7 hours, representing an acceleration of up to 420 times. These results demonstrate that this method significantly improves the operational efficiency and signal response speed of DNA logic circuits, providing a technical foundation for rapid and controllable molecular computing.

[0086] The DNA sequences designed in Application Example 1 and Application Example 2 are shown in Table 2.

[0087] Table 2 DNA sequences in AND and OR logic gates

[0088] Application Example 3: AND-OR gate based on DNA strand substitution This application example provides an AND-OR gate constructed based on a DNA strand substitution reaction. The logic circuit includes three input strands X1, X2, and X3, as well as a switching module, a transmission module, a threshold module, and a reporter module modified with fluorescent and quenching groups. This circuit can implement the Boolean logic operation: (X1 AND X2) OR X3, see [link to relevant documentation]. Figure 6 .

[0089] When X1=1 and X2=1, the input chains X1 and X2 can bind together with the corresponding structural domains of the switching module to form a complex and trigger a chain substitution reaction. The released top chain further binds to the transport module and drives the signal downstream. Under the control of the threshold module, this signal hybridizes with the reporter probe recognition region, causing the separation of the fluorescent group and the quenching group, thereby generating a significant fluorescent signal in the system (output=1).

[0090] When X3=1, even if either X1 or X2 is 0, the input chain X3 can independently act on the switching module and trigger the chain displacement reaction. After the same signal transmission and reporting process, the system still produces a significant fluorescence signal (output=1).

[0091] When X1=1, X2=1, and X3=1, all three input chains exist. The chain substitution reaction can be triggered in the system through two pathways (the synergistic effect of X1 and X2, and the independent effect of X3), thereby further enhancing the signal output (output=1).

[0092] Only when X1=0, X2=0, and X3=0, there is no input chain in the system that can trigger the reaction, no effective complex can be formed, the chain substitution reaction does not occur, the reporting probe remains quenched, and no fluorescence signal is generated in the system (output=0).

[0093] Therefore, this application example implements the logic characteristics of the AND-OR gate: the output is 1 if and only if (X1 and X2 are both 1) or X3 is 1, and X1, X2, and X3 are all 1; otherwise, the output is 0.

[0094] The AND-OR gate was constructed and tested under natural conditions with different volumes of n-butanol added for transient dehydration (specific reaction system as described in Example 1). The results showed that, under natural conditions, even with (X1=1 and X2=1) or X3=1, a complete chain substitution reaction and signal output required at least 10 hours (600 minutes) to produce a correct signal output. Figure 7 Under n-butanol-mediated transient dehydration conditions, the same input state requires only about 1 minute to complete the reaction and generate a significant fluorescence signal. This demonstrates that the reaction rate under n-butanol conditions is approximately 600 times faster than at room temperature, drastically reducing the reaction efficiency of the chain substitution logic circuit from hours to seconds. Under all input combinations, it takes at least 10 hours for the output to reach the threshold, representing an acceleration factor of up to 600 times.

[0095] In summary, the instantaneous DNA strand substitution reaction driven by n-butanol not only successfully realized the logic characteristics of the AND-OR gate, but also significantly improved the reaction speed and time resolution of DNA circuits, providing an effective means for fast and controllable molecular logic computation.

[0096] The DNA sequences involved in this application example are shown in Table 3.

[0097] Table 3 DNA sequences in AND-OR logic gates

[0098] Application Example 4: OR-AND-OR gate based on DNA strand substitution (four inputs) This application example provides a four-input OR-AND-OR gate constructed based on a DNA strand substitution reaction. The logic circuit includes input strands X1, X2, X3, and X4, a switching module, a transmission module, a threshold module, and a reporter module modified with fluorescent and quenching groups. (See attached diagram.) Figure 7 .

[0099] This circuit implements Boolean logic operations: ((X1 OR X2) AND X3) OR X4 When X4=1, regardless of the states of X1, X2, and X3, input chain X4 can independently trigger the switching module, binding with its corresponding structural domain to form a complex. The complex releases the top chain through a chain substitution reaction, which further binds to the recognition sequence of the transport module, transmitting the signal downstream. The signal ultimately hybridizes with the recognition region of the reporter probe, triggering a conformational change in the probe, causing the fluorophore to separate from the quencher group, thereby generating a significant fluorescent signal in the system (output=1).

[0100] When X4=0 and X3=1 and (X1=1 or X2=1), the input chain X1 or X2, together with X3, triggers the switching module, forming a complex and releasing the top chain. The released top chain transmits a signal to the reporter module through the transmission module, binds to the recognition sequence of the reporter probe, and induces a conformational change, causing the fluorescent group to separate from the quenching group, resulting in a significant fluorescent signal in the system (output=1).

[0101] Only when X4=0 and (X1=0 and X2=0) or X4=0 and X3=0, the reaction system cannot form a complete complex, the chain substitution process cannot be effectively initiated, the reporter probe remains quenched, and the fluorescence signal is below the threshold (output=0).

[0102] Therefore, this embodiment implements the logic characteristics of the OR-AND-OR gate: the output is 1 only when ((X1 OR X2) AND X3) = 1 or X4 = 1, and the output is 0 in all other cases.

[0103] The OR-AND-OR gate was constructed and tested under natural conditions with different volumes of n-butanol added for transient dehydration (specific reaction system as described in Example 1). The results showed that under natural conditions, the chain substitution process and signal output required at least 5 hours (300 minutes) to stabilize. In contrast, under n-butanol-mediated transient dehydration conditions, the same input conditions required only 1 minute to complete the chain substitution reaction and generate the correct output signal. The results indicate that the reaction rate under n-butanol conditions is approximately 300 times faster than at room temperature, significantly improving the operational efficiency of the logic gate. Under all input combinations, the output required at least 5 hours to reach the threshold, representing an acceleration of up to 300 times. ​ ).

[0104] In summary, this application example not only successfully implemented the logic operation characteristics of a four-input OR-AND-OR gate, but also shortened the reaction speed from hours to seconds through a n-butanol instantaneous dehydration strategy, providing important technical support for the rapid response and efficient construction of complex molecular logic circuits.

[0105] The DNA sequences involved in this application example are shown in Table 4.

[0106] Table 4. DNA sequences in OR-AND-OR logic gates

[0107] The above embodiments and application examples fully demonstrate the effectiveness of the n-butanol instantaneous dehydration strategy in accelerating DNA strand displacement reactions, and further enable accelerated DNA computation, rapid response of complex molecular logic circuits, and efficient construction. It is understood that the embodiments and application examples provided in this application are merely illustrative; specific logic circuits, calculations, etc., can be designed according to actual needs, and will not be elaborated upon here.

[0108] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for accelerating DNA strand displacement reaction using n-butanol, characterized in that, The method comprises the following steps: providing a DNA solution in aqueous phase containing input strand and duplex complex; adding n-butanol to the DNA solution in aqueous phase, and allowing the n-butanol to contact with the DNA solution in aqueous phase sufficiently; adjusting the effective concentration of DNA molecules and the collision frequency by adjusting the type of anions in the DNA solution in aqueous phase and controlling the volume ratio of n-butanol to the DNA solution in aqueous phase, so that the bottom strand of the input strand and duplex complex is paired complementarily and releases the top strand, and the DNA strand displacement reaction is completed.

2. The method of claim 1, wherein, The sufficient contacting comprises the following steps: (i) adding n-butanol to the DNA solution in aqueous phase, vortexing and centrifuging, and supplementing buffer; (ii) repeating step (i) once, and finally vortexing and centrifuging to remove n-butanol; In step (i), the buffer is used to make up the water phase removed by n-butanol.

3. The method of claim 2, wherein, The volume of the supplemented buffer is added according to the dehydration ratio of 19.6% per volume of n-butanol.

4. The method of claim 1, wherein, The conditions of the DNA strand displacement reaction include temperature, volume ratio of n-butanol to the DNA solution in aqueous phase, type of ions in the DNA solution in aqueous phase, and concentration of the DNA solution in aqueous phase.

5. The method of claim 4, wherein, The temperature is room temperature.

6. The method of claim 4, wherein, The volume of added n-butanol is 1-8 times of the DNA solution in aqueous phase.

7. The method of claim 1, wherein, The DNA solution in aqueous phase further comprises at least one kind of anion electrolyte.

8. The method of claim 7, wherein, The anion species in the anionic electrolyte is at least one of NO3 - , Cl - , Ac - , SO4 2- .

9. The method of claim 1, wherein, The input strand comprises a toe domain, and the length of the toe domain is 1-4 nucleotides.

10. The method according to any one of claims 1-9 in the acceleration of DNA computing, molecular diagnosis, dynamic DNA circuit or nucleic acid molecule logic circuit.

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