CN114164442A

CN114164442A - Electrochemical preparation method and application of framework nucleic acid

Info

Publication number: CN114164442A
Application number: CN202111255391.8A
Authority: CN
Inventors: 沈建磊; 李茜; 翟婷婷; 孙晨蕴; 樊春海
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2022-03-11
Anticipated expiration: 2041-10-27
Also published as: CN114164442B

Abstract

The invention provides an electrochemical preparation method and application of frame nucleic acid, comprising the following steps: construction of electrochemical device: electrochemical device is formed through PDMS layer and glass lamella pasting, includes: the reaction area and the control area take silver/silver chloride as a working electrode, iridium oxide as a reference electrode and iridium as a counter electrode; self-assembly of framework nucleic acids: constructing frame nucleic acid with designable appearance and biochemical properties by an electrochemical strategy, providing a required DNA single strand, adding the frame nucleic acid into a reaction area, applying a certain potential to a working electrode to enable oxidation reaction of water in the reaction area to occur, reducing the pH value of a solution, and performing DNA denaturation; and applying another potential to the working electrode to ensure that the reduction reaction of water occurs in the reaction area, the pH of the solution is increased, DNA renaturation occurs, and the electrochemical self-assembly of the frame nucleic acid is realized. The invention utilizes the characteristic that the pH value of the solution can be quickly and accurately regulated and controlled by controlling the potential of the working electrode, thereby obtaining a series of frame nucleic acids modified by specific functional groups with a series of sizes.

Description

Electrochemical preparation method and application of framework nucleic acid

Technical Field

The invention relates to the technical field of nano materials, in particular to an electrochemical preparation method of frame nucleic acid and application thereof.

Background

The DNA nano technology utilizes the principle of DNA double helix with accurate structure and strict Watson-Click base complementary pairing to realize accurate two-dimensional and three-dimensional static or dynamic DNA nano structure, and has wide application prospect in the fields of medical diagnosis, molecular machines and the like. The DNA nano structure is constructed by mainly utilizing the base complementary principle among DNA single chains and utilizing the binding energy difference among the DNA single chains with different lengths and sequences to realize the most stable configuration of the DNA nano structure. However, in a single DNA strand, some non-specific interactions tend to generate some secondary or tertiary structures (in kinetic traps), so that the folding and assembling of DNA often requires that the secondary or tertiary structures possibly existing in the DNA are opened by heating or other means, and then the base complementary principle among DNAs is utilized to realize the controllable folding, hybridization and assembling of the DNA strand.

Currently, controllable assembly means for realizing DNA nanostructures mainly include thermal denaturation technology and non-ionizing radiation (microwave and terahertz) technology. Among these, heat denaturation is the most common and well-established means for opening up the non-specific interactions between the DNA itself or between different DNAs. Generally, at 90 ℃, DNA undergoes a thermal denaturation process, secondary structures (in kinetic traps) are opened, single DNA strands are restored to an initial loose state, and then DNA renaturation occurs during a cooling process, and complementary paired DNA forms an ordered double-helix structure. Among them, a commercially available nucleic acid amplification instrument for Polymerase Chain Reaction (PCR) can precisely control the temperature of a reaction system, and thus, is widely used for assembling DNA nanostructures. However, commercial PCR is expensive and has poor portability, thus limiting its use in resource-limited situations. Furthermore, it has been reported in recent years that non-ionizing radiation in microwave and terahertz (Thz) systems can achieve assembly/disassembly of DNA nanostructures without any sequence modification. However, this system is not suitable for a miniature DNA nanostructure synthesis system and is costly. In general, the techniques currently available to open up non-specific interactions in DNA and subsequently achieve controlled assembly of DNA nanostructures are limited.

Recent studies have shown that hydrogen bonds in the double helix structure of DNA are very sensitive to solution pH, hydrogen bonds are broken at low pH, DNA double strands are opened, and DNA double strands hybridize again under alkaline conditions. Based on this, a series of works based on pH-controlled DNA molecular machinery and Polymerase Chain reaction were reported successively (Y.Zhang, Q.Li, L.Guo, Q.Huang, J.Shi, Y.Yang, D.Liu and C.Fan, Ion-medical Polymerase Chain reaction per formed with an electronic drive Microfluidic device, Angew.chem., int.Ed.Engl., 2016,55, 12450-. Therefore, a new idea is provided for assembling the DNA nano structure by regulating and controlling the pH of the solution system. While achieving pH regulation in solution systems is a routine laboratory practice, miniaturized DNA nanostructure synthesis systems present new requirements and challenges for pH regulation. The common "volume titration" method introduces exogenous substances during the regulation of pH and changes the total volume of the system, thus is not suitable for the synthesis of DNA nanostructures in a micro scale. The electrochemical method has the characteristics of high sensitivity, quick response and easy control, water is electrolyzed to generate protons by the electrochemical method, the pH value of the system is regulated and controlled, the influence on the volume of the system is small, and no irrelevant accumulated waste is generated, so the electrochemical method is very suitable for regulating and controlling the pH value of the micro system. In 2008, the Hiroaki Suzuki project group in japan reported a non-classical three-electrode electrochemical system. The pH of the micro system is accurately and rapidly adjusted and stabilized by utilizing the pH sensitivity of the iridium oxide electrode and the non-polarizability property of the silver/silver chloride electrode. (K.Morimoto, M.Toya, J.Fukuda and H.Suzuki, Automatic Electrochemical Micro-pH-Stat for biological microsystems. anal.Chem.,2008,80,905-914) the work provides a new opportunity for the precise rapid read rate regulation of the pH of a solution by an Electrochemical method and then the controllable synthesis of a DNA nanostructure.

Disclosure of Invention

The invention aims to provide an electrochemical preparation method of frame nucleic acid and application thereof, so as to solve the problems of expensive equipment, large equipment volume, difficulty in carrying and the like of instrument equipment used for accurately controlling system temperature in the process of preparing the frame nucleic acid by the conventional thermal denaturation technology.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to a first aspect of the present invention, there is provided a method for electrochemical preparation of a framework nucleic acid, the method comprising: construction of electrochemical device: the electrochemical device is formed by sticking a PDMS layer and a glass sheet layer, and comprises: the device comprises a reaction area and a control area which are connected through a salt bridge, wherein silver/silver chloride is used as a working electrode in the reaction area, iridium oxide is used as a reference electrode in the control area, and iridium is used as a counter electrode; self-assembly of framework nucleic acids: constructing frame nucleic acid with designable appearance and biochemical properties by an electrochemical strategy, providing a DNA single chain required by constructing the frame nucleic acid, adding a mixed solution of the DNA single chains into the reaction zone, applying a certain potential to the working electrode to enable oxidation reaction of water in the reaction zone to occur, reducing the pH value of the solution, and performing DNA denaturation; and applying another potential to the working electrode to ensure that the reduction reaction of water occurs in the reaction area, the pH of the solution is increased, DNA renaturation occurs, and finally the electrochemical self-assembly of the frame nucleic acid is realized.

The frame nucleic acid is a three-dimensional DNA nano-structure formed by a plurality of DNA short chains through denaturation and renaturation. It will be appreciated that the number of short chains will depend on the size of the designed framework nucleic acid.

More preferably, the framework nucleic acid is tetrahedral in shape, but it is understood that other shapes of framework nucleic acids, such as cubic, octahedral, triangular prism, triangular bipyramid, and the like, may be used in the present invention.

The self-assembly of the framework nucleic acid further comprises: the modification of functional groups on the structure of the frame nucleic acid is realized by designing a DNA single strand.

More preferably, the functional group modified by the functional framework nucleic acid is a thiol group, but it is understood that other chemical groups and functional molecules such as ferrocene, amine, carboxyl, DBCO, benzene, Cy3, Cy5, Cy7, cholesterol, commercially available fluorescent molecules, and the like modified framework nucleic acids can also be used in the present invention.

More preferably, the stoichiometric ratio of thiol groups in the thiol-modified framework nucleic acid to framework nucleic acid is 3: 1.

The modification sites of the functional groups on the framework nucleic acid include: the apex of the framework nucleic acid, the internal cavity of the framework nucleic acid, or the border of the framework nucleic acid.

In the electrochemical self-assembly process of the framework nucleic acid, the mode of capturing the probe comprises the following steps: base-complementary pairing, high affinity interaction between biotin and avidin, interaction between antigen and antibody, specific recognition between aptamer and specific target.

The reaction system for preparing the framework nucleic acid comprises: NaCl reaction System or MgCl₂A reaction system, wherein the yield of the framework nucleic acid is highest under the condition of 0.6M NaCl in the NaCl reaction system and is at MgCl₂60mM MgCl in the reaction system₂The yield under the conditions is highest.

The self-assembly of the framework nucleic acid further comprises: a biosensor based on the frame nucleic acid is constructed by modifying the frame nucleic acid modified by the functional group at the interface of a gold electrode.

According to a preferred embodiment of the present invention, a DNA biosensor is prepared in which thiol groups are modified at the apexes of frame nucleic acids in order to densely modify the frame nucleic acids on the surface of gold electrodes via Au-S bonds, but it is to be understood that frame nucleic acids at other modification sites such as internal cavities of frame nucleic acids, borders of frame nucleic acids, etc. can also be used in the present invention.

According to the method provided by the invention, the assembly principle of the frame nucleic acid in the electrochemical system is as follows: the potential of the reference electrode has a good linear relationship with the solution pH. Applying a certain potential to the working electrode to enable the reaction area to generate oxidation reaction of water, reducing the pH value of the solution, damaging the hydrogen bond action of the DNA in an acid system and generating DNA denaturation; and applying another potential to the working electrode to enable the reaction area to generate reduction reaction of water, the pH value of the solution is increased, DNA renaturation is generated in an alkaline system, and finally self-assembly of the frame nucleic acid is realized.

It should be understood that the probe of the present invention captures the target molecule by double-strand hybridization, specifically, a certain number of bases extend from the vertex of the frame nucleic acid of the DNA tetrahedron, and the extended portion is a single-stranded DNA, and the single-stranded portion can capture the target molecule by means of base complementary pairing. It will be appreciated that the manner of capture probes described includes, in addition to base complementary pairing, high affinity interactions between biotin and avidin, interactions between antigen and antibody, specific recognition between aptamers and specific targets, etc. The emphasis is on the need for specific recognition with high affinity between the probe and the target. The target molecules detected by the invention are DNA, and other target molecules or ions, such as miRNA, mRNA, protein, glucose, sarcosine, metal ions and the like can also be used in the system.

According to the method provided by the invention, the constant-temperature and rapid assembly of the frame nucleic acid is realized.

According to the method provided by the invention, the assembly of the frame nucleic acid is realized by regulating the pH of the solution in the electrolytic cell through electric potential.

It will be appreciated that different framework nucleic acids require different drive energies, i.e.different concentrations of H are required⁺Or OH^-Denaturation and renaturation are achieved, and therefore the potentials required for the different framework nucleic acids are not identical. According to different frame nucleic acids to be constructed, how to set the potential can be obtained according to the experiment of specific practical conditions.

According to a preferred embodiment of the invention, example 3 of the present invention further improves the yield of tetrahedral production by using multiple gradient potential changes. However, it should be understood that tetrahedrons can also be successfully prepared by merely changing from one potential to another, differing only in the magnitude of the yield. The present invention is not limited to the potential change of the form of embodiment 3.

According to a second aspect of the present invention, there is provided a use of a method for electrochemical preparation of a framework nucleic acid in the fields of medical diagnostics and molecular machines.

As described in the background of the invention, the existing thermal denaturation technology for preparing frame nucleic acid has the problems of expensive equipment, large equipment volume, difficult carrying and the like, and in order to overcome the problems, the invention realizes the regulation and control of the denaturation and renaturation process of DNA by quickly and accurately regulating and controlling the pH value of a solution in an electrochemical system, and simultaneously retains the molecular information and the structural information of the DNA.

Experimental results show that the method is simple, rapid and effective, and a series of size and specific functional group modified framework nucleic acids can be obtained by utilizing the characteristic that the pH of the solution can be rapidly and accurately regulated and controlled by controlling the potential of the working electrode. The frame nucleic acid structure prepared by the invention has the advantages of good dispersibility, complete frame structure and effective regulation and control of a biosensing interface. The reagent medicine and the instrument used by the invention are cheap and portable, and are easy to standardize and commercialize. Therefore, the invention provides a new idea and technical support for constructing the frame nucleic acid by an electrochemical method and breaking through the defects of the traditional thermal denaturation technology.

Drawings

FIG. 1 shows the working principle of the present invention, wherein a shows a schematic diagram of electrochemically regulating pH of a solution, and b shows a schematic diagram of pH control framework nucleic acid formation;

FIG. 2 is a result of characterization of electrochemical properties of an iridium oxide electrode, wherein a is a cyclic voltammogram and b is a result of characterization of electrode stability;

FIG. 3 is the results of experiments of electrochemical performance characterization of the prepared iridium oxide electrode and analysis of relationship between Tm value and pH of the DNA double strand, wherein a is the schematic diagram of the apparatus for electrochemical preparation of frame nucleic acid, b is the results of experiments of linear relationship between potential and pH of iridium oxide, c is the results of experiments of potential regulation and control speed characterization of iridium oxide electrode, and d is the results of experiments of Tm value of double strands with different lengths under different pH conditions;

FIG. 4 shows the results of experiments on the denaturation and renaturation states of DNA double strands controlled by pH, wherein a is a schematic diagram showing the denaturation and renaturation states of DNA double strands under two conditions of

pH

11 and 7 verified by fluorescence, b is the fluorescence spectrum of the system under the conditions of

pH

7 and 11, and c is the result of fluorescence intensity analysis of the system under the conditions of

pH

7 and 11;

FIG. 5 is a diagram showing the specific experimental parameters for preparing a framework nucleic acid in example 3, a is an experimental parameter for an electrochemical method, and b is an experimental parameter for a thermal denaturation method;

FIG. 6 shows the results of gel electrophoresis characterization and yield analysis of framework nucleic acids in salt ions of different species and concentrations, wherein a is different Na⁺Gel electrophoresis characterization of framework nucleic acids in concentration system, b is different Na⁺Yield analysis of framework nucleic acids in the concentration system, c is different Mg²⁺Gel electrophoresis characterization of framework nucleic acids in concentration systems, d is different Mg²⁺Yield analysis experiment results of the frame nucleic acid in the concentration system;

FIG. 7 shows a composition containing Mg²⁺Gel electrophoresis characterization and yield analysis results of three sizes of frame nucleic acids prepared by an electrochemical method and a thermal denaturation method respectively in the system, wherein a is the gel electrophoresis characterization of the frame nucleic acids, and b is the yield analysis results of the frame nucleic acids;

FIG. 8 shows a solution containing Na⁺Gel electrophoresis characterization and yield analysis results of three sizes of frame nucleic acids prepared by an electrochemical method and a thermal denaturation method respectively in the system, wherein a is the gel electrophoresis characterization of the frame nucleic acids, and b is the yield analysis results of the frame nucleic acids;

FIG. 9 is an AFM characterization of three size framework nucleic acids prepared using electrochemical and thermal denaturation methods, respectively;

FIG. 10 shows AFM characterization and size analysis results of three size-framed nucleic acids prepared electrochemically, where c is AFM characterization of three size-framed nucleic acids and d is size analysis results of three size-framed nucleic acids;

FIG. 11 is a gel electrophoresis characterization and an AFM characterization of a thiol-modified framework nucleic acid prepared by an electrochemical method, wherein a is the gel electrophoresis characterization of the thiol-modified framework nucleic acid and b is the AFM characterization of the thiol-modified framework nucleic acid;

FIG. 12 is an experimental principle and performance characterization of a frame nucleic acid biosensor, wherein a is a schematic diagram of a frame nucleic acid biosensor, b is a time-current experimental result of sensors modified by different DNA structures, and c is a current intensity analysis result of sensors modified by different DNA structures;

FIG. 13 is a cyclic voltammogram of sensors modified with different DNA structures.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

According to the present invention, there is provided a method for electrochemically preparing a framework nucleic acid. The invention selects DNA tetrahedrons with different sizes as an example, and then realizes the adjustment of the pH value of the solution in the electrolytic cell by regulating and controlling the potential on the working electrode under the condition of room temperature. Specifically, when a certain potential is applied, a reduction reaction of water occurs in the reaction region, and a large amount of OH is generated^-When the pH value of the reaction area is increased, the single-stranded DNA is denatured; when a certain other potential is applied, the reaction zone undergoes an oxidation reaction of water to generate a large amount of H⁺The pH of the reaction region is lowered, and the complementary paired DNA single strands are hybridized into a double strand, thereby assembling the framework nucleic acid (as shown in FIG. 1). And then, the constructed DNA tetrahedral framework nucleic acid modified by sulfydryl is assembled on the surface of a gold electrode by virtue of Au-S action, and the thermodynamics of hybridization of a nucleic acid probe and a target molecule is regulated, so that the overall performance of the biosensor is improved.

Wherein the DNA is purchased from a living organism (Shanghai); reagents such as potassium chloride, disodium hydrogen phosphate, iridium wire are available from national drug group reagents ltd. Platinum electrodes, silver/silver chloride electrodes were purchased from Wuhan Gaosri-Bisco Ltd. Britton-Robinson buffer (0.01M H)₃PO₄-H₃BO₃-CH₃COOH, 0.1M KCl, adjusted to a specific pH with hydrochloric acid and sodium hydroxide, hereinafter referred to as BR buffer) was purchased from Jiekekang Biotech, Inc., Qingdao.

The DNA sequences are shown in Table 1 below.

TABLE 1

EXAMPLE 1 construction of electrochemical device

In this embodiment, an electrochemical device is constructed, including the steps of:

preparation of iridium oxide electrode: iridium wire as working electrode, silver/silver chloride electrode as reference electrode, platinum electrode as counter electrode, 0.7M Na₂HPO₄The solution was an electrolyte, and cyclic voltammetric oxidation was performed using Chenghua CHI1040c according to the parameters shown in Table 2. And soaking the iridium oxide electrode subjected to cyclic voltammetry oxidation treatment in water, and hydrating and aging for 24 hours.

TABLE 2

Initial potential (V)	-0.5
		Maximum potential (V)	0.8
Lowest potential (V)	-0.5
		Final potential (V)	0
Sweeping speed (V/s)	0.1
		Number of scanning turns	300
Rest time (V)	0
		Sensitivity (A/V)	10^-4

Performance test procedure of the electrode: Britton-Robinson (BR) buffer solutions with pH values of 1.7, 2.7, 3.7, 4.5, 5.5, 6.7, 7.7, 8.7, 9.5, 10.4 and 11.6 are sequentially added into a reaction zone, a silver/silver chloride electrode is used as a working electrode, an iridium oxide electrode is used as a reference electrode, and the potential difference between the iridium oxide electrode and the silver/silver chloride electrode is measured to obtain a correlation curve of the potential difference and the pH value of the solution. After the corresponding relation between the potential difference and the system pH is calibrated, an iridium oxide electrode is set up as a reference electrode, silver/silver chloride is used as a working electrode, an iridium electrode is used as a counter electrode, negative 190mV and negative 178mV are applied in a circulating mode according to data of a relevant curve between the iridium oxide electrode and the solution pH value, and the rapid and reversible regulation and control capacity of an electrochemical method on the solution pH value is investigated.

Preparation of PDMS: PDMS and a curing agent in a mass ratio of 10:1 are weighed in a clean beaker, are uniformly stirred by a glass rod, and are placed in a vacuum drier for vacuumizing for 15min to extract bubbles in the mixture. Then the mixture of PDMS and curing agent is poured into a horizontal glass tank and placed on a horizontal heating table to be cured for 1 hour at 80 ℃ to obtain the PDMS film.

Construction of electrochemical device: silver/silver chloride is used as a working electrode, an iridium oxide electrode is used as a reference electrode, and iridium is used as a counter electrode. An electrolytic cell was assembled by punching two holes with a punch in advance in a distance of about 1.5cm on a PDMS film and then attaching the PDMS film to the surface of a glass slide. mu.L of 0.1M KCl was added to the control zone and 20. mu.L of single-stranded mixture to be prepared into nanostructures were added to the reaction zone, with 0.5% agarose as a salt bridge (as shown in a of FIG. 3).

As a result: the cyclic voltammogram shows that a pair of characteristic peaks of iridium oxide appear around 0.1V and 0.5V (vs. Ag/AgCl), respectively, and the iridium oxide electrode is successfully prepared (shown as a in FIG. 2). The open-circuit potential method shows that the potential of the iridium oxide electrode has good linear relation with the pH value of the solution, and the correlation coefficient R²At 0.999, the slope was 77mV pH^-1(as shown in b in fig. 3). When the operating potential was changed from-190 mV (corresponding to pH 12) to 178mV (corresponding to pH 12)A significant oxidation current was observed and a steady state was reached within 20s (as shown in c in fig. 3), indicating that electrochemical methods can achieve rapid control of solution pH. In addition, the electrochemical regulation system can realize reversible regulation of the pH of the solution for several times (as shown in b in figure 2).

EXAMPLE 2 Regulation of DNA denaturation and renaturation Process by solution pH

The process of measuring the Tm value of the DNA double strand under different pH conditions comprises the following steps: the required single-stranded DNA was quantified using an ultraviolet-visible absorption spectrometer, and all DNA single-stranded concentrations were quantified to a concentration of 100. mu.M. Two complementary paired single-strands with the chain lengths of 13 bp, 20bp, 26bp, 37bp and 47bp respectively are proportionally mixed and added into BR buffer solutions containing 1 xSYBR-Green and with different pH values to prepare samples with the final concentration of the single-strands being 1 mu M. And (3) putting the prepared samples into an RT-PCR instrument, heating at 95 ℃ for 10min, then, rapidly cooling to 25 ℃, slowly heating to 95 ℃, and collecting the fluorescence signals of all groups of samples in the whole temperature changing process in real time.

The process of DNA double strand denaturation and renaturation in different pH systems is verified: two single-stranded DNAs (F1-Alexa488 and F2-BHQ1) modified with Alexa488 and BHQ1 respectively were quantified to 50. mu.M using an ultraviolet-visible absorption spectrometer. Then, the two single-stranded DNAs were mixed in equal amounts and added to BR buffer solutions at

pH

7 and 11, respectively, to give a final concentration of 100nM of single-stranded DNA. The fluorescence test conditions were: the excitation wavelength is 488nm, the emission wavelength is 498-650 nm, and the excitation and emission slits are both 5 nm.

As a result: the DNA melting temperature results show that the Tm value gradually increases as the length of the double-stranded DNA increases. And for the same length of DNA strand, its Tm decreases with increasing pH of the solution (as shown by d in FIG. 3). Thus, the energy of the double-spiral structure is weakened in an alkaline system, and the unwinding behavior is more favorably realized.

In the BR buffer solution with pH 7, F1-Alexa488 and F2-BHQ1 hybridize to form a double-stranded structure, the fluorescent group and the quencher group at both ends are close, the fluorescence of Alexa488 is quenched, and in the alkaline environment with pH 11, the double-stranded structure formed by F1-Alexa488 and F2-BHQ1 is opened, the fluorescent group and the quencher group at both ends are far away, and the fluorescence of Alexa488 is recovered.

Example 3 preparation of DNA tetrahedral framework nucleic acids of different sizes

Experimental procedure for screening salt concentration of electrochemical method for preparing framework nucleic acid: for selection of optimal Mg for DNA tetrahedron preparation²⁺Concentration four single-stranded DNAs forming a DNA tetrahedral nanostructure were mixed in equal proportion to a final concentration of 1. mu.M in TM buffer and 10mM MgCl respectively₂、20mM MgCl₂、40mM MgCl₂And 60mM MgCl₂Heating at 95 deg.C for 10min in a PCR instrument, and rapidly cooling to 4 deg.C for more than 10 min. Optimized Na preparation for screening DNA tetrahedrons⁺Concentration, four single-stranded DNAs forming the DNA tetrahedral nano-structure are mixed in a TM buffer, 0.2M NaCl, 0.3M NaCl, 0.4M NaCl, 0.5M NaCl and 0.6M NaCl in equal proportion according to the final concentration of 1 mu M, respectively, put into a PCR instrument, heated at 95 ℃ for 10min, and then rapidly cooled to 4 ℃ and maintained for more than 10 min.

Electrochemical preparation process of frame nucleic acid: the required single-stranded DNA was quantified using an ultraviolet-visible absorption spectrometer, and the concentration of all DNA single strands was quantified to 100. mu.M. Mixing four or eight single-stranded DNAs for preparing 20bp, 26bp, and 37bp DNA tetrahedrons equally, and adding 0.5M NaCl or 60mM MgCl₂In (3), the final concentration of each single-stranded DNA was 1. mu.M. mu.L of the mixture was added to the reaction zone, and the operating potentials were set to 178mV, 104 mV, 31mV, -43mV, -117mV and-190 mV in this order, and each potential condition was maintained for 1min, to prepare electrochemical framework nucleic acids (E-TDNs) (shown as a in FIG. 5).

The preparation process of the framework nucleic acid by heat denaturation comprises the following steps: the required single-stranded DNA was quantified using an ultraviolet-visible absorption spectrometer, and the concentration of all DNA single strands was quantified to 100. mu.M. Mixing four or eight single-stranded DNAs for preparing DNA tetrahedron equally, and adding into reaction zone containing 1 XTM buffer (20mM Tris,50mM MgCl)₂pH 8.0) at a final concentration of 1. mu.M per single-stranded DNA, heating the resulting mixture to 95 ℃ for 10min, and rapidly cooling to 4 ℃ within 5min for more than 10min to obtain a heat-denatured frameNucleic acids (T-TDNs) (shown as b in FIG. 5).

And (3) gel electrophoresis characterization: characterization was performed using 8% polyacrylamide electrophoresis gel electrophoresis, run at 120V for 2 h.

Atomic force microscopy characterization: the newly torn mica is treated with 0.5% (v/v) APTES solution for 1min, the non-adsorbed APTES on the surface of the mica is removed by washing with a large amount of ultrapure water, and the surface of the mica is dried by ear washing balls for standby. 10 μ L of 1nM sample was dropped onto the mica surface and adsorbed for 5 min. Scanning and imaging under an atomic force microscope, wherein an imaging probe is SCANASYST-FLUID +, and an imaging mode is PeakForce QNM in FLUID.

As a result: the gel electrophoresis result shows that the yield difference is not great under the condition of 0.4-0.6M in the NaCl reaction system, the yield of the frame nucleic acid is the highest under the condition of 0.6M NaCl, and the frame nucleic acid is the highest under the condition of MgCl₂60mM MgCl in the reaction system₂The yield of framework nucleic acids was highest under the conditions, but for ease of formulation, the reaction system chosen in the subsequent framework nucleic acid assembly experiments was 0.5M NaCl or 60mM MgCl₂Solution (fig. 6).

In addition, gel electrophoresis characterization was shown in the NaCl system and MgCl₂Electrochemical methods in the system can successfully prepare 20bp, 26bp and 37bp DNA regular tetrahedrons (FIGS. 7 and 8). And atomic force microscopy characterization showed that the electrochemically prepared DNA tetrahedrons also had good dispersibility and structural integrity compared to the traditional thermal denaturation method (fig. 9), and atomic force image size analysis showed that the lateral dimensions of the 20bp, 26bp, and 37bp DNA tetrahedrons were 8.6nm, 11.7nm, and 17.5nm (fig. 10).

Example 4 preparation of thiol-modified framework nucleic acid

Preparation of thiol-modified framework nucleic acids: the required single-stranded DNA was quantified using an ultraviolet-visible absorption spectrometer, and the concentration of all DNA single strands was quantified to 100. mu.M. Four single-stranded DNAs for DNA tetrahedron preparation were mixed in equal amounts in 1 XTM buffer (20mM Tris,50mM MgCl) containing 3mM TCEP₂pH 8.0) at a final concentration of 1. mu.M, and the framework nucleic acid was synthesized by denaturation. The denaturation process was as in example 3.

As a result: polyacrylamide electrophoresis shows that the electrochemically prepared frame nucleic acid lane has clear bands in target bands, and the preparation of the DNA tetrahedral frame nucleic acid modified with sulfydryl is successful (as shown in a in FIG. 11).

Atomic force microscopy characterization shows that the electrochemically prepared thiol-modified framework nucleic acid has good monodispersity and structural integrity, which indicates that the electrochemical method provided by the invention can be used for preparing the thiol-modified framework nucleic acid (shown as b in fig. 11).

Example 5 preparation of DNA biosensor based on framework nucleic acid Probe

Modification of the tetrahedral DNA nanoprobe on the surface of the gold electrode: referring to the method described in example 4, three frame nucleic acids having thiol groups modified at the vertices were prepared by an electrochemical method and a thermal denaturation method, respectively. And (3) respectively dropwise adding 6 mu L of prepared frame nucleic acid to the surface of the gold electrode, and putting the gold electrode into a wet box for overnight assembly at normal temperature. And 6 mu.L of sulfhydryl modified single-stranded DNA is also dripped on the surface of the gold electrode and assembled overnight at room temperature. Then, the reaction was blocked with 2mM MCH for 1 hour in order to reduce nonspecific adsorption of DNA to the surface and to help the DNA probe to remain in an upright state.

Detection of target DNA: t-probe at a final concentration of 10nM and signal probe R-biotin at a final concentration of 100nM were mixed in 10mM PB hybridization solution, heated at 80 ℃ for 5 minutes, and cooled at room temperature for 20 minutes. Then, soaking the assembled electrode into the mixed solution, and reacting for 2 hours at 37 ℃; the electrode was washed with 0.01M PBS, N₂And (5) drying. Followed by addition of 3. mu.L of avidin-HRP (0.5U/mL) and labeling at room temperature for 15 min. Then 0.01M PBS was washed for electrochemical measurement.

Electrochemical measurement: all electrochemical tests were signal readout by Chenghua CHI660E electrochemical workstation. The three-electrode system comprises a gold electrode as a working electrode, a platinum wire electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode. The sweep rate of cyclic voltammetry was 100mV/s, with a sweep range from-0.25V to 0.4V. The chronoamperometric potential was set at 100mV, and an electrochemical reduction current of 100s was taken as an output electric signal (equilibrium state of HRP catalytic reaction).

As a result: compared with ssDNA sensors, the reduction current of the E-TDNs and the T-TDNs sensors is remarkably increased between-0.3V and 0V (figure 13), and more TMB oxides are reduced on the surfaces of the electrodes modified by the E-TDNs and the T-TDNs. The time-current (i-t) curve shows that the reduction current of the TDNs-modified gold electrode experimental group increases significantly at a constant reduction voltage (-0.1V) (as shown in b in fig. 12). These results show that the DNA hybridization efficiency of the gold electrode interface modified by E-TDNs and T-TDNs is higher. The signal of the single-stranded DNA group was only about 2 times higher than that of the blank group, while the signals of the experimental groups of E-TDNs and T-TDNs were more than 186 times higher than that of the blank group. These results show that the rigid three-dimensional framework structure can precisely adjust the distance between probes, thereby improving the electrochemical interface hybridization efficiency. And the current signal values between the E-TDNs and the T-TDNs are not obviously different (about 2%), which proves that the electrochemical method is consistent with the structure of the frame nucleic acid prepared by the traditional thermal denaturation method (as shown in c in FIG. 12).

The above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the content of the specification of the claims of the present application fall within the scope of the claims of the present patent application. The invention is not exhaustive and is a matter of routine skill.

SEQUENCE LISTING

<110> Shanghai university of transportation

<120> electrochemical preparation method of frame nucleic acid and application thereof

<160> 34

<170> PatentIn version 3.5

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<212> DNA

<213> Artificial sequence

<400> 3

ccgcagcgga gcaggagtcc tcggcc 26

<210> 4

<211> 37

<212> DNA

<213> Artificial sequence

<400> 4

gcgtatgtgt tctgtgcggc ctgccgtccc gtgtggg 37

<210> 5

<211> 47

<212> DNA

<213> Artificial sequence

<400> 5

gcgtatgtgt tctgtgcggc ctgccgtccc gtgtgggcag gccttgg 47

<210> 6

<211> 13

<212> DNA

<213> Artificial sequence

<400> 6

tctgacgtag tgt 13

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

cgaacattcc taagtctgaa 20

<210> 8

<211> 26

<212> DNA

<213> Artificial sequence

<400> 8

ggccgaggac tcctgctccg ctgcgg 26

<210> 9

<211> 37

<212> DNA

<213> Artificial sequence

<400> 9

cccacacggg acggcaggcc gcacagaaca catacgc 37

<210> 10

<211> 47

<212> DNA

<213> Artificial sequence

<400> 10

ccaaggcctg cccacacggg acggcaggcc gcacagaaca catacgc 47

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<400> 11

gctgcaagct tactaatagg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<400> 12

cctattagta agcttgcagc 20

<210> 13

<211> 62

<212> DNA

<213> Artificial sequence

<400> 13

cgattacagc ttgctacacg attcagactt aggaatgttc gacatgcgag ggtccaatac 60

cg 62

<210> 14

<211> 62

<212> DNA

<213> Artificial sequence

<400> 14

cgtatcacca ggcagttgag acgaacattc ctaagtctga aatttatcac ccgccatagt 60

ag 62

<210> 15

<211> 62

<212> DNA

<213> Artificial sequence

<400> 15

cgtgtagcaa gctgtaatcg acgggaagag catgcccatc cactactatg gcgggtgata 60

aa 62

<210> 16

<211> 62

<212> DNA

<213> Artificial sequence

<400> 16

ctcaactgcc tggtgatacg aggatgggca tgctcttccc gacggtattg gaccctcgca 60

tg 62

<210> 17

<211> 84

<212> DNA

<213> Artificial sequence

<400> 17

gcctggagat acatgcacat tacggctttc cctattagaa ggtctcaggt gcgcgtttcg 60

gtaagtagac gggaccagtt cgcc 84

<210> 18

<211> 84

<212> DNA

<213> Artificial sequence

<400> 18

cgcgcacctg agaccttcta atagggtttg cgacagtcgt tcaactagaa tgccctttgg 60

gctgttccgg gtgtggctcg tcgg 84

<210> 19

<211> 84

<212> DNA

<213> Artificial sequence

<400> 19

ggccgaggac tcctgctccg ctgcggtttg gcgaactggt cccgtctact taccgtttcc 60

gacgagccac acccggaaca gccc 84

<210> 20

<211> 84

<212> DNA

<213> Artificial sequence

<400> 20

gccgtaatgt gcatgtatct ccaggctttc cgcagcggag caggagtcct cggcctttgg 60

gcattctagt tgaacgactg tcgc 84

<210> 21

<211> 58

<212> DNA

<213> Artificial sequence

<400> 21

ccctgtactg gctaggaatt cacgttttaa tctgggcttt gggttaagaa actccccg 58

<210> 22

<211> 59

<212> DNA

<213> Artificial sequence

<400> 22

cgctggaggc gcatcaccgt ttgcgtatgt gttctgtgcg gcctgccgtc ccgtgtggg 59

<210> 23

<211> 58

<212> DNA

<213> Artificial sequence

<400> 23

cggtgatgcg cctccagcgc ggggagtttc ttaacccttt ccgacttaca agagccgg 58

<210> 24

<211> 59

<212> DNA

<213> Artificial sequence

<400> 24

gcgagactca ggtggtgcct ttggcattcg accaggagat atcgcgttca gctatgccc 59

<210> 25

<211> 59

<212> DNA

<213> Artificial sequence

<400> 25

cccatgagaa taataccgcc gatttacgtc agtccggttt cccacacggg acggcaggc 59

<210> 26

<211> 58

<212> DNA

<213> Artificial sequence

<400> 26

cgcacagaac acatacgctt tgggcatagc tgaacgcgat atctcctggt cgaatgcc 58

<210> 27

<211> 59

<212> DNA

<213> Artificial sequence

<400> 27

gcccagatta aaacgtgaat tcctagccag tacagggttt ccggactgac gtaaatcgg 59

<210> 28

<211> 58

<212> DNA

<213> Artificial sequence

<400> 28

cggtattatt ctcatgggtt tggcaccacc tgagtctcgc ccggctcttg taagtcgg 58

<210> 29

<211> 104

<212> DNA

<213> Artificial sequence

<400> 29

catcttgcct aaaaaaaaaa gcctggagat acatgcacat tacggctttc cctattagaa 60

ggtctcaggt gcgcgtttcg gtaagtagac gggaccagtt cgcc 104

<210> 30

<211> 84

<212> DNA

<213> Artificial sequence

<400> 30

cgcgcacctg agaccttcta atagggtttg cgacagtcgt tcaactagaa tgccctttgg 60

gctgttccgg gtgtggctcg tcgg 84

<210> 31

<211> 84

<212> DNA

<213> Artificial sequence

<400> 31

ggccgaggac tcctgctccg ctgcggtttg gcgaactggt cccgtctact taccgtttcc 60

gacgagccac acccggaaca gccc 84

<210> 32

<211> 84

<212> DNA

<213> Artificial sequence

<400> 32

gccgtaatgt gcatgtatct ccaggctttc cgcagcggag caggagtcct cggcctttgg 60

gcattctagt tgaacgactg tcgc 84

<210> 33

<211> 30

<212> DNA

<213> Artificial sequence

<400> 33

tttttttttt aggcaagatg cacaacagca 30

<210> 34

<211> 10

<212> DNA

<213> Artificial sequence

<400> 34

tgctgttgtg 10

Claims

1. A method for electrochemically preparing a framework nucleic acid, comprising:

construction of electrochemical device: the electrochemical device is formed by sticking a PDMS layer and a glass sheet layer, and comprises: the device comprises a reaction area and a control area which are connected through a salt bridge, wherein silver/silver chloride is used as a working electrode in the reaction area, iridium oxide is used as a reference electrode in the control area, and iridium is used as a counter electrode; and

self-assembly of framework nucleic acids: constructing frame nucleic acid with designable appearance and biochemical properties by an electrochemical strategy, providing a DNA single chain required by constructing the frame nucleic acid, adding a mixed solution of the DNA single chains into the reaction zone, applying a certain potential to the working electrode to enable oxidation reaction of water in the reaction zone to occur, reducing the pH value of the solution, and performing DNA denaturation; and applying another potential to the working electrode to ensure that the reduction reaction of water occurs in the reaction area, the pH of the solution is increased, DNA renaturation occurs, and finally the electrochemical self-assembly of the frame nucleic acid is realized.

2. The electrochemical production method according to claim 1, wherein the framework nucleic acid is a three-dimensional DNA nanostructure formed by denaturation and renaturation of a plurality of DNA short chains.

3. The electrochemical preparation method of claim 2, wherein the three-dimensional DNA nanostructure comprises: a tetrahedron, cube, octahedron, triangular prism, or triangular bipyramid.

4. The electrochemical preparation method of claim 1, wherein the self-assembly of the framework nucleic acid further comprises: the modification of functional groups on the structure of the frame nucleic acid is realized by designing a DNA single strand.

5. The electrochemical preparation method of claim 4, wherein the functional group comprises: sulfhydryl, ferrocene, amino, carboxyl, DBCO, benzene, Cy3, Cy5, Cy7, cholesterol, or a commercial fluorescent molecule.

6. The electrochemical preparation method of claim 5, wherein the modification site of the functional group on the framework nucleic acid comprises: the apex of the framework nucleic acid, the internal cavity of the framework nucleic acid, or the border of the framework nucleic acid.

7. The electrochemical preparation method of claim 1, wherein the capturing of the probe during the electrochemical self-assembly of the framework nucleic acid comprises: base-complementary pairing, high affinity interaction between biotin and avidin, interaction between antigen and antibody, specific recognition between aptamer and specific target.

8. The electrochemical production method according to claim 1, wherein the reaction system for producing the framework nucleic acid comprises: NaCl reaction System or MgCl₂A reaction system, wherein the yield of the framework nucleic acid is highest under the condition of 0.6M NaCl in the NaCl reaction system and is at MgCl₂60mM MgCl in the reaction system₂The yield under the conditions is highest.

9. The electrochemical preparation method of claim 1, wherein the self-assembly of the framework nucleic acid further comprises: a biosensor based on the frame nucleic acid is constructed by modifying the frame nucleic acid modified by the functional group at the interface of a gold electrode.

10. Use of the method for electrochemical preparation of a framework nucleic acid according to any one of claims 1 to 9 in the fields of medical diagnostics and molecular machines.