Research And Application Of Reghulating DNA Strand Displacement Reaction Based On Conformational Transition Of Hairpin DNA

Based on the unique advantages of DNA strand replacement reaction,constructing strategies for regulating DNA strand replacement reaction and implementing flexible and direct qualitative and quantitative nucleic acid detection methods have important research value for medical clinical testing and environmental pollution prevention and control.This thesis focuses on the regulation of the secondary structure of DNA,the hairpin structure,combining the classical toehold initiated chain displacement reaction and entropy driven strategy,introducing the design of target cycle isothermal amplification,and constructing three nucleic acid sensing strategies.The reaction kinetics and thermodynamics of the three sensing strategies were discussed,and their detection performance and application value were studied.1.A hairpin structure assisted entropy driven isothermal amplification strategy for initiating the toehold chain substitution reaction was designed,which indirectly regulates the chain substitution reaction by changing the conformation of hairpin DNA.In this experiment,we designed a triplet composite probe ABC composed of three DNA strands: A,B,and C.In the free state,the B strand is composed of stable hairpin structure DNA,but in the binding state,it can be bound to the triplet in a single strand state,and the target chain T replaces the chain B from the 3 ’end.When the chain replacement reaction proceeds to a certain extent,the original hairpin structure of the B chain will gradually recover.After forming a hairpin structure,the B chain spontaneously moves away from the A chain.In this chain replacement reaction,the target chain T does not need to replace all the bases on the B chain,and can exhibit the same results as traditional chain replacement reactions.With the addition of the fuel chain,both the C chain and the target chain T will be replaced and detached from the A chain,and the C chain will further release fluorescence signals from the quenched groups;The target chain T will be repeatedly used for the cyclic reaction.This method uses changes in the conformation of hairpin DNA to promote the traditional DNA strand replacement reaction,greatly improving the reaction efficiency,and also has high reliability in the detection of target DNA.This sensing strategy has certain application potential for the detection of actual environments and early diagnosis in the medical field.2.Based on the previous discussion of the role of a single hairpin DNA structure in promoting entropy driven reactions,we further explored the role of two hairpin structures in promoting entropy driven systems.Therefore,in this system,we have designed two DNA strands,Product H(PH)and Signal H(SH),with PH and SH in a stable hairpin structure.PH is a hairpin with seven base pairs in the stem,while SH is a hairpin with six base pairs in the stem.Through hybridization and incubation,the PH and SH chains become straight chains,forming a triplet PHSHS with the S chain.The 3’end of the S chain is modified with a quenching group BHQ1,and the 5’ end of the SH chain is modified with a fluorescent group FAM.After the formation of the triplet,the two groups meet in space,causing fluorescence quenching.The target chain participates in the reaction to replace the PH chain.At this time,four bases are exposed in the middle of the replaced PH,and the fuel chain Fuel starts the chain replacement reaction with these four bases as the support points.Subsequently,both the T and SH chains were replaced,and the F and S chains formed a stable double chain structure.The SH chain leaving the system emits a fluorescence signal again due to its distance from the quenching group on the S chain.The T-chain leaving the system enters the next cycle,triggering the next reaction.Compared with a single hairpin DNA in response time,this research strategy further improves detection efficiency and has a good output in fluorescence signal output.This research method is expected to be further applied in the field of practical detection.3.Based on the previous strategy,we developed a DNA triplet probe(A1A2B)on the surface of a junction alloy electrode by combining double hairpin DNA with DNA strand substitution reaction.A1 is a simple hairpin structure DNA,A2 is a separate hairpin DNA,and B is a straight strand DNA with a 5 ’terminal thiol modified group that is connected to a gold electrode.With the addition of the target chain,the two hairpin DNA drives a DNA chain displacement reaction on the electrode,which occurs in the toehold displacement region.Fuel chain Fuel is a DNA chain that is modified with a methylene blue electrochemical marker at the end of the fuel chain.After adding the fuel chain,the final hairpin A2 is replaced,and the fuel chain Fuel and chain B combine to form a double chain,displaying the electrochemical signal of methylene blue.The designed nucleic acid biosensor visually displays the substitution process and reaction degree of DNA strands through signal conversion,which has great potential in detecting cancer marker mi RNA-21.