Construction Of DNA Strand Displacement System Based On Reversible Reaction And Its Bioanalytical Application

Fluorescent probes have been widely used in many biomedical fields,such as gene sequencing,disease diagnosis,and biological analysis,due to their simple operation,low cost,rapid response,and good adaptability to biological and environmental samples.DNA Strand displacement(SDR)has good sensitivity,specificity,and regulatability,which has received more and more attention in the application of DNA fluorescent probes.On the basis of toehold-mediated strand displacement,many scientific groups have constructed a series of universally applicable fluorescent probes.However,there are still many deficiencies in the toehold-mediated strand displacement mechanism in terms of toehold activation and regulation rate,such as the toehold region is limited by the base sequence,and the reaction rate changes exponentially with the toehold binding strength.Based on the toehold-activated DNA strand displacement mechanism,a novel strategy for detecting the activity of biological enzymes was constructed in this paper,and a reversible reaction was also designed to controllably regulate the DNA strand displacement reaction rate.We used real-time fluorescence monitoring and polyacrylamide gel electrophoresis experiments to verify the feasibility of the scheme.The details are as follows:(1)Endonuclease plays an important role in many important biological processes such as DNA replication,recombination,repair,molecular cloning detection,etc.However,traditional methods for measuring endonuclease activity and inhibition by gel electrophoresis and chromatographic techniques are time-consuming,laborious,insensitive,and expensive.Therefore,this chapter constructed a strategy for detecting Eco R I and RNase A activities based on junction toehold-mediated DNA strand displacement.We designed these two enzyme cleavage sites on two completely independent oligonucleotide chains T-Eco RI and J-A respectively,and then added enzymes for cleavage,thereby activating the DNA strand displacement reaction.The Eco RI and RNase A activities were quantified by monitoring the changes of real-time fluorescence.The results showed that Eco R I has the ability to quantitatively detect in the range of 0-10 U/m L,and the detection limit is 0.02 U/m L;the concentration of RNase A shows a good linear correlation(R~2=0.9929)in the range of 0.4-4.0μg/m L,and the detection limit is 0.2μg/m L.At the same time,the effect of the inhibitor on the enzyme activity was investigated.PP(sodium pyrophosphate)inhibited Eco R I and RNase A in a concentration-dependent manner,while ATP(adenosine5-triphosphate disodium salt)only inhibited Eco R I in a concentration-dependent manner.In addition,the strategy has good selectivity and anti-interference ability in complex environments.(2)The rate of toehold-mediated DNA strand displacement reaction can be adjusted by adjusting the binding strength of the toehold region,but the rate change varies exponentially with the binding strength,which is difficult to fine-tune.In this chapter,the research on DNA strand displacement reaction based on reversible reaction is carried out.According to the instability of short-chain binding,a reversible reaction is constructed to establish equilibrium movement to regulate the rate of strand displacement reaction.First,the upstream reversible reaction was constructed by combining short chains of different lengths with the substrate chain.The experiment showed that the upstream reversible reaction constructed by the 10 nt short-chain(I-1c)could be regulated in a wide range,laying a foundation for the subsequent experiments.The short-chain I-2 is then designed to combine with the target chain T-1 to construct a downstream reversible reaction.By increasing the concentration of I-2 or T-1,the chemical equilibrium is shifted,and the rate of the chain displacement reaction is slowed down or accelerated.Finally,the two-strand(I-3 and I-4)base stacking cooperatively regulates the strand displacement reaction.Therefore,the reversible reaction mechanism designed in this paper provides a new method for slow regulation of DNA strand displacement reaction.