Studies On Hairpin DNA Fluorescent Probes Based On Strands Displacement

Due to their specificity and sensitivity,hairpin nucleic acid probes have made many effective advances in the research of biosensors for many years.The new hairpin DNA fluorescent probes has the advantages of low background signal,high sensitivity,high selectivity,low cost,high signal-to-noise ratio,good quenching efficiency and so on.it is a new type of nucleic acid molecular probe with broad development prospects.Hairpin nucleic acid probes are a kind of DNA detection tools with recognition function widely used in chemistry,biology and medicine.In biological analysis,it can be used for intracellular imaging,small molecule detection,gene detection and therapy,biosensors and biochips,real-time fluorescent quantitative PCR.and in non-biological analysis,it can be used for the detection of cations and heavy metals.In this assay,We will take advantage of the flexibility and programmable features of the toehold-mediated DNA strand displacement reaction design,combined with the instability of the hairpin structure,to construct a new signal conversion method for fluorescent probes.At the same time,the application of hairpin DNA fluorescent probes in regulating the rate of chemical reactions and chemical equilibrium is studied,which provides a basis for the construction of new DNA fluorescent probes.(1)The research work in this chapter proposes a new strategy based on the interaction of toehold and hairpin to activate the chain displacement reaction.In this method,the target DNA loop is located between the toehold and the strand displacement region,which is unstable and will cause the chemical equilibrium to shift to accelerate the strand displacement reaction rate.We used real-time fluorescence monitoring and polyacrylamide gel electrophoresis experiments to verify the feasibility of the design principle and investigated the influence of the length of the toehold and the size of the loop on the DNA reaction rate.The experimental results show that as the toehold is increased by 6-9 bases,the longer the toehold is,the more difficult it is for chain replacement to occur,the slower the reaction rate.When the toehold is 7 bases,the chain replacement reaction is already difficult to occur.In the case of the same toehold,as the loop increases from 0 to 57 bases,the strand replacement is more likely to occur,and the rate of fluorescence decrease is faster.When the toehold is 6 bases,when the loop T base is 30 bases,The fluorescence value decreased from 20 n.u to 2.5 n.u.Then we investigated the tandem reaction of the hairpin toehold DNA.Experiments show that only the key G chain occupies the site to cause the toehold to be exposed,the Q chain can squeeze the hairpin DNA chain to complete the fluorescence quenching,and the greater the concentration of G and Q chains,the greater the chain displacement reaction rate.The design and research of new molecular beacons provide an important research background for the analysis of nucleic acid probes in the future.(2)The research work in this chapter proposes the study of hairpin structure regulation of DNA strand displacement reaction.In this method,the target DNA toehold and the loop are two regions with a certain distance that do not interact.The rigidity of the loop will cause the movement of the chemical equilibrium and determine the speed of the strand displacement reaction.We use real-time fluorescence monitoring and fluorescence detection to verify the feasibility of the design principle and investigate the influence of the size of the loop on the DNA reaction rate.The experimental results show that when the left and right strand replacement regions of the loop are the same,as the loop increases from 0 to 20 bases,the more rigid the loop,the less complete the strand replacement reaction and the slower the fluorescence increase rate.When the loop is 6 bases,the 2 regions are 16 nt,15 nt,12 nt DNA strands I-4,I-b,M-2 with different lengths,and the fluorescence is about 1 n.u,1.8 n.u,5.5 n.u after 1 hour.it shows that the shorter the 2 region,the more complete the chain displacement reaction,and the faster the fluorescence rise.Then we investigated the DNA strand displacement reaction regulated by hairpins of different neck lengths.Experiments show that the hairpin neck grows from 3 to 16 bases.The higher the energy required to open the clip,the more difficult it is for strand replacement to occur and the lower the fluorescence.