Construction And Application Of DNA Fluorescence Probe Based On Cross Toehold-mediated Strand Displacement

Fluorescence biosensors have the advantages of rapid response,high sensitivity and efficiency,low detection limit,low cost and economy.They are often used in gene sequencing,early disease diagnosis,biological analysis,food detection and other fields.The fluorescence biosensors based on the toehold-mediated strand displacement technology have improved the programmability and enhanced the ability to identify complex targets.It can be combined with nucleic acid aptamers,DNA logic gates and biological enzymes to identify more biological analytes.At present,the disadvantage of this scheme is that the fulcrum region has a base sequence dependence,low stability and complicated design steps.To solve these problems,we designed a fluorescent probe based on cross toehold-mediated strand displacement for detection of single-base mismatch and target oligonucleotides.Real-time fluorescence monitoring and polyacrylamide gel electrophoresis proved the feasibility of the scheme.The specific research work is as follows:(1)Construction of toehold-mediated and study of DNA strand displacement: we constructed a new cross toehold-mediated DNA strand displacement mechanism,which is composed of two independent groups of DNA motifs completely,It can activate the next step of DNA strand replacement and regulate the reaction rate of strand replacement.The recognition ability and DNA strand displacement efficiency of the system were optimized by the toehold-mediated length experiment of multiple oligonucleotides.It can be used to distinguish oligonucleotide with slight difference of DNA motifs,and the detection range is 1 n M-100 n M.It is proved that the system has good selectivity and modulability and provides a possibility for the identification of single nucleotide polymorphism.At the same time,the real-time rate control experiment of DNA strand displacement shows that the system has good adjustability and can participate in the design of many kinds of biosensor systems.Enzyme activity was the next experiment,which proves that the sensor can be combined with other strategies,and it has generality,showing the value of application in biological analysis.(2)Research on Advanced DNA fluorescence switch based on cross toehold-mediated: We based on cross toehold-mediated,design a variety of DNA fluorescence switch.First of all,we designed an advanced DNA fluorescence switch based on DNA logic gate technology.The fluorescent switch can catalyze theconversion of "DNA fuel" into fluorescent output signal,and can accurately identify the low-concentration target DNA in complex solution,The fluorescent signal can be amplified by the “DNA fuel”.We also designed a series logic gate sensor based on "AND" logic gate,which is to add two oligonucleotides J-1 and J-2 on the basis of FQJ triplet,and these oligonucleotides will generate a long series DNA lock structure.The sensor consists of multiple input DNA and fluorescence output signals.The input DNA needs to be added to the solution in the right order and exist together,At this time,the system can release fluorescence signal.The fluorescence switch of DNA logic gate shows well detection performance.Next,we investigated the toehold at different positions of the DNA fluorescence switch:The first method is the two side toehold,which is on either side of the tail of the RS double DNA.When both sides have toehold,the fluorescence signal can be released.Another is an advanced DNA fluorescent switch based on double-toehold cooperative,The double-toehold consists of two independent oligonucleotide T1 and T2,TI and T2 work together to replace complete oligonucleotides J,so as to release the new toehold for the next strand displacement.The two DNA fluorescence switches showed excellent recognition of target oligonucleotides with small differences in base sequences.The detection range was 1 n M-100 n M,and the fluorescence switch was proved to be programmable.