| In the outbreak of COVID-19 in 2019,nucleic acid testing became the basis for diagnosing whether people are infected with the virus or not,which played an extremely important role in the fight against the epidemic and was well known by the general public.At present,polymerase chain reaction(PCR)is widely used in nucleic acid detection,which is complicated,time-consuming and costly to operate.Unlike PCR,toehold mediated strand displacement(TMSD)refers that standard displacement is a process in which one or more pre-hybrid strands replace two partially or fully complementary strands.It has the advantages of simple operation,no enzyme involvement and low cost.Therefore,the construction of a new enzyme-free DNA probe for disease diagnosis and detection has become the goal of our research,which is of benefit to the development and application of medicine,diagnostics and practical methods.In this paper,based on the principle of toehold mediated strand displacement,we constructed a DNA strand displacement strategy based on double toeholds,which was verified by fluorescence real-time monitoring and polypropylene gel electrophoresis.The research contents are as follows:(1)Construction of double toeholds and study on DNA strand displacement:The rate of the reaction is usually regulated by a single toehold in enzyme-free DNA strand displacement.When the toehold is not effective as expected.We introduce other toehold to participate in strand displacement,and define it as a double toehold.Here,We divided the target strand into three parts: proximal toehold,interval region and remote toehold.The proximal toehold is short(2 nt),and the strand displacement reaction is slow or almost nonreactive.Therefore,we designed a longer remote toehold(10 nt)on the target strand.Its primary responsibility is to "capture" the fluorescent probe,making the proximal toehold more likely to trigger the strand displacement reaction.We investigated the effects of different lengths of proximal toehold,interval region and remote toehold on the reaction,and the optimal selection was 2 nt,10 nt and 10 nt in turn.At the same time,the reaction temperature was investigated.The fluorescence intensity increased fastest at 25 ℃ for 1 h.On this basis,we hide the proximal toehold in the remote toehold.Only when the remote toehold is untied,exposing the proximal toehold,can the strand displacement reaction can be activated.Reaction background is reduced and selectivity is improved.This strategy has the characteristics of convenience,high sensitivity and low cost,and provides a new tool and a new idea for the development of disease diagnosis.(2)Logic gate operation based on DNA strand displacement strategy using double toeholds: Biologic gate computing can reduce technical problems in the analysis of highly specific biomarkers by analyzing species appearing at much higher concentrations.Compared with traditional single analyte sensing equipment,multi-analyte biosensors with high fidelity can be realized,which is very beneficial to biomedical applications.In the third chapter of this thesis,we construct the "OR" and "AND" logic gates based on the double toehold strand displacement.The "OR" logic gate system consists of two complementary target strands.When both of them exist in solution,they mutually restrict each other to complete strand displacement reaction,and the reaction rate decreases.According to the logic rule of "OR",the existence of two target strands can be judged under certain conditions.We designed two "AND" logic gate schemes with single/double "switch".They are composed of A target strand(A)and A "key"(B)/ two "keys"(B,C).The strand displacement reaction can only be triggered if the condition "A and B" / " A and B and C " is satisfied.In the experiment,fluorescence real-time monitoring and polypropylene gel electrophoresis proved the feasibility of the reaction mechanism.The experimental results show that,within a certain range,the fluorescence intensity of "AND" logic gate is positively correlated with the concentration of input elements.The design enhances the specific recognition of the target,and can preliminarily detect the presence of each target element in the sample,which provides reference and ideas for the development and application of medicine,diagnostics and practical methods,and has a potential application prospect. |
