Strand Displacement Reaction-based Molecule Recognition And Signal Amplification And Its Biosensing Application

Over the past decades, DNA has been adopted as a powerful and intriguing material in the field of nanotechnology that enables programming of the kinetically controlled assembly of DNA nanostructures in a predictable manner. Toehold-mediated strand displacement reaction (TSDR) acted as the basic reaction is applied in the dynamic assembly of DNA nanodevices. It is highly significant for controllable assembly and biomedical application of DNA nanodevices by effectively regulating the reaction rate. The rate of TSDR can be tuned over a factor of 106 through varying the toehold strength (length and sequence composition) and reaches maximum when the base is 6-10, so fine adjustment is difficult. Herein, it is necessary to design a novel strategy of toehold activation which can control the DNA strand displacement rates finely with additional levels.With the development of the research on TSDR, it can not only act as the element material of DNA dynamic assembly, but also function as the recognition element for biosensing directly. Based on the nature of TSDR rate which can be significantly regulated by base change in toehold domain, it can used to effectively distinguish single-base mutation in the gene fragment. Furthermore, the detection of biomolecules such as protein, small molecule and microRNAs can be achieved by introducing recognition elements such as aptamer and complementary sequence in the toehold domain. However, the action of some biomolecules such as nucleases generally aim at the specific DNA sequence or site, and thus lack a specific transducing mechanism to initiate the DNA strand displacement effectively. Therefore, it is desirable to develop a specific transducing mechanism for the detection of nucleases.Due to the advantages of high sensitivity, selectivity and simple operation, the strand displacement amplification has been a common nucleic acid amplification technique, which has been applied in the detection of nucleic acid, protein and small molecules and biomedical research.Based on the above review, we have developed a highly sensitive and selective fluorescent method for the single base mutation in gene fragment on the basis of typical regulation of TSDR rate and strand displacement amplification. In addition, a novel "Sutured Toehold Activation" based on hairpin-reconfiguration has been developed which can regulate the TSDR finely with additional levels. On this basis, we developed a high sensitive biosensor for uracil DNA glycosylase in virtue of molecule recognition of hairpin-reconfiguration and signal amplification of strand displacement amplification. In addition, we proposed a nuclease-responsive (DNA modified enzymes as models) toehold activation strategy through introducing DNA hairpin-reconfiguration or enzyme-blocked digestion mechanism, and achieved the construction of universal dynamic DNA nanodevice successfully.The main contents are as follows:Chapter one is an introduction section of physical and chemical characteristics of DNA and the advantageous nature for DNA nanotechnology. The mechanisms, regulation modes, development history and biomedical application of DNA nanomaterials are summarized in this section. In addition, the problems and the prospects are also stated in the development of DNA nanotechnology. In last, the development and application of the strand displacement amplification are summarized.In chapter two, taking the Kras gene fragment as model, a label-free fluorescent detection method for single-base mutation has been proposed based on high selectivity of toehold-mediated strand displacement reaction and powerful signal amplification capability of isothermal DNA amplification. The key point is the design of a single mutation base on the terminal of the toehold domain in the recognition probe. By comparing with difference of the reaction rate between the perfect target and the mutation target at a set interval, the maximum discrimination ratio is obtained when the discrimination of reaction rate reaches the maximum. The introduction of TSDR allows us to achieve high selectivity between the various types of mutation. The isothermal amplification ensures the high sensitivity of the method with a low detection limit of 1.8 pM.In chapter three, a novel sutured toehold activation strategy is developed based on hairpin-reconfiguration which is initiated by the interaction between the hairpin and different environmental stimuli (Hg2+or ATP as models). In the probes design, the recognition domain of the environmental stimuli is independent of the toehold domain and the branch migration domain which ensures the versatility of the TSDR in the construction of DNA devices. Furthermore, the dynamic rate of the TSDR can be regulated both coarsely by changing length of the toehold and finely by varying the concentrations of target in this strategy.In chapter four, based on the above-mentioned hairpin-reconfiguration mechanism and the strand displacement amplification, we have designed a structure-swich difunctional probe which can convert the action of uracil DNA glycosylase and its substrate to the trigger of the strand displacement amplification directly. Herein, the integration of molecule recognition and signal output are achieved through the design of the difunctional probe. which will provide the strone support for the detection of enzymes in complex system.In chapter five, we have explored a general strategy for rational design of nuclease-responsive toehold activation and fabricated a dynamic DNA device in a ’plug-and-play’ fashion. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we design a trigger module which could convert the action of different nucleases to the recombination of a toehold and branch-migration domain in recognition probe through DNA hairpin-reconfiguration or enzyme-blocked digestion mechanism, and thus the identical trigger sequence was formed for subsequent dynamic assembly. The dynamic assembly module is designed by introducing identical hairpins. Once the trigger module plug in the assembly module, the cascade hybridization of hairpins via strand displacement immediately switch on.