Research On The Regulation Of RNA Activity Based On Orthogonal Activation

RNA is an indispensable part of biological activities.It is a highly complex biological molecule that plays an important role in the growth,development,reproduction,inheritance and mutation of living organisms.Due to the structural characteristics of RNA,it is difficult to break through the bottleneck in the application of RNA technology,and the main biological functions of many types of RNA are still undetermined.The regulation of RNA activity can give new impetus to these problems.We make some changes to the structure of RNA by chemical modification,so as to control the RNA activity in time and space,that is,to be targeted and achieve a multiplier effect.The tool of RNA activity regulation can make people see the different roles of RNA in various environments better and clearer,and can also greatly promote the application of RNA in life sciences and medicine.The specific research contents of the thesis are as follows.In Chapter 2,we developed a new method for the active regulation of DNAzymes based on orthogonal activation.The DNAzyme is an RNA-cleaving DNAzyme,and there is only one RNA on the substrate strand,which has the activity of specifically cleaving RNA substrates with high specificity for a specific metal ion of interest.After the DNAzyme substrate chain reacts with the phenyl borate acylation reagent,the only RNA on the substrate chain is modified,resulting in the inactivated state of the assembled DNAzyme.The phenyl borate group has high sensitivity and high selectivity to H₂O₂,so it can only restore activity in the presence of H₂O₂.In the process of cell imaging,the stability is improved,the background is greatly reduced,and because the concentration of H₂O₂in cancer cells is much higher than that of normal cells,it is possible to spatially regulate the activity of DNAzymes.The strategy developed in this chapter might provide a valuable platform for the regulation of DNAzyme endogenous activity.In Chapter 3,we developed a new method for the active regulation of RNA aptamer based on orthogonal activation.The aptamer is a light-up aptamer which binds to sulforhodamine and separates it from a conjugated contact quencher,and release fluorescence.We use phenylboronate acylation reagents to react with RNA aptamers to modify multiple phenylboronate groups on one RNA strand,thereby inactivating the aptamers.Only in the presence of H₂O₂,the RNA aptamer recovers its activity and releases fluorescence.The strategy developed in this chapter has successfully constructed a uncomplicated method for the regulation of endogenous activity of RNA strands.This method is very simple and easy to operate,and has low requirements for the experimental environment,which is convenient for popularization and application.