Research On Functional Nucleic Acids For Constructing Controllable DNA Molecular Circuit And Optimizing Genome Editing System

Nucleic acids,as essential biomacromolecules in known life forms,carry the critical liability of storing and encoding the genetic information of living bodies.With the development of DNA nanotechnology,the complex structure and function of nucleic acids in natural systems have been widely studied,and their characteristics and advantages have also been accurately analyzed.Therefore,all research has promoted the development of nucleic acid,not only to construct it into nanomaterials,but also into complex nanostructures,devices,and reaction circuits.It can also be used as a designed gene expression network substrate and a powerful specific recognition system to achieve the applications in the fields of synthetic biology,molecular biology,and biophysics.In this paper,what we focus on are the functionalization of nucleic acid structure and its application in the construction of programmable DNA circuits and the optimization of genome editing.Toehold-mediated strand displacement reaction,as the fundamental basis of dynamic DNA nanotechnology,has proven its extraordinary programmability in dynamic molecular systems.Since programmed activation of the toehold in DNA substrate is crucial for building sophisticated DNA devices with digital and dynamic behaviors,we developed a detachable DNA circuit by embedding a pH-controlled intermolecular triplex between the toehold and branch migration domain of the traditional "linear substrate"in chapter 2.The reaction rate and the "on/off" state of the detachable circuit can be regulated by varying the pHs.Similarly,a two-input circuit composed of three pH-responsive DNA modules was constructed subsequently.Most importantly,a resettable self-assembly system of spherical nucleic acids was built by utilizing the high detachability of the intermolecular triplex structure-based DNA circuit in chapter 3.There is the first demonstration of a dynamic DNA device that can be repeatedly operated at a constant temperature without generating additional waste DNA products.Moreover,this strategy showed an example of recycling waste spherical nucleic acids from the self-assembly system of spherical nucleic acids.Our strategy will provide a simple approach for dynamic regulation of complex molecular systems and reprogrammable nanoparticle superlattices.On the other hand,as an essential carrier for storing and encoding the genetic information of the living body,DNA has different effects on the vital signs with its different manifestations and correction states.CRISPR system for targeted genes composed of nucleic acids has brought a more robust application to the field of gene editing,due to its excellent programmability and specific recognition ability.However,its limited gene knock-in capability and high off-target in human mammalian cells(primarily stem cells and primary cells)severely limit the application in more genetic disease diagnosis and treatment technologies.Therefore,in chapter 4,we found that when the 5'-end of the dsDNA containing short homology arms was modified with a small chemical molecule and used as a donor,the efficiency of gene knock-in could be effectively improved.This design combining Cas9-RNP and end modified dsDNA as a donor,has the advantages of high KI efficiency,short test period,high safety,and high precision.Furthermore,it also achieved unprecedented 65/40%KI rates for 0.7/2.5 kb inserts,respectively,in human embryonic kidney 293T cells.The identified 5'-end modification led to up to a fivefold increase in gene KI rates at various genomic loci in human cancer and stem cells.The design of the chemical modification of nucleic acid in this study not only provides a novel optimization direction for this research field but also broadens the application range of gene-editing technology in practical applications.In chapter 5,we applied the concept of dynamic strand displacement reaction of nucleic acids to CRISPR system,which effectively suppressed the big problem of high off-target efficiency in gene editing.We designed a short ssDNA protect strand that is antisense to the sgRNA guide region hybridized with sgRNA.This design successfully reduced off-target efficiency through reasonable structural optimization.Moreover,the off-target efficiency has been reduced by more than 90%at the human endogenous gene locus.This strategy not only achieved a successful combination of biological systems and nucleic acid nanotechnology but also achieved precise editing of gene editing through this simple and effective method.