DNA-based Bio-computing And Super-resolution Imaging

Deoxyribonucleic acid(DNA)is the main carrier of genetic information of life in nature.In recent years,DNA has shown great potential as a unique biomaterial in many fields such as life sciences,medicine,biocomputing and national defense security.Due to the designable sequence and programmable structure,DNA has showed significant potential in fields like bio-computing and data storage.It has has been used for solving various complex computational problems.DNA has good biocompatibility and rich chemical reaction active sites,and it is widely used in the medical industry such as the development of smart drugs.Meanwhile,DNA nanostructures have the advantages of precise addressing and the advantages of precise and multiple modifications,making DNA nanostructures a good platform for the observation and research of multiple chemical reactions.Here we took advantage of the programmability of DNA sequence,the addressability of DNA nanostructure and the reactions between DNA and other types of molecules,and developed the research from the following four parts:1.We designed and constructed dual-rail logic circuits based on DNA strand displacement reaction.Strand displacement reaction between single-stranded DNA(ssDNA)and doublestranded DNA(dsDNA),especially toehold mediated strand displacement reaction,is a basic strategy for many DNA computing systems.So far,the logical operation based on the displacement of DNA strands has been able to solve some basic problems and even used to construct weight neural networks.At the same time,some progress has been made in the intelligent diagnosis system based on DNA logic calculations and used in clinical diagnosis.However,the current DNA computing system still has many problems such as complicated preparation,limited functions,and limited sequence selectivity,which limits the implementation of larger-scale computing circuits.Here,we have studied the structure and sequence-related mechanisms of the DNA strand displacement reaction.We have designed the AND,OR,XOR,and NOT four basic logic operation units modularly using the concept of switching circuits.We used adaptors to achieve signal connection and signal amplification between computing units.Using the mode of computing uints and adaptors,a variety of logic circuits are constructed and functional operations of addition,subtraction,and comparison are realized by changing the connections between computing units.At the same time,we designed the circuit simplification and generation program,which made it possible to automatically generate corresponding DNA computing circuits for the needs of a certain function.2.We designed and constructed linear DNA hydrogel which is responsive to ATP rapidly.Based on the designability of the DNA sequence-encoded structure,the DNA hydrogel has a good ability to absorb and store water.The development of DNA hydrogels has greatly broadened the field of intelligent hydrogel development and its application in the field of drug development.In traditional preparation methods,bifurcated DNA is often used to prepare hydrogels,and the gel formation rate is slow.In recent years,DNA hydrogels prepared from Y-shaped monomers has become a new solution.Here,nucleic acid aptamer was introduced as an intelligent response unit,and the ATP-triggered and rapid-response DNA hydrogel was prepared by utilizing the structural changes caused by its binding to the target sequence.With the advantages of simple preparation and rapid response,it has great potential for application in drug development and target detection3.We performed the study of single-molecule behaviors of CRISPR/Cas9 system on the platform of DNA origami.Nanostructures assembled from DNA have precise spatial addressability and one could place target molecules in specific positions,providing a platform for studying nanoscale spatial characteristics of reactions.In this thesis,the target of CRISPR/Cas9 system was precisely determined on the origami platform,and the target searching mechanism at nanoscale was studied.Through real-time single-molecule dynamic observation of the reaction of CRISPR/Cas9 using total internal reflection fluorescence microscopy(TIRFM),a new target searching mechanism through transfer between PAM neighbors was discovered.Besides,through the design of the guide RNA(gRNA),we achieved local activation of CRISPR/Cas9 system,achieving spatial specificity of the action.4.The optical super-resolution imaging of DNA nanostructures was studied.DNA nanostructures provide a nano-platform which can be used to precisely locate various functional units.The spatial localization and structural characterization is important for the study of functional characteristics.The development of super-resolution fluorescence microscopy provides an in-situ,non-destructive method for observing nanoscale structures in a variety of biological environments for the characterization of nanostructures,allowing the characterization of structures below the wavelength.Using the excitation characteristics of fluorescent dyes,we developed a super-resolution imaging method based on the blinking property of dyes and applied it to the imaging of nanostructures to realize two-dimensional(2D)super-resolution imaging.At the same time,using a stochastic reconstruction method based on single-molecule localization,we achieved super-resolution characteration of a variety of DNA nanostructures.