Research And Application Of Spatiotemporal Properties Of DNA Nanostructures

The essence of life is a series of biochemical reactions with dynamic changes in time and space,such as biological clock,embryonic development,and neural network signal transmission,with precise time evolution function.Inspired by living organisms,it is desirable to use DNA nanotechnology to imitate the spatiotemporal characteristics to improve its application performance in the field of biomedicine.DNA is spatially and temporally.DNA spatiality refers to various spatial structures formed by complementary base pairing and multi-strand programmed self-assembly,which high coding specificity,size controllability and biocompatibility.DNA temporality refers to the assembly rate of DNA and the reaction dynamic characteristics which has extremely high reaction controllability.At present,the related research on DNA nanotechnology still lacks the exploration of the time evolution of the reaction kinetics of the structure itself and its interaction,which is not conducive to the further research and application of the dynamic life activity and the field of in vitro synthetic biology.In this paper,the temporal and spatial properties of DNA are organically linked,and the new concept of "DNA spatiotemporality" is proposed.Through the characteristic and rational design of the DNA spatial structure,the spatial structure guides the specific changes of various reaction kinetics,and the timing characteristics of specific events can be coded and regulated.Using the DNA spatiotemporality under the guidance of various simulation software tools,"DNA timer","DNA molecular switch" and "stimulus-responsive DNA nanotube drug delivery system" were rationally designed and successfully developed high sensitivity,high specificity of target detection,and binding affinity measurement method and the new target tumor treatment strategy.First,the spatial length affects enzymatic reaction kinetics and in turn determines timing of signal generation.Based on the deficiencies of cross light,limited color resolution and high labeling cost caused by fluorescence labeling in multiplexed detection,a "DNA timer" system for multiplexed detection mediated by DNA polymerase was developed.In this system,the target molecules are used as primers through the polymerization elongation process driven by DNA polymerase,and the "timer" of different spatial lengths is used to generate signals after different delay times to bispecific distinguish them.In this work,the multiplexed detection of four bacterial species using a single fluorescence in the same homogeneous solution was achieved by using "clock tags".The system has wild application potential in the field of multiplexed sensing analysis.Second,spatial structure affects single-molecule dynamics and thus determines the temporal characteristics of signal generation.The DNA molecular switch is a versatile and highly programmable toolbox and the binding of the substrate to the switch determines the on-state of a process.However,the strand displacement reactions as the core steps tends to be accompanied by strong non-specific background signals,which seriously limits the development of DNA circuits.In this work,a single-molecule dynamic DNA three-way junction was designed to develop a "zero leakage" robust DNA molecular switching system that can respond to various input signals.The architecture has a recognition domain and a signal transduction domain.The combination of the input target and the recognition domain will induce the enhancement of the stability of the spatial structure,and the fluorescence probe can transient hybridized with signal transduction domain to generate a time-characteristic single-molecule fluorescence dynamics signal.The detection limit of the minimum target can reach 10 f M.Moreover,the system can measure the binding affinities between molecules,which is expected to have great prospects in the field of intelligent artificial component development and biomolecular interactions.Finally,the spatial response site affects the kinetics of the enzymatic reaction and then determines the disintegration and drug release time of the drug delivery system.Based on the base excision repair related enzymes are overexpressed in tumor cells,and the dissociation of DNA nanotube drug carriers containing a special spatial responsive site(uracil)is stimulated with endogenous enzymes in cancer cells Different spatial sites have different dissociation rate of the drug carrier,and the optimal sites for rapid drug release are finally optimized.While much is known about the reactions of such enzymes with their natural substrates,their reactivity with complex DNA nanostructures is poorly understood.Therefore,molecular dynamics simulations based on coarse-grained models were used to predict the reaction between tumor biomarker enzymes and DNA nanotubes.It was found that the accessibility of enzymes and the potential energy of enzymatic hydrolysis products jointly determine the structural changes of DNA nanotubes.The experimental results were in good agreement with the simulation results.Consequently,one optimal site was optimized from 14 spatial site,and the in vivo anti-cancer experiments of various cancer cell lines and tumor-bearing mice were successfully implemented,which not only effectively inhibited tumor growth,but also protects major organs from drug-induced damage.This work has developed stimulus-responsive drug nanocarriers with high sensitivity and selectivity for cancer therapy,which heralds broad applications in nanomedicine.The research results of the dissertation utilize the precise and controllable kinetics of different DNA structures involved in the reaction,and through the spatiotemporal properties of DNA nanostructures,broaden the application of DNA nanotechnology in the fields of biomolecule detection,intelligent response materials and drug delivery.The new systems have the advantages of high sensitivity and high specificity.It is expected to play an important role in clinical diagnosis and treatment as well as precision medicine.