Construction Of DNA Dynamic Assemblies And Their Biological Applications

Self-assembly is the process by which an organized structure spontaneously forms from individual components,and it is widespread in the formation of cellular components,as well as the maintenance of cellular homeostasis and physiological activities.Inspired by intracellular self-assembly,constructing a dynamic assembly system with stimuli-responsiveness is a promising strategy to regulate the cell microenvironment and behaviors.However,there is still a major challenge to construct a dynamic intelligent system coupled with biological activities for regulating cellular functions at the subcellular level.As a kind of biologically active macromolecules,DNA is promising as a building block in constructing assembly with precisely controllable structure and tunable biological function.Specifically,various stimuli-responsive DNA motifs enable the controllable assemble/disassemble in the presence of endogenous molecules.In this dissertation,to explore the effect of intracellular dynamic assembly on cellular functions,we developed a series of DNA dynamic systems coupled with biological activities,and investigated the resulting biological effects.Concretely,we constructed three stimuli-responsive systems to perform the in situ dynamic assembly at the subcellular level,and achieved the interference of different target organelles(lysosomes,mitochondria and cytoskeleton).The specific research contents are as followed:Chapter 2.Construction and dynamic assembly of NIPAM-DNA nanoframework.We constructed polymeric NIPAM-DNA nanoframework by a two-step polymerization reaction,namely the free-radical precipitation polymerization of four acrylamide-derived monomers and the hybridization chain reaction(HCR)of two hairpin monomers.To increase the loading of DNA functional motifs,acrylamide group-modified DNA was incorporated in nanoframework.DNA played a dual role as a cross-linker for bridging polymeric chain,and as an initiator to trigger the tandem hybridization of two hairpins in the second step.NIPAM-DNA nanoframework exhibited a spherical morphology with uniform particle size(～200 nm)and excellent dispersion.To achieve the multifunctional integration on NIPAM-DNA nanoframework,functional DNA motifs were elaborately designed on hairpin monomers.The incorporation of acid-responsive semi-i-motif(5’-CCCCTAACCCC-3’)endowed nanoframework with the reversible aggregation/disaggregation under the trigger of acidic/neutral p H.The introduction of ATP aptamer endowed nanoframework with controllable release of ligated DNA strands in response to high concentration of ATP(>5 m M).In addition,NIPAM-DNA nanoframework protected DNA strands from nuclease degradation up to 24 h.The polymeric NIPAM-DNA nanoframework accomplished the tandem assembly and efficient loading of various DNA functional motifs,thereby achieving the controllable topological transition in response to stimuli.Chapter 3.Proton-driven assembly of NIPAM-DNA nanoframework for lysosome interference.With the assistance of the interactive coupling of precise DNA dynamic assembly and biological processes,the proton-driven assembly of NIPAM-DNA nanoframework(constructed in Chapter 2)interfered with the lysosomal microenvironment and functions.By virtue of lysosome-mediated endocytic pathway and lysosomal mature,NIPAM-DNA nanoframework was endocytosed and entered into acidic lysosomes after 4-h incubation.Since a large number of semi-i-motif were embedded in nanoframework,the abundant H~+in lysosome activated the proton-driven assembly of nanoframework to form micron-scaled aggregates.On the one hand,the aggregates impeded the lysosomal efflux and remained in lysosome for up to 12 h.On the other hand,the consumption of protons decreased the lysosomal acidity and attenuated internal hydrolases activity,thus alleviating the degradation of foreign substance in lysosomes.The process of lysosome interference was conducive to the accumulation of nucleic acid drugs(such as si RNA),thereby increasing the delivery efficiency and gene silencing effect.By coupling ingeniously designed DNA dynamic system with biological processes in lysosome,this work proposed a new concept of“lysosomal interference”,and achieved the rational modulation of lysosomal function.Chapter 4.Targeted dynamic assembly of NIPAM-DNA nanoframeworks for mitochondrial interference.To interfere with mitochondria by site-specific aggregation,we constructed two types of NIPAM-DNA nanoframeworks with mitochondria-targeting and complementary assembly properties.For the precise localization to mitochondria,a mitochondria-targeting group,triphenylphosphine(TPP),was modified on hairpin monomer.In addition,we designed a pair of complementary sequences on two types of nanoframeworks respectively to realize the aggregation.Two nanoframemworks were sequentially endocytosed by cells at 4-h interval,and then aggregated on the mitochondrial surface in a manner of“hand-in-hand”after 8-h incubation.The aggregated nanoframework served as a physical shield to block energy metabolism and further interfered with mitochondrial function,mainly manifested as the elevation of reactive oxygen species and the reversal of mitochondrial membrane potential.This work provided a new strategy for mitochondrion interference by site-specific aggregation of ingenious DNA dynamic assembly.Chapter 5.K~+-responsive assembly of DNA sheet-like nanostructures for cytoskeleton remodeling.To explore the effect of directional assembly of rigid structures on cytoskeleton remodeling,three types of DNA sheet-like nanostructures(rectangular shape of～90 nm×60 nm)with different extension directions were constructed by introducing K~+-responsive semi-G-quadruplex.Semi-G-quadruplex and palindrome sequences were conjugated on the long,short or four sides of DNA sheet to accomplish the extension in different directions.DNA sheet entered cytoplasm at～16h and underwent aggregation driven by high concentration of K~+in cytoplasm.Due to the mechanical strength difference between DNA sheet and actin cytoskeleton,the rigid DNA sheet interfered with the cytoskeletal orientation in local space,and affected the expression of lamellipodia and paxillin(inhibition rate～50%).Furthermore,the dynamic assembly of DNA sheet increased the migration rate of tumor cells(A549 cells)and of normal cells(BEAS-2B cells).Particularly,DNA sheet with four sides extension had a more pronounced effect,increasing the migration rates of tumor and normal cells to 170%and 150%,respectively.This work provided a new mechanistic understanding of cytoskeleton remodeling caused by the dynamic assembly of rigid structure.