Applications Of DNA Dynamic Logic Circuit And Static Structural Self-assembly In Biochemistry

DNA nanotechnology consists of DNA dynamic nanotechnology that focuses on dynamic strand displacement and DNA structural nanotechnology that focuses on creating various DNA structures.DNA nanoengineering can be defined as a project that building dynamic and static nanodevices by taking advantage of DNA nanotechnology.DNA engineers don't pay too much attention to the natural function of DNA as the carrier of genetic information,but use DNA strands as molecular materials.With reasonable design,DNA strands can hybridize or displace in a certain way so that some molecular signal can be transmited and some fancy structures can be formed.The rigid principle of Watson-Crick base pairing,the programmability and the ease of DNA modification allow the creation of series of DNA dynamic/structural nanotechnologybased reaction network or nanodevices.Both the dynamic and structural DNA nanotechnologies have great advanced in fundamental theories and senior applications in the past years.DNA hybridization and strand displacement reaction-based DNA dynamic technology first emerged at the beginning of 21 century after series of dynamics and thermodynamics studies on DNA hybridization in last century.Numerous of impressive DNA dynamic devices was subsequently reported such as DNA logical gates,DNA motors,DNA walkers and DNA logical computing networks.In the field of DNA structural nanotechnology,different kinds of micro or bulk materials structures were created like DNA origami,DNA gel,DNA micelle,DNA dendrimer DNA nanoflower as well as DNA catalyzed selfassembly.Although it is the structure instead of the natural function of DNA molecules that is studied in DNA nanoengineering,the properties of DNA as naturally existed biomolecules make them idea materials in biological applications.DNA is biocompatible and can be used to recognize some biotargets or be recognized by various or enzymes.In the first three projects of this thesis,we try to build DNA dynamic circuits in some bio-or bionic systems.In the last two projects,we focus on the weakness of some DNA self-assembled structures and try to find some solutions.Here are some brief descriptions for each chapter:In Chapter 2,an efficient strategy for enzyme-and hairpin-free nucleic acid detection called an entropy beacon(abbreviated as Ebeacon),was proposed.Different from previously reported DNA hybridization/displacement-based strategies,Ebeacon is driven forward by increases in the entropy of the system,instead of free energy released from new base-pair formation.Ebeacon shows high sensitivity,with a detection limit of 5 p M target DNA in buffer and 50 p M in cellular homogenate.Ebeacon also benefits from the hairpin-free amplification strategy and zerobackground,excellent thermostability,as well as good resistance to complex environments.In particular,based on the huge difference between the breathing rate of a single base pair and two adjacent base pairs,Ebeacon also shows high selectivity toward base mutations,such as substitution,insertion,and deletion and,therefore,is an efficient nucleic acid detection method,comparable to most reported enzyme-free strategies.In Chapter 3,we describe the feasibility of using a DNA network as the computing core of a cell-sized robot,which will perform a mimicked immune response in a concise way when there is a mimicked pathogenic challenge.A DNA network is like a biological algorithm that can respond to ‘molecular input signals,' such as biological molecules,while the artificial cell is like a micron-robot whose function is powered by the encapsulated DNA network.Such a DNA network-powered artificial cell can realize the connection of logical computation and biological recognition due to the natural programmability and biological properties of DNA.Thus,the biological input molecules can be easily involved in the molecular computation.We believe the strategy proposed in the current chapter,i.e.,using DNA circuits to power artificial cells,will lay the groundwork for understanding the basic design principles of DNA algorithm-based nanodevices which will,in turn,inspire the construction of artificial cells,or protocells,that will find a place in future biomedical research.In Chapter 4,DNA dynamic circuits was constructed to detect membrane encounters on live cell membrane.Cells interact with the extracellular environment through molecules expressed on the membrane.Disruption of these membrane-bound interactions(or encounters)can result in disease progression.Advances in superresolution microscopy have allowed membrane encounters to be examined,however,these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules.Here,we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals.The probe,which translocates from one anchor site to another,mimicking motor proteins,is realized through a toehold-mediated DNA strand displacement reaction.Using this probe,we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy.Our results show a preference for encounters within the same lipid domains.In Chapter 5,a catalytic self-assembled DNA dendritic complex was reported and used for si RNA-based gene silencing.This kind of one-pot DNA dendrimer can be conveniently prepared as needed,and it was demonstrated to have better silencing efficiency and lower cytotoxicity than commercial cationic lipid transfection agents.In Chapter 6,a facile and universal method for in situ crosslinking of DNA micelles,using spherically-directed reduction of metal ions,was described.By incorporating a series of specific template domains into lipid-DNA monomers,copper,silver and gold-crosslinked DNA micelles are formed with a hollow or solid core.By combining characterization results and simulation algorithms,accurate structural profiling of DNA micelles is achieved for the first time.The prepared metalcrosslinked DNA micelles show obvious merits,including one-step formation of core structure and ligand corona,facile size-tunability,monodispersity,stability against salt-induced aggregation,high utilization of DNA ligand,mild synthesis condition,as well as less time consumption and better internalization.Based on our sphericallydirected synthesis,metal-crosslinked DNA micelle flares were further prepared for effective intracellular imaging by integrating an aptamer-toehold strategy.In addition,we extended the strategy to cholesterol-based double-stranded DNA micelles,giving strong evidence that our method is not only universal,but flexible,as well.