DNA Strand Displacement Controlled SNA Assembly And DNA Keypad Lock

DNA is a biological macromolecule consisting of four types of repeating units,adenine(A),thymine(T),guanine(G),and cytosine(C),which is generally regarded as gene carriers for storage and expression of genetic information and plays an essential role in the growth,development and genetic processes in living organisms.In the field of DNA nanotechnology,based on the classical Watson-Crick base-pairing principle of the nucleic acid base adenine and thymine(A-T),and cytosine and guanine(C-G),DNA has become a robust building nanomaterial with predictable structure.And depending on its unique programmability,specificity and diversity,DNA has been widely applied to construct various large scale self-assembly nanostructure,dynamic DNA nanodevice and DNA cascade systems with high signal amplification ability.With the development of toehold-mediated DNA strand displacement reactions(TMSDRs),DNA molecule machines have shown outstanding application potential in biological sensing,logic operation,disease diagnosis and treatment.Gold nanoparticles(AuNPs)as a functional material have attracted considerable concerns due to their unique physical and chemical properties,excellent stability and biocompatibility.Since the pioneering work of DNA functionalized AuNPs,named spherical nucleic acid(SNA),was reported by Mirkin et al.in the mid-1990s,SNA conjugates as a polyvalent DNA covalently functionalized gold nanoparticle possessing both the distinct characteristics of the inside gold core and highly oriented oligonucleotide shell,has demonstrated its superiorities in various fields,such as in vivo and in vitro diagnostics,cellular imaging,colorimetric detection,drug delivery and programmed superlattice structure assembly.In this thesis,we firstly constructed a facile strategy for visible disassembly of SNA aggregates programmed by an entropy-driven catalytic DNA circuit.Unlike the traditional direct-linker-addition approach for assembly of SNA conjugates,this integrated system was composed of an upstream catalytic DNA circuit and a downstream SNA aggregates-based disassembly subsystem.These two subsystems can be connected together by trigger strand,which was released from the upstream circuit through successive TMSDRs initiated by a target strand,named disassembler,playing a role of catalyst in the circuit.Subsequently,the released trigger strand will react with the DNA molecules on the surface of SNA to disassemble the SNA aggregates,and the disassembly process of SNA aggregates can be easily detected by the naked eye through a visible color change of the supernatant.This integrated catalytic disassembly system could achieve a low visible detection limit in a short time,which indicated the rapid colorimetric response and good sensitivity.In addition,our integrated system exhibited excellent sequence selectivity in single nucleotide polymorphism(SNP)discrimination,which can not only distinguish correct DNA targets from spurious strands with single-mismatched bases,but also successfully detect a low amount of DNA target under the interference of high concentration of spurious target.For the construction of the downstream system,only one type of SNA molecule and a duplex linker containing a self-complementary region are employed to form the stable SNA aggregates at room temperature.Besides,this delicate design endows the downstream SNA aggregate-based system with the distinctive capability in combining with arbitrary upstream circuits to specifically detect different targets without any need to change the DNA structure of the downstream SNA system,and hence a two-input OR logic gate used for multiplex detection was constructed,proving the flexibility and universality of our strategy.Therefore,this integrated catalytic disassembly strategy have great potential in the fabrication of a facile and portable DNA diagnostic kit and the application of clinical point-of-care testing.As a smart computing device,DNA keypad lock has been intensively developed to address password security issues in combination with sequential logic.It can only complete the processes of logic operation with a correct permutation of inputs,which meet the requirements of information protection at the molecular level.In the research of chapter three,we have developed a novel strategy for building homogeneous DNA-only keypad locks by employing the scalable DNA junction substrate as the RIGHT-proceeding component and a series of double-stranded eliminators as WRONG-eliminating component,which were used for processing correctly-and wrongly-added DNA inputs,respectively,through kinetic differentiation.Only the inputs added in right orders could unlock the device.Supported by the experimental optimizations in structures and kinetic competition of both components,two-,three-,and four-input keypad locks were sequentially constructed and proved to be working with security performance as expected.In contrast to other conventional strategies that necessitated solid-phase platforms for separation or sensing,homogeneous DN A-only keypad locks could process multiple DNA inputs in a one-pot manner through taking advantaging of the powerful programmability and scalability of DNA molecules,promoting their accessibility in building more complicated information security systems.Diverse SNA superlattice structures have been successfully constructed through thermal annealing strategy after the first work reported by Mirkin et al.In chapter four,through coupling the dynamic DNA walker on AuNP surface with the sticky ends-induced SNA assembly strategy,a single-and two-component SNA aggregates were constructed to prepare face-centered cubic(FCC)lattice and CsCl lattice at room temperature,respectively.First,the dynamic walking process of two-legged walker on the surface of gold nanoparticles was proved to be stable with excellent walking efficiency and walking persistence.Second,SNA assembly kinetics results showed that two-legged walker concentrations and DNA linker ratios could greatly influence the aggregation of SNA conjugates.Subsequently,disordered SNA aggregates formed by different kinds of sticky end binding were converted to ordered structures through conformation adjustment using thermal annealing method.Finally,SNA crystalization assembly drived by DNA walker was conducted by the successive walking process with varied concentrations of two-legged walker.When the concentration of walker strand was decreased,the assembly process could have a long residence time to switch between the binding and unbinding of sticky ends and achieve a series of near-equilibrium states.Then,as the interaction between particles grows,the dynamic pathway of AuNP assembly can be programmed to achieve a free-energy minimum.Therefore,two ordered structures(FCC and CsCl lattices)were eventually constructed,which would provide a promising development for the creation of complex and functional nanoscale materials and for the realization of complex phase behaviors.In summary,based on toehold-mediated strand displacement reaction,this thesis constructed various DNA cascade systems controlled SNA assembly and disassembly strategies and demonstrated great applicabilities in nucleic acid detection,SNP discrimination,logic gates and superlattice structure assembly.In addition,homogeneous DNA keypad locks were built with scalable junction substrates,showing potential applications in information security systems.