DNA Reaction Network under Catalysis of Cascade Circuits or Enzyme

Author:Li Xiang

Supervisor:liang hao jun


Degree Year:2019





In the region of life sciences,DNA is commonly considered as the carrier of gene information:it is first transcribed into mRNA,then continually translated into protein.As more and more research is operated on DNA,there comes a new time of DNA nanotechnology,which could be used to construct structural materials in nanoscale,molecular motors,circuit network and so on.The reason nucleic acids could be used as constructing materials is that the complementary pairing of Watson-Crick between bases is predictable(A and T,C and G).Relying on this character,the complex self-assembly could be achieved through the reasonable design of DNA sequence.Besides,DNA could also interact with other materials(such as gold nanoparticles,carbon nanotubes and so on)to construct more complex networks,achieving broader applications.The vigorous development of dynamic DNA nanotechnology benefits from the important concept of toehold-mediated strand displacement reaction(TMSDR):with the aid of toehold,a single strand DNA(input strand)reacts with the pre-hybridized double strand through strand migration,releasing one strand from the bottom strand,and the input strand will hybridize with the bottom strand to form a new double strand.Since the emerging concept of TMSDR,molecular machines in different kinds are increasing in an exponential and explosive way.In this thesis,to the system without enzyme,we have constructed a catalytic chemical amplifier for signal amplification and detection in chemistry and medical diagnosis;to the system with enzyme,we have constructed catalytic renewable circuit and cascade circuits for further development in DNA nanotechnology.In the first chapter of this thesis,we made a brief introduction about DNA nanotechnology,had a careful analysis of the physical and chemical properties in the strand displacement reaction,did a detailed list of the catalytic reactions based on the process of strand displacement,and conducted a classified discuss on the current applications.DNA nanothchnology has become increasingly mature;however,pursuing for technical innovation and effectiveness improvement still haven’t stopped.Then,basing on the two important free-enzyme DNA catalytic circuits which have been successfully fabricated and optimized(the entropy-driven catalytic network(EDCN)and the catalytic hairpin assembly(CHA)),we built a two-layer cascade system with the reporter system of spherical nucleic acid(SNA)assembly.It has following advantages:owing to the relative independence among the three subsystems,we can adjust the involved parameters respectively to get the optimized reaction condition;in the reporter system,the filtration function of SNA assembly signal could split the signal with low concentration of input strand and the leak without input strand to a large extent,finally achieving a high signal amplification of about 100,000 folds;also,besides the kinetic detection in real-time through UV-Vis spectrum,the signal of SNA assembly with target strand in a certain concentration could be detected through naked eye.In the third chapter,basing on EDCN,with the aid of a nicking enzyme(Nt.BbvCI),transforming the waste-substrate into reactant-substrate could solve the two problems of reactant consuming and waste accumulating at the same time in general chemical reactions.In this catalytic circuit,not only the catalytic strand but also the reactant-substrate could be used circularly;and when the fuel strand is used up,adding more fuel strand would induce a new proceeding process,achieving a good renewability.In the research of chapter four,we introduce another exonuclease ExoⅢ into spherical nucleic acid(SNA)assembly to explore the behaviour of catalytic circuit;then we add CHA to the upper stream,constructing a two-layer catalytic system and amplifying the signal to detect a low concentration of target strand.Finally,we proceed a summary and outlook to the above experimental content.In recent years,there are many kinds of research on the mechanisms and corresponding physical and chemical characters of strand displacement reaction;all kinds of DNA nanotechnologies could be ued for assembly,detection,computing,crystallization and so on,solely or combined with other regions.In the meantime,the DNA circulation system with the aid of different enzymes will present distinct features or outstanding advantages,and this needs our further exploration to reveal more scientific secrets in the cell or outside of it.