| The dynamic DNA structures can respond to environmental stimuli and reconfigure their conformations,and are widely used in the research of functional nanodevices,such as engineering nanomachines,biosensors and molecular logic gates.The use of base stacking to drive the reconfiguration of DNA structures is a new way of engineering dynamic DNA structures,but the studies on dynamic DNA structures driven by base stacking are currently limited.Here we designed a DNA nanodevice driven by base stacking:single-stranded DNA was input by hybridizing to the edge of the nanodevice.The reconfiguration at crossover sites was then triggered by base-stacking.In this study,we introduced a fluorescent reporter domain into the DNA nanodevice and fluorescence resonance energy transfer（FRET）signals were measured to characterize the reconfiguration behaviors.We also optimized the response signals of DNA nanodevice and developed molecular logic gates that can perform several Boolean operations.Objective:1.Two kinds of structure of DNA nanodevice（DCA-A and DCA-B）were designed and structural stability of the two designs was compared.We optimized the structures to get the general design principle for such DNA nanodevice.2.The inputs（single-stranded DNA）would interact with the DNA nanodevice to produce reconfiguration behaviors as predicted.Besides,the FRET signals generated by DNA nanodevice would be optimized.3.In terms of application,the significant difference in the fluorescence outputs responded to multiple inputs were used to establish the binary signals,and thus different logic gates were developed.Methods:1.Denaturing PAGE was used to purify single-stranded DNA and determine the purity.2.DNA scaffold was synthesized by ligation:T4polynucleotide kinase（T4 PNK）was used to catalyze the phosphorylation of the 5’end of the single-stranded DNA,and then the helper strands were bound to the DNA strands to be linked.T4 DNA ligase was used as catalyst to ligate DNA fragments into long single-stranded DNA by forming phosphodiester bonds.3.DNA simulation software Tiamat and the website for DNA analysis（https://sg.idtdna.com/pages）were used to design the structure of DNA nanodevice and its sequences.4.DNA strands were mixed in TAE/Mg2+buffer,and were annealed through temperature control program.In a way,they were denaturated at high temperature,and then slowly cooled down and redenatured.These DNA strands self-assembled into designed structures.5.Native PAGE was used to characterize the formation of self-assembled DNA structure.6.AFM imaging was used to characterize the morphology of DNA naonodevice.7.Fluorescence spectrophotometry was used to characterize the reconfiguration of DNA nanodevice which was modified with fluorescent molecules.The D-A distance r was calculated based on F（?）ster theory.Fluorescence responses generated by DNA nanodevice assisted the revisement of initial structures of DNA nanodevice and the optimization of fluorescence response functions.8.By pre-assembling setting strands were pre-assembled with DNA nanodevice at different binding domains to set the initial conformation.According to the significant differences of fluorescence responses to each possible combination of multiple inputs,binary signals were established and thus different kinds of logic gates were developed.Results:1.Denaturing PAGE results showed that the scaffold was successfully synthesized by ligating three different DNA strands and the yield of scaffold reached34.9%after purification.2.Stepwise assembly of initial structures of DNA nanodevice was characterized by native PAGE.However,after adding inputs to DNA nanodevice,undesired base pairings occurred in the DCA-A system,which indicated that the DCA-A structure was not stable and needed to be redesigned.By contrast,target structures formed with high yield when adding inputs to DCA-B system,which indicated that the DNA origami-like structure greatly enhanced the structural stability of the DNA nanodevice.3.Results of atomic force microscope imaging showed that the shape of the DCA was a rhombus with an angle at crossovers.4.The results of fluorescence measurements showed that the input strands in two different directions had opposite effects on DNA nanodevice,indicating that the conformation of the DNA nanodevice changes as desired.However,fluorescent intensity changed by each input was different,and the order was southwest（SW）direction>northwest（NW）direction>southeast（SE）direction.And the FI output change caused by SW direction was much higher than the intensity from the individual input in the NW and SE directions.5.By revising the initial structure of DNA nanodevice and shortening the sequence of input in SW direction,the magnitude provided by each input was corrected to roughly the same Such nanodevice could generate increased,unchanged and decreased FI by the inputs from three directions.6.Based on F（?）ster’s theory,we calculated the distance between the fluorescence donor and the fluorescence acceptor of the DNA nanodevice with inputs to obtain the specific conformations of the DNA nanodevice.7.The fluorescence results showed that we have successfully constructed the signal behaviors of AND gate and NOT gate.Conclusion:In this thesis,we designed and engineered a DNA nanodevice driven by base-stacking,and characterized its controllable reconfiguration of by fluorescence measurement.Such DNA nanodevice was modular,and its signal response function can be adjusted by hybridizing different lengths of DNA strands with the initial structure of DNA nanodevice.In this research,the design of DNA nanodevice was optimized,and different molecular logic gates were designed according to various signal responses.For the potential applications of DNA nanodevice,the sequence of SW,NW or SE region of the DNA nanodevice can be designed to be complementary to target nucleic acid molecules（as analytes）and then biosensors or disease detection devices could be constructed.Besides,different molecular logic gate modules could be intergrated to construct complex molecular computing systems. |
