Engineering Functional Nucleic Acid Structures For DNA Computing,SNP Detection,and RNA Interference

Nucleic acid,the crucial genetic information carrier of living creature,also acts as an excellent nanomaterial.Underpinned by the specific and predictable Watson-Crick base-pairing rule,it can self-assemble into multi-dimensional nanostructure with large size order or specific functionality.Furthermore,nucleic acid structural devices are capable of operating dynamically and autonomously under the stimulation of toehold-mediated strand displacement reaction mechanism.In this thesis,we propose three types of functional nucleic acid structures,which were designed for different applications including DNA computing,SNP detection,and RNA interference.We first designed a DNA "junction substrate" structure to construct a multi-input DNA computing system.A conventional DNA substrate is a multi-stranded DNA complex prepared by hybridizing a long linker chain with several short protector chains via annealing.Due to the high error probability of synthesizing long DNA chains and the technical difficulty in purification,linear substrate has a limited purity,which will cause severe circuit leakage.By contrast,our proposed "junction substrate" has several advantages.First,it is composed of double-stranded DNA combinatorial units,avoiding the usage of long DNA chains and making the substrate scalable.Second,only the double-stranded combinatorial units need to be purified,thus technically facilitating the purification process,which is beneficial to improve the purity of substrate.Third,according to the theoretical analysis,the "junction" architecture can help to reduce the asymptotic leakage.As a proof of concept,we gradually scaled up the optimized junction substrate to successively construct a two-input,a three-input and a four-input DNA computing systems.Compared with the linear-substrate counterparts,the junction substrates exhibited a better resistance to circuit leakages,and provided better kinetics in these systems.As a result,the scalable junction-substrate structure can be used as a reliable chassis for constructing large-scale DNA circuits.In chapter 3,we designed a DNA probe structure to discriminate DNA single nucleotide polymorphism(SNP).The probe consists of two parts,that is,a triple-stranded probe substrate and a single-stranded competition-catalyzing DNA chain.Compared with the conventional toehold-exchange probe,this probe has a better ability to discriminate low concentration SNP.In this chapter,the probe ability to discriminate SNP was measured by yield difference(?Y).By plotting the relationship curves between target concentrations and the corresponding yield differences(that is,the yield difference profiles,?Y profiles)in theoretical calculations,simulations,and experiments,we systematically studied the probe behaviors under different toehold parameters and detection times.The results showed that the SNP discrimination ability(that is,AY)of the competition-catalyzing probe varies with the target concentrations in a parabolic manner.It peak position,which corresponds to the optimal target concentration([I]0,m),would shift to lower concentration regions with the increasing of n(the length of the invading toehold of the probe),the decreasing of m(the length of the incumbent toehold),or the extending of detection time.This result provides a direction for improving the discrimination ability of probes at low concentrations.In addition,the probe also showed an insensitivity to the position of SNP,reflecting the high specificity of the probe.These results facilitate the design of DNA hybridization probes to achieve more efficient SNP differentiation.In chapter 4,we designed a small short hairpin RNA(sshRNA)structure to regulate gene silencing through the toehold-mediated strand displacement mechanism.The sshRNA has a mismatch region in the stem and a 3'-end dangling toehold domain.The mismatches were introduced to break the initiation mechanism of the conventional sshRNA silencing and the toehold region was designed for initiating and regulating the silencing process.Mediated by the toehold binding between the target mRNA and the sshRNA,the mismatch region will be eliminated after the strand displacement,activating the guide strand of sshRNA and thus leading to the Ago2-dependent silencing.Silencing regulation can be achieved by tuning the toehold length.In the experiment,we first constructed a reliable and controllable gene expression pathway of IVT mRNA.Subsequently,on the basis of this gene expression platform,we testified the three-step design strategy.However,the regulatory ability is limited,so the follow-up design and experiment will be carried out to optimize the structural parameters of sshRNA,and finally achieve effective control of gene silencing ability.In summary,this thesis includes the designs and applications of three functional nucleic acid structures,namely,a DNA "junction substrate" for DNA computing,a DNA probe to discriminate low-concentration SNP,and an sshRNA to regulate gene silencing.All these studies demonstrate the advantages of functional nucleic acid structures and their potential values in various applications.