The Research And Application Of Strand Displacement Reactions On Nucleic Acids Biosensing

Strand displacement reactions generally proceed by three-way or four-way branch migration and initially were investigated for their relevance to genetic recombination.Through the use of toeholds,which are single-stranded segments of DNA to which an invader strand can bind to initiate branch migration,the rate with which strand displacement reactions proceed can be varied by more than 6 orders of magnitude.Strand displacement reaction utilizes the thermodynamic properties of double-stranded probe and target gene to achieve the specific detection of the target.Strand displacement reaction probe is partially double-stranded in order to form a toehold domain and consists of a protector strand(the sequence is the same as the target but is shorter than the target)and a complement strand.Thermodynamic properties of the probe would enable near-optimal single-base discrimination and perform robustly across diverse temperatures and different salt conditions.Strand displacement reactions are widely used including nucleic acids detection,aptamer biosensing and DNA nanotechnology.Our work is partially divided in to the following parts:1.An off/on thrombin activated energy driven molecular machine for sensitive detection of human thrombin via non-enzymatic catalyst recycling amplificationIn this part,we report a novel dual on/off thrombin fluorescence aptasensor by combination of energy driven target induced strand displacement and non-enzyme catalyst recycling DNA machine.Firstly,the specific binding of aptamer strand and thrombin induce the release of catalyst which was used as DNA machine trigger.Subsequently,catalyst as trigger initiated the DNA machine through nucleic acid hybridization and branch migration of the DNA machine,resulting in DNA substrate melting and re-hybridized.In such a working model,the DNA machine achieved cooperatively controlling circular strand displacement reaction,realizing catalyst recycling and dual-amplification.The fluorescence signal change of FAM and ROX accumulation had a good linear relationship with thrombin concentration in range of 1 fM to 1 nM.On account of catalyst recycling and dual recognition,detection limit for thrombin was determined to be as low as 0.45 fM(S/N=3).This biosensor also showed good selectivity for thrombin without being affected by some other proteins,such as PSA and lysozyme et al.Moreover,this assay can be applied to the determination of thrombin in diluted human serum.2.Ultrasensitive electrochemical biosensor for attomolar level detection of let 7a based on toehold mediated strand displacement reaction circuits and molecular beacon mediated circular strand displacement polymerizationIn this study,an ultrasensitive electrochemical miRNA biosensor based on toehold mediated strand displacement reaction circuits(SDRCs)and molecular beacon mediated isothermal circular strand displacement polymerization reaction(ICSDPR)has been proposed.During the SDRCs module,the cascade strand displacement reaction induces the recycling of the target let 7a and generation of a large amount of strand A(SA).The SA recognition opens the hairpin capture probe immobilized on the gold electrode,thus,varying the distance between the redox molecules and electrode surface.The primer mediated ICSDPR is observed to further generate a large amount of SA,thus,leading to a reduction in the signal.Considering these merits,the proposed method is observed to exhibit a log-linear linearity from 10 a M to 100 p M and ultrahigh sensitivity towards let 7a down to 6.2 a M,with a capability of distinguishing the let7 a family members,thereby,providing a new electrochemical route for early cancer screening.3.Multiplex real-time PCR using double strand displacing nucleic acids as primers and probes for the detection of nucleic acidsMultiplex PCR encounters difficulties in primers design that all primers pairs working at the same annealing temperature.Multiplex PCR is a widespread molecular biology technique for simultaneously amplification multiple targets in a single PCR tube,which saves sample volume,time and money.Making the annealing temperature suitable for multiple primers and templates simultaneously needs to address the following challenges: primer dimers,primer hairpin structures,primer off-target effects and annealing temperatures compatible with multiple pairs of primers.In this part,we have developed a double strand primer mediated multiple strand displacement reaction for the detection of SARS-COV-2 ORF,N,and E gene as example.The double primer is composed of 5' modified fluorophore strand thus does not impact polymerase extension and 3' modified quencher strand thus cannot elongation.In annealing temperature,fluorophore strand combined template and extended thus resulted fluorescence signal release.Results showed that the double strand primer shows relative wide annealing temperature range,good compatibility between three pairs primers and probes.These merits allow simple and multiplex real-time fluorescent quantification of nucleic acids.The detection limit was 400 copies/m L,and the detection time was approximately 2 hours.In addition to its extreme specificity and simplicity,it has wide range of applications such as multiple PCR and SNPs detection.4.Toehold-mediated nonenzymatic DNA strand displacement coupling UDG mediated PCR and multi-code magnetic beads for DNA genotypingSmall variations,even single-nucleotide variants in nucleic acid sequences may exert significant influence in many diseases.The reliable detection and quantification of DNA can provide a variety of information that has significant clinical value,including disease risk assessment,screening,diagnosis,prognosis,the selection of therapies and monitoring.The coupling efficiency of d NTPs and purification methodology can limit the chemical synthesis of long stranded probes.Here,asymmetric PCR was used to generate single-stranded DNA to improve sensitivity while a strand displacement reaction was adopted to improve selectivity.In this research,we demonstrated a PCR-based method to construct a long-stranded detection probe with high levels of selectivity via a strand displacement reaction with target DNA.This detection probe was enzymatically generated from a double-stranded DNA duplex,containing a single-stranded active toehold domain.This approach was successfully implemented to genotype human glucose-6-phosphate dehydrogenase(G6PD)deficiency,focusing upon the c.1376G>T and c.1388G>A variants.