Molecular Dynamics Simulation-guided Design Of DNA Nanodevice And Probe

Single nucleotide variants(SNVs)occur all the time in biological universe,although the error base can be repaired through base excision repair pathway,there are still some mutant genes which have important influence on biological function and retain.These mutations are usually associated with viruses and cancers,Mutant viruses often appear to be more infectious,and mutant cancers often show higher level of drug resistance.So as emerging biomarkers,it is of great clinical significance to detect SNVs efficiently.Toehold-mediated DNA strand displacement(TMSD)is a powerful tool for detecting SNVs due to the high specificity of DNA itself.However,there is still a lack of comprehensive research on how to design the position of single base mismatch in DNA fluorescent probe to achieve the best discrimination between mutant type and wild type.Here,we utilize oxDNA molecular dynamics simulation to analyze the effects of four typical mismatch positions on forward reaction in TMSD,which provides guidance for rational design of DNA fluorescent probes.These probes show excellent detection performances and are used for detection of mutant SARS-Co V-2 and other quasi-real samples.NUPACK,oxDNA Umbrella Sampling and Forward Flux Sampling were utilized to estimate the strengths and weaknesses of the four mismatch positions in probe from thermodynamics and kinetics.And it was determined that the mismatch at the border of toehold domain and branch migration domain(Toe7)has the best and most stable inhibition ability on the rate of TMSD.Subsequently,nine virus and cancer related mutant gene sequences were selected for experimental verification by four types of probes.The experimental results were confirmed to be identical with the simulation results and the median of DF of the optimal mismatch type reached 124.Then,the detection performance of the DNA fluorescence probe designed under the guidance of simulation was tested,showing rapid detection performance within 5 minutes,high specificity of multiple detection performance,and low VAF of 0.1%detection performance.Finally,λ Exo was successfully introduced to discriminate the mutant type and wild type of amplification in plasmid with DNA fluorescent probe,which realized the detection of clinical samples by probe.In this paper,oxDNA is utilized to provide a description of the mechanism of DNA strand displacement reaction from the level of a single nucleotide,which has a guiding role in the rational design of DNA fluorescent probe.And the ability of highly specific discrimination for SNVs and detection of clinical samples are achieved,that is suitable for biosensor and DNA nanotechnology applications.