The Detection Method Of Single-nucleotide Mutation Based On Nucleic Acid Transient Hybridization And Lambda Exonuclease

Advances in both biophysical methods and dynamic DNA nanotechnology permit the precise engineering of dynamic processes.As the simplest dynamic DNA structure,short DNA duplex(9-12 nt)poses transient hybridization manner,which can be used for superresolution imaging and in vitro detection.Nuclease activity upon nucleic acid substrates enables various applications in analytical chemistry and dynamic DNA nanotechnology.Exonuclease plays a vital role in cellular biology and operates nucleic acid in an accurately predicted way.To study the interaction of dynamic substrate and exonuclease is helpful to create new way to engineer nontrivial molecule behaviors and transfer dynamic DNA nanotechnology into the living cell.Lambda Exonuclease also shows promise for addressing the problem of leakage in DNA based analytical methods and nanotechnology due to its sensitivity to mismatches.Inspired by the use of dynamic probes in DNA nanotechnology to increase specificity,we combined bulk fluorescence and single-molecule fluorescence analysis to investigate the single-base selectivity of Lambda exonuclease for very short double stranded DNA.We characterized the interaction of short double-stranded DNA and Lambda Exonuclease by combining bulk fluorescence and single-molecule kinetics analysis.The results of bulk fluorescence measurement imply that short dsDNA can be effectively digested even the length is less than the enzyme footprint and the degradation rate exhibits high single-nucleotide selectivity.Single-molecule analysis reveals that a perfectly matched(PM)substrate can be selectively stabilized by binding of the enzyme,which combines with the differential stability of transient hybridization of short substrates to yield high single-nucleotide selectivity.Single-molecule analysis reveals that the observable digestion as a non-equilibrium process is dominated by two equilibrium processes,DNA transient hybridization and enzyme binding.The synergistic function of the two processes offers the high single-nucleotide selectivity.Taking advantage of the substrate-enzyme interaction,a highly selective probing for single-nucleotide mutation was developed allowing the detection of KRAS mutation from cell line at low abundance.A highly selective assay for a single-nucleotide mutation in KRAS was developed,which permits detecting this mutation from cell line at as low as 0.1%.