Digestion of Dynamic Substrate by Exonuclease Reveals High Single-Mismatch Selectivity

The distinctive nuclease activity toward nucleic acid substrates enables various applications in analytical chemistry and dynamic DNA nanotechnology. λ Exonuclease is a widely used tool for the processing of PCR products, and DNA sequencing. This enzyme also shows promise for reducing the leakage (i.e., activation in absence of a correct input) in DNA-based analytical methods and nanotechnology due to its sensitivity to mismatches. However, the selectivity of λ exonuclease for single-mismatch in most applications is not high. Inspired by the increased specificity of dynamic probes in DNA nanotechnology, we enhanced the single-mismatch selectivity of λ exonuclease by using very short double-stranded DNA (dsDNA) as the substrate. From the bulk fluorescence measurements, short perfectly matched (PM) substrate which is as a correct input can be effectively digested, but the existence of single-mismatch drastically reduces the digestion rate. Real-time single-molecule kinetics analysis reveals that PM substrate can be selectively stabilized by the binding of λ exonuclease, which combines with the differential stability of transient hybridization of short substrates to yield high single-mismatch selectivity. An excellent selective assay for a single-nucleotide mutation in KRAS was demonstrated, which permits detecting this mutation from cell line at as low as 0.02%, holding potential for detecting rare mutations in circulating tumor DNA of early stage cancers.