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

Abstract

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.

Guiding-Strand-Controlled DNA Nucleases with Enhanced Specificity and Tunable Kinetics for DNA Mutation Detection

“…Compared with other nucleic acid probes, such as molecular beacons 22,23 and strand migration probes, 24−26 the nuclease-assisted probe system has the advantages of fast response and high sensitivity. 27,28 However, λ exonuclease (λ exo), apurinic/apyrimidinic endonuclease-1 (APE-1), and Endo IV, which are widely used in the field, are all prone to unexpected nonspecific cleavage. In the reaction system, nucleic acid probes that do not bind target sequences represent free ssDNA, and their nonspecific cleavage produces a very high background signal, resulting in decreased or even lost discrimination ability between mutant and wild-type DNA.…”

Novel Single-Enzyme-Assisted Dual Recycle Amplification Strategy for Sensitive Photoelectrochemical MicroRNA Assay

“…Enzyme-assisted recycle amplification strategy, a powerful biological amplification technique, has been widely developed in photoelectrochemical (PEC) biosensor. − The application of the enzyme-assisted recycle amplification strategy lays a firm foundation for the improvement of target molecule amplification efficiency and sensitivity of the PEC biosensor. − In the past decades, enzyme-assisted single recycle amplification strategy has been mainly developed to achieve the conversion of bits of the target to the substantial mimic target due to its low cost and easy operation. − For instance, our group has reported a “signal off” PEC biosensor for microRNA-141 (miRNA-141) assay on the basis of a duplex specific nuclease-assisted single recycle procedure . Li and co-workers have constructed a PEC biosensor using T7 exonuclease (T7 Exo), which cleaves the DNA/RNA heteroduplex to release RNA (target) and achieve the target amplification .…”

Improving the sensitivity and selectivity of a DNA probe using graphene oxide-protected and T7 exonuclease-assisted signal amplification

“…In contrast, 5′-end mismatched targets could not trigger the hydrolysis of the probe by T7 Exo. Based on the same principle, Xiao et al achieved highly selective analysis of targets with a singlebase mismatch using λ exonuclease [18].…”

“…However, while these methods achieve highly sensitive and selective detection of target genes, they suffer from limited flexibility when the nuclease activity, amount of nuclease, and length of the probe must be optimized in each case (the length of the target has to be less than 12 nt) [17,18], thus increasing experimental complexity and limiting the scope of application. Therefore, establishing an economical, high selective, and highly sensitive detection method still presents a challenge.…”