Sequence‐Specific Covalent Capture Coupled with High‐Contrast Nanopore Detection of a Disease‐Derived Nucleic Acid Sequence

Abstract

Hybridization-based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but may not provide a durable signal. Sequence-specific covalent cross-link formation can anchor probes to target DNA and also may provide an additional layer of target selectivity. Here we develop a new cross-linking reaction for the covalent capture of specific nucleic acid sequences. This process involves reaction of an abasic site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease-relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap-containing probes are easily prepared and display exquisite specificity for the mutant sequence under isothermal assay conditions. DNA duplexes generated in these studies were quantitatively measured using both denaturing gel electrophoresis and a protein nanopore.

What is the potential of nanolock– and nanocross–nanopore technology in cancer diagnosis?

“…In this review, we describe two different single-molecule platforms, nanolock-nanopore[21] (Fig. 1a) and nanocross-nanopore[22,23] biosensers (Fig. 1b) for the detection of point mutations.…”

“…We have also developed a separate approach that exploits sequence specific cross-linking with a reactive probe strand that covalently captures the mutant BRAF target sequence[22,23] (Fig. 1b).…”

“…In this “nanocross” approach, a DNA abasic site, embedded in a specialized probe sequence, enables highly selective covalent cross-linking to adenine 1799 in the BRAF V600E mutant gene sequence[35]. Importantly, the cross-linked probe-target complex gives an unmistakable, long-lasting current block in the nanopore device[22,23]. This current signature is easily distinguished from the short-duration blocks caused by uncross-linked target ● probe complexes derived from the wild-type sequence.…”

“…This current signature is easily distinguished from the short-duration blocks caused by uncross-linked target ● probe complexes derived from the wild-type sequence. Accordingly, we showed that the fraction of T>A mutant sequence could be quantitatively determined within mixtures containing the high background of the wild-type allele[23].…”

Single Locked Nucleic Acid-Enhanced Nanopore Genetic Discrimination of Pathogenic Serotypes and Cancer Driver Mutations

“…33,35–37 The nanopore-based next-generation sequencing technology is being developed. 36 , 38–40 Through collaboration, we have developed two different single-molecule platforms, nanolock-nanopore 41 and nanocross-nanopore 42,43 biosensers, for the detection of cancerderived point mutations.…”