Chemically modified oligonucleotides that enable sequence-specific recognition of double-stranded (ds) DNA have tremendous potential as tools in diagnostics, gene editing, and molecular therapy. Our laboratory has previously introduced so-called Invader probes, which are short DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized RNA monomers, as a new class of dsDNA-targeting modalities. These energetically activated probes bind to complementary dsDNA target regions via double-duplex invasion, which offers the promise of favorable binding affinity and specificity and straightforward probe design. In the present study, we evaluated Invader probes with single-stranded overhangs, i.e., toeholds, to further improve mixed-sequence dsDNA-recognition efficiency (see illustration below). The stabilities of probe duplexes and recognition complexes were determined from thermal denaturation experiments. Thermodynamic parameters associated with duplex formation were determined from baseline fitting of melting profiles. Electrophoretic mobility shift assays were used to determine C30 values (i.e., the concentration of a probe leading to 30% target recognition). Invader probes with appropriately designed toeholds display faster and more efficient recognition of mixed-sequence dsDNA targets than conventional Invader probes (C30 as low as 20 nM at 37 °C). Toehold Invader probes with locked nucleic acid (LNA)-modified overhangs demonstrate improved binding specificity. These results underscore the potential of these probes for applications in molecular biology, biotechnology, and biomedicine.

Sequence-unrestricted recognition of double-stranded DNA using toehold Invader probes

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.