Selective Nucleic Acid Capture with Shielded Covalent Probes

Abstract

Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets are bound). These difficulties are fundamental, stemming from reliance on base pairing alone to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable molecular conformation change and durable target capture via activatable covalent cross-linking (Vieregg et al, J. Am. Chem. Soc. 2013). In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligonucleotide targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2−3 orders of magnitude. Semi-quantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1−2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications in vitro and in vivo; this talk will present our latest results on SC probe applications.