Enhanced SNP-sensing using DNA-templated reactions through confined hybridization of minimal substrates (CHOMS)
DNA or RNA templated reactions are attractive for nucleic acid sensing and imaging. As for any hybridization-based sensing, there is a tradeoff between sensitivity (detection threshold) and resolution (single nucleotide discrimination). Longer probes afford better sensitivity however compromise single nucleotide resolution due to the small thermodynamic penalty of a single mismatch. Herein we report a design that overcomes this tradeoff. The reaction is leveraged on the hybridization of a minimal substrate (covering 4 nucleotide) that is confined by two guide DNAs functionalized respectively with a ruthenium photocatalyst. The use of a catalytic reaction is essential to bypass exchange of guide DNA while achieve signal amplification through substrate turnover. The guide DNAs restrain the reaction to a unique site and enhance hybridization of short substrates by providing two π-stacking interactions. The reaction was show to enable detection of SNP and SNV down to 50 pM with a discrimination factor ranging from 24-309 (median 82, 27 examples from 3 oncogenes). The clinical diagnostic potential of the technology was demonstrated with the analysis of RAS amplicon obtained directly from cell culture.