Double mismatch interactions in probe design enable improved hybridization-based SNV discrimination
Achieving high selectivity between mutant (MT) and wild-type (WT) sequences at low variant allele frequencies (VAFs) is challenging because the impact of sequence context and mismatch (MM) positioning on hybridization energetics is poorly understood. To establish design principles for single-nucleotide variant discrimination, we used a newly developed high-throughput platform to systematically quantify the energetics of single and double MMs within a controlled synthetic architecture. This unbiased analysis demonstrated that strategically introducing an intentional MM significantly improves MT/WT selectivity, while further characterizing the distance-dependent interactions that govern duplex stability. By applying this framework to the clinically actionable mutations KRAS p.G12C and BRAF p.V600E, we evaluated libraries of 884 probe variants per target, where each probe carried one intentional MM relative to the MT and a dual-MM state relative to the WT. Through over 100,000 measurements, we identified pronouncedpositional effects, particularly at probe termini, that optimize discrimination. Benchmarking on a photoelectrochemical biosensor confirmed that these insights translate into improved MT/WT target selectivity. This study provides a data-driven framework for the predictive design of highly selective hybridization assays for clinical mutation detection.