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Simulation-guided sloppy DNA probe design for mismatch-tolerant hybridization

Bugga, P.; Asthana, V.; Drezek, R. A.

2022-12-21 bioengineering
10.1101/2022.12.20.521289 bioRxiv
Show abstract

The ability to both sensitively and specifically assess the sequence composition of a nucleic acid strand is an ever-growing field. Designing a detection scheme that can perform this function when the sequence of the target being detected deviates significantly from the canonical sequence however is difficult in part because probe/primer design is based on established Watson-Crick base-pairing rules. We present here a robust and tunable toehold-based exchange probe that can detect a sequence with a variable number of SNPs of unknown identity by inserting a series of controlled, sequential mismatches into the protector seal of the toehold probe, in an effort to make the protector seal "sloppy". We show that the mismatch tolerant system follows predicted behavior closely even with targets containing up to four mismatches and thermodynamically deviating from the canonical sequence by up to 15 kcal/mole. The system also performs faithfully regardless of the global mismatch position on either the protector seal or target. Lastly, we demonstrate the generalizability of the approach by testing the increasingly sloppy protectors on HIV clinical samples and show that the system is capable of resolving multiple, iteratively mutated sequences derived from numerous HIV sub-populations with remarkable precision.

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