Single nucleotide variant (SNV) has become an emerging biomarker for various diseases such as cancers and infectious diseases. Toehold-mediated strand displacement (TMSD), the core reaction of DNA nanotechnology, has been widely leveraged to identify SNVs. However, inappropriate choice of mismatch location results in poor discrimination ability. Here, we comprehensively investigate the effect of mismatch location on TMSD kinetics by molecular dynamic simulation tool oxDNA through umbrella sampling and forward flux sampling disclosing that mismatches at the border of the toehold and branch migration domain yield the lowest TMSD reaction rate. Nine disease-related SNVs (SARS-CoV--DG, EGFR-LR, EGFR-TM, KRAS-GR, etc.) were tested experimentally showing a good agreement with simulation. The best choice of mismatch location enables high discrimination factor with a median of 124 for SNV and wild type. Coupling with a probe-sink system, a low variant allele frequency of 0.1% was detected with 3 S/N. We successfully used the probes to detect SNVs with high confidence in the PCR clones of constructed plasmids. This work provides mechanistic insights into TMSD process at the single-nucleotide level and can be a guidance for the design of TMSD system with fine-tuning kinetics for various applications in biosensors and nanotechnology.
Fluorescence‐based PCR and other amplification methods have been used for SARS‐CoV‐2 diagnostics, however, it requires costly fluorescence detectors and probes limiting deploying large‐scale screening. Here, a cut‐price colorimetric method for SARS‐CoV‐2 RNA detection by iron manganese silicate nanozyme (IMSN) is established. IMSN catalyzes the oxidation of chromogenic substrates by its peroxidase (POD)‐like activity, which is effectively inhibited by pyrophosphate ions (PPi). Due to the large number of PPi generated by amplification processes, SARS‐CoV‐2 RNA can be detected by a colorimetric readout visible to the naked eye, with the detection limit of 240 copies mL−1. This conceptually new method has been successfully applied to correctly distinguish positive and negative oropharyngeal swab samples of COVID‐19. Colorimetric assay provides a low‐cost and instrumental‐free solution for nucleic acid detection, which holds great potential for facilitating virus surveillance.
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