Molecular dynamics simulation-guided toehold mediated strand displacement probe for single-nucleotide variants...

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• Single nucleotide variants (SNVs) are crucial biomarkers for diseases like cancer and infections.
• Toehold-mediated strand displacement (TMSD) is a DNA nanotechnology reaction used for SNV identification.
• The precise location of mismatches significantly impacts TMSD's ability to discriminate between variants.

Purpose of the Study:

• To comprehensively investigate how mismatch location affects TMSD kinetics.
• To provide mechanistic insights into TMSD at the single-nucleotide level.
• To guide the design of optimized TMSD systems for biosensing applications.

Main Methods:

• Utilized molecular dynamic simulations (oxDNA) with umbrella sampling and forward flux sampling.
• Investigated TMSD kinetics by systematically varying mismatch locations.
• Experimentally validated simulation findings using nine disease-related SNVs.

Main Results:

• Mismatches at the border of the toehold and branch migration domains result in the lowest TMSD reaction rates.
• Experimental results showed good agreement with simulation predictions.
• Optimized mismatch placement achieved a high discrimination factor (median of 124) between SNVs and wild types.
• A probe-sink system detected low variant allele frequencies (0.1%) with a 3 S/N ratio.
• Successfully detected SNVs in PCR clones with high confidence.

Conclusions:

• Mismatch location critically influences TMSD kinetics and discrimination ability.
• This study offers a mechanistic understanding of TMSD at the single-nucleotide level.
• The findings provide a framework for designing highly specific and sensitive TMSD-based biosensors.