Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Identifying single-nucleotide mutations is crucial for understanding cell biology and disease diagnostics.
• Challenges exist in detecting subtle single-base changes within complex biological systems like living cells.

Purpose of the Study:

• To develop a novel class of prokaryotic riboregulators for ultraspecific RNA detection.
• To enable precise identification of single-nucleotide mutations in vivo and in vitro.

Main Methods:

• De novo design of single-nucleotide-specific programmable riboregulators (SNIPRs).
• Testing SNIPR performance in Escherichia coli (E. coli) for gene expression differences.
• Application of SNIPRs in cell-free transcription-translation systems for in vitro analysis.
• Development of an automated algorithm for designing SNIPRs targeting specific mutations.
• Integration of SNIPRs with paper-based cell-free reactions for isothermal detection.

Main Results:

• SNIPRs achieved over 100-fold differences in gene expression in response to single-nucleotide variations in E. coli.
• SNIPRs demonstrated the ability to resolve single epitranscriptomic marks in vitro.
• Designed SNIPRs for mutations linked to cancer, drug resistance, and genetic disorders.
• Enabled convenient, isothermal detection of cancer mutations and Zika virus strains using colorimetric readouts.

Conclusions:

• De novo-designed SNIPRs offer a powerful tool for ultraspecific RNA detection and single-nucleotide mutation identification.
• SNIPRs facilitate precise molecular diagnostics and biological probing in diverse settings.
• The integration with portable cell-free systems expands the accessibility of advanced molecular detection methods.