μIVC-Seq: A Method for Ultrahigh-Throughput Development and Functional Characterization of Small RNAs

Area of Science:

• Molecular Biology
• Biotechnology
• Microfluidics

Background:

• Systematic Evolution of Ligands by EXponential enrichment (SELEX) is effective for isolating high-affinity RNA binders.
• SELEX is less suitable for optimizing RNAs for complex functions like catalysis or fluorescence.
• Conventional screening assays for complex RNA functions are often cost-prohibitive due to scale limitations.

Purpose of the Study:

• To develop a miniaturized, highly parallelized screening technology for functional RNA optimization.
• To enable cost-effective, ultrahigh-throughput screening of millions of RNA mutants.
• To overcome the limitations of SELEX for complex functional RNA isolation.

Main Methods:

• Development of microfluidic-assisted In Vitro Compartmentalization (μIVC).
• Integration of μIVC with high-throughput sequencing (μIVC-seq pipeline).
• Utilizing droplet-based microfluidics for miniaturized and parallelized screening.

Main Results:

• μIVC allows functional screening of millions of RNA mutants in a single day.
• The μIVC-seq pipeline enables rapid, semi-automated, and low-cost screening.
• This approach significantly enhances the throughput for functional RNA development.

Conclusions:

• Microfluidic-assisted In Vitro Compartmentalization (μIVC) is a powerful tool for functional RNA screening.
• μIVC-seq overcomes the cost and throughput limitations of conventional methods for complex RNA functions.
• This technology facilitates the development of novel RNAs with advanced catalytic or signaling capabilities.