Functional analysis of single enzymes combining programmable molecular circuits with droplet-based microfluidics

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Ensemble-averaged techniques mask single-molecule protein behaviors.
• Traditional digital enzyme quantification relies on linear amplification and is limited to high-turnover enzymes.
• There is a need for sensitive methods to quantify diverse enzymes at the single-molecule level.

Purpose of the Study:

• To develop a novel method for ultrasensitive, single-molecule quantification of DNA-related enzymes.
• To expand the range of enzymes amenable to single-molecule analysis.
• To investigate enzyme activity heterogeneity and inactivation pathways.

Main Methods:

• Integration of an exponential molecular amplifier with DNA-enzyme circuits and droplet microfluidics.
• Development of digital PUMA (Programmable Ultrasensitive Molecular Amplifier) assay.
• Validation across diverse enzymes, including low-turnover enzymes and Cas9 variants.

Main Results:

• Digital PUMA enables specific detection and absolute molar quantification of virtually any DNA-related enzyme at the single-molecule level.
• The method successfully quantified numerous enzymes, including those with slow catalytic rates and Streptococcus pyogenes Cas9 at single turnover.
• Analysis revealed a significant fraction of inactive enzymes in commercial preparations and provided insights into enzyme inactivation pathways.

Conclusions:

• Digital PUMA significantly broadens the scope of enzymes quantifiable at single-molecule resolution.
• The technology offers a versatile framework for accurate enzyme quantification in diagnostics and biotechnology.
• This approach facilitates the study of protein functional heterogeneity and the origins of enzyme inactivation.