Related Experiment Video
Updated: Jul 15, 2026
12:05
A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA
Published on: October 1, 2017
Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA
Sangjin Kim1, Paul C Blainey, Charles M Schroeder
1Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Nature Methods
|April 17, 2007
Summary
We developed a high-resolution single-molecule assay to observe slow enzyme activity on DNA. This method tracks enzymes like DNA polymerase and reverse transcriptase with nanoscale precision over extended periods.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Observing slow or intermittent enzymatic activity at the single-molecule level is challenging.
- Existing assays may lack the necessary spatial resolution or temporal stability for detailed mechanistic studies.
Purpose of the Study:
- To develop a novel single-molecule assay for analyzing nucleic-acid enzymes.
- To achieve high spatial resolution and mechanical stability for detecting subtle enzymatic behaviors.
Main Methods:
- Utilized flow-stretched DNA templates for enzyme immobilization.
- Implemented a single-molecule assay with 15-nm spatial resolution and 1 Hz frame rate.
- Ensured approximately 10 nm mechanical stability over hours for long-term observation.
Main Results:
- Successfully applied the assay to various nucleic-acid enzymes, including phi29 DNA polymerase, HIV-1 reverse transcriptase, lambda exonuclease, and E. coli RNA polymerase.
- Demonstrated the capability to detect slow or intermittent enzymatic activities with high precision.
- Validated the assay's performance through multiplexed data collection.
Conclusions:
- The developed single-molecule assay provides a powerful tool for investigating the kinetics and mechanisms of nucleic-acid enzymes.
- This assay enables detailed characterization of enzyme dynamics previously inaccessible.
- Facilitates advancements in understanding enzyme function and potential therapeutic targets.
