Home›Neuroscience›Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1
NeuroscienceJoVE (Open Access)Citable · DOI
Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1
DOI: 10.3791/60045-v
What you'll learn
✓Set up flow cells and prepare surfaces for single-molecule FRET experiments
✓Perform two-color alternating excitation (ALEX) to determine dissociation constants
✓Analyze real-time smFRET data to obtain dwell time distributions during HJ resolution
✓Interpret single-molecule kinetics of GEN1-mediated Holliday junction cleavage
Protocol
Biopharma Insights Presented here is a protocol for performing single-molecule Förster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.
Difficulty
advanced
Total time
~4–6 hours per experiment (including surface preparation, data acquisition, and analysis)
Steps
1
Prepare flow cells for smFRET experiments
Assemble and prepare flow cells with appropriate surface chemistry for immobilizing Holliday junction substrates and proteins. This includes cleaning, coating, and functionalization of coverslips.
▶ 01:22
2
Conduct single-molecule FRET experiments
Perform smFRET measurements using single-color time-lapse imaging to monitor real-time Holliday junction cleavage by GEN1 and record dwell time distributions.
▶ 03:01
3
Analyze smFRET data for kinetic parameters
Process and analyze single-molecule FRET time series to extract dwell times, transition rates, and kinetic information related to HJ resolution.