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Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers
Published on: September 19, 2017
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DNA G-Quadruplex Recognition In Vitro and in Live Cells by a Structure-Specific Nanobody
Silvia Galli1,2, Larry Melidis1,2, Sean M Flynn1
1Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, U.K.
Journal of the American Chemical Society
|December 9, 2022
Summary
Researchers developed SG4, a nanobody probe for detecting G-quadruplexes (G4s) in DNA. This new tool overcomes limitations of previous probes, enabling intracellular G4 imaging and mapping in live cells.
Area of Science:
- Genomics
- Molecular Biology
- Biophysics
Background:
- G-quadruplexes (G4s) are crucial DNA structures involved in genome regulation.
- Existing G4 probes like the BG4 antibody have limitations in expression and live-cell applications.
Purpose of the Study:
- To develop a novel, high-affinity nanobody probe for G4 detection.
- To enable intracellular imaging and mapping of G4 structures in live cells.
Main Methods:
- Selection of a camelid heavy-chain-only nanobody (SG4) against a human Myc DNA G4.
- In vitro affinity measurements and biophysical characterization.
- AlphaFold and molecular dynamics simulations for structural modeling.
- Intracellular expression and live-cell imaging of endogenous G4s.
Main Results:
- SG4 exhibits low nanomolar affinity for various folded G4 structures.
- Structural modeling accurately predicted key amino acid interactions and binding affinities.
- SG4 can be expressed intracellularly and used to visualize endogenous G4s in live cells.
- SG4 facilitates in situ and chromatin-bound G4 mapping.
Conclusions:
- SG4 is a potent nanobody probe for G4 detection and mapping.
- It overcomes limitations of previous G4 probes, enabling new cellular investigations.
- SG4 represents a valuable tool for studying G4s in their native cellular context.
