Tracking of single quantum dot labeled EcoRV sliding along DNA manipulated by double optical tweezers.
Fluorescence microscopy provides a powerful method to directly observe single enzymes moving along a DNA held in an extended conformation. In this work, we present results from single EcoRV enzymes labeled with quantum dots which interact with DNA manipulated by double optical tweezers. The application of quantum dots facilitated accurate enzyme tracking without photobleaching whereas the tweezers allowed us to precisely control the DNA extension. The labeling did not affect the biochemical activity of EcoRV checked by directly observing DNA digestion on the single molecule level. We used this system to demonstrate that during sliding, the enzyme stays in close contact with the DNA. Additionally, slight overstretching of the DNA resulted in a significant decrease of the 1D diffusion constant, which suggests that the deformation changes the energy landscape of the sliding interaction. Together with the simplicity of the setup, these results demonstrate that the combination of optical tweezers with fluorescence tracking is a powerful tool for the study of enzyme translocation along DNA.
Biebricher
Andreas
A
Wende
Wolfgang
W
Escudé
Christophe
C
Pingoud
Alfred
A
Desbiolles
Pierre
P
D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004058:Diffusion; D008856:Microscopy, Fluorescence / Q000379:methods*; D009690:Nucleic Acid Conformation; D052898:Optical Tweezers; D045663:Quantum Dots
D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases