Single Photon Sensitivity Camera Helps Prove EcoRV Enzymes Can Jump As Well As Slide

A scientific digital camera with single photon sensitivity used in tandem with total internal reflection microscopy (TIRFM) has enabled researchers in Paris to prove that EcoRV enzymes can jump as well as slide along strands of DNA.

It is hoped this discovery will help shed light on how site-specific proteins rapidly find very short DNA sequences in order to preserve cellular integrity and protect cells against viral attacks.

To conduct the research, the group, which was led by Dr Pierre Desbiolles at the Laboratoire Kastler Brossel, CRNS and Université Pierre et Marie Curie, developed a system based on an Andor Technology iXon860 EMCCD capable of detecting single photons originating from fluorescent Cy3 molecules bound to individual enzyme molecules. This challenging level of detection was required as each EcoRV enzyme was only tagged with one of two Cy3 labels and imaging had be undertaken dynamically.

By acquiring extended time series of individual DNA binding events, the team was able to confirm an earlier suggestion that EcoRV could slide along a DNA strand in order to access areas nearby. However, it was unclear whether EcoRV could also jump along DNA between fragments which are spaced further apart. The clear images obtained with their direct microscopic observation technique allowed the researchers to confirm, for the first time, that long-distance excursions of enzyme on DNA were, indeed, possible.

To make this breakthrough, Dr. Desbiolles needed a detection system with an ultra high level of sensitivity. He chose to base it on an Andor Technology iXon860 back-illuminated EMCCD camera because it combined the ability to detect single photons with a frame rate that allowed them to follow the rapidly changing events associated with DNA proof reading and repair.