Watching single molecules in action

Abstract

A fluorescent imaging technique called fastFISH has been used to track the various steps involved in the transcription of a single DNA molecule.

Keywords: fluorescence; in situ hybridization; real-time; single-molecule; transcription; unstructured nucleic acid.

Figure 1.. Tracking the transcription of a DNA molecule with the fastFISH method.

The DNA molecule is labelled with a fluorescent Cy3 molecule and tethered to a glass cover slip; an optical field (not shown) is then used to generate fluorescence, which allows the position of the DNA molecule to be determined. Next, the Cy3 label on the DNA molecule is bleached out so Cy3 can be used to label the DNA probe. The T7 RNA polymerase (RNAP), which is labelled with a fluorescent Cy5 molecule, starts to transcribe the DNA molecule to produce an RNA molecule, and the Cy3 molecule in the DNA probe starts to fluoresce when the probe binds to the nascent RNA molecule. The RNAP and the DNA probe can be monitored independently and simultaneously (with a time resolution of 80 milliseconds) because the Cy5 and Cy3 molecules fluoresce at different wavelengths. It is also possible to distinguish between T7 RNAP bound to a promoter on the DNA and T7 RNAP transcribing the DNA because, in the latter case, the fluorescence from the Cy5 will coincide with fluorescence from the Cy3.