Study and visualize DNA transcription mechanisms at the nanoscale

In this experiment, the exonuclease activity of the T7 polymerase was investigated. This is a DNA polymerase from the T7 bacteriophage that copies DNA strands in the 5’→ 3′ direction, and also features exonuclease activity. For this analysis, an optically trapped double-stranded DNA was held at a constant force that induced exonucleolytic activity of the polymerase. By removing nucleotide after nucleotide from one strand, the polymerase was unwinding the dsDNA. As the length of single-stranded DNA is longer compared to its double-stranded state, the unwinding resulted in a gradual increase in the end-to-end distance of the DNA (Figure a). This change in length was directly translated into the activity of the T7 DNA polymerase and the number of nucleotides it processed over time (Figure b). Specifically, short activity bursts ranging between 3 and 10 nucleotides were revealed, interspersed by frequent pauses of varying duration. This provided deeper insights into the dynamics of T7 exonuclease activity.

The C-Trap Optical Tweezer systems are highly valuable in providing novel insights into the mechanism of DNA-processing enzymes, as they enable performing single-molecule measurements of the stepping behavior of biomolecular motors along nucleic acids.

‍