Employing molecular beacons to assess in vitro transcription with single-molecule resolution.

Transcription is a vital cellular process in which RNA polymerase produces messenger RNAs (mRNA) from a DNA template. Many transcription systems have been developed to reproduce this process in vitro and in the confined environment of giant unilamellar vesicles (GUVs). However, these systems and the studies employing them often use DNA concentrations significantly higher than those found in natural cells, which typically contain a single DNA copy. In this work, we introduce single-molecule in vitro transcription (smIVT) that overcomes this limitation, enabling the visualization and tracking of individual mRNA molecules both in solution and within GUVs. We achieved this by employing a molecular beacon-a quenched RNA or DNA hairpin probe that becomes fluorescent after binding to the transcript mRNA-and template DNA encoding 32 repeats of the molecular beacon binding sequence. With these, we ensured a signal-to-noise ratio sufficient to detect single mRNAs. We use smIVT to compare the performance of various commercially-available in vitro transcription kits, and to quantitatively describe single molecule transcription inside GUVs. We establish smIVT as a remarkable technique for scrutinizing in vitro transcription with single-molecule resolution.