Figure 1

Single-molecule, real-time (SMRT) reverse transcription in zero-mode waveguides (ZMWs). (a) Schematic of the SMRT reverse transcription process. The RNA template (purple) is hybridized to a biotinylated DNA primer (orange) and immobilized at the ZMW bottom (I). Reverse transcriptase (RT, gray) binds to the immobilized hybrid (II). Upon initiation of reverse transcription, a correctly base-paired phospholinked nucleotide binds in the enzyme’s active site (III). The bound nucleotide can either dissociate from the complex (reverse reaction), or become incorporated into the growing DNA chain, accompanied by the release of the labeled pyrophosphate (IV). HIV RT then translocates to the next position and the reaction cycle (II) through (IV) repeats. (b) Hybrid for demonstrating SMRT reverse transcription, showing the RNA template (purple), DNA primer (orange), and resulting DNA (base-specific color-coding). (c) Example trace of SMRT reverse transcription. Fluorescence pulses are color-coded as in (b). Each block of pulses belonging to the same nucleotide is indicated above the trace. The gray block at the end of the trace likely corresponds to non-templated cognate sampling. (d-e) Magnified views of the beginning and end of the trace, respectively.