Figure 1.

A schematic of the in vitro single-round quenched-kinetics transcription assay. The transcription from stable open complexes (RP_ITC=2, prepared in buffer) starts with addition of NTPs under crowding conditions and proceeds until quenched by a reaction quencher. RNA transcripts produced by single-round transcription reactions during the incubation time (t_incubation) are hybridized with the ssDNA probe (black line with two dots) that has sequence complementary to part of the transcript (Supplementary Figure S1). The hybridized fraction can be detected by ALEX-FAMS because the stretched structure of hybridized probe shows lower FRET efficiency compared to that of an unhybridized probe (26,27,36).