Chemical transcription roadblocking for nascent RNA display

Abstract

Site-specific arrest of RNA polymerase is fundamental to several technologies that measure RNA structure and function. Current in vitro transcription ‘roadblocking’ approaches inhibit transcription elongation using a protein blockade bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires the inclusion of a protein factor that is extrinsic to the minimal in vitro transcription reaction. In this work, we show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification efficiently and stably halts Escherichia coli RNA polymerase transcription. To facilitate diverse applications of chemical transcription roadblocking, we establish a simple and sequence-independent method for the preparation of internally modified double-stranded DNA templates by sequential PCR and translesion synthesis. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables nascent RNA molecules to be displayed from RNA polymerase in a minimal in vitro transcription reaction.