Preparation and Characterization of Internally Modified DNA Templates for Chemical Transcription Roadblocking

Site-specific transcription arrest is the basis of emerging technologies that assess nascent RNA structure and function. Cotranscriptionally folded RNA can be displayed from an arrested RNA polymerase (RNAP) for biochemical manipulations by halting transcription elongation at a defined DNA template position. Most transcription "roadblocking" approaches halt transcription elongation using a protein blockade that is non-covalently attached to the template DNA. I previously developed a strategy for halting Escherichia coli RNAP at a chemical lesion, which expands the repertoire of transcription roadblocking technologies and enables sophisticated manipulations of the arrested elongation complexes. To facilitate this chemical transcription roadblocking approach, I developed a sequence-independent method for preparing internally modified dsDNA using PCR and translesion synthesis. Here, I present a detailed protocol for the preparation and characterization of internally modified dsDNA templates for chemical transcription roadblocking experiments. Graphic abstract: Precise transcription roadblocking using functionalized DNA lesions.