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

Overview

Journal Bio Protoc

Specialty Biology

Date 2021 Oct 4

PMID 34604447

Citations 2

Authors

Eric J Strobel

Affiliations

Soon will be listed here.

Abstract

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 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 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.

Citing Articles

Systematic analysis of cotranscriptional RNA folding using transcription elongation complex display.

Kelly S, Strobel E Nat Commun. 2025; 16(1):2350.

PMID: 40064876 PMC: 11894091. DOI: 10.1038/s41467-025-57415-3.

Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing.

Szyjka C, Strobel E Nat Commun. 2023; 14(1):7839.

PMID: 38030633 PMC: 10687018. DOI: 10.1038/s41467-023-43395-9.

Isolation of E. coli RNA polymerase transcription elongation complexes by selective solid-phase photoreversible immobilization.

Strobel E Methods Enzymol. 2023; 691:223-250.

PMID: 37914448 PMC: 10950060. DOI: 10.1016/bs.mie.2023.03.019.

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