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Protocol for forming G-quadruplexes from double-stranded DNA during transcription

Year: 2025

Type: article

Abstract: G-quadruplexes are four-stranded nucleic acid structures that play vital roles in regulating gene expression, maintaining genomic stability, and supporting various biological processes. This protocol details their formation from double-stranded DNA via in vitro transcription. It includes steps for selecting suitable DNA templates, assembling necessary components (such as RNA polymerase, nucleotides, and buffers), setting optimal incubation conditions, and performing dimethyl sulfate (DMS) footprinting to analyze the structures. For complete details on the use and execution of this protocol, please refer to Zhang et al. 1 and Gong et al. 2 • Steps for methylation and cleavage of DNA using DMS and piperidine • Instructions for assembling in vitro transcription components • Guidance on optimizing G-quadruplex formation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. G-quadruplexes are four-stranded nucleic acid structures that play vital roles in regulating gene expression, maintaining genomic stability, and supporting various biological processes. This protocol details their formation from double-stranded DNA via in vitro transcription. It includes steps for selecting suitable DNA templates, assembling necessary components (such as RNA polymerase, nucleotides, and buffers), setting optimal incubation conditions, and performing dimethyl sulfate (DMS) footprinting to analyze the structures.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.103677

Authors: Jiayu Zhang, Jiayuan Gong, Jun Chen, Hanqing Chen