Cotranscriptional RNA Chemical Probing

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Cotranscriptional folding is essential for RNA biogenesis and function.
• Understanding dynamic RNA structures during synthesis is key to RNA-mediated gene regulation.
• Existing methods lack systematic identification of intermediate RNA structures.

Purpose of the Study:

• To present guidelines for cotranscriptional RNA chemical probing.
• To detail a workflow integrating in vitro transcription with high-throughput RNA structure probing.
• To enable systematic identification of intermediate RNA structures formed during transcription.

Main Methods:

• Single-round in vitro transcription using E. coli RNA polymerase.
• High-throughput RNA chemical probing of cotranscriptionally folded intermediates.
• DNA template design and transcription roadblocking strategies.
• RNA purification and 3' adapter ligation for sequencing library preparation.

Main Results:

• A comprehensive protocol for cotranscriptional RNA chemical probing is described.
• The method allows for the study of intermediate RNA structures as they form.
• A purified RNA library suitable for Illumina sequencing is generated.

Conclusions:

• This chapter provides a robust experimental framework for studying cotranscriptional RNA folding.
• The described methodology facilitates a deeper understanding of RNA structure dynamics.
• This approach is vital for elucidating RNA's role in gene regulation.