RNA G-quadruplex (rG4) structure detection using RTS and SHALiPE assays

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RNA G-quadruplexes (rG4s) are non-canonical RNA structures found across diverse species.
• rG4s play crucial roles in fundamental cellular processes.
• Recent advancements in RNA structure probing are essential for understanding rG4s.

Purpose of the Study:

• To present a detailed experimental protocol for RNA G-quadruplex analysis.
• To describe the application of Reverse transcriptase stalling (RTS) and SHALiPE assays for rG4 characterization.
• To enable nucleotide-resolution structural conformation analysis of rG4s in vitro.

Main Methods:

• In vitro transcription for preparing specific RNA molecules.
• Detailed procedures for Reverse transcriptase stalling (RTS) assay.
• Detailed procedures for selective 2'-hydroxyl acylation analyzed by lithium ion-based primer extension (SHALiPE) assay.

Main Results:

• The protocol facilitates the examination of rG4 formation.
• The assays allow for the revelation of rG4 structural conformation at nucleotide resolution.
• Successful characterization of distinct structural features of an rG4 structure of interest.

Conclusions:

• RTS and SHALiPE assays provide a robust method for in vitro rG4 structural analysis.
• The described protocol is accessible to molecular and chemical biologists with basic nucleic acid knowledge.
• The entire procedure, from RNA preparation to assay completion, is time-efficient.