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Updated: Jun 9, 2025
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Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
Published on: December 9, 2022
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Sequential structure probing of cotranscriptional RNA folding intermediates.
Courtney E Szyjka1, Skyler L Kelly1, Eric J Strobel1
1Department of Biological Sciences, The University at Buffalo, Buffalo, NY 14260, USA.
Biorxiv : the Preprint Server for Biology
|October 28, 2024
Summary
We developed a new method, TECprobe-LM, to directly observe RNA folding during transcription. This technique tracks RNA structure rearrangements as new nucleotides are added, offering a clearer view of cotranscriptional RNA folding pathways.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Biology
Background:
- Cotranscriptional RNA folding is crucial for gene regulation and function.
- Current methods infer RNA structure rearrangements indirectly from arrested transcription complexes.
- Direct assessment of dynamic RNA folding during elongation is lacking.
Purpose of the Study:
- To develop and validate a novel method for directly probing cotranscriptional RNA structure rearrangement.
- To overcome the limitations of existing techniques in assessing dynamic RNA folding.
- To provide a tool for detailed analysis of RNA folding pathways as they occur.
Main Methods:
- Developed linked-multipoint Transcription Elongation Complex RNA structure probing (TECprobe-LM).
- Sequentially positioned RNA polymerase (RNAP) at multiple sites on a DNA template.
- Applied chemical probing to nascent RNA at defined positions during transcription.
Main Results:
- TECprobe-LM directly assesses cotranscriptional RNA structure rearrangement.
- Validated the method using known folding events in E. coli SRP RNA, C. beijerinckii pfl ZTP riboswitch, and B. cereus crcB fluoride riboswitch.
- Demonstrated the capability to detect dynamic structural changes during RNA synthesis.
Conclusions:
- TECprobe-LM is a powerful new strategy for directly studying cotranscriptional RNA folding.
- The method enables direct observation of RNA structure dynamics during transcription.
- This technique advances the understanding of RNA folding pathways and their regulation.
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