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Updated: Jul 12, 2025

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RNA Infrastructure Profiling Illuminates Transcriptome Structure in Crowded Spaces.

Lu Xiao1, Linglan Fang1, Eric T Kool1,2

  • 1Department of Chemistry, Stanford University, Stanford, CA 94305, United States.

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Summary
This summary is machine-generated.

Small probes offer enhanced RNA structural analysis within cells. This RNA infrastructure profiling (RISP) method improves understanding of RNA structure, modification, and interactions, especially in crowded cellular environments.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genomics

Background:

  • Cellular RNA exists in compact 3D structures, often interacting with proteins, which limits the effectiveness of traditional structure-probing agents.
  • Understanding RNA folding and modification within the crowded cellular environment is crucial for deciphering RNA biology.
  • Existing methods struggle to access occluded RNA structures and differentiate close molecular interactions.

Approach:

  • Developed and utilized small 2'-OH-reactive probes, such as acetylimidazole (AcIm), to access confined RNA structures within living cells.
  • Compared the performance of small probes against larger, conventional reagents (e.g., NAIN3) using RNA-Seq data to analyze RNA structure and protein proximity.
  • Applied the RNA infrastructure profiling (RISP) method to transcriptome-wide analysis in HEK293 cells, including structurally characterized complexes like human 18S and 28S RNA.

Key Points:

  • Small probes provide significantly broader coverage for intracellular RNA structural analysis compared to larger probes.
  • The smallest probe, acetylimidazole (AcIm), yielded 80% greater structural coverage than the conventional reagent NAIN3.
  • Acetyl probes demonstrated superior signal for identifying N6-methyladenosine (m6A) modification sites, including those inaccessible to larger probes.

Conclusions:

  • RNA infrastructure profiling (RISP) with small probes enhances the analysis of transcriptome structure, modification, and interactions in living cells.
  • This approach is particularly effective in spatially crowded cellular settings, overcoming limitations of previous methods.
  • RISP offers a powerful new tool for detailed investigation of RNA biology within its native cellular context.