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Assaying RNA structure with LASER-Seq.

Boris Zinshteyn1, Dalen Chan2, Whitney England2

  • 1Department of Molecular Biology and Genetics, Johns Hopkins University. Baltimore, MD 21205, USA.

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|November 27, 2018
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Summary
This summary is machine-generated.

We developed Light Activated Structural Examination of RNA by high-throughput sequencing (LASER-Seq) to measure RNA structure in cells. LASER-Seq provides insights into RNA solvent accessibility and ligand binding sites, even in complex structures.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genomics

Background:

  • Chemical probing is essential for understanding RNA structure and function.
  • Existing methods primarily detect Watson-Crick pairing, limiting tertiary structure insights.
  • Solvent accessibility is a key tertiary structure parameter, especially in RNA-protein complexes.

Purpose of the Study:

  • To develop a novel chemical probing method for measuring RNA structure in living cells.
  • To introduce Light Activated Structural Examination of RNA by high-throughput sequencing (LASER-Seq).
  • To assess LASER-Seq's utility for probing solvent accessibility and identifying ligand binding sites.

Main Methods:

  • LASER-Seq utilizes a light-generated nicotinoyl nitrenium ion to adduct C8 of adenosine and guanosine.
  • Reactivity is dependent on the accessibility of the C8 position to the probe.
  • LASER-Seq was integrated with RT-stop and mutational profiling (MaP) for deep sequencing analyses.

Main Results:

  • LASER reactivity demonstrated a strong correlation with solvent accessibility across the ribosome.
  • The method was successfully applied to identify ligand binding sites in an unbiased manner.
  • LASER-Seq effectively modified paired nucleotides, revealing conformational changes in structured RNA.

Conclusions:

  • LASER-Seq is a powerful new tool for high-throughput RNA structure analysis in cellular environments.
  • The method provides valuable information on solvent accessibility and ligand interactions.
  • LASER-Seq offers advantages for studying conformational dynamics and binding events in complex RNA molecules.