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¹H NMR of Conformationally Flexible Molecules: Temporal Resolution00:52

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At room temperature, the chair conformer of cyclohexane undergoes rapid ring flipping between two equivalent chair conformers at a rate of approximately 105 times per second. These two chair conformers are in equilibrium. The rapid ring flipping results in the interconversion of the axial proton to an equatorial proton and an equatorial to the axial proton. Such interconversions are too rapid and cannot be detected on the NMR timescale. Hence, the NMR spectrometer cannot distinguish between the...
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RNA tertiary structure and conformational dynamics revealed by BASH MaP.

Maxim Oleynikov1, Samie R Jaffrey1

  • 1Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, USA.

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|April 22, 2024
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Summary

We developed BASH MaP and DAGGER to reveal hidden RNA tertiary structures and alternative conformations. This method uncovers previously undetectable RNA structures, crucial for understanding RNA function.

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Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • RNA molecules fold into complex three-dimensional tertiary structures that dictate their function.
  • Predicting RNA tertiary structures and identifying alternative conformations remains a significant challenge in molecular biology.

Purpose of the Study:

  • To develop and validate a novel method for identifying alternative RNA tertiary structures.
  • To investigate the conformational diversity and dynamics of RNA G-quadruplexes and the Spinach aptamer.

Main Methods:

  • Development of BASH MaP (dimethyl sulfate footprinting) to probe guanosine N7 accessibility, a key tertiary structure mediator.
  • Integration of BASH MaP with DAGGER, a computational pipeline, for analyzing single-molecule footprinting data.
  • Application of BASH MaP and DAGGER to study RNA G-quadruplexes in vitro and in cells, and the Spinach aptamer.

Main Results:

  • BASH MaP successfully identified diverse conformational states and dynamics of RNA G-quadruplexes.
  • Analysis of the Spinach aptamer revealed alternative tertiary conformations influencing its fluorescence.
  • The study demonstrated the ability of BASH MaP to detect previously inaccessible RNA tertiary structures.

Conclusions:

  • BASH MaP coupled with DAGGER provides a powerful approach for structural analysis of RNA.
  • This method enables the discovery of alternative RNA tertiary structures and conformational heterogeneity.
  • Understanding RNA conformational dynamics is essential for elucidating RNA function and regulation.