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Related Concept Videos

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

¹H NMR of Conformationally Flexible Molecules: Temporal Resolution

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...
¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR01:15

¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
2D NMR: Overview of Homonuclear Correlation Techniques01:16

2D NMR: Overview of Homonuclear Correlation Techniques

Homonuclear correlation spectroscopy (COSY) is a powerful technique used in Nuclear Magnetic Resonance (NMR) spectroscopy to study the correlations between nuclei of the same type within a molecule. It provides information about scalar couplings between adjacent nuclei, which helps determine connectivity and structural information. There are several COSY variants, each with its unique strengths and experimental parameters.
COSY90 is the standard two-dimensional (2D) COSY experiment that...
¹H NMR: Complex Splitting01:13

¹H NMR: Complex Splitting

A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
Two-Dimensional (2D) NMR: Overview01:12

Two-Dimensional (2D) NMR: Overview

The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily interpreted using 2-dimensional (2D) NMR. Unlike 1D NMR, 2D NMR has two frequency axes that provide the coupling information between the nucleus A and nucleus B in a molecule. The process from which 2D spectra are obtained has four steps.
The first step is the preparation period, during which nucleus A is excited with a radiofrequency pulse.
Applications Of NMR In Biology01:25

Applications Of NMR In Biology

Nuclear magnetic resonance (NMR) spectroscopy is a very valuable analytical technique for researchers. It has been used for more than 50 years as an analytical tool. F. Bloch and E. Purcell formulated NMR in 1946 and won the 1952 Nobel Prize in Physics  for their work. Biological macromolecules such as proteins, nucleic acids, lipids, and organic molecules including pharmaceutical compounds, can be studied using this versatile tool that exploits the magnetic properties of certain nuclei.
The...

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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
14:55

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Published on: September 17, 2017

RNA structure determination by NMR.

Lincoln G Scott1, Mirko Hennig

  • 1Cassia, LLC, San Diego, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 20, 2008
PubMed
Summary
This summary is machine-generated.

This study details methods for determining RNA structure using liquid-state nuclear magnetic resonance (NMR). It covers RNA sample preparation, resonance assignment, and structure calculation for detailed molecular insights.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for determining the three-dimensional structure of biomolecules in solution.
  • Accurate RNA structure determination is crucial for understanding its diverse biological functions, including catalysis, regulation, and scaffolding.
  • Challenges in RNA structure determination include obtaining sufficient quantities of pure, structurally intact RNA and assigning NMR resonances.

Purpose of the Study:

  • To provide a comprehensive review of methodologies for RNA structure determination using liquid-state NMR.
  • To outline a practical strategy for RNA structure studies in solution, from sample preparation to structure calculation.
  • To highlight key steps and considerations for successful NMR-based RNA structure analysis.

Main Methods:

  • Review of in vitro transcription methods using T7 RNA polymerase for producing milligram quantities of isotopically labeled RNA.
  • Discussion of purification techniques including denaturing polyacrylamide gel electrophoresis (PAGE) and anion-exchange chromatography.
  • Outline of the NMR-based RNA structure determination workflow: resonance assignment, restraint collection, and structure calculation.

Main Results:

  • Demonstration of the critical role of routine production of milligram quantities of isotopically labeled RNA for NMR success.
  • Presentation of a basic strategy for studying RNA in solution by NMR, covering essential steps.
  • Inclusion of selected NMR spectra examples for a correctly folded 30-nucleotide RNA to illustrate the process.

Conclusions:

  • Liquid-state NMR is a viable and powerful method for high-resolution RNA structure determination.
  • Standardized protocols for RNA sample preparation and purification are essential for successful NMR studies.
  • The presented strategy provides a framework for researchers to pursue NMR-based RNA structure elucidation.