Skip to main content
construction release_alert
Scholars@Duke will be undergoing maintenance April 11-15. Some features may be unavailable during this time.
cancel

Characterizing RNA Excited States Using NMR Relaxation Dispersion.

Publication ,  Journal Article
Xue, Y; Kellogg, D; Kimsey, IJ; Sathyamoorthy, B; Stein, ZW; McBrairty, M; Al-Hashimi, HM
Published in: Methods Enzymol
2015

Changes in RNA secondary structure play fundamental roles in the cellular functions of a growing number of noncoding RNAs. This chapter describes NMR-based approaches for characterizing microsecond-to-millisecond changes in RNA secondary structure that are directed toward short-lived and low-populated species often referred to as "excited states." Compared to larger scale changes in RNA secondary structure, transitions toward excited states do not require assistance from chaperones, are often orders of magnitude faster, and are localized to a small number of nearby base pairs in and around noncanonical motifs. Here, we describe a procedure for characterizing RNA excited states using off-resonance R1ρ NMR relaxation dispersion utilizing low-to-high spin-lock fields (25-3000 Hz). R1ρ NMR relaxation dispersion experiments are used to measure carbon and nitrogen chemical shifts in base and sugar moieties of the excited state. The chemical shift data are then interpreted with the aid of secondary structure prediction to infer potential excited states that feature alternative secondary structures. Candidate structures are then tested by using mutations, single-atom substitutions, or by changing physiochemical conditions, such as pH and temperature, to either stabilize or destabilize the candidate excited state. The resulting chemical shifts of the mutants or under different physiochemical conditions are then compared to those of the ground and excited states. Application is illustrated with a focus on the transactivation response element from the human immune deficiency virus type 1, which exists in dynamic equilibrium with at least two distinct excited states.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

Methods Enzymol

DOI

EISSN

1557-7988

Publication Date

2015

Volume

558

Start / End Page

39 / 73

Location

United States

Related Subject Headings

  • Transcriptional Activation
  • Thermodynamics
  • Temperature
  • RNA, Viral
  • RNA Folding
  • Nucleic Acid Conformation
  • Nitrogen
  • Mutation
  • Molecular Sequence Data
  • Molecular Dynamics Simulation
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Xue, Y., Kellogg, D., Kimsey, I. J., Sathyamoorthy, B., Stein, Z. W., McBrairty, M., & Al-Hashimi, H. M. (2015). Characterizing RNA Excited States Using NMR Relaxation Dispersion. Methods Enzymol, 558, 39–73. https://doi.org/10.1016/bs.mie.2015.02.002
Xue, Yi, Dawn Kellogg, Isaac J. Kimsey, Bharathwaj Sathyamoorthy, Zachary W. Stein, Mitchell McBrairty, and Hashim M. Al-Hashimi. “Characterizing RNA Excited States Using NMR Relaxation Dispersion.Methods Enzymol 558 (2015): 39–73. https://doi.org/10.1016/bs.mie.2015.02.002.
Xue Y, Kellogg D, Kimsey IJ, Sathyamoorthy B, Stein ZW, McBrairty M, et al. Characterizing RNA Excited States Using NMR Relaxation Dispersion. Methods Enzymol. 2015;558:39–73.
Xue, Yi, et al. “Characterizing RNA Excited States Using NMR Relaxation Dispersion.Methods Enzymol, vol. 558, 2015, pp. 39–73. Pubmed, doi:10.1016/bs.mie.2015.02.002.
Xue Y, Kellogg D, Kimsey IJ, Sathyamoorthy B, Stein ZW, McBrairty M, Al-Hashimi HM. Characterizing RNA Excited States Using NMR Relaxation Dispersion. Methods Enzymol. 2015;558:39–73.

Published In

Methods Enzymol

DOI

EISSN

1557-7988

Publication Date

2015

Volume

558

Start / End Page

39 / 73

Location

United States

Related Subject Headings

  • Transcriptional Activation
  • Thermodynamics
  • Temperature
  • RNA, Viral
  • RNA Folding
  • Nucleic Acid Conformation
  • Nitrogen
  • Mutation
  • Molecular Sequence Data
  • Molecular Dynamics Simulation