Multiple segmental and selective isotope labeling of large RNA for NMR structural studies

Nucleic Acids Res. 2008 Aug;36(14):e89. doi: 10.1093/nar/gkn397. Epub 2008 Jun 26.

Abstract

Multiple segmental and selective isotope labeling of RNA with three segments has been demonstrated by introducing an RNA segment, selectively labeled with (13)C(9)/(15)N(2)/(2)H((1', 3', 4', 5', 5''))-labeled uridine residues, into the central position of the 20 kDa epsilon-RNA of Duck Hepatitis B Virus. The RNA molecules were produced via two efficient protocols: a two-step protocol, which uses T4 DNA ligase and T4 RNA ligase 1, and a one-pot protocol, which uses T4 RNA ligase 1 alone. With T4 RNA ligase 1 all not-to-be-ligated termini are usually protected to prevent formation of side products. We show that such labor-intensive protection of termini is not required, provided segmentation sites can be chosen such that the segments fold into the target structure or target-like structures and thus are not trapped into stable alternate structures. These sites can be reliably predicted via DINAMelt. The simplified NMR spectrum provided evidence for the presence of a U28 H(3)-imino resonance, previously obscured in the fully labeled sample, and thus of the non-canonical base pair U28:C37. The demonstrated multiple segmental labeling protocols are generally applicable to large RNA molecules and can be extended to more than three segments.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Carbon Isotopes
  • DNA Ligases
  • Deuterium
  • Electrophoresis, Polyacrylamide Gel
  • Hepatitis B Virus, Duck / genetics
  • Isotope Labeling / methods*
  • Molecular Sequence Data
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular*
  • RNA / chemistry*
  • RNA Ligase (ATP)
  • RNA, Viral / biosynthesis
  • RNA, Viral / chemistry
  • Viral Proteins

Substances

  • Carbon Isotopes
  • Nitrogen Isotopes
  • RNA, Viral
  • Viral Proteins
  • RNA
  • Deuterium
  • DNA Ligases
  • RNA Ligase (ATP)
  • bacteriophage T4 RNA ligase 1