title: Multiple segmental and selective isotope labeling of large RNA for NMR structural studies.
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.
authors.affiliation: Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.
authors.forename: Frank H T
authors.initials: FH
authors.lastname: Nelissen
authors.forename: Adriaan J
authors.initials: AJ
authors.lastname: van Gammeren
authors.forename: Marco
authors.initials: M
authors.lastname: Tessari
authors.forename: Frederic C
authors.initials: FC
authors.lastname: Girard
authors.forename: Hans A
authors.initials: HA
authors.lastname: Heus
authors.forename: Sybren S
authors.initials: SS
authors.lastname: Wijmenga
mesh_terms: D001483:Base Sequence; D002247:Carbon Isotopes; D011088:DNA Ligases; D003903:Deuterium; D004591:Electrophoresis, Polyacrylamide Gel; D015611:Hepatitis B Virus, Duck / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D012313:RNA / Q000737:chemistry*; D011130:RNA Ligase (ATP); D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D014764:Viral Proteins
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012367:RNA, Viral; D014764:Viral Proteins; D012313:RNA; D003903:Deuterium; D011088:DNA Ligases; D011130:RNA Ligase (ATP); C477285:bacteriophage T4 RNA ligase 1