Multiple segmental and selective isotope labeling of large RNA for NMR structural studies.
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.
Nelissen
Frank H T
FH
Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.
van Gammeren
Adriaan J
AJ
Tessari
Marco
M
Girard
Frederic C
FC
Heus
Hans A
HA
Wijmenga
Sybren S
SS
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
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