Site-Specific Isotope-Labeling of Inosine Phosphoramidites and NMR Analysis of an Inosine-Containing RNA Duplex.
Structural features and internal dynamics of inosine-containing RNAs are poorly understood. NMR studies of such RNAs require13C,15N-labeling, which cannot be achieved using in vitro transcription as inosine and guanosine are not distinguished by RNA polymerase. Herein, we report the synthesis of an inosine phosphoramidite with selective13C8 and15N7-isotope incorporation in the base and uniform13C-labeling of the ribose. Chemical synthesis of an RNA duplex containing four consecutive IU base pairs with this optimized isotope-labeling scheme greatly simplifies NMR spectra and resolves signal overlap. The absence of detectable NMR signals of imino protons and unusual inter-residue NOE correlations in this RNA indicate deviations from standard A-form geometry, consistent with reduced stability of this duplex seen in UV melting studies compared to its nonedited RNA counterparts. These studies indicate that the introduction of IU base pairs distorts and destabilizes RNA helices significantly compared to the also noncanonical GU base-pairs. Our optimized isotope-labeling scheme enables high-resolution NMR studies of inosine-edited RNAs.
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany.|Center for Integrated Protein Science Munich at Chair Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85747, Garching, Germany.|Department of Chemistry, Humboldt Universität zu Berlin, 12489, Berlin, Germany.
Andre
A
Dallmann
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany.|Center for Integrated Protein Science Munich at Chair Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85747, Garching, Germany.
Alexander V
AV
Beribisky
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany.
Felix
F
Gnerlich
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany.|Center for Integrated Protein Science Munich at Chair Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85747, Garching, Germany.
Martin
M
Rübbelke
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany.
Stefan
S
Schiesser
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377, Munich, Germany. thomas.carell@cup.lmu.de.
Thomas
T
Carell
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany. sattler@helmholtz-muenchen.de.|Center for Integrated Protein Science Munich at Chair Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85747, Garching, Germany. sattler@helmholtz-muenchen.de.
Michael
http://orcid.org/0000-0002-1594-0527
M
Sattler
D020029:Base Pairing; D006151:Guanosine / Q000737:chemistry*; D007288:Inosine / Q000737:chemistry*; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D019906:Nuclear Magnetic Resonance, Biomolecular; D009943:Organophosphorus Compounds / Q000737:chemistry*; D011522:Protons; D012313:RNA / Q000737:chemistry*; D012266:Ribose / Q000737:chemistry*
D009943:Organophosphorus Compounds; D011522:Protons; C434331:phosphoramidite; D006151:Guanosine; D007288:Inosine; D012313:RNA; D012266:Ribose
NMR spectroscopy; RNA structures; biosynthesis; isotopic labeling; structural biology