NMR spectroscopic characterization of a model RNA duplex reflecting the core sequence of hammerhead ribozymes.

Hammerhead ribozymes are a model system for studying molecular mechanism of RNA catalysis. Physicochemical data-driven mechanistic studies are an indispensable step towards understanding the catalysis of hammerhead ribozymes. Here we characterized a model RNA duplex with catalytically important sheared-type G12-A9 base pair and A9-G10.1 metal ion-binding motif in hammerhead ribozymes. By using high magnetic field NMR, all base proton signals, including catalytic residues, were unambiguously assigned. We further characterized structural features of this RNA molecule and found that it reflects the structural features of the A9-G10.1 motif of hammerhead ribozymes. Therefore, this RNA molecule is suitable for extracting an intrinsic physicochemical properties of catalytically important residues.

Structure Determination of an Ag(I) -Mediated Cytosine-Cytosine Base Pair within DNA Duplex in Solution with (1) H/(15) N/(109) Ag NMR Spectroscopy.

The structure of an Ag(I) -mediated cytosine-cytosine base pair, C-Ag(I) -C, was determined with NMR spectroscopy in solution. The observation of 1-bond (15) N-(109) Ag J-coupling ((1) J((15) N,(109) Ag): 83 and 84 Hz) recorded within the C-Ag(I) -C base pair evidenced the N3-Ag(I) -N3 linkage in C-Ag(I) -C. The triplet resonances of the N4 atoms in C-Ag(I) -C demonstrated that each exocyclic N4 atom exists as an amino group (-NH2 ), and any isomerization and/or N4-Ag(I) bonding can be excluded. The 3D structure of Ag(I) -DNA complex determined with NOEs was classified as a B-form conformation with a notable propeller twist of C-Ag(I) -C (-18.3±3.0°). The (109) Ag NMR chemical shift of C-Ag(I) -C was recorded for cytidine/Ag(I) complex (δ((109) Ag): 442 ppm) to completed full NMR characterization of the metal linkage. The structural interpretation of NMR data with quantum mechanical calculations corroborated the structure of the C-Ag(I) -C base pair.

Direct detection of the mercury-nitrogen bond in the thymine-Hg(II)-thymine base-pair with (199)Hg NMR spectroscopy.

We have observed the 1-bond (199)Hg-(15)N J-coupling ((1)J((199)Hg,(15)N) = 1050 Hz) within the Hg(II)-mediated thymine-thymine base pair (T-Hg(II)-T). This strikingly large (1)J((199)Hg,(15)N) is the first one for canonical sp(2)-nitrogen atoms, which can be a sensitive structure-probe of N-mercurated compounds and a direct evidence for N-mercuration.

Site-specific isotope labeling of long RNA for structural and mechanistic studies.

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.