Effects of nucleotide substitution and modification on the stability and structure of helix 69 from 28S rRNA.

The helix 69 (H69) region of the large subunit (28S) rRNA of Homo sapiens contains five pseudouridine (Psi) residues out of 19 total nucleotides (26%), three of which are universally or highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine. The role of a loop nucleotide substitution from A in bacteria (position 1918 in Escherichia coli 23S rRNA) to G in eukaryotes (position in 3734 in H. sapiens) was also examined. The thermodynamic parameters were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by 1H NMR and circular dichroism spectroscopy. In addition, a [1,3-15N]Psi phosphoramidite was used to generate H69 analogs with site-specific 15N labels. By using this approach, different Psi residues can be clearly distinguished from one another in 1H NMR experiments. The effects of pseudouridine on H. sapiens H69 are consistent with previous studies on tRNA, rRNA, and snRNA models in which the nucleotide offers stabilization of duplex regions through PsiN1H-mediated hydrogen bonds. The overall secondary structure and base-pairing patterns of human H69 are similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved. Nonetheless, pseudouridine-containing RNAs have subtle differences in their structures and stabilities compared to the corresponding uridine-containing analogs, suggesting possible roles for Psi such as maintaining translation fidelity.

Solution conformations of two naturally occurring RNA nucleosides: 3-methyluridine and 3-methylpseudouridine.

The conformations of 3-methyluridine and 3-methylpseudouridine are determined using a combination of sugar proton coupling constants from 1D NMR spectra and 1D NOE difference spectroscopy. Both C2'-endo and C3'-endo conformations are observed for 3-methyluridine (59:41, 37 degrees C, D2O) and 3-methylpseudouridine (51:49, 37 degrees C, D2O). 3-Methyluridine preferentially adopts an anti conformation in solution, whereas 3-methylpseudouridine is primarily in a syn conformation. anti/syn-Relationships are deduced by 1D NOE difference spectroscopy.

NMR conformational analysis of p-tolyl furanopyrimidine 2'-deoxyribonucleoside and crystal structure of its 3',5'-di-O-acetyl derivative.

The conformation of a representative molecule of a new, potent class of antiviral-active modified nucleosides is determined. A bicyclic nucleoside, 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one, shows C2'-endo and C3'-endo ribose conformations in solution (63:37, 37 degrees C; DMSO-d6), as determined by 1H NMR studies. The crystal structure of a 3',5'-di-O-acetyl-protected derivative (monoclinic, P21, a/b/c= 6.666(1)/12.225(1)/24.676(2) A, beta=90.24(1) degrees , Z=4) shows exclusively C2'-endo deoxyribose puckering. The base is found in the anti position both in solution and in crystalline form.

Synthesis of helix 69 of Escherichia coli 23S rRNA containing its natural modified nucleosides, m(3)Psi and Psi.

The synthesis of 3-methylpseudouridine (m(3)Psi) phosphoramidite, 5'-O-[benzhydryloxybis(trimethylsilyloxy)silyl]-2'-O-[bis(2-acetoxyethoxy)methyl]-3-methylpseudouridine-3'-(methyl-N,N-diisopropyl)phosphoramidite, is reported. Selective pivaloyloxymethyl protection of the Psi N1 followed by methylation at N3 was used to generate the naturally occurring pseudouridine analogue. The m(3)Psi phosphoramidite was used in combination with pseudouridine (Psi) and standard base phosphoramidites to synthesize a 19-nucleotide RNA representing helix 69 of Escherichia coli 23S ribosomal RNA (rRNA) (residues 1906-1924), containing a single m(3)Psi at position 1915 and two Psi's at positions 1911 and 1917. Our synthesis of the fully modified helix 69 RNA demonstrates the ability to make milligram quantities of RNA that can be used for further high-resolution structure studies. Site-selective introduction of the methyl group at the N3 position of pseudouridine at position 1915 causes a slight increase in the thermodynamic stability of the RNA hairpin relative to pseudouridine; RNAs containing either uridine or 3-methyluridine at position 1915 have similar stability. One-dimensional imino proton NMR and circular dichroism spectra of the modified RNAs reveal that the methyl group does not cause any substantial changes in the RNA hairpin structure.

Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin.

The synthesis of a 5'-O-BzH-2'-O-ACE-protected-3-methyluridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The phosphoramidite was employed in solid-phase RNA synthesis to generate a series of RNA hairpins containing single or multiple modifications, including the common nucleoside pseudouridine. Three 19-nucleotide hairpin RNAs that represent the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S ribosomal RNA were generated. Modifications were present at positions 1911, 1915, and 1917. The stabilities and structures of the three RNAs were examined by using thermal melting, circular dichroism, and NMR spectroscopy