O-methyl Modification Research Articles

Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.

A systematic, ligation-based approach to study RNA modifications

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.

New Insights into the Glycosylation of the Surface Layer Protein SgsE from Geobacillus stearothermophilus NRS 2004/3a

The surface of Geobacillus stearothermophilus NRS 2004/3a cells is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. To this S-layer glycoprotein, elongated glycan chains are attached that are composed of [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-L-Rhap-(1-->] repeating units, with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain and a core saccharide as linker to the S-layer protein. On sodium dodecyl sulfate-polyacrylamide gels, four bands appear, of which three represent glycosylated S-layer proteins. In the present study, nanoelectrospray ionization time-of-flight mass spectrometry (MS) and infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry were adapted for analysis of this high-molecular-mass and water-insoluble S-layer glycoprotein to refine insights into its glycosylation pattern. This is a prerequisite for artificial fine-tuning of S-layer glycans for nanobiotechnological applications. Optimized MS techniques allowed (i) determination of the average masses of three glycoprotein species to be 101.66 kDa, 108.68 kDa, and 115.73 kDa, (ii) assignment of nanoheterogeneity to the S-layer glycans, with the most prevalent variation between 12 and 18 trisaccharide repeating units, and the possibility of extension of the already-known -->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1--> core by one additional rhamnose residue, and (iii) identification of a third glycosylation site on the S-layer protein, at position threonine-590, in addition to the known sites threonine-620 and serine-794. The current interpretation of the S-layer glycoprotein banding pattern is that in the 101.66-kDa glycoprotein species only one glycosylation site is occupied, in the 108.68-kDa glycoprotein species two glycosylation sites are occupied, and in the 115.73-kDa glycoprotein species three glycosylation sites are occupied, while the 94.46-kDa band represents nonglycosylated S-layer protein.

Retention of Core Catalytic Functions by a Conserved Minimal Ribonuclease E Peptide That Lacks the Domain Required for Tetramer Formation

Ribonuclease E (RNase E) is a multifunctional endoribonuclease that has been evolutionarily conserved in both Gram-positive and Gram-negative bacteria. X-ray crystallography and biochemical studies have concluded that the Escherichia coli RNase E protein functions as a homotetramer formed by Zn linkage of dimers within a region extending from amino acid residues 416 through 529 of the 116-kDa protein. Using fragments of RNase E proteins from E. coli and Haemophilus influenzae, we show here that RNase E derivatives that are as short as 395 amino acid residues and that lack the Zn-link region shown previously to be essential for tetramer formation (i.e. amino acid residues 400-415) are catalytically active enzymes that retain the 5' to 3' scanning ability and cleavage site specificity characteristic of full-length RNase E and that also confer colony forming ability on rne null mutant bacteria. Further truncation leads to loss of these properties. Our results, which identify a minimal catalytically active RNase E sequence, indicate that contrary to current models, a tetrameric quaternary structure is not required for RNase E to carry out its core enzymatic functions.

X-Ray analyses of hybrid duplexes between antisense oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine and their target RNAs

Incorporation of 5-(N-aminohexyl)carbamoyl-2'-O-methyluridine ((N)Um) into oligonucleotides increases antisense properties such as RNA binding affinity, nuclease resistance and RNase H activity. The present X-ray studies on hybrid duplexes formed between antisense oligonucleotides containing (N)Um and their target RNAs have revealed the structural basis for such properties. The terminal ammonium groups of the aminohexyl chains interact with the phosphate oxygen anions. The 2'-O-methyl modification induces the ribose group to adopt the C3'-endo conformation. Comparisons with the structure of unmodified duplex show that the (N)Um incorporation narrows the minor grooves and alters their hydration structures. These structural changes are well correlated to the favorable properties for useful antisense molecules.

X-Ray analyses of DNA duplexes containing 5-(N-aminohexyl)carbamoyl modifications

Oligonucleotides containing polyamines are currently being evaluated as potential antigene compounds for therapeutic purposes. Among them, 5-(N-aminohexyl) carbamoyl-2'-deoxyuridine ((N)U) and 5-(N-aminohexyl) carbamoyl-2'-O-methyluridine ((N)Um) substituted oligonucleotides have higher resistance against nuclease degradation compared to native DNA. Furthermore, oligonucleotides containing (N)U stabilizes duplex formation with the complementary DNA. To elucidate the mechanisms behind these improved antigene properties, we synthesized and crystallized two Dickerson-Drew-type DNA duplexes containing (N)U and (N)Um. The 2'-O-methyl modification in (N)Um was found to induce the ribose ring to adopt the C3'-endo conformation. Electron density maps show possible interactions of the terminal ammonium ion of the aminohexyl groups with the phosphate oxygen anions.

An oligonucleotide complementary to the SL-B1 domain in the 3′-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase

We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3′-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC 50 values lower than 1 μM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template. The introduction of a 2′- O-methyl modification increased the inhibitor activity 11-fold (IC 50 = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3′-end of the minus-strand RNA.

Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.

Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.

2'-O-methyl-RNA hairpins generate loop-loop complexes and selectively inhibit HIV-1 Tat-mediated transcription.

The interaction of the TAR RNA element of human immunodeficiency virus type 1 (HIV-1) with a 2'-O-methyl analogue of an RNA hairpin aptamer previously identified by in vitro selection [Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614] was characterized by UV-monitored thermal denaturation and surface plasmon resonance experiments. The complex between TAR and this aptamer derivative displays stability (K(d) = 9.9 +/- 1.0 nM) and kinetic properties [k(on) = 9.0 +/- 0.3 M(-1) s(-1), k(off) = (8.9 +/- 0.6) x 10(-4) s(-1)] close to those of the parent RNA aptamer. The modified aptamer forms a "kissing" complex with TAR driven by the same key elements as the TAR-RNA aptamer one. In particular, the G and A residues closing the aptamer loop remain crucial for TAR-2'-O-methyl aptamer complexes. Moreover, the 2'-O-methyl aptamer analogue specifically inhibits Tat-mediated transcription in an in vitro assay more efficiently than the RNA aptamer. This is likely due to the increased lifetime of the former oligonucleotide in the cell-free extract. The 2'-O-methyl modification extends the range of molecules that can be used to target viral hairpin RNA through loop-loop interactions. More generally, this demonstrates the interest of SELEX for targeting RNA hairpins and understanding nucleic acid interactions.

The Nucleotide Sequence of the GGG-specific Glycine Transfer Ribonucleic Acid of Escherichia coli and of Salmonella typhimurium

Abstract The primary nucleotide sequence of tRNAggggly (glycine tRNA specific for the codon GGG) from Escherichia coli and Salmonella typhimurium has been determined. The E. coli tRNA sequence is [see PDF for sequence] where U at position 8 is most likely 4-thiouracil. The sequence of the S. typhimurium tRNAggggly is identical with that of E. coli except that no evidence for the modification of U at position 8 has been found. In addition, a portion of the S. typhimurium tRNAggggly has a 2'-O-methyl modification of the G at position 17. Some unusual features of the tRNA structure are discussed.