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Influence of temperature on tRNA modification in archaea: Methanococcoides burtonii (optimum growth temperature [Topt], 23 degrees C) and Stetteria hydrogenophila (Topt, 95 degrees C)
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Noon KR Guymon R Crain PF McCloskey JA Thomm M Lim J Cavicchioli R 《Journal of bacteriology》2003,185(18):5483-5490
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转移核糖核酸(tRNA)的转录后修饰对tRNA正常行使生物学功能具有重要意义,这些功能包括tRNA的正确折叠和维持其稳定性、在核糖体上正确解码。虽然tRNA转录后大部分核苷酸修饰形式在20世纪70年代已被鉴定出,但最近才在大肠杆菌及酵母中鉴定出催化这些tRNA核苷酸修饰的酶的绝大部分基因。这些修饰酶基因的鉴定为研究tRNA转录后修饰的生物功能开启了新的大门。人胞质tRNA和线粒体tRNA(mt tRNA)都存在大量核苷酸修饰,这些修饰的缺陷常常与多种人类疾病相关。因此,研究tRNA核苷酸修饰有助于我们了解相关疾病的发病机理。 相似文献
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The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing North-East conformationally constrained 1',2'-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2',4'-ethylene-bridged (aza-ENA-T) nucleoside, and 2'-alkoxy modified nucleosides (2'-O-Me-T and 2'-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (DeltaTm = 2.5-4 degrees C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4-5.5 degrees C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of approximately 5-6 degrees C per modification. The 2'-O-Me-T and 2'-O-MOE-T modifications, on the other hand, showed an increased of Tm by 0.5 C per modification in their AON/RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The Km and Vmax values obtained from the RNA concentration-dependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (Kcat) of RNase H were found to be greater (approximately 1.4-2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/Km) showed a decrease (approximately 1.7-8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (approximately 1.1-3.2-fold) enzyme activity (Kcat/Km) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2'-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2'-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3'-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3'-exonuclease preferentially hydrolyzed the 3'-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3'-exonuclease cleaved at the 3'-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5'-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5'-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2'-O-Me-T, and 2'-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs. 相似文献
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Template nucleotide moieties required for de novo initiation of RNA synthesis by a recombinant viral RNA-dependent RNA polymerase
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The recombinant RNA-dependent RNA polymerase of the bovine viral diarrhea virus specifically requires a cytidylate at the 3' end for the de novo initiation of RNA synthesis (C. C. Kao, A. M. Del Vecchio, and W. Zhong, Virology 253:1-7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2'-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2'-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2'-amino modifications are tolerated better after the +11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases. 相似文献
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Fauster K Hartl M Santner T Aigner M Kreutz C Bister K Ennifar E Micura R 《ACS chemical biology》2012,7(3):581-589
Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2'-azido cytidine and 2'-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2'-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2'-azido uridine and 2'-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2'-azido group supports the C3'-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2'-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2'-azido modifications are compatible with 2'-fluoro and/or 2'-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2'-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2'-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification. 相似文献
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A systematic, ligation-based approach to study RNA modifications 总被引:2,自引:1,他引:1
Saikia M Dai Q Decatur WA Fournier MJ Piccirilli JA Pan T 《RNA (New York, N.Y.)》2006,12(11):2025-2033
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. 相似文献
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Syntheses of 4'-thioribonucleosides and thermodynamic stability and crystal structure of RNA oligomers with incorporated 4'-thiocytosine
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A facile synthetic route for the 4'-thioribonucleoside building block (4'S)N (N = U, C, A and G) with the ribose O4' replaced by sulfur is presented. Conversion of l-lyxose to 1,5-di-O-acetyl-2,3-di-O-benzoyl-4-thio-d-ribofuranose was achieved via an efficient four-step synthesis with high yield. Conversion of the thiosugar into the four ribonucleoside phosphoramidite building blocks was accomplished with additional four steps in each case. Incorporation of 4'-thiocytidines into oligoribonucleotides improved the thermal stability of the corresponding duplexes by approximately 1 degrees C per modification, irrespective of whether the strand contained a single modification or a consecutive stretch of (4'S)C residues. The gain in thermodynamic stability is comparable to that observed with oligoribonucleotides containing 2'-O-methylated residues. To establish potential conformational changes in RNA as a result of the 4'-thio modification and to better understand the origins of the observed stability changes, the crystal structure of the oligonucleotide 5'-r(CC(4'S)CCGGGG) was determined and analyzed using the previously solved structure of the native RNA octamer as a reference. The two 4'-thioriboses adopt conformations that are very similar to the C3'-endo pucker observed for the corresponding sugars in the native duplex. Subtle changes in the local geometry of the modified duplex are mostly due to the larger radius of sulfur compared to oxygen or appear to be lattice-induced. The significantly increased RNA affinity of 4'-thio-modified RNA relative to RNA, and the relatively minor conformational changes caused by the modification render this nucleic acid analog an interesting candidate for in vitro and in vivo applications, including use in RNA interference (RNAi), antisense, ribozyme, decoy and aptamer technologies. 相似文献
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Eckstein F 《Biochimie》2002,84(9):841-848
This review describes some of the contributions of chemistry to the RNA field with a personal bias towards the phosphorothioate modification and the derivatives at the ribose 2'-position. The usefulness of these modifications is discussed and documented with some examples. 相似文献
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Investigation of some properties of oligodeoxynucleotides containing 4'-thio-2'-deoxynucleotides: duplex hybridization and nuclease sensitivity. 总被引:2,自引:2,他引:0
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The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration. 相似文献
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Hyperthermus butylicus, a hyperthermophilic sulfur-reducing archaebacterium that ferments peptides 总被引:6,自引:0,他引:6
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W Zillig I Holz D Janekovic H P Klenk E Imsel J Trent S Wunderl V H Forjaz R Coutinho T Ferreira 《Journal of bacteriology》1990,172(7):3959-3965
The hyperthermophilic peptide-fermenting sulfur archaebacterium Hyperthermus butylicus was isolated from the sea floor of a solfataric habitat with temperatures of up to 112 degrees C on the coast of the island of S?o Miguel, Azores. The organism grows at up to 108 degrees C, grows optimally between 95 and 106 degrees C at 17 g of NaCl per liter and pH 7.0, utilizes peptide mixtures as carbon and energy sources, and forms H2S from elemental sulfur and molecular hydrogen as a growth-stimulating accessory energy source but not by sulfur respiration. The same fermentation products, CO2, 1-butanol, acetic acid, phenylacetic acid, and a trace of hydroxyphenylacetic acid, are formed both with and without of S0 and H2. Its ether lipids, the absence of a mureine sacculus, the nature of the DNA-dependent RNA polymerase, and phylogenetic classification by DNA-rRNA cross-hybridization characterize H. butylicus as part of a novel genus of the major branch of archaebacteria comprising the orders Thermoproteales and Sulfolobales, representing a particularly long lineage bifurcating with the order Sulfolobales above the branching off of the genus Thermoproteus and distinct from the genera Desulfurococcus and Pyrodictium. 相似文献
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Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets. 总被引:1,自引:1,他引:0
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Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section. 相似文献