共查询到20条相似文献,搜索用时 31 毫秒
1.
Current studies of lariat RNA structure and function are hindered by the lack of access to synthetic lariats. A novel approach to the synthesis of both DNA and RNA lariats is presented here. Noteworthy features of the methodology are the regiospecific formation of the 2'-5'-phosphodiester linkage, the unusual parallel stranded DNA/RNA hybrid (or parallel RNA/RNA duplex) that forms between an RNA template and a folded 22-nt DNA (or RNA) substrate, and the efficiency of the chemical ligation step at an adenosine branchpoint (50-80%). The DNA and RNA lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition were confirmed by MALDI-TOF mass spectrometry. Thermal denaturation as well as enzymatic and chemical hydrolysis fully supported the proposed lariat structures. Characterization of control parallel duplexes was conducted by gel shift assays and enzymatic degradation with RNase H. The successful synthesis of the lariat molecules described here will allow structural and biochemical studies aimed at better understanding the splicing and debranching mechanisms in which these unusual nucleic acids are involved. 相似文献
2.
Krivos KL Addepalli B Limbach PA 《Rapid communications in mass spectrometry : RCM》2011,25(23):3609-3616
RNase mapping by nucleobase-specific endonucleases combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a powerful analytical method for characterizing ribonucleic acids (RNAs). Endonuclease digestion of RNA yields products that contain a 3'-terminal phosphate group. MS/MS via collision-induced dissociation (CID) of these digestion products on a linear ion trap generates fragmentation pathways that include the loss of phosphoric acid (-H(3)PO(4); -98 u), which does not provide information about the sequence of the digestion products and can reduce ion abundance from other pathways that provide sequence information. Here we investigate the use of bacterial alkaline phosphatase (BAP) after RNase digestion to remove the 3'-terminal phosphate from all RNase digestion products prior to LC/MS/MS analysis. RNase digestion products lacking the 3'-phosphate were found to produce CID spectra with more consistent, high-abundance c- and y-type fragment ions as well as significantly more a-Base and w-type ions than digestion products retaining the 3'-phosphate. In this manner, RNase mapping with LC/MS/MS can provide more complete RNA sequence information from fragment ions of higher abundance that are easier to interpret and identify. 相似文献
3.
DNA.RNA hybrid duplexes are biologically important molecules and are shown to have potential therapeutic properties. To investigate the relationship between structures, energetics, solvation and RNase H activity of hybrid duplexes in comparison with pure DNA and RNA duplexes, a molecular dynamics study using the CHARMM27 force field was undertaken. The structural properties of all four nucleic acids considered are in very good agreement with the experimental data. The backbone dihedral angles and the puckering of the (deoxy)ribose indicate that the purine rich strands retain their A-/B-like properties but the pyrimidine rich DNA strand undergoes A-B conformational transitions. The minor groove widths of the hybrid structures are narrower than those in the RNA duplex, a requirement for RNase H binding. In addition, sampling of noncanonical phosphodiester backbone dihedrals by the DNA strands, differential solvation properties and helical properties, most notably rise, are suggested to contribute to hybrids being RNase H substrates. Differential RNase H activity toward hybrids containing purine versus pyrimidine rich RNA strands is suggested to be due to sampling of values of the phosphodiester backbone dihedrals in the DNA strands. Notably, the present results indicate that hybrids have decreased flexibility as compared to RNA, in contrast to previous reports. 相似文献
4.
5.
Dr. Jacek Cieślak Dr. Cristina Ausín Dr. Andrzej Grajkowski Dr. Serge L. Beaucage 《Chemistry (Weinheim an der Bergstrasse, Germany)》2013,19(14):4623-4632
The reaction of 2‐cyano‐2‐methyl propanal with 2′‐O‐aminooxymethylribonucleosides leads to stable and yet reversible 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl)ribonucleosides. Following N‐protection of the nucleobases, 5′‐dimethoxytritylation and 3′‐phosphitylation, the resulting 2′‐protected ribonucleoside phosphoramidite monomers are employed in the solid‐phase synthesis of three chimeric RNA sequences, each differing in their ratios of purine/pyrimidine. When the activation of phosphoramidite monomers is performed in the presence of 5‐benzylthio‐1H‐tetrazole, coupling efficiencies averaging 99 % are obtained within 180 s. Upon completion of the RNA‐chain assemblies, removal of the nucleobase and phosphate protecting groups and release of the sequences from the solid support are carried out under standard basic conditions, whereas the cleavage of 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl) protective groups is effected (without releasing RNA alkylating side‐products) by treatment with tetra‐n‐butylammonium fluoride (0.5 m) in dry DMSO over a period of 24–48 h at 55 °C. Characterization of the fully deprotected RNA sequences by polyacrylamide gel electrophoresis (PAGE), enzymatic hydrolysis, and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry confirmed the identity and quality of these sequences. Thus, the use of 2′‐O‐aminooxymethylribonucleosides in the design of new 2′‐hydroxyl protecting groups is a powerful approach to the development of a straightforward, efficient, and cost‐effective method for the chemical synthesis of high‐quality RNA sequences in the framework of RNA interference applications. 相似文献
6.
The purine nucleotides pdAp, pdGp, (dA)2, (dA-dG), (dG-dA), (dG)2, (dA)3, (dA-dG-dA), (dA-dA-dG), (dG-dA-dA), (dG-dA-dG) and known mixtures of purine nucleotide sequence isomers were separated by preparative scale chromatography of partial hydrolysates of depyrimidinated herring sperm DNA. Herring sperm DNA is first partially hydrolysed to a mixture of purine nucleotides. The low-molecular-weight oligonucleotides are then separated by column chromatography on DEAE-cellulose at pH 7.5, and fractionated by chromatography on QAE-Sephadex. Impurities which are not fully removed by column chromatography are separated by paper chromatography. The sequence of the isolated DNA fragments and the composition of the mixtures of sequence isomers were determined from the chromatographic data, absorption characteristics and by enzymatic degradation. 相似文献
7.
Höbartner C Rieder R Kreutz C Puffer B Lang K Polonskaia A Serganov A Micura R 《Journal of the American Chemical Society》2005,127(34):12035-12045
The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1,4-dimercapto-2,3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives. 相似文献
8.
Powner MW Sutherland JD Szostak JW 《Journal of the American Chemical Society》2010,132(46):16677-16688
The recent development of a sequential, high-yielding route to activated pyrimidine nucleotides, under conditions thought to be prebiotic, is an encouraging step toward the greater goal of a plausible prebiotic pathway to RNA and the potential for an RNA world. However, this synthesis has led to a disparity in the methodology available for stepwise construction of the canonical pyrimidine and purine nucleotides. To address this problem, and further explore prebiotically accessible chemical systems, we have developed a high-yielding, aqueous, one-pot, multicomponent reaction that tethers masked-sugar moieties to prebiotically plausible purine precursors. A pH-dependent three-component reaction system has been discovered that utilizes key nucleotide synthons 2-aminooxazole and 5-aminoimidazoles, which allows the first divergent purine/pyrimidine synthesis to be proposed. Due to regiospecific aminoimidazole tethering, the pathway allows N9 purination only, thus suggesting the first prebiotically plausible mechanism for regiospecific N9 purination. 相似文献
9.
Yongkang Ye Wen Wen Yun Xiang Xiaodong Qi Jeffrey T. La Belle Julian J. L. Chen Joseph Wang 《Electroanalysis》2008,20(8):919-922
Here we present a highly sensitive, rapid and simple electrochemical assay of RNase based on coupling magnetic separation of the enzymatically treated RNA with stripping potentiometric detection of the purine nucleobases. A detection limit of 1×10?8 U RNase (ca. 4 pg/mL) is obtained in connection to a 60 min enzymatic digestion. The attractive performance of this direct indicator‐free electrochemical assay offers great promise for a wide range of molecular biology and water quality applications. 相似文献
10.
Shkrob IA Marin TM Adhikary A Sevilla MD 《The journal of physical chemistry. C, Nanomaterials and interfaces》2011,115(8):3393-3403
Photocatalytic oxidation of nucleic acid components on aqueous metal oxides (TiO(2), α-FeOOH, and α-Fe(2)O(3)) has been studied. The oxidation of purine nucleotides results in the formation of the purine radical cations and sugar-phosphate radicals, whereas the oxidation of pyrimidine nucleotides other than thymine results in the oxidation of only the sugar-phosphate. The oxidation of the thymine (and to a far less extent for the 5-methylcytosine) derivatives results in deprotonation from the methyl group of the base. Some single stranded (ss) oligoribonucleotides and wild-type ss RNA were oxidized at purine sites. In contrast, double stranded (ds) oligoribonucleotides and DNA were not oxidized. These results account for observations suggesting that cellular ds DNA is not damaged by exposure to photoirradiated TiO(2) nanoparticles inserted into the cell, whereas ss RNA is extensively damaged. The astrobiological import of our observations is that the rapid degradation of monomer nucleotides make them poor targets as biosignatures, whereas duplex DNA is a better target as it is resilient to oxidative diagenesis. Another import of our studies is that ds DNA (as opposed to ss RNA) appears to be optimized to withstand oxidative stress both due to the advantageous polymer morphology and the subtle details of its radical chemistry. This peculiarity may account for the preference for DNA over RNA as a "molecule of life" provided that metal oxides served as the template for synthesis of polynucleotides, as suggested by Orgel and others. 相似文献
11.
BACKGROUND: The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. RESULTS: Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by approximately 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chemical step. CONCLUSIONS: These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions. 相似文献
12.
The possible role of catalysis in forming a limited number of RNAs from activated monomers is investigated by examining the sequence- and regioselectivity in the montmorillonite-catalyzed formation of RNA dimers and trimers. The reactivity of A was similar to that of G, and C was comparable in reactivity to U. Yet the reactivity of the purine nucleotides differed from that of the pyrimidines. In the reaction of nucleotides (pN) with activated monomers (ImpN), the sequence- and regioselectivity was Pu(3')Py > Pu(3')Pu = Pu(2')Py > Pu(2')Pu. The 5'-pyrimidine initiated dimers formed less efficiently than the 5'-purine initiated dimers. Trimer formation was investigated by the synthesis of 8 dimers (pNpN) and measuring the yields of trimers formed in the reaction of each dimer with a mixture of equal molar amounts of four activated monomers. The reactivity of the dimers depended on the nucleotide attached to the 3'-end of the RNA and the regiochemistry of the phosphodiester bond. Rules based on these studies are proposed to predict the sequence- and regioselectivity of the RNAs formed in montmorillonite-catalyzed reactions. These rules are consistent with the structures of the 2-5-mers formed in the reaction of equimolar amounts of ImpA and ImpC. This research establishes that the montmorillonite catalyst limits the number of RNA oligomer isomers formed. The potential significance of these findings to the origins of life is discussed. 相似文献
13.
Structural states of dictyostelium myosin 总被引:7,自引:0,他引:7
Myosin purified from Dictyostelium amoebae has approximately 10% by weight of RNA associated with it, unless specific steps (DEAE cellulose chromatography or RNase digestion) are taken to remove it. This RNA has significant effects on the structural states formed by the myosin at low ionic strength in the presence of Mg2+. Rapid precipitation of RNA-free myosin by dilution generates bipolar thick filaments (540 nm long, 33 nm thick), often with a bare zone and a 15-nm transverse repeat. Rapid precipitation of myosin with copurified RNA yields linear aggregates of bipolar filaments, showing some lateral association. Slow precipitation of RNA-free myosin by dialysis yields very long filaments or ribbons (greater than 5 micrometer, 30--60 nm wide) in which the myosin may be packed diagonally across the filament, similar to the "side-polar" aggregates formed by other nonmuscle myosins and by smooth muscle myosin (Craig R, Megerman J: J Cell Biol 75:990, 1977; Hinssen H, D'Haese J, Small JV, Sobieszek A: J Ultrastruct Res 64:282, 1978). Slow precipitation of myosin with copurified RNA generates linear filaments with repeat intervals of 290 and 650 nm. Other polyanions were tested for their effects on myosin aggregation. Total RNA and ribosomal RNA from Dictyostelium, when added to RNA-free myosin, also induced the extensive linear aggregation seen with the copurified RNA/myosin complex, although higher concentrations of RNA were required to obtain quantitatively the same effect. DNA and heparin were also effective inducers of linear aggregation, whereas homopolymers of nucleotides and of acidic or basic amino acids were poorly effective. 相似文献
14.
乙酰鸟氨酸脱乙酰酶固定化细胞拆分D,L-缬氨酸 总被引:3,自引:1,他引:3
报道了一种利用具有乙酰鸟氨酸脱乙酰酶活性的固定化细胞拆分D,L-缬氨酸的新方法. 该酶促反应最适条件: pH=6, 反应温度50 ℃, 底物N-乙酰-D,L-缬氨酸浓度200 mmol/L, 固定化细胞用量0.2 g/mL(或100 U/mL). 0.1 mmol/L CoCl2条件对该酶促反应有显著的激活作用. 在以上条件下反应2~3 h, 测得产物L-缬氨酸浓度95 mmol/L. 该固定化细胞连续10次使用, 平均转化率为90.8%(以N-乙酰-L-缬氨酸计), 显示出了良好的工业化应用前景. 相似文献
15.
Bondensgaard K Petersen M Singh SK Rajwanshi VK Kumar R Wengel J Jacobsen JP 《Chemistry (Weinheim an der Bergstrasse, Germany)》2000,6(15):2687-2695
We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation. 相似文献
16.
Aminoacylation of tRNA in cells involves activation of the amino acid as an aminoacyl adenylate, a mixed anhydride with AMP, which reacts with tRNA. We have now established that aminoacyl phosphate esters in the presence of lanthanide ions in water will acylate hydroxyls at the 3'-terminus of RNA or a simple nucleotide. By extension, this will permit synthetically aminoacylated tRNA to be produced in a single-step biomimetic process. The reactions of Boc-4-fluorophenylalanyl ethyl phosphate were followed by HPLC separation, MS, and 19F NMR analysis. In stoichiometric combination with lanthanum salts in aqueous buffer, Boc-4-fluorophenylalanyl ethyl phosphate rapidly produces 2'- and 3'-monoesters of cytidine and cytidine monophosphate. Reaction of the reagent with RNA in the presence of lanthanum and magnesium salts introduces a specifically detectable signal into the RNA, which is evidence of formation of the aminoacyl ester. When the same RNA is initially oxidized with periodate to convert the 3'-terminal vicinal diol to the cleaved dialdehyde, reaction with the aminoacyl phosphate no longer occurs as evidenced by the lack of a signal in the 19F NMR spectrum. The results are consistent with a requisite chelation mechanism in which lanthanum serves as a template for both the aminoacyl phosphate and the 3'-terminal diol of RNA and nucleotides. The coordinated diol will then react through specific base-catalyzed intramolecular addition of the alkoxide nucleophile to the acyl group of the aminoacyl phosphate. Assessment of the method with a single tRNA was also achieved using the fluorescent reagent N-dansyl-glycyl ethyl phosphate. Lanthanide-promoted aminoacylation at the 3'-terminus of tRNAPhe is detected by the introduction of fluorescence (detected directly and by antibody-enhanced emission). This does not occur if the 3'-terminus is converted to the dialdehyde by reaction with periodate. 相似文献
17.
Immobilization of RNase in PVC ultrafiltration membranes was carried out. The obtained membranes were used for concentration of BSA solution, RNA being simultaneously removed. The yield of RNA hydrolysis was found to be controlled by the initial concentration of RNA in feed solution. The protein affected enzyme action as a result of its adsorption on the membrane surface at the beginning of ultrafiltration, whereas it did not inhibit RNase activity during the process. 相似文献
18.
F. Cramer 《Angewandte Chemie (International ed. in English)》1966,5(2):173-184
Problems and results of the synthesis of oligonucleotides are reviewed. The central role of the nucleic acids in biochemistry is a challenge to synthesize nucleic acids of known base sequence and chain length. Oligomers with various sequences of up to 12 members and homo-oligomers with a maximum chain length of 30 nucleotides can be obtained by chemical synthesis. The enzymatic synthesis of ribonucleic acids can be directed in such a way that polynucleotides with definite sequences are produced. 相似文献
19.
Hidenori Okamura Antony Crisp Sarah Hübner Sidney Becker Petra Rov Thomas Carell 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2019,131(51):18864-18869
The RNA world hypothesis assumes that life on Earth began with nucleotides that formed information‐carrying RNA oligomers able to self‐replicate. Prebiotic reactions leading to the contemporary nucleosides are now known, but their execution often requires specific starting materials and lengthy reaction sequences. It was therefore proposed that the RNA world was likely proceeded by a proto‐RNA world constructed from molecules that were likely present on the early Earth in greater abundance. Herein, we show that the prebiotic starting molecules bis‐urea (biuret) and tris‐urea (triuret) are able to directly react with ribose. The urea‐ribosides are remarkably stable because they are held together by a network of intramolecular, bifurcated hydrogen bonds. This even allowed the synthesis of phosphoramidite building blocks and incorporation of the units into RNA. Investigations of the nucleotides’ base‐pairing potential showed that triuret:G RNA base pairs closely resemble U:G wobble base pairs. Based on the probable abundance of urea on the early Earth, we postulate that urea‐containing RNA bases are good candidates for a proto‐RNA world. 相似文献
20.
We recently used in vitro selection to identify 7S11, a deoxyribozyme that synthesizes 2',5'-branched RNA. The 7S11 DNA enzyme mediates the nucleophilic attack of an adenosine 2'-hydroxyl group at a 5'-triphosphate, forming 2',5'-branched RNA in a reaction that resembles the first step of in vivo RNA splicing. Here, we describe 7S11 characterization experiments that have two important implications for nucleic acid chemistry and biochemistry. First, on the basis of a comprehensive analysis of its substrate sequence requirements, 7S11 is shown to be generally applicable for the synthesis of a wide range of 2',5'-branched RNAs. Strict substrate sequence requirements are found at the two RNA nucleotides that directly form the branched linkage, and these requirements correspond to those nucleotides found most commonly at these two positions in natural spliced RNAs. Outside of these two nucleotides, most substrate sequences are tolerated with useful ligation activity, although rates and yields vary. Because chemical synthesis approaches to branched RNA are extremely limited in scope, the deoxyribozyme-based route using 7S11 will enable many experiments that require branched RNA. Second, comprehensive nucleotide covariation experiments demonstrate that 7S11 and its RNA substrates adopt a three-helix-junction structure in which the branch-site nucleotide is located at the intersection of the three helices. Because many natural ribozymes have multi-helix junctions, 7S11 is an interesting model system for catalytic nucleic acids. 相似文献