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1.
P W Trown 《Archives of biochemistry and biophysics》1973,156(2):694-702
[γ-32P]GTP-Labeled Qβ 6S RNA yielded only one major radioactive oligonucleotide after digestion with pancreatic ribonuclease. Nearest neighbor analysis of this 5′-oligonucleotide demonstrated that approximately 95% of the molecules terminate with the same sequence, pppGpGpCp. This sequence is the complement of the only major 3′-sequence found in this RNA. Both strands of 6S RNA therefore appear to have identical 3′- and 5′-terminal trinucleotide sequences. 相似文献
2.
Yueh-Hsiu Chien Chun-Tsan Deng Peter Chandler Norman Davidson 《Analytical biochemistry》1980,102(2):281-287
A method for the isolation of segments of any desired length from the 5′ end of retrovirus RNA has been tested. The method is based on selection of 5′-specific segments by hybridizing suitably fragmented genomic (35 S) RNA to mercurated strong stop cDNA followed by chromatography on sulfhydryl-agarose. The method has been shown to be effective for Akv viral RNA by observing the T1 oligonucleotide fingerprints of a 5′-enriched fraction. This fingerprint pattern is of lower complexity than that of total 35 S RNA, contains oligonucleotide spots that have previously been assigned as 5′ specific by conventional fingerprinting methods, and does not overlap with the pattern from 3′-specific RNA. 相似文献
3.
H Wippo F Reck R Kudick M Ramaseshan G Ceulemans M Bolli R Krishnamurthy A Eschenmoser 《Bioorganic & medicinal chemistry》2001,9(9):2411-2428
The (L)-alpha-lyxopyranosyl-(4'-->3')-oligonucleotide system-a member of a pentopyranosyl oligonucleotide family containing a shortened backbone-is capable of cooperative base-pairing and of cross-pairing with DNA and RNA. In contrast, corresponding (D)-beta-ribopyransoyl-(4'-->3')-oligonucleotides do not show base-pairing under similar conditions. We conclude that oligonucleotide systems can violate the 'six-bonds-per-backbone-unit' rule by having five bonds instead, if their vicinally bound phosphodiester bridges can assume an antiperiplanar conformation. An additional structural feature that seems relevant to the cross-pairing capability of the (L)-alpha-lyxopyranosyl-(4'-->3')-oligonucleotide system is its (small) backbone/basepair axes inclination. An inclination which is similar to that in B-DNA seems to be a prerequisite for an oligonucleotide system's capability to cross-pair with DNA. 相似文献
4.
5.
Enzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens. 总被引:12,自引:7,他引:5
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Synthetic oligonucleotides, complementary to unique sequences in the heat stable enterotoxin gene of Escherichia coli specific for humans, were prepared with a 30-atom spacer arm and a 3' terminal sulfhydryl group which was coupled to bromoacetyl-derivatized alkaline phosphatase. The resulting direct enzyme-linked oligonucleotide probes, containing one enzyme molecule per oligonucleotide, successfully diagnosed enterotoxigenic Escherichia coli in clinical specimens by using a modified colony hybridization method and a colorimetric assay. The procedure is rapid, simple and reliable with a sensitivity equivalent to that using 5'-terminally labelled [32p]-oligonucleotide probes. The results indicate that the enzyme-labelled oligonucleotide probes should be applicable to the routine diagnosis of enterotoxigenic Escherichia coli and possess the potential for the detection of other microbial pathogens. 相似文献
6.
Yu. N. Vorobjev 《Molecular Biology》2005,39(5):777-784
Three-dimensional atomic models of complexes between yeast tRNAPhe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNAPhe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNAPhe destruction and formation of the final tRNAPhe-oligonucleotide complementary complex. The primary complex formation and the following tRNAPhe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element. 相似文献
7.
A method is described for the rapid analysis of the nucleotides, guanosine 5′-diphosphate-3′-diphosphate (ppGpp) and guanosine 5′-triphosphate-3′-diphosphate (pppGpp), by high-performance liquid chromatography. It has been found that the inclusion of magnesium acetate in the potassium phosphate buffer facilitates elution of these highly phosphorylated compounds from the Partisil strong anion-exchange resin and allows their determination under isocratic conditions. The application of this methodology to formic acid extracts of bacterial cells is demonstrated. 相似文献
8.
Christina B. Nielsen Sanjay K. Singh Jesper Wengel Jens Peter Jacobsen 《Journal of biomolecular structure & dynamics》2013,31(2):175-191
Abstract LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2′-0, 4′-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional'H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32Å. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3Å. The average twist over the sequence are 35.9° ± 0.3°. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5′-CTLAG-3′ site than in the unmodified 5′-CTLAG-3′ site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex. 相似文献
9.
Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA 总被引:28,自引:0,他引:28
Methods for the direct determination of nucleotide sequences in DNA have been developed and used to determine the complete primary structure of a fragment of bacteriophage φX174 DNA which is 48 residues in length. This fragment was liberated from φX DNA by digestion at low temperature and high ionic strength with the T4 phage-induced endonuclease IV. The fragment was redigested with endonuclease IV under vigorous conditions and the products fractionated two-dimensionally providing a characteristic endonuclease IV “fingerprint” of the fragment. The Burton (Burton &; Petersen, 1960) depurination reaction was used to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini. These oligonucleotide products were then fully sequenced by partial exonuclease digestion with spleen and snake venom phosphodiesterase and analysis of the fractionated digests by base composition, depurination, and 5′ end-group analysis using exonuclease I. Rules for the interpretation of two-dimensional fingerprints of partial exonuclease digests, which rapidly provide sequence information by simple inspection, were also deduced. To derive the complete structure of the fragment, the fully sequenced oligonucleotides were ordered by characterizing large, overlapping, partial endonuclease IV digestion products by means of the depurination reaction. The sequencing methods described are general and may be used for the direct determination of the primary structures of other fragments of DNA. 相似文献
10.
Sebastian Engmann Felicia A. Bokel Hyun Wook Ro Dean M. DeLongchamp Lee J. Richter 《Liver Transplantation》2016,6(10)
A method to study the structural evolution of organic bulk heterojunctions via real‐time, in‐situ, steady‐state photoluminescence (PL) is presented. In‐situ PL, in combination with real‐time transmission and reflection measurements, allows us to quantitatively describe the progression of intimate mixing during blade coating of two OPV systems: the common model system poly(3‐hexylthiophene‐2,5‐diyl)/phenyl‐C61‐butyric‐acid‐methyl ester (P3HT/PCBM), and the higher power conversion efficiency system 7,7′‐(4,4bis(2‐ethylhexyl)‐4H‐silolo[3,2‐b:4,5‐b′]dithiophene‐2,6‐diyl)bis(6‐fluoro‐5‐(5′‐hexyl‐[2,2′‐bithiophen]‐5‐yl)benzo[c][1,2,5]thiadiazaole), p‐DTS(FBTTh2)2/[70]PCBM. Evaluating the time dependence of the PL intensity during drying using a 3D‐random‐walk diffusion model allows for the quantitative determination of the ratio of characteristic domain size to exciton diffusion length during solidification in the presence of the processing additives 1‐chloronaphtalene (CN), 1,8‐octanedithiol (ODT), and 1,8‐diiodooctane (DIO). In both cases, the obtained results are in good agreement with the typically observed fibril widths and grain sizes, for P3HT and p‐DTS(FBTTh2)2, respectively. 相似文献
11.
《Nucleosides, nucleotides & nucleic acids》2013,32(4):359-371
Abstract A strategy was developed for the synthesis of 3′-O-β-D-ribofuranosyl 2′-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5′-O- and 5″-O-phosphorylated analogues were synthesized and their conformation was determined using NMR spectroscopy. 相似文献
12.
Abstract Oligonucleotide having 2′-5′ phosphodiester linkage has been synthesised on solid support using indigenously prepared 3′-deoxy-2′-phosphoramidites. The 2′-5′ oligonucleotide showed higher half-life when subjected to 3′-exonuclease, SVPD, digestion. This oligonucleotide formed a stable duplex with complementary RNA but not with DNA. Similarly, it did not form triplex as well either with DNA or RNA duplex. 相似文献
13.
Damage to DNA can result in strand breaks with 5′-hydroxyl and 3′-phosphate termini. Before DNA polymerases and ligases can rejoin the broken strands, such termini have to be restored to 5′-phosphate and 3′-hydroxyl groups. Polydeoxynucleotide kinase is an enzyme that may fulfil this function. We have purified the kinases from calf thymus and rat liver to near homogeneity. Based on SDS-polyacrylamide gel electrophoresis and activity gels, the enzymes from both sources are ∼60-kDa polypeptides. Both enzymes have an acidic pH optimum (5.5–6.0) for kinase activity, and similar pl values (8.5–8.6), and a specificity for DNA. The calf thymus kinase possesses a 3′-phosphatase activity, as has previously been shown for the rat liver enzyme. The minimum size of oligonucleotide that can be labelled is 7–8 nucleotides in length, but the optimal size appears to be >18 nucleotides. Comparison of phosphorylation of oligo(dA)24 and oligo(dT)24 with oligonucleotides containing a varied nucleotide sequence indicated that the homopolymers are poorer substrates. Unlike the bacteriophage T4 polynucleotide kinase, the mammalian kinases exhibit no preference for 5′-overhanging termini when acting at DNA termini produced by restriction enzymes. With double-stranded oligonucleotide complexes designed to model single-strand gaps and nicks, the mammalian kinases preferentially phosphorylate the 5′-terminus associated with the gap or nick, in keeping with the idea that the kinases are involved in the repair of DNA single-strand breaks. J. Cell. Biochem. 64:258–272. © 1997 Wiley-Liss, Inc. 相似文献
14.
Lahja Uitto Jussi Halleeen Päivi Remes Tapio Kesti Juhani E. Syväoja 《Chromosoma》1992,102(1):S142-S146
The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity. 相似文献
15.
Julie Moreau Nabil Dendane Bernd Schöllhorn Nicolas Spinelli Claire Fave Eric Defrancq 《Bioorganic & medicinal chemistry letters》2013,23(4):955-958
Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3′ or 5′ end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3′ end and an electrochemical label at 5′ end. 相似文献
16.
Robert Van Ostrand Casey Jacobsen Alicia Delahunty Carley Stringer Ryan Noorbehesht Haidi Ahmed 《Nucleosides, nucleotides & nucleic acids》2017,36(3):181-197
Reported is an efficient synthesis of adenyl and uridyl 5′-tetrachlorophthalimido-5′-deoxyribonucleosides, and guanylyl 5′-azido-5′-deoxyribonucleosides, which are useful in solid-phase synthesis of phosphoramidate and ribonucleic guanidine oligonucleotides. Replacement of 5′-hydroxyl with tetrachlorophthalimido group was performed via Mitsunobu reaction for adenosine and uridine. An alternative method was applied for guanosine which replaced the 5′-hydroxyl with an azido group. The resulting compounds were converted to 5′-amino-5′-deoxyribonucleosides for oligonucleotide synthesis. Synthetic intermediates were tested as antimicrobials against six bacterial strains. All analogs containing the 2′,3′-O-isopropylidine protecting group demonstrated antibacterial activity against Neisseria meningitidis, and among those analogs with 5′-tetrachlorophthalimido and 5′-azido demonstrated increased antibacterial effect. 相似文献
17.
A concise synthesis of oligonucleotide 5′-peptide-conjugates via copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition in aqueous solution is described. Synthesis of reagents was accomplished by on-column derivatization of corresponding peptides and oligonucleotides. This method is well suited for the preparation of peptide–oligonucleotide conjugates containing 1,2,3-triazole linkage between the 5′-position of an oligonucleotide and the N-terminus of a peptide. 相似文献
18.
Abstract 2′-Deoxy-5′-0-(4,4′-dimethoxytrityl)-5-methyl-N 4-(1-pyrenylmethyl)-α-cytidine (5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(l,2,4-triazolyl)-α-thymidine derivative 3 which was synthesized from 5-O-DMT protected α-thymidine 1. Aminolysis of 3 afforded 3′-O-acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (8). Benzoylation of 8 and removal of acetyl afforded N 4-benzoyl-2-deoxy-5–0-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (10). The amidites of compounds 5and 10 were prepared and used in α-oligonucleotide synthesis. DNA three-way junction (TWJ) is stabilized when an α-ODN is used for targeting the dangling flanks of the stem in a DNA hairpin. Further stabilization of the TWJ is observed when 5 is inserted into the α-ODN at the junction region. 相似文献
19.
Grajkowski A Cieślak J Kauffman JS Duff RJ Norris S Freedberg DI Beaucage SL 《Bioconjugate chemistry》2008,19(8):1696-1706
The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions. 相似文献
20.
Morris J. Robins Bogdan Doboszewski Bradley L. Nilsson Matt A. Peterson 《Nucleosides, nucleotides & nucleic acids》2013,32(1-2):69-86
Abstract We report syntheses of new amide-linked (di-penta)nucleoside analogues of antisense oligonucleotide components. Solution-phase coupling of 3′-(carboxymethyl)-3′-deoxy- and 5′-amino-5′-deoxynucleoside derivatives provides amide dimers. Activated [3′-(carboxymethyl)-3′-deoxy] units with a 5′-azido-5′-deoxy function provide “masked” 5′-amino-5′-deoxy residues for chain extension, and a 5′-O-DMT-protected unit provides the 5′-terminus for attachment to a phosphodiester linkage. 相似文献