Solid-phase synthesis of oligo(2'-deoxyxylonucleotides) and PCR amplification of base-modified DNA fragments.

1-(2'-Deoxy-beta-D-threo-pentofuranosyl)thymine (xTd) and -adenine (xAd) were converted into their appropriately protected 3'-phosphonates 1a, 2a as well as their 2-cyanoethyl phosphoramidites 1b, 2b. These compounds were used for solid-phase syntheses of the oligo(2'-deoxy-beta-D-xylonucleotides) 5-8. Structural properties and behavior against nucleases is described. Apart from oligo(2'-deoxyxylonucleotides) the PCR-amplification of a pUC18 DNA fragment with Taq polymerase was studied in the presence of the 7-deazapurine derivatives of dGTP, dATP, and dITP. The incorporation efficiency of the modified compounds was compared with those of the parent nucleotides. 7-Deaza-2'-deoxyguanosine protected the DNA-fragment from hydrolysis by the restriction endodeoxyribonuclease Eco RI, Pst I, Bam HI, and Sma I if the nucleoside was located within the recognition site.     

Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group.

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.     

Specific labelling of the active site of T7 RNA polymerase.

We describe a method for specifically labelling T7 RNA polymerase at (or near) the active site. Enzyme molecules that have been modified by covalent attachment of a benzaldehyde nucleotide derivative in the presence of template DNA are subsequently incubated with radioactively labelled nucleoside triphosphates. Labelling of the enzyme occurs as a result of the formation of the first phosphodiester bond. The labelling is template-directed and the expected specificity of initiation at individual T7 promoters is observed. The label has been localized to an 80 kd tryptic fragment that contains the carboxy-terminal portion of the enzyme.     

Excision in vitro of the DNA bound carcinogen, 4-nitroquinoline 1-oxide.

It has been shown that 4-hydroxyaminoquinoline 1-oxide, the proximate form of the carcinogen 4-nitroquinoline 1-oxide, binds covalently to the purine bases of DNA. Here we report that carcinogen-bound nucleotides can be excised from DNA by a 5' leads to 3' exonuclease associated with DNA polymerase I of E. coli in the forms of either mononucleotides or oligonucleotides. Beef spleen phosphodiesterase II (5' leads to 3') also split carcinogen-bound nucleotides, while a 3' leads to 5' exonuclease of DNA polymerase I and E. coli exonuclease III (3' leads to 5') could not excise the modified nucleotide.     

A unique region in bacteriophage t7 DNA polymerase important for exonucleolytic hydrolysis of DNA

A crystal structure of the bacteriophage T7 gene 5 protein/Escherichia coli thioredoxin complex reveals a region in the exonuclease domain (residues 144-157) that is not present in other members of the E. coli DNA polymerase I family. To examine the role of this region, a genetically altered enzyme that lacked residues 144-157 (T7 polymerase (pol) Delta144-157) was purified and characterized biochemically. The polymerase activity and processivity of T7 pol Delta144-157 on primed M13 DNA are similar to that of wild-type T7 DNA polymerase implying that these residues are not important for DNA synthesis. The ability of T7 pol Delta144-157 to catalyze the hydrolysis of a phosphodiester bond, as judged from the rate of hydrolysis of a p-nitrophenyl ester of thymidine monophosphate, also remains unaffected. However, the 3'-5' exonuclease activity on polynucleotide substrates is drastically reduced; exonuclease activity on single-stranded DNA is 10-fold lower and that on double-stranded DNA is 20-fold lower as compared with wild-type T7 DNA polymerase. Taken together, our results suggest that residues 144-157 of gene 5 protein, although not crucial for polymerase activity, are important for DNA binding during hydrolysis of polynucleotides.     

Synthesis of new modified DNAs by hyperthermophilic DNA polymerase: substrate and template specificity of functionalized thymidine analogues bearing an sp3-hybridized carbon at the C5 alpha-position for several DNA polymerases

Novel thymidine analogue triphosphates, which have an sp3-hybridized carbon at the C5 alpha-position with amino-linker arms, a methyl ester, or a carboxyl group at the C5 sidearm, were good substrates for primer-extension reactions by DNA polymerase from Pyrococcus kodakaraensis (KOD Dash DNA polymerase), yielding exclusively full-length products. The resulting modified DNA was further allowed to react with a functional molecule such as fluorescein isothiocyanate. By contrast, only truncated products were formed from the thymidine analogue substrate bearing the amino-linker arm or the negatively charged carboxyl group using Taq, Tth DNA polymerase, or DNA polymerase I from E. coli (Klenow fragment). The results indicate either that the thymidine analogue was not accepted by the enzymes, or that the polymerases could not extend the products, once the analogue had been incorporated, depending on the type of the analogue. A conventional thymidine analogue bearing an aminopropenyl group at the C5-position was accepted by all enzymes, among which KOD Dash DNA polymerase showed the highest activity for the polymerization with this analogue. Templates bearing the thymidine analogues in place of one thymidine residue were read by KOD Dash, Taq, Tth DNA polymerases, and the Klenow fragment giving the full-length product. KOD Dash DNA polymerase could expand structural diversities of substrates that can be used to prepare modified DNAs.     

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase.

We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a single round of DNA synthesis of the lac Z alpha gene by Taq polymerase responds to changes in dNTP concentration, pH, and the concentration of MgCl2 relative to the total concentration of deoxynucleotide triphosphates present in the reaction. Both base substitution and frameshift error rates of less than 1/100,000 were observed at pH 5-6 (70 degrees C) or when MgCl2 and deoxynucleotide triphosphates were present at equimolar concentrations. These high fidelity reaction conditions for DNA synthesis by the Taq polymerase may be useful for specialized uses of DNA amplified by the polymerase chain reaction.     

Characterization of the B6.61 polymerase ribozyme accessory domain

The "RNA world" hypothesis rests on the assumption that RNA polymerase ribozymes can replicate RNA without the use of protein. In the laboratory, in vitro selection has been used to create primitive versions of such polymerases. The best variant to date is a ribozyme called B6.61 that can extend a RNA primer template by 20 nucleotides (nt). This polymerase has two domains: the recently crystallized Class I ligase core, responsible for phosphodiester bond formation, and the poorly characterized accessory domain that makes polymerization possible. Here we find that the accessory domain is specified by a 37-nt bulged stem-loop structure. The accessory domain is positioned by a tertiary interaction between the terminal AL4 loop of the accessory and the J3/4 triloop found within the ligase core. This docking interaction is associated with an unwinding of the A3 and A4 helixes that appear to facilitate the correct positioning of an essential 8-nt purine bulge found between the two helices. This, together with other constraints inferred from tethering the accessory domain to a range of sites on the ligase core, indicates that the accessory domain is draped over the vertex of the ligase core tripod structure. This geometry suggests how the purine bulge in the polymerase replaces the P2 helix in the Class I ligase with a new structure that may facilitate the stabilization of incoming nucleotide triphosphates.     

Folding of the cocaine aptamer studied by EPR and fluorescence spectroscopies using the bifunctional spectroscopic probe Ç

The cocaine aptamer is a DNA molecule that binds cocaine at the junction of three helices. The bifunctional spectroscopic probe Ç was incorporated independently into three different positions of the aptamer and changes in structure and dynamics upon addition of the cocaine ligand were studied. Nucleoside Ç contains a rigid nitroxide spin label and can be studied directly by electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy after reduction of the nitroxide to yield the fluoroside Ç^f. Both the EPR and the fluorescence data for aptamer 2 indicate that helix III is formed before cocaine binding. Upon addition of cocaine, increased fluorescence of a fully base-paired Ç^f, placed at the three-way junction in helix III, was observed and is consistent with a helical tilt from a coaxial stack of helices II and III. EPR and fluorescence data clearly show that helix I is formed upon addition of cocaine, concomitant with the formation of the Y-shaped three-way helical junction. The EPR data indicate that nucleotides in helix I are more mobile than nucleotides in regular duplex regions and may reflect increased dynamics due to the short length of helix I.     

Hydrolysis of oligoribonucleotides: influence of sequence and length.

The chemical stability of phosphodiester bonds of some oligoribonucleotides in the presence of a cofactor like polyvinylpyrolidine (PVP) is sequence dependent. It was found that pyrimidine-A (YA) and pyrimidine-C (YC) are especially susceptible to hydrolysis. The hydrolyzability of this same phosphodiester bond is dependent on its position in the oligomer. The presence of 3' and 5'-adjacent nucleotides enhances hydrolysis of the UA phosphodiester bond. The acceleration of the hydrolysis of UA by a 5'-adjacent nucleotide is not base dependent. However, a 3'-adjacent purine increases hydrolysis of a UA phosphodiester bond more than a 3'-pyrimidine. The presence of the exoamino group on the 3'-side base (on 6 and 4 position for adenosine and cytidine, respectively) of YA or YZ phosphodiester bond is required for hydrolysis.     

8-Aza-7-deazapurine DNA: Synthesis and Duplex Stability of Oligonucleotides Containing 7-Substituted Bases

Abstract

The 7-substituted 8-aza-7-deazapurine phosphoramidites 1a – 3c as well as the phosphoramidite 4a were synthesized. In comparison to the parent purine oligonucleotide duplexes, the 7-substituted 8-aza-7-deazapurine residues lead to a significant duplex stabilization.     

RNA hydrolysis and inhibition of translation by a Co(III)-cyclen complex

Metal ion-chelator catalysts based on main-group, lanthanide, or transition metal complexes have been developed as nonenzymatic alternatives for the hydrolysis of the phosphodiester bonds in DNA and RNA. Cobalt (III), with its high-charge density, is known for its ability to hydrolyze phosphodiesters with rate constants as high as 2 x 10(-4) s(-1). We have developed a kinetically inert Co(III)-cyclen-based complex, Co(III)-cycmmb that is very potent in inhibiting the translation of RNA into protein. Contact time as short as 10 min is sufficient to achieve the complete inhibition of the translation of a concentrated luciferase RNA solution into the enzyme in a cell-free translation system. The inhibition appears to proceed through two pathways. The first pathway involves the kinetic or substitutional inertness of Co(III) for the RNA template at short contact times. This interaction is mediated through the kinetic inertness of Co(III) for the phosphate groups of the nucleotides, as well as coordination of Co(III) to the nitrogenous bases. The second pathway occurs at longer contact times and is mediated by the hydrolysis of the phosphodiester backbone. This report represents the first demonstrated use of a metal-chelate complex to achieve the inhibition of the translation of RNA into protein. This Co(III) system can be useful in its present nonsequence-specific form as a novel viral decontamination agent. When functionalized to recognize specific nucleic acid sequences, such a system could potentially be used in gene-silencing applications as an alternative to standard antisense or RNAi technologies.     

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides

In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2',4'-bridged nucleotides and three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2'-O,4'-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2',4'-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2',4'-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2',4'-bridged nucleotides into extending strands using 2',4'-bridged nucleoside-5'-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.     

长春和北京地区引起婴幼儿肺炎的3型与7型腺病毒的分子流行病学研究

对长春和北京地区连续12年(1976年冬至1988年春)引起小儿肺炎的3、7型腺病毒102株标本,进行了限制性内切酶核酸电泳图谱分析。56株7型腺病毒经BamHⅠ、BclⅠ、BglⅠ、XbaⅠ、SmaⅠ、HindⅢ分析后,表现为两个基因组型——Ad7 b和Ad7 d。46株3型腺病毒被Bg1 Ⅱ、BamHⅠ酶解后,表现为 3个基因组型——Ad 3Ⅰ、Ad 3Ⅱ、Ad 3Ⅲ。各基因组型的分布情况是:56株7型腺病毒中,43株为Ad 7 b(76.8％),流行于1976年冬至1986年春;13株是Ad 7 d(23.2％),出现于1982年,与Ad 7 b共同流行;1986年～1988年分析的5株病毒都是Ad 7d。43株3型腺病毒中,Ad3Ⅰ42株(91.0％),分布于12年中;Ad 3Ⅱ、Ad 3Ⅲ各2株,散在分布。此结果表明,国内这12年中引起小儿肺炎的3型腺病毒至少有3个基因组型,7型腺病毒至少有两个基因组型。Ad3Ⅰ和Ad7 b是流行优势基因组型。但自80年代初开始出现Ad7 d以来,有逐年增多的趋势,最近两年的标本又都是Ad7 d,很可能它将取代Ad7 b而成为流行的优势基因组型.