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1.
Nucleic Acids Res ; 24(11): 2044-52, 1996 Jun 01.
Article in English | MEDLINE | ID: mdl-8668534

ABSTRACT

We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA synthesis. Standard 'runoff' RNA synthesis by T7 RNA polymerase gives RNA products which have ragged ends as a result of transcription which often extends beyond the end of the template DNA strand. Similarly, the Klenow fragment of Escherichia coli DNA polymerase I tends to run past the end of the template strand during DNA synthesis. We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5'-end of a template DNA strand, cause the polymerases to stop more abruptly at the last coding nucleotide. This results in a considerably more homogeneous oligonucleotide being produced. Three novel nucleosides are tested as potential terminators: 4-methylindole beta-deoxynucleoside (M), 1-naphthyl alpha-deoxynucleoside (N) and 1-pyrenyl alpha-deoxynucleoside (P). Comparison is made to an abasic nucleoside (phi) and to unterminated synthesis. Of these, M is found to be the most efficient at terminating transcription, and both P and M are highly effective at terminating DNA synthesis. It is also found that the ability of a nucleoside to stall synthesis when it is internally placed in the template strand is not necessarily a good predictor of terminating ability at the end of a template. Such terminator nucleosides may be useful in the preparative enzymatic synthesis of RNA and DNA, rendering purification simpler and lowering the cost of synthesis by preventing the uptake of potentially costly nucleotides into unwanted products.


Subject(s)
DNA Polymerase I/metabolism , DNA-Directed RNA Polymerases/metabolism , DNA/biosynthesis , Deoxyribose/analogs & derivatives , Hydrogen Bonding , Indoles/pharmacology , Naphthalenes/pharmacology , Nucleosides/pharmacology , Pyrenes/pharmacology , RNA/biosynthesis , Base Sequence , DNA/chemistry , Deoxyribose/chemical synthesis , Deoxyribose/pharmacology , Escherichia coli/enzymology , Indoles/chemical synthesis , Molecular Sequence Data , Naphthalenes/chemical synthesis , Nucleosides/chemical synthesis , Nucleosides/chemistry , Pyrenes/chemical synthesis , RNA/chemistry , Templates, Genetic , Viral Proteins
2.
Nucleic Acids Res ; 23(17): 3547-53, 1995 Sep 11.
Article in English | MEDLINE | ID: mdl-7567468

ABSTRACT

We report a novel convergent approach to the construction of circular DNA oligonucleotides from two smaller linear precursors. Circular DNAs 34-74 nucleotides (nt) in size are constructed non-enzymatically in a single step from two half-length oligomers. A DNA template is used to assemble the constituent parts into a triple helical complex which brings the four reactive ends together for chemical ligation with BrCN/imidazole/Ni2+. A homodimerization reaction strategy is successfully used on a small scale to construct circles 42, 58 and 74 nt in size. In addition, a heterodimerization strategy is successfully used in two cases to construct circular 34mers from different 16mer and 18mer precursors. Measurement of preparative yields for one biologically active 34mer circle shows that the dimerization strategy gives a yield higher than that from conventional cyclization and nearly as high as that for a normally synthesized linear DNA, establishing that there is not necessarily a yield penalty for circle construction. Six additional preparative circle constructions, giving conversions of approximately 33-85% from precursors to circular product, are also described. Convergent strategies allow the construction of medium and large size DNA molecules in higher yields than can be achieved by standard linear synthesis alone.


Subject(s)
DNA, Circular/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Base Sequence , Molecular Sequence Data , Nucleic Acid Hybridization
3.
J Med Chem ; 38(14): 2582-95, 1995 Jul 07.
Article in English | MEDLINE | ID: mdl-7629798

ABSTRACT

Purine-based analogs of SDZ 211-500 (5) were prepared and evaluated as inactivation modifiers of guinea pig or human cardiac sodium (Na) channels expressed in Xenopus oocytes. Substances which remove or slow the Na channel inactivation process in cardiac tissue are anticipated to prolong the effective refractory period and increase inotropy and thus have potential utility as antiarrhythmic agents. Heterocyclic substitution at the 6-position of the purine ring resulted in compounds with increased Na activity and potency, with 5-membered heterocycles being optimal. Only minor modifications to the benzhydrylpiperazine side chain were tolerated. Selected compounds which delayed the inactivation of Na channels were found to increase refractoriness and contractility in a rabbit Langendorff heart model, consistent with the cellular mechanism. Activity in both the oocyte and rabbit heart assays was specific to the S enantiomers. Preliminary in vivo activity has been demonstrated following intravenous infusion. The most promising compound on the basis of in vitro data is the formylpyrrole (S)-74, which is 25-fold more potent than DPI 201-106 (1) in the human heart Na channel assay.


Subject(s)
Heart/drug effects , Heterocyclic Compounds/chemical synthesis , Myocardium/chemistry , Purines/chemical synthesis , Sodium Channel Blockers , Animals , Cats , Guinea Pigs , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Humans , In Vitro Techniques , Magnetic Resonance Spectroscopy , Male , Myocardial Contraction/drug effects , Purines/chemistry , Purines/pharmacology , Rabbits , Structure-Activity Relationship , Xenopus
5.
Health Visit ; 53(3): 98, 1980 Mar.
Article in English | MEDLINE | ID: mdl-6898590
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