Search PubMed⌕ Search

Biomedical subjects

N Usman

Publications and source records attributed to N Usman.

62 records · Page 4Linked to original sources
PubMed1990

Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites.

The preparation of fully protected diisopropylamino-beta-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity greater than 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-iBu (Gua), beta-cyanoethyl for phosphate, in conjunction with TBDMS for 2'-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Further, it is shown that the deprotected oligoribonucleotides are free from 5'-2' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic 'Hammerhead Ribozyme'. These oligoribonucleotides were directly compared in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.

Base Sequence↗
PubMed1989

The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNA(fMet).

Stepwise, solid-phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNA(fMet). The 34-ribonucleotide oligomer corresponding to the sequence of the 5'-half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis. This 34-nucleotide oligomer serves as an acceptor in the RNA-ligase-catalyzed reaction with a phosphorylated 43-ribonucleotide oligomer corresponding to the sequence of the 3'-half molecule of tRNA(fMet). The DNA molecule having the sequence of tRNA(fMet) is a 76-deoxyribonucleotide oligomer with a 3'-terminal riboadenosine residue and all U residues replaced by T. These polymers have been compared with an oligodeoxyribonucleotide lacking all 2'-hydroxyl groups except for the 3'-terminal 2'-OH, an oligoribonucleotide lacking modified nucleosides and E. coli tRNA(fMet). The all-RNA 77-nucleotide oligomer can be aminoacylated by E. coli methionyl-tRNA synthetase preparation from E. coli with methionine and threonylated in the A37 position using a yeast extract. In agreement with work by Khan and Roe using tDNA(Phe) and tDNA(Lys), the rA77-DNA(fMet) can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent. Kinetic data support the notion that aminoacylation of tRNA(fMet) does not depend on the presence of 2'-hydroxyl groups with the exception of that in the 3'-terminal nucleotide.

Acylation↗
PubMed1988

The molecular structure of the complex of Hoechst 33258 and the DNA dodecamer d(CGCGAATTCGCG).

The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution. The crescent-shaped Hoechst compound is found to bind to the central four AATT base pairs in the narrow minor groove of the B-DNA double helix. The piperazine ring of the drug has its flat face almost parallel to the aromatic bisbenzimidazole ring and lies sideways in the minor groove. No evidence of disordered structure of the drug is seen in the complex. The binding of Hoechst to DNA is stabilized by a combination of hydrogen bonding, van der Waals interaction and electrostatic interactions. The binding preference for AT base pairs by the drug is the result of the close contact between the Hoechst molecule and the C2 hydrogen atoms of adenine. The nature of these contacts precludes the binding of the drug to G-C base pairs due to the presence of N2 amino groups of guanines. The present crystal structural information agrees well with the data obtained from chemical footprinting experiments.

Benzimidazoles↗
PubMed1988

Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity.

Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichia coli Ado-47-containing tRNA(fMet) species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoram idites. p-Nitro-phenylethyl groups were used to protect the O6 of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated that the synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species, tRNA(fMet) containing 7-methylguanosine at position 47.

Base Sequence↗
PubMed1988

Derivatization of controlled pore glass beads for solid phase oligonucleotide synthesis.

An improved and simplified procedure for the attachment of nucleosides onto long chain alkylamine controlled pore glass beads (LCAA-CPG) is presented. This procedure uses 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide (DEC) to couple nucleoside 3'-succinates directly to the LCAA-CPG. The preparation of nucleoside 3'-succinate anhydrides, p-nitrophenyl, or pentachlorophenyl esters and the use of highly toxic dicyclohexylcarbodiimide (DCC) is no longer required. Procedures involving acidic activation of the LCAA-CPG before derivatization and a pre-synthesis capping are also described, which prevent the formation of oligonucleotides linked by 3'-phosphates to the LCAA-CPG. Evidence is presented indicating that this type of linkage is responsible for the apparently greater than 100% coupling yields observed for the first coupling cycle.

Colorimetry↗
PubMed1986

Prevention of guanine modification and chain cleavage during the solid phase synthesis of oligonucleotides using phosphoramidite derivatives.

Phosphoramidite reagents can phosphitylate guanine bases at the O6-position during solid phase synthesis and serious chain cleavage occurs if the base phosphitylation is not eliminated before the iodine/water oxidation step. This can be accomplished by blocking the O6-position with a 2-cyanoethyl protecting group for deoxyribonucleotides or with a p-nitrophenylethyl group for ribonucleotides, regenerating the guanine base with water or acetate ions, or using N-methylanilinium trifluoroacetate (TAMA) as the phosphoramidite activator. The effectiveness of these methods was demonstrated by both 31P NMR studies and by the synthesis of d(Gp)23G, (Gp)14G, and d-(Gp)13rG sequences.

Amides↗
PubMedDate not supplied

Autoimplantation technique in the treatment of anogenital warts: a clinico-immunological study.

An autoimplantation technique was adopted in the treatment of 50 cases of anogenital warts and was compared with the conventionally used podophyllin regimen in a matched group of 50 patients. They were assessed with 15 untreated subjects in a control group for the rate of clinical cure after 6 weeks, recurrence after 1 year follow up and for humoral and cell mediated immune responses before and after treatment. In the podophyllin group, 70% of patients were cured after 6 weeks while in autoimplantation, only 44% of patients were cured, and none in the control group had natural remission of warts without any treatment. After 1 year all the cured cases (100%) that completed follow up had recurrence of warts with podophyllin treatment, while none had recurrence of lesions in the autoimplantation group. Results of the humoral and cell mediated immune (CMI) response studies revealed that autoimplantation technique significantly augmented both humoral and CMI responses while there was not significant change in the immune status after podophyllin treatment (P > 0.001).

Anus Diseases↗