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Biomedical subjects

N Usman

Publications and source records attributed to N Usman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Endoscopic ultrasound miniprobe-guided steroid injection for treatment of refractory esophageal strictures.

Local injection of corticosteroids into refractory esophageal strictures to decrease the restenosis rate has been reported. Here we report our efforts in three patients to render the delivery of steroids more precise, by injecting them in the thickest segment of the stricture with the guidance of a high frequency ultrasound miniprobe passed through a regular upper endoscope. Steroid injection under ultrasound miniprobe guidance may be indicated for patients who do not respond to a "blind" steroid injection without miniprobe guidance.

Aged↗
PubMed1997

Cellular uptake properties of a 2'-amino/2'-O-methyl-modified chimeric hammerhead ribozyme targeted to the epidermal growth factor receptor mRNA.

Catalytic RNA or ribozymes have important potential applications as molecular biological tools in the study of gene expression and as therapeutic inhibitors of disease-causing genes. Very little is known, however, about the cellular uptake mechanisms of exogenously delivered synthetic ribozymes. In this study, we have characterized the uptake properties of a synthetic, 2'-O-methyl-modified ribozyme containing U4/U7 amino groups within the catalytic core of the hammerhead motif. The cellular uptake of the internally [32P]-radiolabeled hammerhead ribozyme in U87-MG glioma cells was temperature, energy, and pH dependent and involved an active process that could be competed with cold ribozyme of the same chemistry and sequence, an all 2'-O-methyl-modified ribozyme of the same sequence, antisense PS-ODNs, and a variety of other polyanions (salmon sperm DNA, spermidine, dextran sulfate, and heparin). Subcellular distribution studies of fluorescently labeled ribozymes confirmed an extranuclear, punctate localization similar to that observed for an endosomal marker, dextran. Our study highlights that hammerhead ribozymes, despite exhibiting a defined secondary structure, enter cells by an endocytic mechanism that appears to be similar to that reported for a variety of antisense ODNs. These observations should facilitate the development of more efficient delivery systems.

Base Sequence↗
PubMed1996

Optimizing the cell efficacy of synthetic ribozymes. Site selection and chemical modifications of ribozymes targeting the proto-oncogene c-myb.

Expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5'-end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2'-modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.

Animals↗
PubMed1996

Chemically modified hammerhead ribozymes with improved catalytic rates.

A site-specific chemical modification strategy has been employed to elucidate structure-function relationships at the only phylogenetically nonconserved position within the core of the hammerhead ribozyme (N7). Four different base substitutions at position 7 resulted in increased catalytic rates. A pyridin-4-one base substitution increased the rate of the chemical step up to 12-fold. These results are the first examples of chemical modifications within a catalytic RNA that enhance the rate of the chemical step. Four base substitutions resulted in decreased catalytic rates. The results do not correlate with proposed hydrogen bond interactions (Pley et al., 1994; Scott et al., 1995). This study demonstrates the utility of using unnatural nucleotide analogs-rather than mutagenesis with the four standard nucleotides alone-to elucidate structure-function relationships of small RNAs.

Catalysis↗
PubMed1996

Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases.

We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.

2-Aminopurine↗
PubMed1996

RNA hydration: a detailed look.

The crystal structure of the RNA duplex [r(CCCCGGGG)]2 has been refined to 1.46 A resolution with room temperature synchrotron diffraction data. This represents the highest resolution reported to date for an all-RNA oligonucleotide and is well beyond the best resolution ever achieved with an A-form DNA duplex. The analysis of the ordered hydration around the octamer duplex reveals conserved regular arrangements of water molecules in both grooves. In the major groove, all located first shell water molecules can be fitted into a pattern that is repeated through all eight base pairs, involves half the phosphate oxygens, and joins the two strands. In the minor groove, roughly across its narrowest dimension, tandem water molecules link the 2'-hydroxyl groups of adjacent nucleotides in base-pair steps in a similarly regular fashion. The structure provides evidence for an important role of the 2'-hydroxyl groups in the thermodynamic stabilization of RNA, beyond their known functions of locking the sugar pucker and mediating 3' --> 5' intrastrand O2'...O4' hydrogen bonds. The ribose 2'-hydroxyls lay the foundation for the enthalpic stability of the RNA relative to the DNA duplex, both as a scaffold for the water network in the minor groove and through their extensive individual hydration.

Crystallography, X-Ray↗
PubMed1996

Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure.

The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage in trans and has potential as a therapeutic agent. We have used a chemical synthesis approach to study the requirements of hairpin RNA cleavage for sugar and base moieties in residues of internal loop B, an essential region in one of the two ribozyme domains. Individual nucleosides were substituted by either a 2'-deoxy-nucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we propose a new model for the potential secondary structure of internal loop B of the hairpin ribozyme.

Base Composition↗
PubMed1996

Hammerhead ribozyme engineering.

Of all the catalytic RNAs the hammerhead ribozyme is the most chemically modified and structurally studied. Such studies have resulted in improvements in the nuclease resistance of ribozymes, reductions in their size, and improvements in their catalytic efficiency. These improvements have facilitated the use of ribozymes for therapeutic applications, and have allowed us to study how the three-dimensional structure of the enzyme and its array of functional groups interact to create a catalytic site where a phosphodiester bond is cleaved.

Base Sequence↗
PubMed1996

Template-directed ligation of peptides to oligonucleotides.

BACKGROUND: Oligonucleotide-peptide conjugates have several applications, including their potential use as therapeutic agents. We developed a strategy for the chemical ligation of unprotected peptides to oligonucleotides in aqueous solution. The two compounds are joined via a stable amide bond in a template-directed reaction. RESULTS: Peptides, ending in a carboxy-terminal thioester, were converted to thioester-linked oligonucleotide-peptide intermediates. The oligonucleotide portion of the intermediate binds to a complementary oligonucleotide template, placing the peptide in close proximity to an adjacent template-bound oligonucleotide that terminates in a 3' amine. The ensuing reaction results in the efficient formation of an amide-linked oligonucleotide-peptide conjugate. CONCLUSIONS: An oligonucleotide template can be used to direct the ligation of peptides to oligonucleotides via a highly stable amide linkage. The ligation reaction is sequence-specific, allowing the simultaneous ligation of multiple oligonucleotide-peptide pairs.

Amides↗
PubMed1996

Crystal structures of an A-form duplex with single-adenosine bulges and a conformational basis for site-specific RNA self-cleavage.

BACKGROUND: Bulged nucleotides are common secondary structural motifs in RNA molecules and are often involved in RNA-RNA and RNA-protein interactions. RNA is selectively cleaved at bulge sites (when compared to other sites within stems) in the presence of divalent metal cations. The effects of bulge nucleotides on duplex stability and topology have been extensively investigated, but no detailed X-ray structures of bulge-containing RNA fragments have been available. RESULTS: We have crystallized a self-complementary RNA-DNA chimeric 11-nucleotide sequence containing single-adenosine bulges under two different conditions, giving two distinct crystal forms. In both lattices the adenosines are looped out, leaving the stacking interactions in the duplex virtually unaffected. The bulges cause the duplex to kink in both cases. In one of the structures, the conformation of the bulged nucleotide places its modeled 2'-oxygen in line with the adjacent phosphate on the 3' side, where it is poised for nucleophilic attack. CONCLUSIONS: Single adenosine bulges cause a marked opening of the normally narrow RNA major groove in both crystal structures, rendering the bases more accessible to interacting molecules compared with an intact stem. The geometries around the looped-out adenosines are different in the two crystal forms, indicating that bulges can confer considerable local plasticity on the usually rigid RNA double helix. The results provide a conformational basis for the preferential, metal-assisted self-cleavage of RNA at bulged sites.

Crystallography, X-Ray↗
PubMed1996

Inhibition of vascular smooth muscle cell proliferation by ribozymes that cleave c-myb mRNA.

Proliferation of injured smooth muscle cells contributes to the reocclusion or restenosis of coronary arteries that often occurs following angioplasty procedures. We have identified and optimized nuclease-resistant ribozymes that efficiently cleave c-myb RNA. Three ribozymes targeting different sites in the c-myb mRNA were synthesized chemically and delivered to rat aortic smooth muscle cells with cationic lipids; all three inhibited serum-stimulated cell proliferation significantly. RNA molecules with two base substitutions in the catalytic core that render the ribozyme catalytically inactive had little effect on smooth muscle cell proliferation. Ribozymes with scrambled binding arm sequences also failed to affect cell cycle progression of vascular smooth muscle cells. Furthermore, inhibition of rat smooth muscle cell proliferation correlated with a reduction in intact c-myb mRNA. Efficacy of the chemically-modified ribozyme was compared directly to phosphorothioate antisense oligodeoxynucleotides targeting the same site in the c-myb RNA; the ribozyme had superior efficacy and showed greater specificity than the antisense molecules. Exogenously delivered ribozymes also inhibited porcine and human smooth muscle cell proliferation effectively. Ribozymes targeting c-myb or other regulators of smooth muscle cell proliferation may represent novel therapeutics for the treatment of restenosis after coronary angioplasty.

Animals↗
PubMed1995

Synthesis of 2'-modified nucleotides and their incorporation into hammerhead ribozymes.

Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereoselective synthesis of the R isomer of 2'-deoxy-2'-C-allyl uridine and cytidine phosphoramidites, based on the Keck allylation procedure, were developed. Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was optimized and a method for the convenient preparation of 5'-O-dimethoxytrityl-2'-deoxy-2'-phthalimidouridine 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was developed. During the attempted preparation of the 2'-O-t-butyldimethylsilyl-3'-O-phosphoramidite of arabinouridine a reversed regioselectivity in the silylation reaction, compared with the published procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclization is proposed. The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2'-difluoromethylene uridine phosphoramidites is described. Based on a '5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modified monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule.

Cytidine↗
PubMed1995

Crystallization and preliminary X-ray diffraction analysis of double-helical RNA octamers.

Single crystals of a chemically synthesized self-complementary RNA octamer with sequence r(CCCCGGGG) have been obtained by screening 50 different conditions at room temperature using a standard sparse-matrix sampling method. Two crystal forms with different morphologies grew under diverse crystallization conditions within days by hanging-drop vapor diffusion. Hexagonal crystals with space group P6(1)22 (one strand per asymmetric unit) and unit-cell dimensions a = b = 39.73 and c = 58.55 A, diffracted to 2.6 A. Rhombohedral crystals with space group R32 (one duplex per asymmetric unit) and unit-cell dimensions a = b = 42.38 and c = 131.70 A, (hexagonal setting) diffracted beyond 1.5 A. Data sets for both crystal forms were collected on image-plate/rotating-anode generator equipment and structure determinations and refinements are under way.

Journal Article↗
PubMed1995

An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer.

Base Sequence↗
PubMed1995

Synthesis and biological activities of a phosphorodithioate analog of 2',5'-oligoadenylate.

To enhance the resistance of 2-5A (pppA2'p5'A2'p5'A) to degradation by exo- and endonucleases, a phosphorodithioate analog was synthesized using a solid-phase phosphite triester approach with N6-benzoyl-5'-O-dimethoxytrityl-3'-O-t-butyldimethylsilyladenosine 2'-[S-(beta-thiobenzoylethyl)-pyrrolidinophosphorothioamidit e]. 5'-Monophosphorylation was accomplished with 2-[2-(4,4'-dimethoxytrityloxy)-ethylsulfonyl]ethyl-(2-cyanoe thyl)-(N,N- diisopropyl)-phosphoramidite. The resulting product, p5'A2'(s2p)- 5'A2'(s2p)5'A, was approximately 10-fold less effective as an activator of purified human recombinant 2-5A-dependent RNase than was 2-5A itself. This loss of activation ability was related directly to the loss of binding ability of the phosphorodiothioate analog. As predicted, p5'A2'(s2p)5'A2' (s2p)5'A was stable to snake venom phosphodiesterase and the nucleolytic activities of both human lymphoblastoid CEM cell extracts and human serum, under conditions that led to facile degradation of parent 2-5A. This nuclease stability permitted the observation of the CEM cell extracts and human serum phosphatase activity which led to 5'-dephosphorylation of p5'A2'(s2p)5'A2'(s2p)5'A.

Adenine Nucleotides↗
PubMed1995

Synthesis, deprotection, analysis and purification of RNA and ribozymes.

Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.

Base Sequence↗
PubMed1995

The crystal structure of r(CCCCGGGG) in two distinct lattices.

The structure of the RNA octamer r(C4G4) has been determined in two different crystal forms. The conformations of the RNA duplex in the rhombohedral and hexagonal lattices display only minimal deviations, demonstrating that the RNA double helix is considerably less deformable than the DNA double helix. For the first time the crystal structures of an A-RNA and an A-DNA with identical sequence can now be compared. The large number of ordered water molecules observed in the minor groove of the RNA duplex suggests that an important contribution to its higher rigidity derives from the improved hydration due to the presence of the 2'-hydroxyl groups. Our finding that the conformation of the RNA double helix is virtually unaffected by different crystal packing modes provides evidence that proteins may not alter the conformation of RNA stem regions in a significant way.

Chemical Phenomena↗