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Biomedical subjects

N Usman

Publications and source records attributed to N Usman.

At least 19 recordsLinked to original sources
PubMed1998

Structural variation induced by different nucleotides at the cleavage site of the hammerhead ribozyme.

The hammerhead ribozyme is capable of cleaving RNA substrates at 5' UX 3' sequences (where the cleavage site, X, can be A, C, or U). Hammerhead complexes containing dC, dA, dI, or rG nucleotides at the cleavage site have been studied by NMR. The rG at the cleavage site forms a Watson-Crick base pair with C3 in the conserved core of the hammerhead, indicating that rG substrates inhibit the cleavage reaction by stabilizing an inactive conformation of the molecule. Isotope-edited NMR experiments on the hammerhead complexes show that there are different short proton-proton distances between neighboring residues depending upon whether there is a dC or dA at the cleavage site. These NMR data demonstrate that there are significant differences in the structure and/or dynamics of the active-site residues in these hammerhead complexes. Molecular dynamics calculations were used to model the conformations of the cleavage-site variants consistent with the NMR data. The solution conformations of the hammerhead ribozyme-substrate complexes are compared with the X-ray structure of the hammerhead ribozyme and are used to help understand the thermodynamic and kinetic differences among the cleavage-site variants.

Binding Sites↗
PubMed1998

The structural basis of hammerhead ribozyme self-cleavage.

We have captured an 8.7 A conformational change that takes place in the cleavage site of the hammerhead ribozyme during self-cleavage, using X-ray crystallography combined with physical and chemical trapping techniques. This rearrangement brings the hammerhead ribozyme from the ground state into a conformation that is poised to form the transition state geometry required for hammerhead RNA self-cleavage. Use of a 5'-C-methylated ribose adjacent to the cleavage site permits this ordinarily transient conformational change to be kinetically trapped and observed crystallographically after initiating the hammerhead ribozyme reaction in the crystal. Cleavage of the corresponding unmodified hammerhead ribozyme in the crystal under otherwise identical conditions is faster than in solution, indicating that we have indeed trapped a catalytically relevant intermediate form of this RNA enzyme.

Binding Sites↗
PubMed1998

Inductive interactions regulating body patterning in planarian, revealed by analysis of expression of novel gene scarf.

Subtractive hybridization was used to search for the early difference in gene expression between anteriorly and posteriorly regenerating tissues of the same region of the planarian body. A sequence (named scarf) specific for posteriorly regenerating tissue was isolated, encoding a soluble C-type lectin consisting of two slightly different carbohydrate-recognition domains. Such an unusual bivalent structure allows attribution of the protein to a novel subfamily of C-type lectins. scarf expression in intact planarian is rather abundant and follows a characteristic pattern not linked to any known morphological structure. We performed a series of experiments using scarf as a molecular marker. Its expression was monitored during different types of regeneration by whole-mount in situ hybridization and reverse-transcription polymerase chain reaction. The obtained data suggest that scarf expression is positively regulated by anterior tissues closely adjacent to the scarf-expressing region, so that their surgical removal results in rapid scarf switch-off. In turn, tissues expressing scarf seem to inhibit its activation anteriorly. This indicates that at least some of the body patterning events in planarians are based on a system of reciprocal inductive interactions rather than on a global morphogen gradient.

Amino Acid Sequence↗
PubMed1998

1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes.

The incorporation of 1-deazaadenosine (c1A, 1b) into a hammerhead ribozyme and the resulting catalytic activity is described. For this purpose the phosphoramidite 2a and the 3'-phosphonate 2b as well as Fractosil-linked 1-deazaadenosine (3b) were prepared. The methoxyacetyl group was used for the 6-amino group protection and the triisopropylsilyl residue was introduced as the 2'-OH protecting group. Replacement of residues A14and A15.1 of the hammerhead ribozyme by 1-deazaadenosine resulted in a significantly reduced catalytic activity. Substitution of the A6, A9 and A13 residues has only a minor influence. The findings observed on ribozymes modified with 1-deazaadenosine were compared with those containing other adenosine analogues.

Base Composition↗
PubMed1997

Ordered differential display: a simple method for systematic comparison of gene expression profiles.

A method for display of 3'-end restriction fragments of cDNAs is proposed, extending the idea reported recently. First, representative pools of such fragments are selectively amplified using PCR suppression effect. Then, simplified subsets of these fragments suitable for comparison by PAGE are amplified by adapter-specific primers extended by two randomly picked bases at their 3'ends. By testing all possible combinations of extended primers the whole mRNA pool may be systematically investigated. The method was applied to search for molecular regional markers of freshwater planarian Dugesia tigrina .

Animals↗
PubMed1997

Effect of structural modifications on the activity of the leadzyme.

The structure/function properties of functional groups in the leadzyme have been studied by assaying the activity of analog ribozymes generated by the systematic substitution of modified nucleotides in the internal loop region of the ribozyme. Guanosine analogs introduced at positions 4 and 7 occupied by guanosine in the wild-type molecule severely diminished cleavage. The substitution of deoxycytidine for cytidine at the cleavage site completely eliminated the activity of the leadzyme, as expected if the adjacent 2'-OH were the nucleophile in the cleavage reaction. On the other hand, substitution of an abasic nucleotide for adenosine at position 8 did not affect the activity of the ribozyme. An analysis of the activity of these analogs gives rise to the proposal of a triple-base pair motif implicating C1, G4, and G7.

Base Sequence↗
PubMed1997

Quantitating tertiary binding energies of 2' OH groups on the P1 duplex of the Tetrahymena ribozyme: intrinsic binding energy in an RNA enzyme.

Binding of the Tetrahymena ribozyme's oligonucleotide substrate (S) involves P1 duplex formation with the ribozyme's internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The overall binding energies provided by 2' OH groups on S and IGS have been measured previously. To obtain the energetic contribution of each of these 2' OH groups in the docking step, we have separately measured their contribution to the stability of a model P1 duplex using "substrate inhibition". This new approach allows measurement of duplex stabilities under conditions identical to those used for ribozyme binding measurements. The tertiary binding energies from the individual 2' OH groups include a small destabilizing contribution of 0.7 kcal/mol and stabilizing contributions of up to -2.9 kcal/mol. The energetic contributions of specific 2' OH groups are discussed in the context of considerable previous work that has characterized the tertiary interactions of the P1 duplex. A "threshold" model for the open and closed complexes is presented that provides a framework to interpret the energetic effects of functional group substitutions on the P1 duplex. The sum of the tertiary stabilization provided by the conserved G x U wobble at the cleavage site and the individual 2' OH groups on the P1 duplex is significantly greater than the observed tertiary stabilization of S (11.0 vs 2.2 kcal/mol). It is suggested that there is an energetic cost for docking the P1 duplex into the active site that is paid for by the "intrinsic binding energy" of groups on the P1 duplex. Substrates that lack sufficient tertiary binding energy to overcome this energetic barrier exhibit reduced reactivities. Thus, the ribozyme appears to use the intrinsic binding energy of groups on the P1 duplex for catalysis. This intrinsic binding energy may be used to position reactants within the active site and to induce electrostatic destabilization of the substrate, relative to its interactions in solution.

Animals↗
PubMed1997

Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect.

Here we describe a method for preparing high-quality cDNA libraries from total RNA. By this method, double-stranded (ds) cDNA ligated with a specially designed ds adaptor is amplified by PCR using a modified T-primer and another primer corresponding to the outer part of the adaptor. The suppression PCR effect strongly inhibits the amplification of poly(A) RNA, thereby reducing background. This method leads to amplification of high-quality cDNA, facilitating the construction of representative cDNA libraries from as little as 10-100 ng of total RNA.

DNA, Complementary↗
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↗