Biomedical subjects

L Beigelman

Publications and source records attributed to L Beigelman.

At least 37 records · Page 2Linked to original sources

PubMed1998

Site-specific cross-linking of amino acids in the basic region of human immunodeficiency virus type 1 Tat peptide to chemically modified TAR RNA duplexes.

The Human Immunodeficiency Virus type 1 Tat protein interacts specifically with a U-rich bulge within an RNA stem-loop known as the trans-activation responsive region (TAR) that occurs in all viral transcripts. We have photochemically cross-linked to Tat peptide (37-72), a model TAR duplex substituted at U23 in the bulge by 4-thioU. We have identified the cross-linked amino acid as Arg55 in the basic region of the Tat peptide by use of a combination of proteolytic digestions and MALDI-TOF mass spectrometric analysis. The identification also required use of a synthetic Tat peptide containing a site-specific, uniformly 13C and 15N isotopically labeled arginine. We also describe a new chemical procedure for obtaining site-specific cross-links to Tat via the use of 2'-beta-alanyl U-substituted TAR and the amino-specific reagent dithiobis(succinimidyl propionate). U23-2'-functionalized TAR was shown to cross-link uniquely to Lys51 in the basic region of Tat, whereas other sites in the upper and lower stems of TAR (U35, U38, and U42) showed cross-linking only to the N-terminus of Tat peptide (37-72). U40 cross-linked to both Lys51 and the N-terminus of the peptide. The results help to establish a preliminary model of the binding of Tat peptide to the major groove of TAR RNA.

Affinity Labels↗

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↗

PubMed1998

Ribozyme catalysis from the major groove of group II intron domain 5.

The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

Binding Sites↗

PubMed1998

Structure-function relationships in the hammerhead ribozyme probed by base rescue.

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

Adenine↗

PubMed1998

Discrete roles of hepatocytes and nonparenchymal cells in uridine catabolism as a component of its homeostasis.

Previous studies indicated that uridine is essentially cleared in a single pass through a rat liver and replaced in a highly regulated manner by uridine formed presumably by de novo synthesis. We report a cellular basis for the catabolic component of this apparent paradox by dissociation of the liver with collagenase into two cell fractions, hepatocytes and a nonparenchymal cell population. Suspensions of the nonparenchymal cells rapidly cleave uridine to uracil, whereas in hepatocytes this activity was <5% of that in nonparenchymal cells. Conversely, hepatocytes cause extensive degradation of uracil to -alanine. These differences correlate with the uridine phosphorylase and dihydrouracil dehydrogenase activity in cell-free extracts of each cell type. We have documented the existence of a Na+-dependent, nitrobenzylthioinosine-insensitive transport system for uridine in the parenchymal cells (Michaelis constant 46 +/- 5 microM) that achieves a three- to fourfold concentration gradient in hepatocytes. A similar system is present in the nonparenchymal cell population. In addition, a highly specific and active Na+-dependent transport system for beta-alanine, the primary catabolic metabolite of uracil, has been demonstrated in hepatocytes.

Animals↗

PubMed1997

Specific recognition of an rU2-N15-rU motif by VP55, the vaccinia virus poly(A) polymerase catalytic subunit.

VP55, the vaccinia poly(A) polymerase catalytic subunit, interacts with oligonucleotide primers via two uridylate recognition sites (Deng, L., and Gershon, P. D. (1997) EMBO J. 16, 1103-1113). Here, we show that the cognate RNA sequence comprises a 5'-rU2-N15-rU-3' motif (where N = any deoxyribo or ribonucleotide), embedded within oligonucleotide primers 29-30 nucleotides (nt), or greater, in length. Nine residues separate the 3'-most ribouridylate of the optimally positioned motif from the primer 3'-OH. A ribose sugar at the extreme 3'-terminal nucleotide of the primer is absolutely required for VP55's adenylyltransferase activity, but not for stable VP55-RNA interaction. A ribose at position -3 markedly stimulates both adenylyltransferase activity and stable binding. The use of uridine analogs indicated (i) those functional groups of the uracil base which contribute to stable VP55-primer interaction, and (ii) that VP55's ability to discriminate uracil from cytosine stems largely from the requirement for a protonated N3 nitrogen within the pyrimidine ring. The rU2-N15-rU motif was identified within the uridylate-rich 3' end of a naturally occurring vaccinia mRNA. However, oligonucleotides whose only internal uridylates comprised the motif supported only a 3-5-nt processive burst of oligo(A) tail addition, as opposed to the approximately 30-35-nt burst observed with the naturally occurring 3' end.

Binding Sites↗

PubMed1997

Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation.

Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by the pro-Rp oxygen of phosphate 9 and by N7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The Rp-, but not Sp-, phosphorothioate reduces the cleavage rate by 10(3)-fold, and the rate can be fully restored by addition of low concentrations of Cd2+, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location approximately 20 A from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd2+ binds with a Kd of 25 microM in the ground state and a Kd of 2.5 nM in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.

Base Sequence↗

PubMed1997

Branch-site selection in a group II intron mediated by active recognition of the adenine amino group and steric exclusion of non-adenine functionalities.

The 2'-hydroxyl on a specific bulged adenosine is the nucleophile during the first step of splicing by group II introns. To understand the means by which the ribozyme core recognizes this adenosine, it was mutagenized and effects on catalytic activity were quantified. The results indicate that a low level of mutational variability is tolerated at the branch-site of group II introns, with no apparent loss of fidelity. Analyses of mutant and modified nucleotides at the branch-site reveal that adenine is recognized primarily through the N6 amino group and by steric exclusion of functionalities found on other bases. The mutational and single atom effects reported here contrast with those observed during spliceosomal processing, suggesting that there are important differences in adenosine recognition by the two systems.

Adenine↗

PubMed1997

Functional groups on the cleavage site pyrimidine nucleotide are required for stabilization of the hammerhead transition state.

The role of individual functional groups on cytidine 17 in the hammerhead ribozyme was assessed by introducing modified pyrimidines into two kinetically well-characterized hammerheads. As long as the pyrimide ring size was maintained, the modifications had no effect on substrate binding, suggesting that the C17-C3 hydrogen bond observed in the X-ray structure is energetically neutral. However, modification of the exocyclic amino group and the carbonyl of C17 reduced the cleavage rate significantly, indicating that these groups are important in stabilizing the transition-state structure. C17 modifications did not affect the ratio of the forward and reverse reaction rates. Thus, unlike that believed previously, C17 is another one of many hammerhead residues critical in maintaining its active structure.

Cytidine↗

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

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

Nuclease-resistant ribozymes decrease stromelysin mRNA levels in rabbit synovium following exogenous delivery to the knee joint.

Catalytic RNA molecules, or ribozymes, have generated significant interest as potential therapeutic agents for controlling gene expression. Although ribozymes have been shown to work in vitro and in cellular assays, there are no reports that demonstrate the efficacy of synthetic, stabilized ribozymes delivered in vivo. We are currently utilizing the rabbit model of interleukin 1-induced arthritis to assess the localization, stability, and efficacy of exogenous antistromelysin hammerhead ribozymes. The matrix metalloproteinase stromelysin is believed to be a key mediator in arthritic diseases. It seems likely therefore that inhibiting stromelysin would be a valid therapeutic approach for arthritis. We found that following intraarticular administration ribozymes were taken up by cells in the synovial lining, were stable in the synovium, and reduced synovial interleukin 1 alpha-induced stromelysin mRNA. This effect was demonstrated with ribozymes containing various chemical modifications that impart nuclease resistance and that recognize several distinct sites on the message. Catalytically inactive ribozymes were ineffective, thus suggesting a cleavage-mediated mechanism of action. These results suggest that ribozymes may be useful in the treatment of arthritic diseases characterized by dysregulation of metalloproteinase expression.

Animals↗

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

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

Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2'-O-Me nucleotides, 1-2 other 2'-modified nucleotides at positions U4 and U7, and a 3'-3'-linked nucleotide "cap." Eight 2'-modified uridine residues were introduced at positions U4 and U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2'-NH2 substitutions at U4 and U7, or 2'-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only approximately 0.1 min. The addition of a 3'-3'-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to > 260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.

Animals↗