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T W Bruice

Publications and source records attributed to T W Bruice.

14 recordsLinked to original sources
PubMed2001

A novel dicationic polyamide ligand binds in the DNA minor groove as a dimer.

We have investigated DNA binding properties of a dicationic polyamide molecule (GL020924) that has exhibited unique protein displacement and gene regulation activities. Fluorescence, thermal melting and electrospray ionization mass spectrometry experiments showed that the binding stoichiometry of GL020924 is 2:1 to various DNA oligomers with 8-11 contiguous A/T bp. In accordance with those findings, circular dichroism experiments showed GL020924 binds as a partially staggered side-by-side dimer spanning 10-12 bp. These observations and molecular modeling studies demonstrate that the 2:1 GL020924-DNA complex may exhibit a novel form of stacking orientation involving at least partially parallel peptide groups.

Cations↗
PubMed2001

Allosteric interaction of minor groove binding ligands with UL9-DNA complexes.

The herpes simplex virus type 1 origin binding protein (UL9) is a sequence-specific DNA binding protein. Several studies have demonstrated that UL9 binds to the 11-base pair sequence 5'-CGTTCGCACTT-3' primarily, or solely, through interaction with the major groove. Minor groove binding ligands, such as distamycin, netropsin, and GLX, an indole-linked dimer of netropsin, can effectively disrupt the UL9-DNA complex only when their DNA binding sites are coincident with the right side of the DNA binding site of the protein and overlap with the protein binding site by two (TT) base pairs. These results suggest that the right side of the UL9-DNA complex has a unique structure that is sensitive to minor groove ligand binding. In addition, a biphasic displacement curve was observed with GLX, which suggests two modes of ligand binding which have different effects on UL9-DNA complexes. Using a fluorescence-based hybridization stabilization assay, we determined that GLX can bind to its binding site as an overlapping dimer (i.e., 2:1 stoichiometry). Footprinting of UL9-DNA complexes with the minor groove directed chemical nuclease 1,10-phenanthroline copper confirms that the DNA conformation at the position of the right-side ligand binding site of GLX is altered and has a widened minor groove. In contrast, it is well established that at 1:1 stoichiometries, AT sequence specific ligands, such as netropsin, distamycin, and GLX, prefer uniform, narrow minor grooves. The opposing conformational requirements of UL9 and lower concentrations of GLX at the ligand binding A-tract overlapping the right side of the protein binding site indicate that allosteric inhibition, rather than direct steric competition, contributes to ligand-induced protein displacement. At higher GLX concentrations, giving 2:1 binding in a widened minor groove, co-binding with UL9 is allowed. A model is presented that is consistent with these observations, and implications for targeted regulation of gene transcription are discussed.

Allosteric Regulation↗
PubMed2001

The hybridization-stabilization assay: a solution-based isothermal method for rapid screening and determination of sequence preference of ligands that bind to duplexed nucleic acids.

The gene-to-drug quest will be most directly served by the discovery and development of small molecules that bind to nucleic acids and modulate gene expression at the level of transcription and/or inhibit replication of infectious agents. Full realization of this potential will require implementation of a complete suite of modern drug discovery technologies. Towards this end, here we describe our initial results with a new assay for identification and characterization of novel nucleic acid binding ligands. It is based on the well recognized property of stabilization of hybridization of complementary oligonucleotides by groove and/or intercalation binding ligands. Unlike traditional thermal melt methodologies, this assay is isothermal and, unlike gel-based footprinting techniques, the assay also is performed in solution and detection can be by any number of highly sensitive, non-radioisotopic modalities, such as fluorescence resonance energy transfer, described herein. Thus, the assay is simple to perform, versatile in design and amenable to miniaturization and high throughput automation. Assay validation was performed using various permutations of direct and competitive binding formats and previously well studied ligands, including pyrrole polyamide and intercalator natural products, designed hairpin pyrrole-imidazole polyamides and furan-based non-polyamide dications. DNA specific ligands were identified and their DNA binding site size and sequence preference profiles were determined. A systematic approach to studying the relationship of binding sequence specificity with variation in ligand structure was demonstrated, and preferred binding sites in longer DNA sequences were found by pseudo-footprinting, with results that are in accord with established findings. This assay methodology should promote a more rapid discovery of novel nucleic acid ligands and potential drug candidates.

Base Pairing↗
PubMed1997

Control of complexity constraints on combinatorial screening for preferred oligonucleotide hybridization sites on structured RNA.

We have explored the use of short (10-mer), fully sequence-randomized oligonucleotide libraries for affinity-based screening in solution for energetically preferred sites of hybridization of a model 47-nucleotide (nt) mutant Ha-ras mRNA stem-loop fragment. In characterizing the model, binding studies using either a gel mobility-shift assay or an RNase ONE footprinting assay indicated the presence of a greatly preferred hybridization site for individual antisense RNA oligonucleotides on the 5'-most side of the ras RNA 19-nt loop. However, initial attempts to affinity-titrate combinatorial uniform 2'-O-methyl-substituted oligonucleotide libraries for selective binding to this 5'-loop site using an RNase ONE footprinting assay that can discriminate between binding to different sites on ras RNA were unsuccessful. By reducing the complexity of the library to a mix of seven RNA oligonucleotides complementary to a range of sites on ras RNA and with no self-complements, footprinting evidence for binding was obtained but was characterized by ras RNA site-specific binding constants differing dramatically from binding constants for individual oligonucleotides. The library complexity was reduced further to three different cases of two RNA oligonucleotides, one of which for all cases was the highest affinity 5'-loop complement. Detailed kinetic and thermodynamic binding analyses revealed a good fit of the data to independent (5'-loop and ascending stem sites), competitive (overlapping 5'-loop sites), or mutually allosteric (5'-loop and 3'-loop sites) formalisms and an energetics description showed that ras 5'-loop site-specific binding could be achieved by affinity titration only for the independent case. Reconstruction of events with the full complexity library suggested that there was the emergence of multiple, linked binding interactions and implied that successful hybridization affinity screening would be achieved only if all possible bimolecular binding interactions of individual library oligonucleotides with target RNA could be made mutually independent. Accordingly, by holding the calculated concentration of unique oligonucleotide sequences of a full complexity DNA library well below the value for the dissociation constant for binding of individual complement to the 5'-loop site and then titrating the concentration of ras RNA through this value, hybridization specific to the 5'-side of the ras loop was demonstrated as assayed either by sequential gel mobility-shift resolution of bimolecular complexes and RNase ONE footprinting in situ in gel slices or by RNase H cleavage of complexes in solution. Because this strategy uses an unbiased oligonucleotide library it should combinatorially identify energetically preferred hybridization sites on folded RNA targets of any sequence and of undetermined structure. This should enable a focused in vitro optimization of antisense oligonucleotide length, sequence, and chemical composition for preferred site binding affinity and specificity which, in turn, may be expected to provide for enhanced biological potency and specificity (Lima et al., 1996). Finally, the complexity constraints encountered and the fundamental requirement to control them presented here also should be applicable to interactions with any biomolecule target of any chemical class of combinatorial library when screened in solution in pooled mixes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Combinatorial screening and rational optimization for hybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity.

We describe our initial application of a biochemical strategy, comprising combinatorial screening and rational optimization, which directly identifies oligonucleotides with maximum affinity (per unit length), specificity, and rates of hybridization to structurally preferred sites on folded RNA, to the problem of design of antisense oligonucleotides active against the hepatitis C virus (HCV). A fully randomized sequence DNA oligonucleotide (10-mer) library was equilibrated with each of two folded RNA fragments (200 and 370 nucleotides (nt)), together spanning the 5' 440 nt of an HCV transcript (by overlapping 130 nt), which were varied over a range of concentrations. The equilibrations were performed in solution under conditions determined to preserve RNA structure and to limit all RNA-DNA library oligonucleotide interactions to 1:1 stoichiometry. Subsequent Escherichia coli RNase H (endoribonuclease H: EC 3.1.26.4) cleavage analysis identified two preferred sites of highest affinity heteroduplex hybridization. The lengths and sequences of different substitute chemistry oligonucleotides complementary to these sites were rationally optimized using an iterative and quantitative analysis of binding affinity and specificity. Thus, DNA oligonucleotides that hybridized with the same affinity to the preferred sites in the folded RNA fragments found by screening as to short (< or = 25 nt) RNA complements were identified but were found to vary in length (10-18 nt) from site to site. Phosphorothioate (P=S) and 2'-fluoro (2'-F) uniformly substituted oligonucleotides also were found, which hybridized optimally to these sites, supporting the design of short (10-15-nt) and maximally specific oligonucleotides that are more nuclease-resistant (via P=S) and have higher affinity (via 2'-F) than DNA. Finally, the affinities of DNA and uniform 2'-F-, P=S-substituted 10-20-mer oligonucleotide complements for the best hybridization site, from HCV nt 355 to nt 364-374, closely corresponded to antisense mechanism inhibition activities in an in vitro translation assay and in a human cell-based HCV core protein expression assay, respectively. These results validate our strategy for the selection of hybridization-optimized and biologically active antisense oligonucleotides targeting HCV RNA and support the potential for utility in further applications.

Gene Expression Regulation, Viral↗
PubMed1992

Pseudo--half-knot formation with RNA.

A pseudo--half-knot can be formed by binding an oligonucleotide asymmetrically to an RNA hairpin loop. This binding motif was used to target the human immunodeficiency virus TAR element, an important viral RNA structure that is the receptor for Tat, the major viral transactivator protein. Oligonucleotides complementary to different halves of the TAR structure bound with greater affinity than molecules designed to bind symmetrically around the hairpin. The pseudo--half-knot--forming oligonucleotides altered the TAR structure so that specific recognition and binding of a Tat-derived peptide was disrupted. This general binding motif may be used to disrupt the structure of regulatory RNA hairpins.

Base Sequence↗
PubMed1986

Energetics of proline racemase: transition-state fractionation factors for the two protons involved in the catalytic steps.

The isotope effects for the interconversion of L-proline and D-proline, catalyzed by proline racemase, have been determined in the saturated region with both [2-2H]proline and [2-3H]proline. The deuterium fractionation factors for each of the protons in flight have been obtained from two kinds of experiment: by measuring the rate of racemization of one [2-2H]proline enantiomer as it racemizes into an equilibrated pool of unlabeled proline and by measuring the deuterium content of a proline sample at the optical rotation maximum that occurs when an equimolar mixture of one deuterium-labeled enantiomer and the other unlabeled enantiomer runs to equilibrium. The tritium fractionation factors for each of the protons in flight have been determined from measurements of the rate of loss of tritium to the solvent as one [2-3H]proline enantiomer runs to equilibrium. Good agreement is found among the fractionation factors determined by each method. The deuterium fractionation factors for the two protons are not identical: that for the proton derived from L-proline is 0.375 and that for the proton derived from D-proline is 0.44. This difference has been confirmed by a double-competition experiment in which the optical rotation of a mixture of DL-[2-2H]proline and unlabeled DL-proline is followed with time. The rotation (initially zero) passes through a maximum, from which the ratio of the two fractionation factors (0.86) is obtained. These data, coupled with the equilibrium fractionation factor for the 2-position of proline (which has been determined to be 1.17), provide the transition-state factors for each of the in-flight protons, and delineate the nature of the transition state(s) for the enzyme-catalyzed racemization.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: fractionation factors for the essential catalytic groups in the enzyme-substrate complexes.

The fractionation factors of protons bound to the essential catalytic groups in proline racemase have been determined by comparison of the time courses of two competitive deuterium washout experiments. The rate of achievement of the maximum perturbation in the optical rotation has been measured in the oversaturated region (that is, at high substrate concentrations) under two conditions: in the first, we start with an equimolar mixture of deuterated substrate S' and of unlabeled product P; in the second, we again start with equal concentrations of substrate and product, but the concentration of the deuterated material S' is less than 20% that of S. The different concentrations of deuterated substrate produce different levels of deuteration of the enzyme's catalytic groups, the kinetic consequence of which allow the fractionation factors of these enzymic groups to be determined. The observed values for the fractionation factors of the enzyme's groups of 0.55 +/- 0.1 are only consistent with these groups' being thiols. This conclusion is supported by results of measurements of the solvent isotope effect determined in the unsaturated regime. These findings confirm the earlier suggestion of Abeles and his group that two cysteine residues mediate the catalysis of proline racemization by this enzyme.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: rates, fractionation factors, and buffer catalysis in the oversaturated region. Nature of the interconversion of the two forms of free enzyme.

To probe the nature of the interconversion of the two unliganded forms of proline racemase, a number of experiments have been performed under oversaturating conditions where the rate of the enzymic reaction is mainly limited by the rate of this interconversion. Competitive deuterium washout experiments, where an equimolar mixture of D- and L-proline (in which some or all of one enantiomer is specifically deuterated at the 2-position) is allowed to reach chemical and isotopic equilibrium mediated by the enzyme, have been followed in four ways. The size and the rate of achievement of the maximum perturbation in the optical rotation have been measured, the deuterium content of the substrate at this maximum has been determined, and the final approach to equilibrium after the perturbation maximum has been followed. Further, the enzyme-catalyzed rate of tritium loss from [2-3H]proline has been established. Finally, it has been shown that the enzyme interconversion reaction is catalyzed by several buffers (such as ammonium, hydrazinium, and hydrogen sulfide). These data are discussed in terms of Marcus' theory, which allows a rather detailed picture of the mechanism of free enzyme interconversion to be drawn. This process nicely parallels the mechanism of the enzyme-catalyzed interconversion of the proline enantiomers, and it is evident that substrate racemization (with the concomitant switch of the enzyme-bound protons) is mirrored by the water-mediated switch of the enzyme-bound protons that effects the interconversion of the free enzyme forms. The results favor a stepwise reaction for the interconversion of the free enzyme forms in which a proton is abstracted from a bound water molecule to give a reaction intermediate having a hydroxide ion bound to the diprotonated form of the enzyme.

Amino Acid Isomerases↗
PubMed1980

Use of alpha-secondary isotope effects in nucleophile-promoted reactions of pyrimidine derivatives; evidence for transient 5,6-dihydropyrimidine intermediates.

alpha-Secondary isotope effect provides a useful tool for detection of transient 5,6-dihydropyrimidine intermediates which have been indirectly implicated in a number of chemical and enzymic conversions of pyrimidine heterocycles. The method involves the use of kinetic secondary alpha-hydrogen isotope effects which are expected to accompany sp2 to sp3 rehybridization of C-6 of the pyrimidine ring if they occur prior to or at the rate determining step. Thus, using 6-tritiated pyrimidines, and measurement of the isotopic ratio of reactant and products, kT/kH values of 1.15 or greater would be indicative of rehybridization. Using this method, we have studied the inhibition of thymidylate synthetase by 5-fluorodeoxyuridylate and 5-nitro-deoxyuridylate as well as the chemical and enzymic dehalogenation of 5-bromodeoxyuridine and 5-bromodeoxyuridylate. Large secondary isotope effects observed in all of these reactions provided strong evidence for the formation of 5,6-dihydropyrimidine intermediates.

Bromodeoxyuridine↗