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Biomedical subjects

David G Gorenstein

Publications and source records attributed to David G Gorenstein.

At least 19 recordsLinked to original sources
PubMed2005

The effects of thiophosphate substitutions on native siRNA gene silencing.

RNA mediated interference has emerged as a powerful tool in controlling gene expression in mammalian cells. We investigated the gene silencing properties of six thiophosphate substituted siRNAs (all based on a commercial luciferase medium silencer) compared to that of unmodified siRNA. We also examined the cytotoxicity and dose-response using several thiophosphate modified siRNAs with unmodified siRNA. Our results show that two thiophosphate siRNA sequences convert from medium to high silencers with the addition of four randomly placed thiophosphates. Both thiophosphate siRNAs have a statistically significant difference in luciferase gene silencing (5% and 6% activity) relative to the unmodified native medium silencer referred to as siRNA-2 (18% activity) and four other thiophosphate siRNAs that maintain their medium silencing capability. This indicates that specific thiophosphate substitutions may alter native siRNA function. Further, this shows that thiophosphate siRNAs with the same nucleotide sequence but with different sulfur modification positions have different silencing effects. Both the native siRNA and the thio siRNAs showed a concentration dependent relationship, i.e., with concentration increase, the luciferase gene silencing effect also increased. Confirming cytotoxicity experiments showed no significant changes when HeLa cells were treated with 10nM thiophosphate siRNAs over the course of several days. These results suggest that specific placement of thiophosphates could play an important role in the development of siRNAs as therapeutics by engineering in properties such as strength of binding, nuclease sensitivity, and ultimately efficacy.

Gene Expression Regulation↗
PubMed2004

5-hydroxyuracil can form stable base pairs with all four bases in a DNA duplex.

NMR studies, UV-monitored melting experiments, and ab initio calculations show that 5-hydroxyuracil, produced by the oxidative de-amination of cytosines by reactive oxygen species, can form stable base-pairs with dA, dG, dC and dT residues in a DNA duplex, providing a basis for the in-vivo incorporation of 5-hydroxyuracil during DNA replication.

Base Pairing↗
PubMed2004

TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain.

A number of transcription factor proteins contain domains that are fully or partially unstructured. The means by which such proteins acquire naturally folded conformations are not well understood. When they encounter their proper binding partner(s), several of these proteins adopt a folded conformation through an induced-fit mechanism. The glucocorticoid receptor (GR) is a ligand-activated transcription factor. Expressed independently as a recombinant peptide, the N-terminal transactivation domain (AF1) of the GR shows little structure and appears to exist as a collection of random coil configurations. The GR AF1 is known to interact with other transcription factors, including a critical component of the general transcription machinery proteins, the TATA box binding protein (TBP). We tested whether this interaction can lead to acquisition of structure in the GR AF1. Our results show that recombinant GR AF1 acquires a significant amount of helical content when it interacts with TBP. These structural changes were monitored by Fourier transform infrared and NMR spectroscopies, and by proteolytic digestions. Our results support a model in which TBP binding interaction with the GR AF1 induces significantly greater helical structure in the AF1 domain. This increased helical content in the GR AF1 appears to come mostly at the expense of random coil conformation. These results are in accordance with the hypothesis that an induced-fit mechanism gives structure to the GR AF1 when it encounters TBP.

Circular Dichroism↗
PubMed2004

Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites.

Translation of the open reading frames (ORF) of the hepatitis C virus (HCV) and closely related GB virus B (GBV-B) genomes is driven by internal ribosome entry site (IRES) elements located within the 5' non-translated RNA. The functioning of these IRES elements is highly dependent on primary and higher order RNA structures. We present here the solution structures of a common, critical domain within each of these IRESs, stem-loop IIIc. These ten-nucleotide hairpins have nearly identical sequences and similar overall tertiary folds. The final refined structure of each shows a stem with three G:C base-pairs and a novel tetraloop fold. Although the bases are buckled, the first and fourth nucleotides of both tetraloops form a Watson-Crick type base-pair, while the apical nucleotides are located in the major groove where they adopt C(2)-endo sugar puckering with B-form geometry. No hydrogen bonding interactions were observed involving the two apical residues of the tetraloop. Stability of the loops appears to be derived primarily from the stacking of bases, and the hydrogen bonding between the fourth and seventh residues. Mutational analysis shows that the primary sequence of stem-loop IIIc is important for IRES function and that the stem and first and fourth nucleotides of the tetraloop contribute to the efficiency of internal ribosome entry. Base-pair formation between these two positions is essential. In contrast, the apical loop nucleotides differ between HCV and GBV-B, and substitutions in this region of the hairpin are tolerated without major loss of function.

Base Sequence↗
PubMed2004

High-resolution structure of a picornaviral internal cis-acting RNA replication element (cre).

Picornaviruses constitute a medically important family of RNA viruses in which genome replication critically depends on a small RNA element, the cis-acting replication element (cre), that templates 3D(pol) polymerase-catalyzed uridylylation of the protein primer for RNA synthesis, VPg. We report the solution structure of the 33-nt cre of human rhinovirus 14 under solution conditions optimal for uridylylation in vitro. The cre adopts a stem-loop conformation with an extended duplex stem supporting a novel 14-nt loop that derives stability from base-stacking interactions. Base-pair interactions are absent within the loop, and base substitutions within the loop that favor such interactions are detrimental to viral RNA replication. Conserved adenosines in the 5' loop sequence that participate in a slide-back mechanism of VPg-pUpU synthesis are oriented to the inside of the loop but are available for base templating during uridylation. The structure explains why substitutions of the 3' loop nucleotides have little impact on conformation of the critical 5' loop bases and accounts for wide variation in the sequences of cres from different enteroviruses and rhinoviruses.

Base Pairing↗
PubMed2004

Combinatorial selection and edited combinatorial selection of phosphorothioate aptamers targeting human nuclear factor-kappaB RelA/p50 and RelA/RelA.

Nuclear factor-kappaB (NF-kappaB) transcription factors are important in regulating the immune response and play critical roles in the pathogenesis of chronic inflammatory diseases and a variety of human cancers. Agents that target specific NF-kappaB dimers may serve as therapeutic agents for the prevention of pathogenic immune responses. We have selected monothiophosphate-modified aptamers, or "thioaptamers", to the NF-kappaB p50/RelA heterodimer using combinatorial selection techniques. We also utilized a "double sieve" or editing approach for the generation of thioaptamers with enhanced selectivity to the RelA/RelA homodimer. The thioaptamers from these selections and our previous selections on the p50/p50 and RelA/RelA homodimers all had unique sequences and bound tightly to the recombinant NF-kappaB dimers against which they were selected. The selected thioaptamers also appear to maintain their selectivity and specificity among other cellular proteins, because they have the ability to bind NF-kappaB proteins within nuclear extracts from lipopolysaccharide (LPS)-induced macrophages and B cells.

B-Lymphocytes↗
PubMed2004

Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virus.

The solution structure of domain III from the New York West Nile virus strain 385-99 (WN-rED3) has been determined by NMR methods. The West Nile domain III structure is a beta-barrel structure formed from seven anti-parallel beta-strands in two beta-sheets. One anti-parallel beta-sheet consists of beta-strands beta1 (Phe(299)-Asp(307)), beta2 (Val(313)-Tyr(319)), beta4 (Arg(354)-Leu(355)), and beta5 (Lys(370)-Glu(376)) arranged so that beta2 is flanked on either side by beta1 and beta5. The short beta4 flanks the end of the remaining side of beta5. The remaining anti-parallel beta-sheet is formed from strands beta3 (Ile(340)-Val(343)), beta6 (Gly(380)-Arg(388)), and beta7 (Gln(391)-Lys(399)) arranged with beta6 at the center. Residues implicated in antigenic differences between different West Nile virus strains (and other flaviviruses) and neutralization are located on the outer surface of the protein. Characterization of the binding of monoclonal antibodies to WN-rED3 mutants, which were identified through neutralization escape experiments, indicate that antibody neutralization directly correlates with binding affinities. These studies provide an insight into theoretical virus-receptor interaction points, structure of immunogenic determinants, and potential targets for antiviral agents against West Nile virus and highlight differences between West Nile virus and other flavivirus structures that may represent critical determinants of virulence.

Antibodies, Monoclonal↗
PubMed2004

Progress in thioaptamer development.

Thioaptamers are thiophosphate ester modified nucleic acids that are isolated via in vitro or bead-based thioaptamer selection against a target molecule such as a protein. Thioaptamers offer advantages over traditional aptamers in their enhanced affinity and specificity and higher stability, largely due to the properties of the sulfur backbone-modifications. An in vitro thioaptamer selection procedure that simultaneously selects for sequence and optimized hybrid phosphoromonothioate or phosphate backbone substitutions is outlined. A novel bead-based thioaptamer selection protocol that can produce mixed phosphorodithioate, phosphoromonothioate or phosphate hybrid backbones is also described. Several examples of thioaptamers targeting specific protein are provided. Such thioaptamers are shown to modulate protein activity in vivo.

Combinatorial Chemistry Techniques↗
PubMed2003

Initiation of repair of A/G mismatches is modulated by sequence context.

The efficiency of DNA glycosylases to initiate base excision repair (BER) has been demonstrated to be modulated by the precise sequence context in which the lesion or mismatch is located. In the case of DNA containing an A/G mismatch, in which the recognition and excision of adenine from the mismatch is mediated by the Escherichia coli MutY enzyme, not only does the local sequence context affect the strength of base stacking interactions, but it also modulates the syn/anti conformation around the glycosyl bond of the bases in the mispair. Utilizing prior NMR data to identify DNA sequence contexts that adopt either an anti/anti or a syn/anti configuration at an A/G mismatch, we tested the hypothesis that the initial equilibrium of the mismatched base orientations would modulate the overall efficiency of glycosyl bond scission. By systematically varying the sequence context around a central A/G mismatch within a 30-mer duplex DNA, significant kinetic differences were observed that were consistent with this hypothesis. Since the relative efficiency of the kinetics fell into only two groupings, a NMR study was conducted on a DNA sequence context of unknown syn/anti conformation. These data established that the relative syn/anti conformation did not correlate with the excision efficiency, as well as there being a lack of correlation between kinetics and thermal stability of these DNAs.

Base Pair Mismatch↗
PubMed2003

Solution structure, dynamics, and thermodynamics of the native state ensemble of the Sem-5 C-terminal SH3 domain.

Although the high-resolution structure of a protein may provide significant insight into which regions are important for function, it is well-known that proteins undergo significant conformational fluctuations, even under native conditions. This suggests that the static structure alone may not provide sufficient information for elucidation of the thermodynamic determinants of biological function and that an accurate molecular-level description of function requires knowledge of the nature and energetics of the conformational states that constitute the native state ensemble. Here the native state ensemble of the C-terminal src homology domain-3 (C-SH3) from Caenorhabditis elegans Sem-5 has been studied using a variety of high-resolution biophysical techniques. In addition to determining the first solution structure of the unliganded protein, we have performed (15)N relaxation and native state hydrogen-deuterium exchange. It is observed that the regions of greatest structural variabilility also show low protection and order parameters, suggesting a higher degree of conformational diversity. These flexible regions also coincide with those regions of Sem-5 that have been predicted by the COREX algorithm to be unfolded in many of the most probable conformational states within the native state ensemble. The implications of this agreement and the potential role of conformational heterogeneity of the observed biophysical properties are discussed.

Algorithms↗
PubMed2003

Immunofluorescence assay and flow-cytometry selection of bead-bound aptamers.

An immunofluorescence assay was developed to identify proteins specifically binding to oligonucleoside phosphorodithioate (ODN) aptamers from a bead-bound ODN library. Accordingly, NF-kappaB p50 protein was incubated with either bead-bound NF-kappaB consensus sequence or a bead-bound ODN combinatorial library and adsorption was then assessed using a specific primary antibody and a secondary antibody conjugated with Alexa 488 fluorescent dye. This assay avoids any problems related to fluorescently labeling target proteins. The method is straightforward and readily applicable to other transcription factors and proteins, and the feasibility of its application for high-throughput screening of large aptamer bead-based libraries by flow cytometry is demonstrated.

Binding, Competitive↗
PubMed2003

Solution structure of a cis-opened (10R)-N6-deoxyadenosine adduct of (9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a DNA duplex.

The solution structure of an 11-mer DNA duplex, d(CGGTCA*CGAGG) x d(CCTCGTGACCG), containing a 10R adduct at dA* that corresponds to the cis addition of the N(6)-amino group of dA(6) to (+)-(9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied by 2D NMR methods. The NOESY cross-peak patterns indicate that the hydrocarbon is intercalated on the 5'-side of the modified base. This observation is the same as that observed for other oligonucleotides containing (10R)-dA adducts but opposite to that observed for the corresponding (10S)-dA adducts which are intercalated on the 3'-side of the modified base. The hydrocarbon is intercalated from the major groove without significant disruption of either the anti glycosidic torsion angle of the modified residue or the base pairing of the modified residue with the complementary residue on the opposite strand. The ensemble of 10 structures determined exhibits relatively small variations (6-15 degrees) in the characteristic hydrocarbon-base dihedral angles (alpha' and beta') as well as the glycosidic torsion angle chi. These angles are similar to those in a previously determined cis-opened benzo[a]pyrene diol epoxide-(10R)-dA adduct structure. Comparison of the present structure with the cis-opened diol epoxide adduct suggests that the absence of the 7- and 8-hydroxyl groups results in more efficient stacking of the aromatic moiety with the flanking base pairs and deeper insertion of the hydrocarbon into the helix. Relative to normal B-DNA, the duplex containing the present tetrahydroepoxide adduct is unwound at the lesion site, whereas the diol epoxide adduct structure is more tightly wound than normal B-DNA. Buckling of the adducted base pair as well as the C(5)-G(18) base pair that lies immediately above the hydrocarbon is much less severe in the present adducted structure than its cis-opened diol epoxide counterpart.

Base Pairing↗
PubMed2003

Solution structure of a DNA duplex containing 8-hydroxy-2'-deoxyguanosine opposite deoxyguanosine.

Deoxyguanosine residues are hydroxylated by reactive oxygen species at the C-8 position to form 8-hydroxy-2'-deoxyguanosine (8-OG), one of the most important mutagenic lesions in DNA. Though the spontaneous G:C to C:G transversions are rare events, the pathways leading to this mutation are not established. An 8-OG:G mispair, if not corrected by DNA repair enzymes, could lead to G:C to C:G transversions. NMR spectroscopy and restrained molecular dynamics calculations are used to refine the solution structure of the base mismatch formed by the 8-OG:G pair on a self complementary DNA dodecamer duplex d(CGCGAATT(8-O)GGCG)(2). The results reveal that the 8-OG base is inserted into the helix and forms Hoogsteen base-pairing with the G on the opposite strand. The 8-OG:G base-pairs are seen to be stabilized by two hydrogen bonding interactions, one between the H7 of the 8-OG and the O6 of the G, and a three-center hydrogen bonding between the O8 of the 8-OG and the imino and amino protons of the G. The 8-OG:G base-pairs are very well stacked between the Watson-Crick base-paired flanking bases. Both strands of the DNA duplex adopt right-handed conformations. All of the unmodified bases, including the G at the lesion site, adopt anti glycosidic torsion angles and form Watson-Crick base-pairs. At the lesion site, the 8-OG residues adopt syn conformations. The structural studies demonstrate that 8-OG(syn):G(anti) forms a stable pair in the interior of the duplex, providing a basis for the in vivo incorporation of G opposite 8-OG. Calculated helical parameters and backbone torsional angles, and the observed 31P chemical shifts, indicate that the structure of the duplex is perturbed near lesion sites, with the local unwinding of the double helix. The melting temperature of the 8-OG:G containing duplex is only 2.6 deg. C less than the t(m) of the unmodified duplex.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2002

Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing.

Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S2-ODNs) with sulfurs replacing one or both of the non-bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one-bead one-S-ODN and one-bead one-S2-ODN libraries. Binding and selection of specific beads to the transcription factor NF-kappaB p50/p50 protein were demonstrated. Sequencing both the nucleic acid bases and the positions of any 3'-O-thioate/dithioate linkages was carried out by using a novel PCR-based identification tag of the selected beads. This approach allows us to rapidly and conveniently identify S-ODNs or S2-ODNs that bind to proteins.

Base Sequence↗
PubMed2002

Combinatorial selection and binding of phosphorothioate aptamers targeting human NF-kappa B RelA(p65) and p50.

Previously, we reported the in vitro combinatorial selection of phosphorothioate aptamers or "thioaptamers" targeting the transcription factor NF-IL6. Using the same approach and purified recombinant human NF-kappa B proteins RelA(p65) and p50, duplex thioaptamers have been selected that demonstrate high-affinity, competitive binding with the duplex 22-mer binding site, Ig kappa B. Binding energetics of RelA(p65) and p50 homodimers were studied using a quantitative electrophoretic mobility shift assay or EMSA. As a reference system for competitive aptamer binding, the duplex 22-mer phosphoryl binding site known as Ig kappa was determined to bind each p65 and p50 homodimer with a 1:1 stoichiometry and with affinities, determined by global analysis, K(d) = 4.8 +/- 0.2 nM for p65 and K(d) = 0.8 +/- 0.2 nM for p50. A global analysis tool for competitive NF-kappa B/Ig kappa binding was developed and utilized to measure the affinity of thioaptamers selected by both p65 and p50. The competition results indicate that the thioaptamers bind and compete for the same NF-kappa B site as the known promoter element Ig kappa B (K(d) = 78.9 +/- 1.9 nM for a p65-selected aptamer and 19.6 +/- 1.3 nM for a p50-selected thioaptamer). Qualitative gel shift binding experiments with p50 also demonstrate that the nature of enhanced affinity and specificity can be attributed to the presence of sulfur. Collectively, these results demonstrate the feasibility of the thioaptamer in vitro combinatorial selection technology as a method for producing specific, high-affinity ligands to proteins.

Base Sequence↗
PubMed2002

Separation of synthetic oligonucleotide dithioates from monothiophosphate impurities by anion-exchange chromatography on a mono-q column.

A method using a strong anion-exchange liquid-chromatography column, Mono-Q, has been developed for high-resolution analysis and purification of oligonucleotide dithioates, which were synthesized by an automated, solid-phase, phosphorothioamidite chemistry. High-resolution separation of oligonucleotide phosphorodithioates from monothiophosphate impurities was obtained. High-resolution separation was also demonstrated at pH 8. The separation of oligonucleotide dithioates was found to be linearly dependent on the number of sulfurs for the same sequence length. Thiocyanate, SCN-, as eluting anion, can be used to purify oligonucleotides containing a high percentage of phosphorodithioate linkages in lower salt concentrations and provides better separation than chloride as eluting anion.

Anion Exchange Resins↗