Bridging the gap between proteins and nucleic acids: a metal-independent RNAseA mimic with two protein-like functionalities.

Two synthetically modified nucleoside triphosphate analogues (adenosine modified with an imidazole and uridine modified with a cationic amine) are enzymatically polymerized in tandem along a degenerate DNA library for the combinatorial selection of an RNAse A mimic. The selected activity is consistent with both electrostatic and general acid/base catalysis at physiological pH in the absence of divalent metal cations. The simultaneous use of two modified nucleotides to enrich the catalytic repertoire of DNA-based catalysts has never before been demonstrated and evidence of general acid/base catalysis at pH 7.4 for a DNAzyme has never been previously observed in the absence of a divalent metal cation or added cofactor. This work illustrates how the incorporation of protein-like functionalities in nucleic acids can bridge the gap between proteins and oligonucleotides underscoring the potential for using nucleic acid scaffolds in the development of new materials and improved catalysts for use in chemistry and medicine.

Oligoribonucleotide-based gene-specific transcription inhibitors that target the open complex.

We have demonstrated that oligoribonucleotides that lack a 3'-OH group and cannot be extended by RNA polymerase can hybridize to the single-stranded DNA formed inside the transcription initiation bubble (or open complex) and inhibit transcription. Using the lacUV5/Escherichia coli RNA polymerase or trpEDCBA/E. coli RNA polymerase transcription system as a model, we have found that effective inhibitors are five nucleotides in length and must be complementary to the DNA template strand in the region from -5 to +2 about the transcription start site (designated +1). We have used the DNA cleavage activity of 1,10-phenanthroline-copper to confirm that the mechanism of inhibition is via oligoribonucleotide hybridization to the open complex and have used this cleavage chemistry to demonstrate that these oligonucleotide inhibitors hybridize in an antiparallel orientation to their DNA target. Systematic modification of the parent phosphodiester oligoribonucleotide pentamer revealed that the phosphorothioate backbone-containing analogs have increased open complex binding affinity and are more effective transcription inhibitors than their phosphodiester counterparts.

Artificial nucleases.

The oxidation of DNA and RNA provides a facile approach for investigating the interaction of nucleic acids with proteins and oligonucleotides. In this article, we have outlined our understanding of the mechanism of DNA scission by 1,10-phenanthroline-copper(I) in the presence of hydrogen peroxide. We also discuss results obtained by using 1,10-phenanthroline-oligonucleotide conjugates in probing the size of the transcriptionally active open complex. Finally, we outline an effective method for converting DNA-binding proteins into site-specific modification agents by using 1,10-phenanthroline-copper(I).

Nucleic acids for recognition and catalysis: landmarks, limitations, and looking to the future.

Combinatorial selection of nucleic acids has led to the discovery of novel ligands and catalysts that have implications for both chemistry and medicine. In the context of combinatorial chemistry, degenerate syntheses of nucleic acid libraries readily generate as many as 10(15) different molecules in which a small percentage exhibit interesting binding and/or catalytic properties. The primary advantage of nucleic acids is that library coding is an intrinsic property; sequential composition directly determines the activity. At low temperatures, the sequential composition of single stranded nucleic acids governs folding into irregular tertiary structures resulting in interesting activities. At higher temperatures, the same structures are unfolded and decoded by polymerases to reveal sequential information. The use of PCR (polymerase chain reaction) permits amplification and thus enrichment of the selected activity which is then regenerated chemi-enzymatically. Iterative selection and amplification result in one of the highest throughput screens conceivable whereby each molecule encodes its own activity permitting the ultimate in parallel sampling. Finally, sequence information, and by extension the chemical composition, is obtained by simple sequencing techniques obviating the need for mass spectrometric deconvolution, parallel tagging, and/or large volumes needed for viral and cell culture. This review begins with an introduction of general concepts and considerations. The potential for nucleic acids to generate tight-binding ligands is of interest to structural biologists and medicinal chemists. The therapeutic implications to medicine are also touched upon. Since combinatorially selected nucleic acids and antibodies share many conceptual similarities, their respective advantages and limitations are compared. Theoretical and practical limitations for catalyst discovery are discussed along with the use of other chemical and physical approaches to address some current catalytic shortcomings. Finally some future directions are suggested.

An approach to gene-specific transcription inhibition using oligonucleotides complementary to the template strand of the open complex.

The single-stranded region of DNA within the open complex of transcriptionally active genes provides a unique target for the design of gene-specific transcription inhibitors. Using the Escherichia coli lac UV5 and trp EDCBA promoters as in vitro models of open complex formation, we have identified the sites inside these transcription bubbles that are accessible for hybridization by short, nuclease-resistant, non-extendable oligoribonucleotides (ORNs). Binding of ORNs inside the open complex was determined by linking the chemical nuclease bis(1,10-phenanthroline) cuprous chelate [(OP)(2)Cu(+)] to the ORN and demonstrating template-specific DNA scission. In addition, these experiments were supported by in vitro transcription inhibition. We find that the most effective inhibitors are 5 nt long and have sequences that are complementary to the DNA template strand in the region near the transcription start site. The ORNs bind to the DNA template strand, forming an antiparallel heteroduplex inside the open complex. In this system, RNA polymerase is essential not only to melt the duplex DNA but also to facilitate hybridization of the incoming ORN. This paradigm for gene-specific inactivation relies on the base complementarity of the ORN and the catalytic activity and sequence specificity of RNA polymerase for the site- and sequence-specific recognition and inhibition of transcriptionally active DNA.

Expanding the catalytic repertoire of nucleic acid catalysts: simultaneous incorporation of two modified deoxyribonucleoside triphosphates bearing ammonium and imidazolyl functionalities.

Two nucleoside triphosphates, a pyrimidine modified with an ammonium functionality and a purine modified with an imidazolyl functionality are compatible with all conditions for a combinatorial selection of nucleic-acid catalysts. We believe that this work is the first to demonstrate the potential for using not one but two modified nucleotides in tandem. The potential for an enriched catalytic repertoire is envisioned.

Recognition of tetrahedral 1,10-phenanthroline-cuprous chelates by transcriptionally active complexes does not depend on the sequence of the promoter.

BACKGROUND: The open complex formed at the initiation site of transcription within the active site of RNA polymerase is unique to actively transcribing genes and is thus an ideal target for the design of transcription inhibitors. Many redoxactive tetrahedral cuprous chelates of 1,10-phenanthroline (OP) or derivatives cleave the single-stranded template, principally at sequence positions -7 to -3, whereas the redox-inactive tetrahedral cuprous chelate of 2, 9-dimethyl-OP (neocuproine) blocks transcription, but does not cleave. The octahedral (OP)3-Fe2+ chelate has no effect. Different promoters can give different cleavage patterns. We therefore searched for structural determinants of the open complex that are important in the cleavage reaction. RESULTS: Using site-directed mutagenesis, we systematically altered the nucleotides at the cleavage sites of the Escherichia coli lac UV-5-RNA polymerase open complex (positions -6 to -4), which are highly variable in E. coli promoters. Surprisingly, these changes had little effect on catalytic activity, on transcription inhibition by the cuprous complex of neocuproine and on the cleavage patterns generated by the cuprous chelates of OP derivatives. The scission pattern of a lac UV-5 promoter mutant in which the cleavage sites have the sequence of the trp EDCBA promoter is that of the lac UV-5 promoter, not the trp EDCBA promoter. CONCLUSIONS: Nucleotide-specific interactions are not responsible for the observed cleavage patterns. The recognition of the tetrahedral OP chelate must be due to a specific structure of the single-stranded regions, determined by RNA polymerase-DNA interactions in the upstream regulatory region.

Optimizing the targeted chemical nuclease activity of 1,10-phenanthroline-copper by ligand modification.

Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1,10-phenanthroline, we have compared the activity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-OP). Tethering MOP derivatives to the Escherichia coli Fis protein enhances DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP. However, scission efficiency is not increased at the stronger cleavage sites, or when scission is targeted to single-stranded DNA by a complementary oligonucleotide. These results are consistent with a change in the rate-determining step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant. Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neocuproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complementary DNA sequence. These results establish that the nucleolytic species in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-oxo intermediate rather than a metal-coordinated peroxy species. However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of the phenanthroline-copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for cleavage of single-stranded regions and the failure of neocuproine-DNA-binding protein chimeras and a C2-tethered chimera to cleave DNA.

Inhibitors of Escherichia coli RNA polymerase specific for the single-stranded DNA of transcription intermediates. Tetrahedral cuprous chelates of 1,10-phenanthrolines.

Single-stranded DNA of the lacUV-5 promoter formed at the active site of Escherichia coli RNA polymerase during transcription is specifically cleaved by the redox active tetrahedral cuprous chelates of 1,10-phenanthroline and its derivatives. The cleavage sites are observed in the open, initiating, and elongating complexes. Redox-inert, tetrahedral cuprous chelates of neocuproine (2,9-dimethyl-1,10- phenanthroline) and its 5-phenyl and 4-phenyl derivatives protect the template strand of DNA from scission within these steady state intermediates and inhibit transcription. Although these cuprous chelates of neocuproine bind at multiple sites within three distinct enzyme intermediates, the highest affinity site is within the elongation complex. The I50 of 5 microM for the 2:1 5-phenylneocuproine cuprous complex ((5 phi NC)2Cu+) in runoff transcription therefore primarily reflects its intermediate. The neocuproine cuprous chelates are novel transcription inhibitors because they bind to single-stranded DNA sites generated during the course of catalysis by RNA polymerase.

Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.

Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP(Cu)]. Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody. Remarkably, the same cleavage products are observed with permease embedded in the native membrane. Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI. Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies. Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa. The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII. When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a single product is observed at 19 kDa, suggesting fragmentation at the cytoplasmic end of helix VII. However, when the reagent is moved 100 degrees in the other direction to position 147 (Gly-147 --> Cys permease), cleavage is not observed. The results suggest that helix V is in close proximity to helices VII and VIII with position 148 in the interface between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer.

Inhibition of human immunodeficiency virus type 1 integrase by a hydrophobic cation: the phenanthroline-cuprous complex.

The human immunodeficiency virus type 1 integrase (HIV-1 integrase) is required for integration of a double-stranded DNA copy of the viral RNA genome into a host chromosome and for HIV replication. We have examined the effects of 2:1 1,10-phenanthroline-cuprous complexes on purified HIV-1 integrase. Although the uncomplexed phenanthrolines are not active below 100 microM, four of the cuprous complexes (neocuproine, 4-phenyl neocuproine, 2,3,4,7,8,9-hexamethyl phenanthroline, and 2,3,4,7,8-pentamethyl phenanthroline) have a 50% inhibitory concentration (IC50) for integration ranging between 1 and 10 microM. Disintegration is also inhibited by these phenanthroline-cuprous complexes at slightly higher concentrations (between 10 and 40 microM). Dialysis experiments showed that the inhibition is reversible and kinetic analyses revealed that the mode of inhibition by these cuprous complexes appears to be noncompetitive with respect to the substrate DNA. Consistent with these findings, binding assays demonstrate that, although these complexes can inhibit binding to DNA at high concentrations, they do not inhibit binding of integrase to the DNA substrate at their IC50 values. Because these complexes do not bind to B-DNA below 50 microM, inhibition via binding to a specific region on the enzyme was examined. Using deletion mutants of integrase, it was determined that neither the amino-terminal (zinc finger) nor the carboxy-terminal (DNA-binding) integrase domain is required for inhibition by the phenanthroline-cuprous complexes. Therefore, inhibition via binding to the enzyme catalytic core or to the interface between the enzyme and a noncanonical DNA structure generated during the enzymatic reaction is the probable mechanism. These results suggest the utility of neocuproine-cuprous complexes in developing inhibitors of HIV-1 integrase as well as probes for drug-binding sites and enzymatic reaction mechanism.

Inhibition of prokaryotic and eukaryotic transcription by the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex, a ligand specific for open complexes.

The redox-stable, tetrahedral cuprous chelate of neocuproine (2,9-dimethyl-1,10-phenanthroline) binds to the single-stranded DNA formed in open complexes and is an effective inhibitor of eukaryotic and prokaryotic transcription. Despite the many kinetic and structural differences between prokaryotic and eukaryotic transcription systems, they are all similarly inhibited by neocuproine copper, suggesting that all open complexes may share a homologous structure.

Hybridization of a complementary ribooligonucleotide to the transcription start site of the lacUV-5-Escherichia coli RNA polymerase open complex. Potential for gene-specific inactivation reagents.

An ribooligonucleotide, UGGAA, complementary to the template strand of the lacUV-5 promoter can hybridize to the transcription "bubble" of the open complex formed by Escherichia coli RNA polymerase. Its site-specific binding, measured by gel retardation, enzyme inhibition, and chemical nuclease footprinting, is dependent on catalysis by RNA polymerase and the sequence of the hybridizing ribooligonucleotide. When UGGAA is linked to the chemical nuclease 1,10-phenanthroline copper, site-specific scission of the template strand of the transcriptionally active gene is observed. The formation of single-stranded DNA at transcription start sites by RNA polymerases provides a target for antigene strategies.

Interactions of transcription inhibitors with the Escherichia coli RNA polymerase-lacUV5 promoter open complex.

The interactions of transcription inhibitors with the open complex composed of Escherichia coli RNA polymerase and the lacUV5 promoter have been studied using gel retardation, the chemical nuclease activity of the cuprous complexes of 1,10-phenanthroline (OP) and its derivatives, and steady-state kinetics. Gel retardation shows that two inhibitors, the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex [(2,9-Me2OP)2Cu+] and rifampicin, bind stably to the open-complex. (2,9-Me2OP)2Cu+ blocks scission by the chemical nuclease by interfering with the binding of its redox-active isosteres. Rifampicin does not block scission by the cuprous complexes of 3,4,7,8-tetramethyl-OP, 4-phenyl-OP, and OP but does perturb scission by the cuprous complex of 5-phenyl-OP. Organic ligands including intercalating agents and groove binders (e.g., daunomycin, di(amidinophenyl)indole (DAPI), actinomycin D, distamycin, 9-aminoacridine, mithramycin, and chromomycin A3), which bind to free DNA with high affinity, do not form stable ternary complexes with the open-complex. Gel retardation experiments demonstrate that they promote dissociation of the enzyme from the promoter. The greater sensitivity of enzymatic catalysis to inhibitor concentration relative to polymerase binding suggests that these ligands form metastable, catalytically inactive ternary complexes with RNA polymerase and the promoter.

A transcription inhibitor specific for unwound DNA in RNA polymerase-promoter open complexes.

The kinetically component open complexes formed at prokaryotic and eukaryotic transcription start sites are efficiently nicked by the chemical nuclease activity of the 2:1 1,10-phenanthroline-copper(I) complex [(OP)2Cu+] and hydrogen peroxide. This reaction specificity has been attributed to the creation of a binding site(s) for redox-active tetrahedral (OP)2Cu+ when RNA polymerase form productive complexes with promoters. This proposal has been confirmed for the Escherichia coli lac UV-5 promoter by the demonstration that the 2:1 2,9-dimethyl-1,10-phenanthroline-copper(I) complex [(Me2OP)2Cu+], a redox-inactive isostere of (OP)2-Cu+, protects the transcription start site from scission by the chemical nuclease activity. (Me2OP)2Cu+ is also an effective inhibitor of transcription. The inhibition of transcription and the protection from scission of the open complex by (OP)2Cu+ exhibit the same dependence on the concentration of (Me2OP)2Cu+. This redox- and exchange-stable species is a previously undescribed transcription inhibitor that binds to a site generated by the interaction of RNA polymerase with the promoter. Unlike the intercalating agent proflavine, which is also an effective transcription inhibitor, it does not displace the enzyme from the promoter. The ability of (Me2OP)2Cu+ to inhibit transcription may be partially responsible for its potent cytotoxicity.

Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase.

The TIS10 cDNA was cloned as a primary response gene transcript whose mRNA rapidly accumulates in 3T3 cells treated with serum, polypeptide growth factors, or phorbol esters. The sequence of the TIS10 cDNA suggested that the gene encodes a protein with strong similarities to prostaglandin G/H synthase/cyclooxygenase (EC 1.14.99.1). Transient transfection into COS-1 cells of an expression vector driving the TIS10 cDNA leads to production and secretion of prostaglandin E2. Microsomes prepared from COS-1 cells transfected with this construct demonstrate both hydroperoxidase and cyclooxygenase activities similar to that demonstrated by cells transfected with a vector encoding the ovine prostaglandin G/H synthase. These data demonstrate that the TIS10 gene encodes a functional prostaglandin synthase/cyclooxygenase distinct from the prostaglandin synthase/cyclooxygenase whose cDNAs and/or genes have previously been cloned from sheep, mouse, and man. The structure of the TIS10 gene, determined by a combination of sequencing of genomic clones and polymerase chain reactions from genomic clones, demonstrates remarkable exon-intron conservation with the human prostaglandin synthase/cyclooxygenase gene. A 1-kilobase sequence located immediately proximal to the start site of transcription of the TIS10 gene can confer phorbol ester and serum inducibility to a luciferase reporter gene following transient transfection into NIH 3T3 cells, suggesting that this region of the gene is responsible for transcriptional regulation of the TIS10 gene by mitogens in fibroblasts.