Small molecule-RNA recognition: Binding of the benzophenanthridine alkaloids sanguinarine and chelerythrine to single stranded polyribonucleotides.

Single stranded RNAs are biologically potent as they participate in various key cellular processes. The binding efficacy of two potent anticancer alkaloids, sanguinarine (here after SANG) and chelerythrine (here after CHEL), with single-stranded ribonucleic acids poly(rI), poly(rG), and poly(rC) were studied using spectroscopic and thermodynamic tools. Results reveal that both SANG and CHEL binds well with single stranded RNAs with affinity in the order poly(rI)>poly(rG)>poly(rC). CHEL showed slightly higher affinity compared to SANG with all the single stranded RNAs. Both SANG and CHEL showed association affinity of the lower 106 order with poly(rI), higher 105 order binding with poly(rG) and lower 105 order with poly(rC). The binding mode was partial intercalation due to the staking interaction between the bases and the alkaloids. The complexation of both the SANG and CHEL to the RNAs were mainly enthalpy driven and also favoured by entropy changes. Perturbation was observed in the RNA conformation due to binding of the alkaloids. In this present study we have deciphered the fundamental structural and calorimetric aspects of the interaction of the natural benzophenanthridine alkaloids with single stranded RNAs and these results may help to develop new generation alkaloid based therapeutics targeting single stranded RNAs.

Experimental and density functional theory (DFT) studies on the interactions of Ru(II) polypyridyl complexes with the RAN triplex poly(U)˙poly(A)*poly(U).

There is renewed interest in investigating triple helices because these novel structures have been implicated as a possible means of controlling cellular processes by endogenous or exogenous mechanisms. Due to the Hoogsteen base pairing, triple helices are, however, thermodynamically less stable than the corresponding duplexes. The poor stability of triple helices limits their practical applications under physiological conditions. In contrast to DNA triple helices, small molecules stabilizing RNA triple helices at present are less well established. Furthermore, most of these studies are limited to organic compounds and, to a far lesser extent, to metal complexes. In this work, two Ru(II) complexes, [Ru(bpy)2(btip)](2+) (Ru1) and [Ru(phen)2(btip)](2+) (Ru2), have been synthesized and characterized. The binding properties of the two metal complexes with the triple RNA poly(U)˙poly(A)*poly(U) were studied by various biophysical and density functional theory methods. The main results obtained here suggest that the slight binding difference in Ru1 and Ru2 may be attributed to the planarity of the intercalative ligand and the LUMO level of Ru(II) complexes. This study further advances our knowledge on the triplex RNA-binding by metal complexes, particularly Ru(II) complexes.

Binding properties of Ru(II) polypyridyl complexes with poly(U)·poly(A)*poly(U) triplex: the ancillary ligand effect on third-strand stabilization.

The binding properties of [RuL(2)(mip)](2+) {where L is 1,10-phenanthroline (phen) or 4,7-dimethyl-1,10-phenanthrollne (4,7-dmp) and mip is 2'-(3",4"-methylenedioxyphenyl)imidazo[4',5'-f][1,10]phenanthroline} with regard to the triplex RNA poly(U)·poly(A)*poly(U) were investigated using various biophysical techniques and quantum chemistry calculations. In comparison with [Ru(4,7-dmp)(2)(mip)](2+), remarkably higher binding affinity of [Ru(phen)(2)(mip)](2+) for the triplex RNA poly(U)·poly(A)*poly(U) was achieved by changing the ancillary ligands. The stabilization of the Hoogsteen-base-paired third strand was improved by about 10.9 °C by [Ru(phen)(2)(mip)](2+) against 6.6 °C by [Ru(4,7-dmp)(2)(mip)](2+). To the best of our knowledge, [Ru(phen)(2)(mip)](2+) is the first metal complex able to raise the third-strand stabilization of poly(U)·poly(A)*poly(U) from 37.5 to 48.4 °C. The results reveal that the ancillary ligands have an important effect on third-strand stabilization of the triplex RNA poly(U)·poly(A)*poly(U) when metal complexes contain the same intercalative ligands.

First ruthenium(II) polypyridyl complex as a true molecular "light switch" for triplex RNA structure: [Ru(phen)2(mdpz)]2+ enhances the stability of Poly(U)·Poly(A)*Poly(U).

Stabilization of triple helical structures is extremely important for carrying out their biological functions. Nucleic acid triple helices may be formed with DNA or RNA strands. In contrast to many studies in DNA, little has been reported concerning the recognition of the RNA triplex by transition-metal complexes. In this article, [Ru(phen)(2)(mdpz)](2+) (Ru1) is the first metal complex able to enhance the stability of the RNA triplex Poly(U)·Poly(A)*Poly(U) and serve as a prominent molecular "light switch" for the RNA triplex.

The leader sequence of tobacco mosaic virus RNA devoid of Watson-Crick secondary structure possesses a cooperatively melted, compact conformation.

The 5'-untranslated region (5'-UTR) of RNA of tobacco mosaic virus (TMV), called omega sequence, is known as an mRNA leader promoting efficient initiation of translation. The central part of the sequence consists of many CAA repeats, which were reported to be mainly responsible for the enhancing activity of the omega leader. In this work we synthesized the polyribonucleotides containing either the natural omega sequence or the regular (CAA)(n) sequence, and studied them using UV spectrophotometry and analytical ultracentrifugation methods. It was demonstrated that the polyribonucleotides manifest significant hypochromicity, cooperative melting of their structures upon heating, high melting temperature, and the sedimentation coefficients typical of compactly folded RNAs of this size. Thus, the omega leader and its core (CAA)(n) repeat sequence devoid of secondary structure of the Watson-Crick type seem to be well structured elements of mRNA.

Chiral selection in oligoadenylate formation in the presence of a metal ion catalyst or poly(U) template.

The lead ion-catalyzed oligomerization of 5'-phosphorimidazolides of D-, L- or racemic DL-adenosine (D-ImpA, L-ImpA and DL-ImpA) gave oligoadenylates up to a pentamer. The oligomers resulting from racemic ImpA were comparable in yields and length to those from chiral D- or L-ImpA. A complex mixture of homochiral and heterochiral oligomers was formed in the reaction from racemic ImpA. Total dimer product from racemic ImpA by the lead ion catalyst showed homochiral selectivity. The reaction catalyzed by uranyl ion yielded oligoadenylates up to 15mer from chiral D- or L-ImpA in over 95% yield. A complex mixture of isomeric oligoadenylates was formed from racemic DL-ImpA in the presence of uranyl ion catalyst in comparable yields to those from D- or L-ImpA. The analysis of the dimer product from DL-ImpA showed that the homochiral 2' -5' linked dimer was selectively formed. D-ImpA polymerized effectively on a poly(U) template, which is exclusively composed of D-uridine, yielding oligoadenylates up to a pentamer. In contrast, L-ImpA or racemic DL-ImpA polymerized far less efficiently on the poly(U) template, demonstrating that chiral selection takes place in the poly(U) template-directed oligoadenylate formation.

Failure to generate atheroprotective apolipoprotein AI phenotypes using synthetic RNA/DNA oligonucleotides (chimeraplasts).

BACKGROUND: Elevated plasma high-density lipoprotein (HDL), and its major constituent apolipoprotein AI (apoAI), are cardioprotective. Paradoxically, two natural variants of apoAI, termed apoAI(Milano) and apoAI(Paris), are associated with low HDL, but nevertheless provide remarkable protection against heart disease for heterozygous carriers and may even lead to longevity. Both variants arise from point mutations and have Arg(173) and Arg(151) to Cys substitutions, respectively, which allow disulphide-linked dimers to form. Potentially, synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct single point mutations in genomic DNA. Here, we use a variation of such targeted gene repair technology, 'gain-of-function chimeraplasty', and attempt to enhance the biological activity of apoAI by altering a single genomic base to generate the atheroprotective phenotypes, apoAI(Milano) and apoAI(Paris). METHODS: We targeted two cultured cell lines that secrete human apoAI, hepatoblastoma HepG2 cells and recombinant CHO-AI cells, using standard 68-mer chimeraplasts with polyethyleneimine (PEI) as carrier and then systematically varied several experimental conditions. As a positive control we targeted the dysfunctional APOE2 gene, which we have previously converted to wild-type APOE3. RESULTS: Conversion of wild-type apoAI to apoAI(Milano) proved refractory, with limited correction in CHO-AI cells only. However, a successful conversion to apoAI(Paris) was achieved, as demonstrated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct genomic sequencing. Unexpectedly, attempts with a new batch of 68-mer chimeraplast to enhance conversion, by using different delivery vehicles, including chemically modified PEI, failed to show a base change; nor could conversion be detected with an 80-mer or a 52-76-mer series. In contrast, when a co-culture of CHO-E2 and CHO-AI cells was co-targeted, a clear conversion of apoE2 to apoE3 was seen, whereas no apoAI(Paris) could be detected. When the individual chimeraplasts were analysed by denaturing electrophoresis only the active apoE2-to-E3 chimeraplast gave a sharp band. CONCLUSIONS: Our findings suggest that different batches of chimeraplasts have variable characteristics and that their quality may be a key factor for efficient targeting and/or base conversion. We conclude that, although an evolving technology with enormous potential, chimeraplast-directed gene repair remains problematical.

Indolocarbazole glycosides in inactive conformations.

Indolocarbazole glycosides related to rebeccamycin represent a promising category of antitumor agents targeting DNA and topoisomerase I. These drugs prefer to adopt a closed conformation with an intramolecular hydrogen bond between the indole NH group and the pyranose oxygen atom. Three pairs of indolocarbazole monoglycosides bearing an NH or an N-methyl indole moiety were synthesized and their biological properties investigated at the molecular and cellular level. Replacing the indole NH proton with a methyl group reduces DNA interaction and abolishes activity against DNA topoisomerase I. Surface plasmon resonance studies performed with a pair of water-soluble indolocarbazole glycosides and two hairpin oligonucleotides containing an [AT]4 or a [CG]4 sequence indicate that both the NH and the N-methyl derivative maintain a relatively high affinity for DNA (Keq = 2 - 6 x 10(5) M(-1)) but the incorporation of the methyl group restricts access to the DNA. The number of ligand binding sites (n) on the oligonucleotides is about twice as high for the NH compound compared to its N-methyl analogue. Modeling and 1H NMR studies demonstrate that addition of the N-methyl group drives a radical change in conformation in which the orientation of the aglycone relative to the beta-glucoside is reversed. The loss of the closed conformation by the N-methyl derivatives perturbs thir ability to access DNA binding sites and prevents the drug from inhibiting topoisomerase I. As a consequence, the NH compounds exhibit potent cytotoxicity against CEM leukemia cells with an IC50 value in the 1 microM range, whereas the N-methyl analogues are 10 to 100 times less cytotoxic. These studies offer circumstantial evidence supporting the importance of the closed conformation in the interaction of indolocarbazole glycosides with their molecular targets, DNA and topoisomerase I.

Functional domains of the yeast splicing factor Prp22p.

The essential Saccharomyces cerevisiae PRP22 gene encodes a 1145-amino acid DEXH box RNA helicase. Prp22p plays two roles during pre-mRNA splicing as follows: it is required for the second transesterification step and for the release of mature mRNA from the spliceosome. Whereas the step 2 function of Prp22p does not require ATP hydrolysis, spliceosome disassembly is dependent on the ATPase and helicase activities. Here we delineate a minimal functional domain, Prp22(262-1145), that suffices for the activity of Prp22p in vivo when expressed under the natural PRP22 promoter and for pre-mRNA splicing activity in vitro. The biologically active domain lacks an S1 motif (residues 177-256) that had been proposed to play a role in RNA binding by Prp22p. The deletion mutant Prp22(351-1145) can function in vivo when provided at a high gene dosage. We suggest that the segment from residues 262 to 350 enhances Prp22p function in vivo, presumably by targeting Prp22p to the spliceosome. We characterize an even smaller catalytic domain, Prp22(466-1145) that suffices for ATP hydrolysis, RNA binding, and RNA unwinding in vitro and for nuclear localization in vivo but cannot by itself support cell growth. However, the ATPase/helicase domain can function in vivo if the N-terminal region Prp22(1-480) is co-expressed in trans.

Biomesogenic matrix systems.

The bifunctionally reactive nucleoside and distant nucleoside analogs adenosine (Ado), S-[(adenine-9-yl)methoxyethyl]-L-cysteine (Na-salt) (cysA) and 9-vinyladenine (vA) in aqueous solutions assemble on complementary polyuridylic acid templates to form complex lyomesophases. The systems are investigated by polarizing microscopy, differential scanning calorimetry (DSC) and 1H- and 31P-nmr spectroscopies, assisted by molecular modeling studies. The results indicate the importance of biomesogenic (pre)ordering in nucleic acid native and artificial matrix reactions.

Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA.

The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90-103, which regulates osteogenesis and hematopoiesis. By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA. Indeed, cells transfected with a construct encoding [His102]H4 secreted the corresponding [His13]OGP. These results suggest production of OGP from H4 genes. Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both "long" and "short" CAT proteins. The short CAT was retained following an ATG --> TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation. These results suggest that a PreOGP is translated starting at AUG85. The translational initiation at AUG85 could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG. Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic [His102]H4-(85-103) as substrate. Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide.

Photochemistry of DNA and related biomolecules: quantum yields and consequences of photoionization.

The reactions of nucleic acids and constituents, which can be induced by laser UV irradiation, are described. Emphasis is placed on the quantum yields of various stable photoproducts of DNA and model compounds upon irradiation at 193, 248, 254 or 266 nm. In particular, those quantum yields and processes are discussed which involve photoionization as the initial step and occur in aqueous solution under well defined conditions, e.g. type of atmosphere. The efficiencies of some photoproducts, with respect to photoionization using irradiation at 193 or 248 nm, are presented. Radical cations of nucleobases are important sources of damage of biological substrates since they can cause lesions other than dimers and adducts, e.g. strand breakage, abasic sites, crosslinks or inactivation of plasmid and chromosomal DNA. While competing photoreactions, such as hydration, dimerization or adduct formation, diminish the selectivity of the photoionization method, a combination with model studies on pyrimidine- and purine-containing constituents of DNA has brought about an enhanced insight into the reaction mechanisms. The knowledge concerning the lethal events in plasmid and cellular DNA has been greatly improved by correlation with the chemical effects obtained by gamma-radiolysis, vacuum-UV (< 190 nm) and low-intensity irradiation at 254 nm.

Interaction between glutathione and Cu(II) in the vicinity of nucleic acids.

GSH interacts with Cu(II) in the vicinity of DNA (pH approximately 7) to form the DNA-Cu(I) complex, which can be quantified by characteristic absorption changes [e.g. delta epsilon 295 = 4516 cm-1.M-1 Cu(I)]. Under initial conditions of Cu(II)/GSH >> 1 and DNA(base)/Cu(II) >> 5, the stoichiometry is 1 DNA-Cu(I) per SH group (also for other thiols). Stopped-flow kinetics show that the complex is formed with half-lives of 1-30 s, depending on the environment, but independent of O2. DNA-Cu(I) generation is much slower, less efficient, and O2-dependent at Cu(II)/GSH < 1, or when GSH interacts with Cu(II) before the addition of DNA. Interaction of GSH with Cu(II) in the presence of DNA [at Cu(II)/GSH > 1] leads to DNA-associated transients, probably DNA-GS(-)-Cu(I); DNA-Cu(I) formation under these conditions is proposed to occur by ligand exchange: DNA-GS(-)-Cu(I)+Cu(II)<-->DNA-Cu(I)+GS(-)-Cu(II). There is no evidence for generation of free thiyl radicals (GS.) on reaction of Cu(II) with GSH. Formation of DNA-Cu(I) is, in our opinion, a primary step involved in DNA-strand cleavage by GSH in the presence of Cu(II) [Reed and Douglas (1991) Biochem. J. 275, 601-608]. In this context the question of the pro-oxidative and/or antioxidative activity of GSH, when combined with copper, is discussed. GSH also generates Cu(I) complexes with other nucleic acids. An updated order of affinities of various nucleic acids for Cu(I) is presented. Cu(I) exhibits a high preference for alternating dG-dC sequences and might even be a Z-DNA inducer. The poly(C)-Cu(I) complex seems to form a base-paired structure at pH approximately 7, as demonstrated by intercalation of ethidium bromide.

Polyethenoadenosine phosphate as a fluorogenic substrate for barnase.

Polyethenoadenosine phosphate is rapidly hydrolyzed by barnase with an accompanying large increase in fluorescence. At pH 8, 0.2 M ammonium acetate, the initial rate of fluorescence increase is proportional to barnase concentration and thus provides an excellent quantitative assay for its activity. The preparation and use of this substrate in the barnase assay is presented in detail.

[Biological actions and therapeutic perspectives of double stranded polyribonucleotides: a reappraisal]. / Actions biologiques et perspectives thérapeutiques des polyribonucléotides double-brins: une réévaluation.

Double-stranded polynucleotides, which are composed of two complementary homopolyribonucleotides containing no genetic information, are synthetic molecules capable of mimicking the action of natural double-stranded RNA or viral RNA on cells. Double-stranded polyribonucleotides act as an alarm system alerting the cell to the presence of an external aggression, e.g. a viral attack. In addition, polyribonucleotides have a more active function in that they trigger cell defense processes through activation of a family of genes, of which some encode cytokines, activation of cytoplasmic enzymes involved in antiviral mechanisms or signal transduction, and activation of nonspecific immune responses. Double-stranded polyribonucleotides containing one mismatched base pair per helix have been found to be especially interesting. The best known example is poly(I).poly(C12U), also called ampligen. Poly(I).poly(C12U) is capable, in experimental models, of limiting the development of viruses (including HIV), reducing tumor growth, eliminating metastases, and, according to one report, preventing steady declines in T-cell counts in HIV-positive patients. Therapeutic doses used in the USA as an experimental drug induced little toxicity. In vitro, poly(I).poly(C12U) acts synergistically with interferon, interleukin 2 or AZT, suggesting that these latter drugs may be effective in lower, less toxic doses when used in combination with poly(I).poly(C12U). The therapeutic activity of poly(I).poly(C12U) holds promise. More extensive prospective studies of this agent are warranted.