Theoretical calculations of base-base interactions in nucleic acids: I Stacking interactions in free bases

Stacking interactions in free bases were computed on the basis of molecular association. The results of the calculations were compared with the stacking patterns observed in a few single crystals of nucleic acid components as examples. The following are the conclusions: (i) there can be two types of stacking pattern classified as normal and inverted types for any two interacting bases and both can be energetically favourable (ii) in both the types the stacking interaction is a combined effect of the overlap of the interacting bases and relative positions and orientations of the atomic centres of the two bases (iii) crystal symmetry and H-bonding interaction may influence stacking patterns.

Interaction of metal ions with nucleic acids and related compounds. II. Studies on Au(III)-nucleic acid system

The nature of interaction of Au(III) with nucleic acids was studied by using methods such as uv and ir spectrophotometry, viscometry, pH titrations, and melting-temperature measurements. Au(III) is found to interact slowly with nucleic acids over a period of several hours. The uv spectra of native calf-thymus DNA 9pH 5.6 acetate buffer containing (0.01M NaCIO4) showed a shift in λ max to high wavelengths and an increase in optical density at 260 nm. There was a fourfold decrease in viscosity (expressed as ηsp/c). The reaction was faster at pH 4.0 and also with denatured DNA (pH 5.6) and whole yeast RNA (pH 5.6). The order of preference of Au(III) (as deduced from the time of completion of reaction) for the nucleic acids in RNA > denatured DNA > DNA. The reaction was found to be completely reversible with respect KCN. Infrared spectra of DNA-Au(III) complexes showed binding to both the phosphate and bases of DNA. The same conclusions were also arrived at by melting-temperature studies of Au(III)-DNA system. pH titrations showed liberation of two hydroxylions at r = 0.12 [r = moles of HAuCl4 added per mole of DNA-(P)] and one hydrogen ion at r = 0.5. The probable binding sites could be N(1)/N(7) of adenine, N(7) and/or C(6)O of guanine, N(3) of cytosine and N(3) of thymine. DNAs differing in their (G = C)-contents [Clostridium perfingens DNA(G = C, 29%), salmon sperm DNA (G + C, 42%) and Micrococcus lysodeikticus DNA(G + C, 29%), salmon sperm DNA (G = C, 72%)] behaved differently toward Au(III). The hyperchromicity observed for DNAs differing in (G + C)-content and cyanide reversal titrations indicate selectivity toward ( A + T)-rich DNA at lw values of r. Chemical analysis and job's continuous variation studies indicated the existence of possible complexes above and below r = 1. The results indicate that Au(III) ions probably bind to hte phosphate group in the initial stages of the reaction, particularly at low values of r, and participation of the base interaction also increases. Cross-linking of the two strands by Au(III) may take place, but a complete collapse of the doulbe helix is not envisaged. It is probable that tilting of the bases or rotaiton of the bases around the glucosidic bond, resulting in a significant distrotion of the double helix, might take place due to binding of Au(III) to DNA.

Kinetic studies on the interaction of gold(III) with nucleic acids. I. Native DNA-Au(III) system-spectrophotometric studies

Kinetics of the interaction of Au(III) with native calf thymus DNA has been studied spectrophotometrically to determine the kinetic parameters and to examine their dependency on the concentrations of DNA and Au(III), temperature, ionic strength and pH. The reaction is of the first order with respect to both the nucleotide unit of DNA and Au(III) in the stoichiometry of 2∶1 respectively. The rate constants vary with the initial ratio of DNA to Au(III) and is attributed to the effect of free chloride ions and the existence of a number of reaction sites with slight difference in the rate constants. The activation energies of this interaction have been found to be 14–16 kcal/mol. From the effect of ionic strength the reaction is found to occur between a positive and a negative ion in the rate-limiting step. The logarithm of rate constants are the linear function of pH and the slopes are dependent on ther-values. A plausible mechanism has been proposed which involves a primary dissociation of the major existing species (AuCl2(OH)2)−, to give (AuCl2)+ which then reacts with a site in the nucleotide unit of DNA in the rate-liminting step followed by a rapid binding to another site on the complementary strand of the DNA double helix. There exist a number of binding sites with slight difference in reactivity.

Stereochemistry of 2'-5' linked nucleic acids: crystal and molecular structure of ammonium adenylyl-2',5'-adenosine tetrahydrate: a core fragment of 2'-5' oligo A's produced by interferon induced adenylate synthetase

The preponderance of 3'-5' phosphodiester links in nucleic acids is well known. Albeit less prevalent, the 2'-5' links are specifically utilised in the formation of 'lariat' in group II introns and in the msDNA-RNA junction in myxobacterium. As a sequel to our earlier study on cytidylyl-2',5'-adenosine we have now obtained the crystal structure of adenylyl-2',5'-adenosine (A2'p5'A) at atomic resolution. This dinucleoside monophosphate crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 7.956(3) A, b = 12.212(3) A and c = 36.654(3) A. CuK alpha intensity data were collected on a diffractometer. The structure was sloved by direct methods and refined by full matrix least squares methods to R = 10.8%. The 2' terminal adenine is in the commonly observed anti (chi 2 = 161 degrees) conformation and the 5' terminal base has a syn (chi 1 = 55 degrees) conformation more often seen in purine nucleotides. A noteworthy feature of A2'p5'A is the intranucleotide hydrogen bond between N3 and O5' atoms of the 5' adenine base. The two furanose rings in A2'p5'A show different conformations - C2' endo, C3' endo puckering for the 5' and 2' ends respectively. In this structure too there is a stacking of the purine base on the ribose O4' just as in other 2'-5' dinucleoside structures, a feature characteristically seen in the left handed Z DNA. In having syn, anti conformation about the glycosyl bonds, C2' endo, C3' endo mixed sugar puckering and N3-O5' intramolecular hydrogen bond A2'p5'A resembles its 3'-5' analogue and several other 2'-5' dinucleoside monophosphate structures solved so far. Striking similarities between the 2'-5' dinucleoside monophosphate structures suggest that the conformation of the 5'-end nucleoside dictates the conformation of the 2' end nucleoside. Also, the 2'-5' dimers do not favour formation of miniature classical double helical structures like the 3'-5' dimers. It is conceivable, 2-5(A) could be using the stereochemical features of A2'p5'A which accounts for its higher activity.

Interaction of nucleic acids with carbon nanotubes and dendrimers

Nucleic acid interaction with nanoscale objects like carbon nanotubes (CNTs) and dendrimers is of fundamental interest because of their potential application in CNT separation, gene therapy and antisense therapy. Combining nucleic acids with CNTs and dendrimers also opens the door towards controllable self-assembly to generate various supra-molecular and nano-structures with desired morphologies. The interaction between these nanoscale objects also serve as a model system for studying DNA compaction, which is a fundamental process in chromatin organization. By using fully atomistic simulations, here we report various aspects of the interactions and binding modes of DNA and small interfering RNA (siRNA) with CNTs, graphene and dendrimers. Our results give a microscopic picture and mechanism of the adsorption of single- and double-strand DNA (ssDNA and dsDNA) on CNT and graphene. The nucleic acid-CNT interaction is dominated by the dispersive van der Waals (vdW) interaction. In contrast, the complexation of DNA (both ssDNA and dsDNA) and siRNA with various generations of poly-amido-amine (PAMAM) dendrimers is governed by electrostatic interactions. Our results reveal that both the DNA and siRNA form stable complex with the PAMAM dendrimer at a physiological pH when the dendrimer is positively charged due to the protonation of the primary amines. The size and binding energy of the complex increase with increase in dendrimer generation. We also give a summary of the current status in these fields and discuss future prospects.

GFT projection NMR for efficient H-1/C-13 sugar spin system identification in nucleic acids

A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify H-1/C-13 sugar spin systems in C-13 labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of C-13-H-1 groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1'- and 3'-CH signal dispersion for complete spin system identification including 5'-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3'-untranslated region of the pre-mRNA of human U1A protein.

Specificity of protein - Nucleic acid interaction and the biochemical evolution

The water soluble carbodiimide mediated condensation of dipeptides of the general form Gly-X was carried out in the presence of mono- and poly-nucleotides. The observed yield of the tetrapeptide was found to be higher for peptide-nucleotide system of higher interaction specificity following mainly the anticodon-amino acid relationship (Basu, H.S. & Podder, S.K., 1981, Ind. J. Biochem. Biophys.,19, 251-253). The yield of the condensation product of L-peptide was more because of its higher interaction specificity. The extent of the racemization during the condensation of Gly-L-Phe, Gly-L-Tyr and Gly-D-Phe was found to be dependent on the specificity of the interaction -the higher the specificity, the lesser the racemization. The product formed was shown to have a catalytic effect on the condensation reaction. These data thus provide a mechanism showing how the specific interaction between amino acids/dipeptides and nucleic acids could lead to the formation of the lsquoprimitiversquo translation machinery.

Natively unfolded nucleic acid binding P8 domain of SeMV polyprotein 2a affects the novel ATPase activity of the preceding P10 domain

Open reading frame (ORF) 2a of Sesbania mosaic virus (SeMV) codes for polyprotein 2a (Membrane anchor-protease-VPg-P10-P8). The C-terminal domain of SeMV polyprotein 2a was cloned, expressed and purified in order to functionally characterize it. The protein of size 8 kDa (P8) domain, like viral protein genome linked (VPg), was found to be natively unfolded and could bind to nucleic acids.Interestingly, P10-P8 but not P8 showed a novel Mg2+ dependent ATPase activity that was inhibited in the presence of poly A. In the absence of P8, the ATPase activity of the protein of size 10 kDa (P10) domain was reduced suggesting that the natively unfolded P8 domain influenced the P10 ATPase.

Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

Influence of clofibrate administration on the rate of synthesis of macromolecules in regenerating rat liver

Administration of the antihypercholesterolaemic drug clofibrate stimulates the rates of synthesis of nucleic acids and proteins in rat liver. The biosynthesis of mitochondrial proteins also is enhanced by the drug. In drug-fed animals, the rates of incorporation in vivo of radioactive precursors into DNA, RNA and proteins are stimulated even when the liver undergoes regeneration following partial hepatectomy. The rate of synthesis of mitochondrial proteins in the regenerative phase is higher in clofibrate-fed animals. These effects are consistent with the hepatomegalic and mitochondria-proliferating property of the drug.

Characterization of protoporphyrin as the physiological regulator of delta-aminolevulinate dehydratase in Neurospora crassa

In Neurospora crassa, the activity of δ-aminolevulinate dehydratase, the second and rate-limiting enzyme of the heme-biosynthetic pathway, is low in normal cells compared to the activity detected in plants, animals and bacteria. The activity is almost undetectable when Neurospora crassa is grown under iron-deficient conditions. The enzyme activity increases strikingly on addition of iron to iron-deficient cultures. This increase can be blocked by the addition of protoporphyrin, the penultimate product of the heme-biosynthetic pathway, to the cultures. The question whether iron directly acts at the genetic level or acts merely by removing protoporphyrin, converting the latter into heme prosthetic groups of hemoproteins, has been investigated by studying the effect of inhibition of heme synthesis on the induction of δ-aminolevulinate dehydratase. It has been found that treatments with levulinic acid or cyanide which inhibit the formation of the porphyrin moiety, induce δ-aminolevulinate dehydratase, whereas treatments which inhibit at a step after protoporphyrin formation (iron-deficiency and cobalt treatment) repress the enzyme. The endogenous levels of protoporphyrin are strictly controlled: a decrease below the optimum level causing induction and an increase above the optimum level leading to repression of δ-aminolevulinate dehydratase. Levulinic acid and cyanide can induce the enzyme in iron-deficient cultures in the absence of added iron, indicating that the metal iron acts only by converting protoporphyrin to heme fixed in hemoproteins in Neurospora crassa. Therefore it is suggested that protoporphyrin is the physiological regulator of δ-aminolevulinate dehydratase in Neurospora crassa.

3-Amino-1,2,4-triazole is an inhibitor of protein synthesis on mitoribosomes in Neurospora crassa

The effects of the herbicide, 3-amino-1,2,4-triazole, an inhibitor of heme synthesis in rat liver, have been examined in the mold Neurospora crassa. The drug is a potent inhibitor of the growth of the mold and produces biochemical changes identical to those produced by chloramphenicol. 3-Amino-1,2,4-triazole, like chloramphenicol, is a direct and specific inhibitor of protein synthesis on mitoribosomes. A decrease in the levels of mitochondrial proteins which are completely or partly made on mitoribosomes and an accumulation in the levels of mitochondrial proteins of cytosolic origin have been observed. Both drugs depress porphyrin and heme levels, but there is actually an elevation in the levels of δ-aminolevulinate dehydratase, the rate-limiting enzyme of the heme-biosynthetic pathway in Neurospora crassa. In liver the enzyme is present in non-limiting amounts and the levels are depressed under conditions of 3-amino-1,2,4-triazole treatment. In Neurospora crassa the ‘derepression’ of δ-aminolevulinate dehydratase under conditions of 3-amino-1,2,4-triazole or chloramphenicol treatment is only partial because the drugs inhibit protein synthesis on mitoribosomes. It is concluded that an optimal rate of protein synthesis on mitoribosomes is necessary to maintain an adequate rate of heme synthesis.