Immobilization of nucleoside on silica gel and specific separation of oligonucleotides.

Deoxyadenosine was immobilized on silica gel, in order to use as HPLC resins for selective separation of oligonucleotides. The longest retention time was observed for the complementary pd(T)4, and increased with decrease of temperature. This fact suggested that the main separation factor was the base pairing between complementary nucleic acid bases. These resins may be useful for separation of components of nucleic acids and polynucleotides as a specific separation system.     

Affinity chromatography of nucleosides and nucleic acid base derivatives with nucleic acid bases or nitrobenzeneboronic acid substituted silicas

Nucleic acid bases such as adenine and uracil, and nitrobenzeneboronic acid substituted silicas were prepared by the reaction of chloromethylbenzene substituted silica with adenine sodium salt and trimethylsilylated uracil, and nitration of benzeneboronic acid substituted silica, respectively. From the results of HPLC of nucleosides and N-ethyl derivatives of nucleic acid bases using modified silicas, hydrophobic base stacking interaction, selective hydrogen bonding interaction between purine and pyrimidine bases, and reversible cyclic boronate ester formation between diols of nucleosides with boronic acid were effective for the separation of nucleic acid related compounds. Moreover, association constants for hydrogen bonding formation of nucleic acid bases were estimated.     

Mixed monolayers of amphiphile-modified nucleic bases and diynoic acids. I. Phase states at air-water interface and in Langmuir-Blodgett films

Monolayers of amphiphile-modified nucleic bases with diynoic acid were obtained and characterized. The synthesized nucleic bases contained in the monolayer complementarily bind the nucleotide molecules contained in the aqueous subphase, and the structure of the resulting monolayers can be fixed by the photopolymerization of diynoic acid. The resulting monolayer exemplifies a novel type of model systems for investigating molecular recognition at the surface of biological membranes. Procedures for the transfer of the monolayers onto solid substrates and photopolymerization of the diynoic acid in mixtures with the derivatives of nucleic bases were developed. The films obtained were structurally characterized using atomic force microscopy. Compression isotherms of the mixed monolayers as well as individual components of monolayers at the air-water interface allowed one to determine the concentration range at which the diynoic acid form true mixtures or domain structures with the derivatives of nucleic base. A study of the films transferred to the solid substrate by atomic force microscopy indicated that this concentration dependence of miscibility behavior was conserved in the transferred films.     

A theoretical study of the sequence specificity in binding of lexitropsins to B-DNA

A theoretical study is presented on the binding to B-DNA of a series of lexitropsins, these ligands being netropsin derivatives in which one or both of the pyrrole rings have been replaced by imidazoles. The best complexes have been located by energy minimisation taking into account nucleic acid flexibility, ligand flexibility, explicit, mobile counterions and solvent dielectric effects. Calculations have been performed for two homopolymeric DNA receptor sequences, AT base sequence, which only decreases in the imidazole derivatives. These results emphasize the decisive role of the molecular electrostatic potential of the nucleic acid in determining the sequence selectivity of these ligands, as opposed to the postulated role of adenine C2 - pyrrole beta hydrogen contacts.     

Nucleic acid unwinding by hepatitis C virus and bacteriophage t7 helicases is sensitive to base pair stability

Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.     

Nucleotide,nucleoside and base nutritional requirements of the mosquito Culex pipiens

Nucleic acid subcomponents needed to satisfy the dietary nucleic acid requirement of Culex pipiens were studied in growth experiments using synthetic media in which nucleosides, bases and alternative nucleotides were variously substituted in mixtures of 3 nucleotides (adenylic acid, thymidylic acid, and either cytidylic or uridylic acid) previously shown to be adequate replacements for whole nucleic acid. Any or all 3 nucleotides could be replaced by corresponding nucleosides without adverse effect, except that adenosine substitution moderately delayed pupation. All base substitutions were unsatisfactory: substitution of thymine for thymidylic acid allowed development to the adult stage but at a greatly reduced rate; single substitution of adenine, cytosine or uracil for the corresponding nucleotides allowed scarcely more development than in the total absence of nucleic acid derivatives. Inosinic acid or inosine were adequate substitutes for adenylic acid, but orotic acid or orotidine were ineffective in place of the pyrimidine ribonucleotides, cytidylic or uridylic acids. Deoxyadenylic acid could take the place of adenylic acid, though inefficiently, but deoxycytidylic and deoxyuridylic acids were very poor replacements for the corresponding ribonucleotides. The minimal required nucleic acid derivatives thus appear to be a purine ribonucleotide (adenylic or inosinic acids), a pyrimidine ribonucleoside (either uridine or cytidine), and the pyrimidine deoxyribonucleoside, thymidine.     

Prominent stacking interaction with aromatic amino acid by N-quarternization of nucleic acid base: X-ray crystallographic characteristics and biological implications

In order to investigate the mode of interaction between the N-quarternized cytosine base and the aromatic amino acid, the crystal structure of the 3-methyl-cytidine-5'-monophosphate:tryptamine complex was analyzed by X-ray diffraction. The complex crystals were stabilized by extensive hydrogen bond formations in which eight independent water molecules per complex pair participated. A prominent stacking interaction, characterized by a parallel alignment of both rings with a separation distance of ca. 3.4 A, was observed between the cytosine base and the indole ring. Combining the present results with X-ray crystallographic data on the adenine--and guanine--aromatic amino acid interactions, we summarize the structural characteristics observed in the stacking interaction of the N-quarternized nucleic acid base with the aromatic amino acid and discuss their biological implications, especially in connection with the significance of N-protonation of nucleic acid base for selective recognition by protein.     

A Theoretical Study of the Sequence Specificity in Binding of Lexitropsins to B-DNA

Abstract

A theoretical study is presented on the binding to B-DNA of a series of lexitropsins, these ligands being netropsin derivatives in which one or both of the pyrrole rings have been replaced by imidazoles. The best complexes have been located by energy minimisation taking into account nucleic acid flexibility, ligand flexibility, explicit, mobile counterions and solvent dielectric effects. Calculations have been performed for two homopolymeric DNA receptor sequences, AT and GC. All the compounds studied exhibit an overall binding preference for the AT base sequence, which only decreases in the imidazole derivatives. These results emphasize the decisive role of the molecular electrostatic potential of the nucleic acid in determining the sequence selectivity of these ligands, as opposed to the postulated role of adenine C2 - pyrrole β hydrogen contacts.     

Acetic anhydride requirement in the colorimetric determination of tryptophan

Nucleic acid research frequently necessitates the analytical resolution of nucleic acid derivatives. Thin-layer chromatography (tlc), for its simplicity, short development time, and superior resolving power, is often preferable to other methods (1–3). Although the literature contains a large number of methods that have been devised for the separation of purine and pyrimidine derivatives (4,5) no tlc technique has hitherto been described for the concomitant separation of bases, nucleosides, nucleoside 5′-monophosphates, nucleoside 3′-monophosphates, nucleoside diphosphates, and nucleoside triphosphates.The present communication deals with methods devised for the simultaneous separation of the above-mentioned pyrimidine derivatives. They enable the resolution of either the six uracil derivatives or the six cytosine derivatives, on commercial cellulose tlc sheets. Alternatively, the six pyrimidine derivatives can be separated on cellulose layers 0.75 mm thick. Since formic acid extracts of bacterial cells do not interfere with the separation, these methods can be used for the direct estimation of extracts of biological materials.     

Magnetic particles for the separation and purification of nucleic acids

Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.     

Evaluation of the hydrogen bond energy of base pairs formed between substituted 9-methyladenine derivatives and 1-methyluracil by use of molecular orbital theory.

Systematic substituent effects on the stability of the hydrogen bonding between substituted 9-methyladenine derivatives (Ax) and 1-methyluracil (U) were studied by ab initio molecular orbital theory. Predicted substituent effects on the hydrogen bond energies of Ax-U base pairs were in good agreement with those observed for experimental binding constants. Ab initio calculation is effective for evaluation of the stability of the hydrogen-bonding pairs of chemically modified nucleic acid base analogues. In contrast to the substitution effect of uracil on hydrogen bond energies of A-Ux base pairs, it is difficult to systematically interpret the substitution effect of adenine derivatives for Ax-U base pairs.     

A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis

In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant K(i) = 20 to 35 microM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.     

The reaction of 14 C-labelled platinum ethylenediamine dichloride with nucleic acid constituents

The anti-tumour agent platinum ethylenediamine dichloride [Pt(en)Cl₂] reacts in 0.1 M NaClO₄ with adenine, guanine, cytosine and their nucleosides and nucleotides, but not with thymine and its derivatives. Such reactions have been followed by spectral changes and by chromatographic separation of reactants and products, using Pt(¹⁴C-en)Cl₂.In general, guanine derivatives reacted the most rapidly, notably guanosine. From double-labelling experiments using tritiated nucleic acid components, reaction products showed a Pt(en)Cl₂: base ratio of close to unity. An exception was ATP where an additional product was observed with Pt: base = 2. Here the phosphate moiety may also have been involved. A comparison of guanine and derivatives in which the N-7, N-9 or both positions were blocked indicates that probably more than one site is available for reaction with Pt(en)Cl₂.     

Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA.

Stacking between aromatic amino acids and nucleic acid bases may play an important role in the interaction of enzymes with nucleic acid substrates. In such circumstances, disruption of base aromaticity would be expected to decrease enzyme activity on the modified substrates. We have examined the requirement for DNA base aromaticity of five enzymes that act on single-stranded DNA, T4 polynucleotide kinase, nucleases P1 and S1, and snake venom and calf spleen phosphodiesterases, by comparing their kinetics of reaction with a series of dinucleoside monophosphates containing thymidine or a ring-saturated derivative. The modified substrates contained either cis-5R,6S-di-hydro-5,6-dihydroxythymidine (thymidine glycol) or a mixture of the 5R and 5S isomers of 5,6-dihydrothymidine. It was observed that for all the enzymes, except snake venom phosphodiesterase, the parent molecules were better substrates than the dihydrothymidine derivatives, while the thymidine glycol compounds were significantly poorer substrates. Snake venom phosphodiesterase acted on the unmodified and dihydrothymidine molecules at almost the same rate. These results imply that for all the remaining enzymes base aromaticity is a factor in enzyme-substrate interaction, but that additional factors must contribute to the poorer substrate capacity of the thymidine glycol compounds. The influence of the stereochemistry of the dihydrothymidine derivatives was also investigated. We observed that nuclease P1 and S1 hydrolysed the molecules containing 5R-dihydrothymidine approximately 50-times faster than those containing the S-isomer. The other enzymes displayed no measurable stereospecificity.     

Peptide synthesis on a phenolic resin support

Protected peptides assembled on a phenolic resin support were cleared by peroxide-catalysed hydrolysis. In genenal peptide phenyl ester resins were more labile to nucleophiles than were corresponding Merrifield resin derivatives; transesterification with dimethylaminoethanol providing on alternative cleavage method for peroxide-sensitive peptides. Losses of radiolabelled peptide from both Merrifield and phenolic resins were determined during acid deprotection, base wash and coupling steps in the synthesis of a tetrapeptide. Using 40% (v/v) trifluoroacetic acid in dichloromethane for Boc-deprotection the phenolic resin gave improved results compared to the Merrifield resin. The merits of the procedure for the preparation of protected peptide acids suitable for subsequent condensation reactions were exemplified by the synthesis of an octapeptide sequence of a modified lysozyme.     

Synthesis of polyacrylamides N-substituted with PNA-like oligonucleotide mimics for molecular diagnostic applications.

Two types of oligonucleotide mimics relative to peptide nucleic acids (PNAs) were tested as probes in nucleic acid hybridisation assays based on polyacrylamide technology. One type of mimic oligomers represented a chimera constructed of PNA and phosphono-PNA (pPNA) monomers, and the other one contained pPNA residues alternating with PNA-like monomers on the base of trans -4-hydroxy-L-proline (HypNA). A chemistry providing efficient and specific covalent attachment of these DNA mimics to acrylamide polymers using a convenient approach based on the co-polymerisation of acrylamide and some reactive acrylic acid derivatives with oligomers bearing 5'- or 3'-terminal acrylamide groups has been developed. A comparative study of polyacrylamide conjugates with oligonucleotides and mimic oligomers demonstrated the suitability and high potential of PNA-pPNA and HypNA-pPNA chimeras as sequence-specific probes in capture and detection of target nucleic acid fragments to serve current forms of DNA arrays.     

Liquid-liquid partition chromatography of biopolymers in aqueous two-phase systems.

The theoretical and practical principles of liquid-liquid partition chromatography (LLPC) applying aqueous two-phase polymer systems are presented. The method is based on support materials which bind one of the two aqueous phases with high preference and reject the other. This selectivity is obtained by making use of incompatibilities between polymers grafted on support particles and polymers in solution. Applications of the separation technique to the fractionation of protein and nucleic acid mixtures are shown. For the DNA-fractionation according to base composition an affinity partition chromatography using polyethylene glycol-bound base-specific complexing agents has been developed which exhibits a resolution superior to all other methods known.     

2'-deoxyadenosine bearing hydrophobic carborane pharmacophore

Modification of 2'-deoxyadenosine at position 8 with para-carborane boron cluster is described. Incorporation of boron cluster into nucleic base has been accomplished using Sonogashira palladium-catalyzed cross-coupling reaction or, alternatively, Huisgen "click" type reaction. These are the first examples of adenosine derivatives with hydrophobic carborane pharmacophore attached to purine base.     

The mutagenicity of benzimidazole and benzimidazole derivatives. II. Incorporation of benzimidazole into the nucleic acids of Escherichia coli

The mutagenicity of benzimidazole has been attributed to base substitutions. These were believed to be the result of a small incorporation of benzimidazole—instead of a natural base—into nucleic acids. By using a new indirect method of radioactive labelling it has been possible to prove that benzimidazole is incorporated as such. This method involves growing bacteria in a medium containing radioactive phosphate and sufficient benzimidazole to guarantee optimal incorporation. Upon electrophoretic separation of the nucleotides resulting from the hydrolysis of the nucleic acids an additional phosphorus-containing compound was found and subsequently identified by comparison with an enzymatically synthesized benzimidazole mononucleotide.