Ondřej Páv

Theoretical and Experimental Study of Charge Transfer through DNA: Impact of Mercury Mediated T-Hg-T Base Pair

DNA-Hg complexes may play an important role in sensing DNA defects or in detecting the presence of Hg in the environment. A fundamental way of characterizing DNA-Hg complexes is to study the way the electric charge is transferred through the molecular chain. The main goal of this contribution was to investigate the impact of a mercury metal cation that links two thymine bases in a DNA T-T mismatched base pair (T-Hg-T) on charge transfer through the DNA molecule. We compared the charge transfer efficiencies in standard DNA, DNA with mismatched T-T base pairs, and DNA with a T-Hg(II)-T base pair. For this purpose, we measured the temperature dependence of steady-state fluorescence and UV-vis of the DNA molecules. The experimental results were confronted with the results obtained employing theoretical DFT methods. Generally, the efficiency of charge transfer was driven by mercury changing the spatial overlap of bases.

5′-O-Methylphosphonate nucleic acids—new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes

Several oligothymidylates containing various ratios of phosphodiester and isopolar 5′-hydroxyphosphonate, 5′-O-methylphosphonate and 3′-O-methylphosphonate internucleotide linkages were examined with respect to their hybridization properties with oligoriboadenylates and their ability to induce RNA cleavage by ribonuclease H (RNase H). The results demonstrated that the increasing number of 5′-hydroxyphosphonate or 5′-O-methylphosphonate units in antisense oligonucleotides (AOs) significantly stabilizes the heteroduplexes, whereas 3′-O-methylphosphonate AOs cause strong destabilization of the heteroduplexes. Only the heteroduplexes with 5′-O-methylphosphonate units in the antisense strand exhibited a significant increase in Escherichia coli RNase H cleavage activity by up to 3-fold (depending on the ratio of phosphodiester and phosphonate linkages) in comparison with the natural heteroduplex. A similar increase in RNase H cleavage activity was also observed for heteroduplexes composed of miRNA191 and complementary AOs containing 5′-O-methylphosphonate units. We propose for this type of AOs, working via the RNase H mechanism, the abbreviation MEPNA (MEthylPhosphonate Nucleic Acid).

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase.

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC(50) values below 1 microM were selected. Out of them, four compounds strongly inhibited the enzyme with IC(50) values lying in a range of 11-45 nM. These most potent compounds might be bi-substrate analogues.

Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.

Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair.

Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: Complex theoretical and experimental studies

Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings.

Nucleoside‐O‐Methyl‐(H)‐Phosphinates: Novel Monomers for the Synthesis of Methylphosphonate Oligonucleotides Using H‐Phosphonate Chemistry

This unit comprises the straightforward synthesis of protected 2′‐deoxyribonucleoside‐O‐methyl‐(H)‐phosphinates in both 3′‐ and 5′‐series. These compounds represent a new class of monomers compatible with the solid‐phase synthesis of oligonucleotides using H‐phosphonate chemistry and are suitable for the preparation of both 3′‐ and 5′‐O‐methylphosphonate oligonucleotides. The synthesis of 4‐toluenesulfonyloxymethyl‐(H)‐phosphinic acid as a new reagent for the preparation of O‐methyl‐(H)‐phosphinic acid derivatives is described.

Hybridization Properties of 4′-Branched Oligonucleotides

The positive results of our previous work encouraged the continuation of preparation, and evaluation of the properties of specific DNA=RNA analogs. The findings that 40-methoxy substituent (unlike 40-methyl) in modified oligonucleotides enhances hybridization with RNA and in lesser extent with DNA prompted us to investigate this phenomenon in detail. The 40-substituents of oligonucleotide strand in a duplex are oriented into the minor groove, and could interfere with a hydration and=or with groups of a complementary strand. The detailed view of the image obtained from the molecular mechanics calculation shows the 40-substituents as the bridging across the minor groove. In addition, the 40-substitution itself influences significantly the ratio of individual sugar ring conformers so that it could lead to thermodynamically more stable duplexes eventually. 40-Modified monomers (40-N3, 40-CH3O, 40-CH3OCH2CH2O, . . . ) were prepared, in spite of chemical difficulties, and significant decomposition of the compound during the key reaction steps. Olefin, 40,50-didehydro-20,50-dideoxythymidine, was prepared from 50-iodo-20,50-dideoxythymidine using sodium methoxide,

Ribo-, xylo-, and arabino-configured adenine-based nucleoside phosphonates: synthesis of monomers for solid-phase oligonucleotide assembly.

Abstract Adenine-based, regioisomeric nucleoside phosphonates with ribo, xylo and arabino configuration were synthesized in the protected form suitable for the phosphotriester-like, solid-phase synthesis of oligonucleotides. Phosphonate moiety was protected by 4-methoxy-1-oxido-2-picolyl group and the furanose hydroxyl by the dimethoxytrityl group.