Influence of conformationally restricted pyrimidines on the activity of 10-23 DNAzymes.

The catalytic core of a 10-23 DNAzyme was modified introducing conformationally restricted nucleosides such as (2'R)-, (2'S)-2'-deoxy-2'-C-methyluridine, (2'R)-, (2'S)-2'-deoxy-2'-C-methylcytidine, 2,2'-anhydrouridine and LNA-C, in one, two or three positions. Catalytic activities under pseudo first order conditions were compared at different Mg(2+) concentrations using a short RNA substrate. At low Mg(2+) concentrations, triple modified DNAzymes with similar kinetic performance to that displayed by the non-modified control were identified. In the search for a partial explanation of the obtained results, in silico studies were carried out in order to explore the conformational behavior of 2'-deoxy-2'-C-methylpyrimidines in the context of a loop structure, suggesting that at least partial flexibility is needed for the maintenance of activity. Finally, the modified 2'-C-methyl DNAzyme activity was tested assessing the inhibition of Stat3 expression and the decrease in cell proliferation using the human breast cancer cell line T47D.

10-23 DNAzyme modified with (2'R)- and (2'S)-2'-deoxy-2'-C-methyluridine in the catalytic core.

The catalytic core of a 10-23 DNAzyme was modified using (2'R), (2'S)-2'-deoxy-2'-C-methyluridine and LNA-T. Catalytic activities under pseudo first order conditions were compared at different Mg(2+) concentrations, indicating that certain 2'-C-methyl modified DNAzymes have significant activities. Resistance against MCF-7 cell lysate and endonuclease RQ1 was also measured, showing that the introduction of 2'-C-methyl-2'-deoxynucleosides increased the stability.

Activity of core-modified 10-23 DNAzymes against HCV.

The highly conserved untranslated regions of the hepatitis C virus (HCV) play a fundamental role in viral translation and replication and are therefore attractive targets for drug development. A set of modified DNAzymes carrying (2'R)-, (2'S)-2'-deoxy-2'-C-methyl- and -2'-O-methylnucleosides at various positions of the catalytic core were assayed against the 5'-internal ribosome entry site element (5'-IRES) region of HCV. Intracellular stability studies showed that the highest stabilization effects were obtained when the DNAzymes' cores were jointly modified with 2'-C-methyl- and 2'-O-methylnucleosides, yielding an increase by up to fivefold in the total DNAzyme accumulation within the cell milieu within 48 h of transfection. Different regions of the HCV IRES were explored with unmodified 10-23 DNAzymes for accessibility. A subset of these positions was tested for DNAzyme activity using an HCV IRES-firefly luciferase translation-dependent RNA (IRES-FLuc) transcript, in the rabbit reticulocyte lysate system and in the Huh-7 human hepatocarcinoma cell line. Inhibition of IRES-dependent translation by up to 65 % was observed for DNAzymes targeting its 285 position, and it was also shown that the modified DNAzymes are as active as the unmodified one.

Conformational states of 2'-C-methylpyrimidine nucleosides in single and double nucleic acid stranded structures.

The hybridization performance of a set of 12-mer RNA:RNA duplexes containing 2'-C-methyluridine, 5-bromo-2'-C-methyluridine, or (2'S)-2'-deoxy-2'-C-methyluridine was analyzed. Melting point temperatures of the modified duplexes showed an important ΔT(m) decrease (-8.9 to -12.5 °C), while circular dichroism experiments indicated that the helix was still A-type, suggesting a localized disturbance disorder. Molecular dynamics simulations using AMBER were carried out in order to gain structural knowledge about the effect of the 2'-C-methyl modification in double stranded environments. On the other hand, in an attempt to explain the behavior of the 2'-deoxy-2'-C-methyl nucleosides in single stranded environments, like the 10-23 DNAzyme core, molecular dynamic simulations were performed, incorporating the modified analogues into single stranded reported stem-loop structures, studding the sugar conformations along the MD trajectories. It was observed that, despite their preferential conformational states, the 2'-C-methyl analogues are flexible enough to adopt a different puckering in single stranded environments.

DNAzymes and ribozymes carrying 2'-C-methyl nucleotides.

DNAzymes and Ribozymes find applications as inhibitors of gene expression and in detection systems such as biosensors, among others. In particular, we are interested in the properties of hammerhead ribozymes carrying 2'-C-methylnucleotides and 10-23 DNAzymes containing (2'R) or (2'S)- 2'-C-methyl 2'-deoxynucleotides. In this work a new synthesis of 2'-C-Methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2'-hydroxyl group. Comparison to previous protecting strategies as well as the stability under oligonucleotide synthesis conditions is discussed.2'-C-methyl-2'-deoxynucleosides show differential preferred conformations depending on the configuration of the 2'-carbon. The influence of these modifications on the activity of 10-23 DNAzymes is also assessed.