DNAzymes with peroxidase and nuclease activities

MARÍA DELLAFIORE; LAURA ROBALDO; JAVIER MONTSERRAT; ADOLFO IRIBARREN

Poznam

Simposio; XXI International Roundtable on Nucleosides, Nucleotides and Nucleic Acids; 2014

International Society of Nuclesides, Nucleotides and Nucleic Acids

10-23 DNAzymes carrying 2?-C-methyl-2?-deoxynucleosides  were synthesized and their biological properties assayed. In addition, the use of deoxyribozymes as useful catalyst for oxygen transfer reactions to organic compounds was explored. INTRODUCTION 2?-C-methyl-2?-deoxynucleosides show differential preferred sugar conformations depending on the absolute configuration at the 2?-C.(2?R)-2?-deoxy-2?-C-methyluridine, (2?S)-2?-deoxy-2?-C-methyluridine, (2?R)-2?-deoxy-2?-C-methylcytidine and (2?S)-2?-deoxy-2?-C-methylcitidine were synthesized and introduced in one, two and three positions of the 10?23 DNAzyme catalytic core. In the case of DNAzyme Dz-4S7R8R, 72% of the activity was maintained at 1 mM Mg2+. When conformationally locked nucleosides, like LNA-C or anhydrouridine, were introduced at the same positions, no catalytic activity was observed1. Preliminary in silico studies showed that these results could be partially explained by the conformational flexibility showed by 2?-C-methylnucleosides when incorporated into loop environments2. DNAzymes with peroxidase-mimicking activity have great potential in bioanalytical chemistr1. These functional nucleic acids have not only the ability of binding to hemin but also the capability of enhancing its catalytic activity, therefore combining aptamers specific target recognition with DNAzymes catalytic properties3. Several applications of peroxidase mimicking DNAzymes in biosensor designs have been reported using colorimetric substrates3. In this work we focused on the use of these deoxyribozymes as useful catalyst for oxygen transfer reactions to organic compounds. Considering biological fluids as possible reaction media, we also tested the efficiency of a fully modified peroxidase analog using L-DNA, the mirror image of natural D-DNA. RESULTS AND DISCUSSION In relation to modified 10-23DNAzyme stability, some triple modified molecules were three times more resistant against endonuclease degradation and have four to five times higher activity/resistance ratios than non-modified sequences. Different regions of HCV IRES have been tested for DNAzyme activity using a HCV IRES-Firefly luciferase translation dependent RNA (IRES-FLuc) transcript, in Rabbit Reticulocyte Lysate System and in Huh-7 human cells. Inhibition of IRES dependent translation up to 65% were obtained for DNAzymes targeting its 285 position, finding that the modified DNAzymes were as active as the unmodified one. In addition, onemodified DNAzyme carrying three 2?-C-methylpyrimidines was tested against Stat3, a promoter of human breast cancer, showing a complete abrogation of progestin-induced proliferation 4. For the study of DNA- peroxidases, reactions were carried out incubating hemin with DNA in HEPES-NH4 buffer (pH=8) at 25°C for 15 minutes. Then substrate and hydrogen peroxide were added to start the reaction. Products were determined using RP-C18 HPLC. Both D and L peroxidases were able to oxidize different sulfides with comparable efficiency, obtaining the sulfoxide as main product. When tested using the colorimetric substrate Tetramethylbenzidine (TMB) comparable activities were also found between D and L Peroxidases. Reactions were also performed using D-DNA inmovilized on polystyrene beads obtaining comparable absorbance values when using TMB as substrate and a 69 % of conversion when using thioanisole as substrate. InmovilizedDNAzyme allowed the recuperation and reuse of the oligonucleotide observing no decrease in activity after several uses. CONCLUSION 10-23 DNAzymesmodified with 2?-C-nucleosides were more resistant to nuclease degradation thah their corresponding natural controls, being active against HCV IRES and Stat3 targest. In relation to peroxidase DNAzyme, it was usefull in the transformation of varios organic sulfides to sulfoxides, in solution or immobilized on polystyrene beads. REFERENCES 1.       Robaldo, L. , Montserrat, J., Iribarren, A., Bioorg. Med. Chem. Letters, 2010, 4367-4370 2.       Robaldo, L. , Pontiggia, R., Di Lella, S., Estrin, D., Engels, J., Iribarren, A., Montserrat, J., J. Phys. Chem B, 2012, 57-59 3.       Yingfu, L., Yi, L. (Eds). 2009, Functional Nucleic Acids for Analytical Applications. Springer. 4.       Robaldo, L. , Izzo, F., Dellafiore, M., Proietti, C., Elizalde, P., Montserrat, J., Iribarren, A., Bioorg. Med. Chem. Letters, 2012, 2581-2586