10-23 DNAzyme catalytic core modified with 2´-C-methyl-2´-deoxyuridine and thymidine LNA nucleotides

LAURA ROBALDO, JAVIER MONTSERRAT, ADOLFO IRIBARREN

Odense, Dinamarca

Simposio; Nucleic Acid Chemical Biology (NACB) Symposium; 2009

10-23 DNAzyme catalytic core modified with 2´-C-methyl-2´-deoxyuridine and thymidine LNA nucleotides.   Laura Robaldoa, Javier Montserrarta,b and Adolfo Iribarrena,c   a) INGEBI Vuelta de Obligado 2490 (1428) Buenos Aires - Argentina; b) Universidad de General Sarmiento, J.M. Gutierrez 1150 Los Polvorines, Pcia de Buenos Aires - Argentina; c)Laboratorio de Biotransformaciones, Universidad Nacional de Quilmes, Roque Saenz Peña 180, Bernal, Pcia de Buenoa Aires - Argentina. e-mail: airibarren@unq.edu.ar   At present, the active conformation, cleavage mechanism and three dimensional structure of the substrate-deoxyribozyme 10-23 complex remains unknown[1]. Although numerous efforts have been performed to establish the role of each nucleotides in the catalytic core[2], the potential conformational requirements of the sugar moiety has not been investigated. On the other hand, there are several modified deoxyribozymes which have been designed to improve the substrate cleavage reaction and to increase the stability against degradation by nucleases.[3] The 2´-C-methyl-2´-desoxynucleosides show differential preferred sugar conformations depending on the absolute configuration of the 2´-carbon. The (2´S)-2´-C-methyl-2´-deoxynucleosides mainly adopt the C3´endo conformation while those with (2´R) configuration adopt the C2´endo conformation[4]. Besides, these nucleosides have shown enhanced nuclease resistance when they are incorporated into oligonucleotides[5]. X4 X8 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N  X xi = Thymidine       (2´R) 2´C-methyl 2´deoxi uridine                     (     (2´S) 2´C-methyl 2´deoxi uridine       Thymidine lock nucleic acid Figure 1: modified DNAzymes In this work, we substituted the thymidine positions in the catalytic core of the 10-23 DNAzyme by either (2´S) or (2´R)-2´-C-methyl-2´-deoxyuridine.  We evaluated the kinetic activity, the nuclease resistance and magnesium dependence of punctual or double mutations on the catalytic core (Figure). Finally, we also compared the activity of 2´-C-methyl modified DNAzymes with analogs which have been substituted at the same positions with LNA modifications.     [1] a)Nowakowski, Shin, Prasad, Stout, Joyce Nat. Struct Biol 1999 6, 151-156 b) Nowakowski, Shin, Stout, Joyce J. Mol Biol. 2000 300, 93-102 [2] a) Zaborowska, Furste, Erdmann, Kurreck J Biol. Chem. 2002 277 43 40617 b) Zaborowska, Schubert, Kurreck, Erdmann  FEBS Letters 2005 579 554-558 c) Nawrot, Widera, Wojcik, Rebowska, Nowak, Stec FEBS Journal 2007 274 1062-1072 [3] a)Vester, Hansen, Lundberg, Babu, Soresen, Wengel, Douthwaite BMC Mol. Biol. 2006 7:19 b) Schubert, Gul, Grunert, Zeichhardt, Erdmann, Kurrect Nucleic Acids Research 2003 31 No.20 [4] Cicero, Iribarren, Bazzo Appl. Magn. Reson. 1994 7, 95105 [5] Iribarren, Cicero, Neuner  Antisense Research and Development 1994 4:95-98