Javadi-Zarnaghi, Fatemeh

English

Javadi Zarnaghi, F.

MPG.PuRe

Functional characterization and application of 2',5'- branched RNA forming deoxyribozymes using lanthanides as cofactors.

Deoxyribozymes or DNA catalysts are synthetic single stranded DNA molecules capable of catalysis for a wide range or reactions. Deoxyribozymes are discovered by in vitro selection and are mostly dependent on bivalent metal ions for their activity. Very little is known about the structure and function of deoxyribozymes. The role of metal ions in the catalytic mechanism of deoxyribozymes is not well understood. The research presented in this thesis describes functional characterization of RNA ligating deoxyribozymes. In particular, the class of deoxyribozymes which form 2’,5’ branched-RNA has been studied in detail. 
To investigate necessary nucleotides and functional groups for DNA catalysis, combinatorial mutation interference analysis and nucleotide analogue interference mapping of DNA have been developed. Additional biochemical and biophysical techniques including DMS interference, kinetic studies in the presence of combinations of monovalent and bivalent metal ions and trivalent lanthanides, phosphorothioate rescue experiments, DMS and DNase footprinting assays and spectroscopic methods  (i.e. CD and fluorescence spectroscopy) have been employed to achieve better understanding about the mechanism of DNA catalysis.
Lanthanides and specifically terbium have been shown to be effective accelerators of several 2’,5’- branched RNA forming deoxyribozymes (e.g. 9F7, 9F13, 7S11 and 10DM24), when present in the appropriate concentrations. For 9F7, 100 µM terbium in combination with 7 mM magnesium accelerated the enzyme up to 10000-fold. The optimal terbium concentration for manganese-dependent reactions of 9F7, 9F13 and 7S11 was 10 µM. In contrast to the beneficial effect of lanthanides for DNA-catalyzed branching reactions, a deoxyribozyme that synthesizes lariat RNA (6BX22) was inhibited by terbium. 
The 9F7 deoxyribozyme was studied in most detail. We have shown that presence of optimal concentration of terbium increases the affinity of the enzyme to the active bivalent metal ion. Additional investigations e.g. phosphorothioate rescue experiments, foot printing and terbium binding assays pointed toward activation of the 2’-OH at the branch site. Simultaneous coordination of the active metal ions with the 2’-OH electrophile and oxygen atoms of the nucleophile is conceivable.  
The ratio of the metal ions (terbium and the bivalent metal ion) to nucleic acid components of the reaction was crucial for fast ligation. For 9F7 deoxyribozyme 100-fold excess of terbium to oligonucleotides was required for preparative reactions reaching high yield within a short time. For 10DM24 deoxyribozyme, terbium to DNA ratio was required to be only slightly above 10.
The results of the fundamental studies on deoxyribozyme mechanism and folding reported in this thesis, expanded practical applications of 2’,5’ branched-RNA forming deoxyribozymes. Three immediate in vitro applications of branch forming deoxyribozymes have been introduced. 
Terbium mediated acceleration of 10DM24 was employed for post synthetic RNA labeling. The site specific attachment of a single guanosine to the RNA molecule in presence of terbium was efficient and fast. Compared to the original report on using GTP as substrate for10DM24, terbium accommodated the reaction at more neutral conditions (pH 7.5). The deoxyribozyme apparent affinity for GTP was significantly increased. The optimum GTP concentration for labeling was decreased from 3 mM to 10 µM in kinetic scale. In preparative scale only 4-fold excess of GTP to RNA substrate was sufficient to reach high yields in less than 1 hour. 
Terbium mediated acceleration of 10DM24 deoxyribozyme was also employed to ligate adaptor RNAs to an mRNA as scaffold RNA to form a branched mRNA for in vitro translation experiments, in which the ribosome response to the branched mRNA was studied. The results suggested that ribosome is not able to bypass the branch point. The ribosomal interference has potential applications in the field of cellular and molecular biology. 
Preparative scale syntheses of branched RNAs with sequence similarities to the yeast actin mRNA have been achieved using 6BX22 deoxyribozyme. Preliminary binding assays have been performed with 2’,5’ branched RNA products (resembling part of the lariat RNA) and spliceosomal proteins and snRNA fragments.2014-04-0

Georg-August-University Göttingen