Faculté des sciences de base SB, Section de chimie, Institut de chimie moléculaire et biologique ICMB

Synthese und Paarungseigenschaften von pDNA und deren Anwendung bei der Entwicklung funktionalisierter Ribozyme

Ackermann, Damian ; Pitsch, Stefan (Dir.)

Thèse sciences Ecole polytechnique fédérale de Lausanne EPFL : 2004 ; no 2920.

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    Summary
    Within this project methods for the preparation of the unnatural nucleic acids 3'-O-Methyl-pRNA and pDNA (3'-Desoxy-pRNA) were developed. The pairing properties of these two systems have been examined and compared with that of pRNA. All of the three systems do follow the same pairing rules. Pairing has been observed only on the Watson-Crick side forming duplexes in antiparallel direction. All three systems are autonomous pairing systems that do not interact with RNA or DNA. In this series pRNA forms the most stable duplexes. Nucleobase 9H-purin-2,6-diamin instead of adenin and 5-methyl-cytosine instead of cytosine were introduced to increase the stability of pDNA duplexes. A solution structure of the self-complementary pDNA sequence [MGAATTMG]2 was elucidated by NMR. It shows an antiparallel duplex twisted against the major groove with sligthly lefthanded helicity. In analogy to pRNA the interstrand stacking predominates with a inclination angel of approximately 45°. Proceeding form the NMR structure a MD simulation was performed, in which the duplex has strongly bent after a regeneration time of 1.5 ns; this bent shape was conserved during the remaining simulation time of 4.0 ns. Using automated solid phase synthesis pDNA/RNA hybrid sequences were prepared. Examining the pairing strength, the RNA-2'—>4'-pDNA linkage at the pDNA/RNA interface was observed to lead to more stable duplexes than the RNA-3'—>4'-pDNA linkage did. This kind of pDNA/RNA structures have been used to replace structure defining elements like hairpins or stem motifs by pDNA-duplexes in complex RNA structures. From the FMN binding RNA aptamer an analogous pDNA/RNA construct has been prepared, that was built of two hybrid sequences. The binding of FMN in the pDNA/RNA construct was shown by fluorescence spectroscopy and the dissociation constant K0 = 2.6·10-7 M was calculated. Using the same strategy a pDNA/RNA construct of the hammerhead ribozyme was prepared, having substrate cleavage activity in analogy to the natural ribozyme. Cleavage properties of the pDNA/RNA construct at different Mg2+ and Mn2+ concentrations and at different reaction temperatures showed the same trend as those from the natural ribozyme did. Under all measured conditions, the natural ribozyme was about 1.5 times as active as the pDNA/RNA construct. Additional functional groups were introduced at different positions of the pDNA/RNA hybrid sequences using a pre-functionalized 6'-bromopentyl allofuranosyl building bloc on which the bromide was substituted with MeNH2 resp. EtSH after solid phase synthesis. 25 different functionalized pDNA/RNA ribozyme constructs could be built up in a combinatorial way. From 17 out of the 25 constructs, the cleavage kinetics was measured and the cleavage rate evaluated. The substitution pattern of the most active pDNA/RNA ribozyme analogue was applied on the hammerhead RNA sequence and the doubly methylamino-substituted construct prepared. Compared to the natural hammerhead ribozyme, the cleavage rates of this construct were higher with a factor of 2.0 - 3.3 under all measured conditions.