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

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.

3'-Deoxyribopyranose (4' -> 2')-oligonucleotide duplexes (=p-DNA duplexes) as substitutes of RNA hairpin structures

Damian Ackermann

By automated synthesis, we prepd. hybrid oligonucleotides consisting of covalently linked RNA and p-DNA sequences (p-DNA = 3'-deoxyribopyranose (4'->2')-oligonucleotides). The pairing properties of corresponding hybrid duplexes formed from fully complementary single strands were investigated. An uninterrupted p-p-stacking at the p-DNA/RNA interface and cooperative pairing between the two systems was achieved by connecting them via a 4'-p-DNA-2'->5'-RNA-3' and 5'-RNA-2'->4'-p-DNA-2' phosphodiester linkage, resp. The RNA 2'-phosphoramidites required for the formation of the RNA-2'->4'-p-DNA phosphodiester linkage were synthesized from the corresponding, 3'-O-tom-protected ribonucleosides (tom = [(triisopropylsilyl)oxy]methyl). Analogs of the FMN binding aptamer and the hammerhead ribozyme were prepd. Each of these analogs consisted of two p-DNA/RNA hybrid single strands with complementary p-DNA sequences, designed to substitute stem/loop and stem motifs within the parent compds. By comparative binding and cleavage studies, it was found that mixing of the two complementary p-DNA/RNA hybrid sequences resulted in the formation of the fully functional analogs of the FMN-binding aptamer and of the hammerhead ribozyme, resp. [on SciFinder (R)]

2002

The yeast C/D box snoRNA U14 adopts a "weak" K-turn like conformation recognized by the Snu13 core protein in solution

Benjamin Marcel Daniel Rothé

Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment. This structural motif has an internal three-nucleotide bulge flanked on its 3' side by a G.A/A.G tandem pairs followed by one or two non-Watson-Crick pairs, and on its 5' side by a classical RNA helix. This peculiar arrangement induces a strong curvature of the phosphodiester backbone, which makes it conducive to multiple tertiary interactions. SNU13/Snu13p (Human/Yeast) binds specifically the U14 C/D box snoRNA K-turn sequence motif. This event is the prerequisite to promote the assembly of the RNP, which contains NOP58/Nop58 and NOP56/Nop56 core proteins and the 2'-O-methyl-transferase, Fibrillarin/Noplp. The U14 small nucleolar RNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation and ribose methylation. Here, we report the solution structure of the free U14 snoRNA K-turn motif (kt-U14) as determined by Nuclear Magnetic Resonance. We demonstrate that a major fraction of free kt-U14 adopts a pre-folded conformation similar to protein bound K-turn, even in the absence of divalent ions. In contrast to the kt-U4 or tyrS RNA, kt-U14 displays a sharp bent in the phosphodiester backbone. The U.U and G.A tandem base pairs are formed through weak hydrogen bonds. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. The structure of the free U14 RNA is the first reference example for the canonical motifs of the C/D box snoRNA family. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER2019

The yeast C/D box snoRNA U14 adopts a "weak" K-turn like conformation recognized by the Snu13 core protein in solution

Benjamin Marcel Daniel Rothé

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER2019