This thesis is divided into two parts based on a total of 8 papers: Part 1: Synthesis, physicochemical and biochemical studies of chemically modified oligonucleotides and their duplexes and triplexes. Potency of the chromophore conjugated DNA oligonucleotides as antigene and antisense gene repressors was evaluated. The effect of geometry, bulk and ¥ð-electron density of a series of chromophores, tethered at the 5'-end of oligonucleotides, as well as the effect of the linker nature, length and the attachment site of the chromophore to the oligo were explored based on the stability of the duplexes and triplexes. A dramatic improvement in the triplex stability with ara-U linked phenazine oligo (potent antigene) was achieved (¥ÄTm = 16.5¢ª C). A number of selected phenazine and dipyridophenazine tethered antisense oligos (AONs) and their phosphorothioate analogues were shown to form the AON/RNA hybrid duplexes with enhanced thermal stability. CD experiments revealed that these duplexes have the global structure unaltered from that of the native counterpart. RNase H degradation studies on three RNA targets having different degrees of folded structures showed that tethering of phenazine and dipyridophenazine increases the hydrolysis rates (potent antisense) of the target RNA, and that chemical nature of the chromophore influences the RNase H cleavage pattern. Further investigation at the RNA saturated conditions revealed that 3'-tethered chromophores influence the substrate recognition, and the kinetics of the cleavage by RNase H. Conjugation of different chromophores, charged polyaromatic systems and metal complexes with polyaromatic ligands at different sites of the AON revealed that RNase H is very sensitive to any modifications in the middle region of the AON/RNA duplex. On the contrary, any modification at the 3'-end of the AON regardless of the bulk of the substituent or presence of positive charge can be easily tolerated by the enzyme. Sensitivity of the RNase H towards the local structural changes in the AON/RNA hybrid was probed with a number of AONs containing a single 1-(1',3'-O-anhydro-©¬-<u>D</u>-psicofuranosyl)thymine with locked 3'-endo sugar conformation at different sites of AON. RNase H degradation studies revealed that the local conformational changes brought by the constrained nucleoside, although invisible by CD, span in the hybrid as far as 5 nucleotides toward the 5'-end of the AONs (3'-end of RNA), showing the unique transmission of the structural distortion from a single modification site. The results also showed that the structural requirements for the substrate binding and substrate cleavage by RNase H appear to be different. Part 2: Preparation of biologically important isotope labelled oligo-RNAs for the NMR structure determination in solution. Synthesis of the non-uniformly 13C5 labelled 29mer HIV-1 TAR RNA was achieved by solid-phase synthesis using 13C5 labelled ribonucleosides from 13C6-<u>D</u>-glucose). Two hammerhead forming RNAs (16mer and 25mer) were synthesized according to the Uppsala NMR-window strategy, where the sugar residues of the nucleosides forming stem I, II and the loop of the stem III of the resulting hammerhead complex were deuterated. UV melting and high resolution NMR structural studies showed that the 16mer RNA under quasiphysiological condition folds to a very stable hairpin structure, which prevents formation of a hammerhead RNA with the 25mer, primarily owing to thermodynamic reasons.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-1195 |
| Date | January 2000 |
| Creators | Zamaratski, Edouard |
| Publisher | Uppsala universitet, Institutionen för bioorganisk kemi, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1104-232X ; 589 |
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