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-©¬-D-psicofuranosyl)thymine with locked 3′-endo sugar conformation at different sites of AON…
Contents
1. Synthesis, physicochemical and biochemical studies of chemically modified oligonucleotides and their duplexes and triplexes.
1.1 Introduction
1.2 Present work
1.2.1 Synthesis of 5′-tethered ONs (Paper I)
1.2.2 Thermal denaturation studies on duplexes and triplexes formed by the 5′-tethered ONs (Paper I)
1.2.3 Synthesis of the ONs tethered with phenazine through the arabino- and xylo-nucleotides (Paper II)
1.2.4 Thermal denaturation and fluorescence studies on duplexes and triplexes formed
by the phenazine-tethered ONs (Paper II)
1.2.5 Synthesis of dipyridophenazine-tethered ONs (Paper III)
1.2.6 Thermal stability of the duplexes and triplexes formed by the dipyridophenazine-tethered ONs (Paper III)
1.2.7 Physicochemical characterization of the RNA targets used in evaluation of the antisense potency of the phenazine and dipyridophenazine tethered AONs(Paper IV)
1.2.8 Thermodynamic characteristics of the AON/RNA hybrid duplexes formed by phenazine and dipyridophenazine tethered AONs with phosphodiester and thiophosphodiester backbone (Paper IV)
1.2.9 Circular dichroism (CD) studies on the AON/RNA hybrid duplexes formed by phenazine and dipyridophenazine tethered AONs (Paper IV)
1.2.10 RNase H degradation studies on the AON/RNA hybrid duplexes formed by phenazine and dipyridophenazine tethered AONs (Paper IV)
1.2.11 Nuclease resistance of the 3′-phenazine and 3′-dipyridophenazine modified AONs (Paper IV)
1.2.12 Influence of the 3′ tether of the AON on the kinetics of the RNase H cleavage reaction (Paper V)
1.2.13 RNase H sensitivity towards the modifications at the different sites of the AON/RNA hybrid duplex (Paper IV)
1.2.14 RNase H sensitivity to the local conformational changes in the AON strand of the AON/RNA hybrid duplex (Paper VI)
2. Preparation of biologically important isotope labelled oligo-RNAs for the NMR structure determination in solution.
2.1 Introduction
2.2 Present work
2.2.1 Synthesis of sequence-specific non-uniformly
C5 labelled HIV-1 TAR RNA (Paper VII)
2.2.2 Influence of the RNA secondary structure on the hammerhead ribozyme formation in the solution at physiological conditions (Paper VIII)
3. Acknowledgements
4. References
Author: Zamaratski, Edouard
Source: Uppsala University Library
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