Conformationally Constrained Oligonucleotides for RNA Targeting
Abstract: A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems.In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7?-Me-carba-LNA-A, -G, -MeC and -T as well as 6?, 7?-substituted ?-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application.AONs modified with 7?-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing ?-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart. It has been demonstrated in Papers I-III that 7?-methyl substitution in ?-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7?-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7?-Me-carba-LNA and ?-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as ?-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6? of ?-L-carba-LNAs and methyl group in C7? of 7?-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3?-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3?-phosphate.Additionally, AONs modified with 7?-Me-carba-LNAs and ?-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).
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