Chemical synthesis and biological evaluation of circular, branched and lariat oligonucleotides

This thesis highlights novel synthetic routes towards the facile synthesis of lariat DNA and RNA oligonucleotides, and the utilities of branched (bNAs) and circular (i.e. dumbbell-shaped) nucleic acids for targeting biologically relevant processes (i.e. HIV proliferation, alternative RNA splicing) with potential therapeutic applications.An innovative synthetic strategy for the synthesis and cyclization of a medium-sized (21-nucleotide) DNA lariat starting from a CPG-tethered, convergently synthesized branched DNA (bDNA) molecule was devised in Chapter 2. This synthetic route exploited the differential cleavage rates of two CPG-oligonucleotide tethers, namely the base-labile hydroquinone-O,O'-diacetate (Q-linker) and the more robust succinate (S-linker ) linkages, as well as phosphitylation of the 5'-oligonucleotide terminus and cyclization under standard phosphoramidite coupling conditions to effect new phosphodiester bond construction. The results clearly indicate a disadvantageous correlation between high branching efficiency on a densely loaded CPG and the production of dendrimeric (i.e. hyperbranched) oligonucleotide species rather than effective cyclization.Given the entropic disadvantage of synthesizing medium-sized DNA lariats on solid-support using the method described in Chapter 2, unique intermolecular (i.e. DNA dumbbells) and intermolecular template-mediated approaches for lariat cyclization commencing with convergently and divergently synthesized bDNAs and bRNAs were developed in Chapter 3. Both methods lead to the exclusive and high-yielding formation of medium sized (46--57 nucleotides) DNA and RNA lariats. Parameters for successful phosphodiester bond construction were also elucidated in both systems.A novel class of highly specific and potent oligonucleotide-based HIV-1 reverse transcriptase inhibitors, RNA dumbbells, comprising of a 10 base-pair stem and two flanking UUCG hairpin-loop motifs are described in Chapter 4. Explicitly, such constructs were capable of selectively hampering the RNase-H mediated activity of the retroviral enzyme without consequence to its DNA polymerase function with an IC50 in the 3 muM range. Its precise interaction with the RNase H domain of RT was authenticated via a UV-crosslinking assay. Furthermore, the RNA dumbbells did not inflict any effect on mammalian RNase H activity, suggesting that such compounds would not obstruct cellular RNase H function.Chapter 5 describes the utility of synthetic bRNA for the inhibition and modulation of pre-mRNA splicing in yeast and mammalian in vitro systems. Most notably, synthetic bNAs can be suitably exploited as agents for the study of branchpoint recognition during in vitro splicing of a pre-mRNA transcript. The results clearly indicate the requirement for a fully formed branchpoint (i.e. 5 '-, 2'- and 3'-extensions; Y-shaped molecules) off the conserved branchpoint adenosine for efficient splicing inhibition. Specific methods for stabilizing bNAs against ubiquitous cellular exo- and endonucleases as well as the 2'-scissle (2'-debranching) activity present in the HeLa extract milieu are also described