Synthesis and characterization of oligonucleotides containing 2'-0-substitued adenosines

Tremendous interest has been shown in small oligonucleotides as antisense inhibitors of gene expression in the last few years. Considerable efforts have been made to stabilize the duplex formed between the antisense oligonucleotide and its target. The work presented in this dissertation is concerned with the same problem. However, a somewhat different approach was considered to address this issue. The primary goal of the research was to incorporate a 2'-O-aralkyl nucleoside into DNA as well as a DNA:RNA hybrid and to study in detail the manner in which 2'-O-aralkyl substituent would influence the structure of the ribonucleotide to which it is attached and the nucleic acid duplexes with which it interacts. This could, in principle, lead to duplex stabilization or destabilization. The information gained from this study will enable evaluation of the criteria required for helix stabilization by intercalation. These studies will also be useful in elucidating the nature of non-intercalative modes of interaction between duplexes and an aromatic ring. 2'-O-Phenethyladenosine was synthesized and incorporated in DNA:DNA and DNA:RNA duplexes. The ultraviolet (UV) melting temperature studies (Tm) indicated that the modification destabilized both duplexes. The detailed conformation of the modified duplexes was studied using high resolution nuclear magnetic resonance (NMR) and molecular modeling. These studies revealed that the benzene ring preferred the major groove of the duplex over the minor groove or the intercalation inside the duplex. The hydrophobicity of the benzene was the driving force for this location of benzene ring. These studies indicated that the benzene ring was too small for stable intercalation. Consequently, an anthraquinone was conjugated to the 2'-hydroxyl of adenosine via a methylene bridge. This compound, which incorporated into the oligonucleotides, showed significant stabilization of the duplex. Detailed NMR studies and molecular modeling studies revealed the anthraquinone was intercalated inside the helix despite a short methylene linker. The observation made in the dissertation research are unique and will have significant impact on the design of the next generation of nucleic acid binding molecules like intercalator and groove binders.