Triplex-forming branched obligonucleotides: sythesis and interactions with ribo- and deoxyribo-oligonucleotides

The goal of this thesis project was to design a branched oligodeoxynucleotide (odn) using novel synthetic approaches that can form stable triplex with target single stranded DNA or RNA. This triplex approach may lead toward a potential antisense construct that will target mRNA sequences. The project is based upon the previous observation that when a single 2'-O-modified adenosine is incorporated within a DNA strand and that strand is subsequently used to form either DNA:DNA or DNA:RNA duplex, the aromatic substituent of the modified adenosine lies within the major groove of the duplex. Therefore, logically, the extension of this substituent into an oligonucleotide sequence may result in alignment of the oligonucleotide along the major groove of a target duplex to form a triplex. Therefore, synthesis of 2'-O-(thymidin-5-yl)adenosme ( 16 ) was accomplished. The branched odn was created by incorporating 16 into a purine rich sequence and then extending the thymidine substituent into a pyrimidine rich sequence. This branched odn was designed to allow the purine sequence to form a duplex with a single-stranded pyrimidine target and then to have the pyrimidine branch subsequently form a triplex by aligning within the major groove of the duplex. Previous results in this laboratory and preliminary molecular modeling studies indicated that the methylene linker between the 2'-O- of adenosine (of 16 ) and the C-5 of pyrimidine would be sufficient for introduction of the branch into major groove. Different sequences of branched odns were designed and triplex formation with the targets was evaluated using UV-melting, gel electrophoresis, molecular modeling and CD spectroscopy. The results showed that the branched odns were effective in forming stable triplexes with DNA and RNA targets. The branched odns provided a three order of magnitude increase in the association rate of the triplex as compared to unmodified triplexes. The stabilization was effective when nucleoside 16 was incorporated at the 5' or 3' terminal of the branched odns. However, when the modified adenosine was introduced internally, the constraint at the branch point resulted in poor stabilization of the complex. The branched odn model explored in this thesis contributed to the understanding of some of the parameters involved in formation of stable triplexes by branched odns with single stranded DNA and RNA targets.