| A general procedure for the solid-phase synthesis of branched oligonucleotide analogs, including some with similar base sequence to the yeast ( Saccharomyces cerevisiae) rp51 mRNA, is described using the automated solid-phase phosphite triester approach. Using readily available phosphoramidite reagents, branch-points were introduced by sequential removal of the phosphate (beta-cyanoethyl or methyl) and silyl protecting groups without detaching the nascent oligonucleotide from the solid-support. This then allowed the extension from the branch-point in the conventional 3',5 ', or the unconventional 5',3' directions, thus affording complete control over the base composition and direction of strand around the branch point.;The protocol was modified for the synthesis of msDNA, a DNA/RNA branched chimera. Using a combination of silyl and Fpmp groups for the protection of 2'-hydroxyl functions, a linear oligonucleotide was assembled. Branching was then introduced using the methodology developed for branched DNA.;Utilization of capillary electrophoresis (CE) in the analysis of branched nucleic acids was also touched upon. CE proved to be a very powerful technique in the separation and identification of a mixture of four branched oligonucleotides. This technique, CE, was also seen to be an attractive alternative for following the debranching reaction, thus eliminating the need for radioactively labeled substrates.;The unique structural properties of branched nucleic acids were exploited in investigation of the association properties of bNAs. By synthesizing branch molecules, capable of forming T:A/T triplexes in the parallel (Hoogsteen) or the antiparallel (reverse Hoogsteen) T:A/T motifs allowed, for the first time, a direct comparison of their relative stabilities. Most of the 0.25 kcal mol-1/TAT base triplet difference in stabilities was seen to come from differences in enthalpy, with the parallel (Hoogsteen) base triplet being more stable.;Studies to explore structural effects in the use of a branched adenosine as replacement for nucleotide loops in duplex DNA are also described. Branched oligonucleotides of the type rA[2',5'dC ndA10-5']3',5dC ndT10-3' and rA[2',5' dCn3',3' dA10-5']3', 5dCndT10-3' are shown to form hairpin duplexes with thermal stabilities comparable to or better than that of one with a natural deoxynucleotide loop. Molecular modeling was used to study the conformation of branched hairpin loops. In certain cases branched hairpins adopted conformations similar to the ones seen in naturally occurring super stable RNA hairpins.;The branched nucleic acids (bNAs) also proved to be invaluable tools for studying in vitro splicing and the substrate specificity of the yeast debranching enzyme. |
