Regiospecific Solid-phase Synthesis of Branched Oligonucleotides. Effect of Vicinal 2',5'- (or 2',3'-) and 3',5'-phosphodiester Linkages on the Formation of Hairpin DNA

A general procedure for the solid-phase regiospecific synthesis of branched oligonucleotides (bNA) analogues using readily available phosphoramidite reagents has been developed. The key feature of this method is use of the solid-phase phosphoramidite procedure to assemble linear oligonucleotide sequences and sequential removal of the phosphate (beta-cyanoethyl or methyl) and silyl protecting groups without detaching the nascent oligonucleotide from the solid support. Conversion of the phosphate backbone into the more stable phosphodiester linkages allows for removal of the 2'-O-tert-butyldimethylsilyl protecting group without cleavage or isomerization at the branch point. This method allows for the formation of branched oligonucleotides with sequences of arbitrary base composition, length, and orientation around the branch point junction, including a "Y"-shaped octadecamer d(TACTA)-rA[2',5'd(GTATGT)]3',5'd(CAAGTT). Studies to explore structural effects in the use of a branched adenosine as replacement for nucleotide loops in duplex and triplex DNA are also described. Branched oligonucleotides of the type rA[2',5'dCndA10-5']3',5'dCndT10-3' and rA[2',5'dCn3',3'dA10-5']3',5'dCnT10-3' form hairpin duplexes with thermal stability comparable to or better than that of one with a natural deoxynucleotide loop.