Solid-phase synthesis of branched oligonucleotides

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single-stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2',5'- and 3',5'-phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre-mRNA splicing. The protocols described herein allow for the facile solid-phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion-exchange HPLC and PAGE are also illustrated.