Enzymatic oligonucleotide assembly using hairpins and enzymatic cleavage

Abstract

Sequential assembly of oligonucleotide hairpins is used to create oligonucleotides with specific sequences. Each oligonucleotide hairpin includes a payload region containing one or more nucleotides that are added to the end of an anchor strand. Overhang regions on the oligonucleotide hairpins hybridize to anchor strands attached to a substrate. The hybridized oligonucleotide hairpins are covalently attached to the anchor strands by the activity of ligase. The oligonucleotide hairpins include an enzyme cleavage region which, when cleaved, separates the payload region from the remainder of the oligonucleotide hairpin. The oligonucleotide hairpin is separated from the anchor strand by denaturation and washed away. This process is repeated with additional oligonucleotide hairpins to add additional nucleotides to the ends of the anchor strands. A microelectrode array may be used to control the location of hybridization and create multiple oligonucleotides in parallel. Fully assembled oligonucleotides can be separated from the substrate and stored.