Enzymatic oligonucleotide assembly using hairpins and enzymatic cleavage

A method of assembling an oligonucleotide having an arbitrary sequence, the method comprising:

• identifying a binary string to encode in the oligonucleotide;
• a. contacting an anchor strand attached to a microelectrode array comprising a plurality of individually addressable electrodes with an oligonucleotide hairpin under conditions such that an overhang region of the oligonucleotide hairpin hybridizes to the anchor strand, the oligonucleotide hairpin comprising:
• a loop region;
• and
• a stem region having an overhang side and a non-overhang side, wherein an end of the overhang side includes the overhang region of at least 3 nucleotides, an end of the non-overhang side includes a payload region with a length of 2-5 nucleotides, and the non-overhang side includes an enzyme cleavage region directly adjacent to the payload region, the enzyme cleavage region cleaved by contact with a cleavage enzyme;
• b. contacting the oligonucleotide with ligase under conditions such that the ligase catalyzes formation of a phosphodiester bond between the payload region of the oligonucleotide hairpin and the anchor strand;
• c. contacting the oligonucleotide with the cleavage enzyme under conditions such that the cleavage enzyme cleaves the enzyme cleavage region thereby separating the payload region from a remainder of the oligonucleotide hairpin and the payload region remains attached to the anchor strand;
• d. denaturing a double-stranded structure formed from hybridization of the oligonucleotide hairpin to the anchor strand;
• e. washing the oligonucleotide following the denaturing to remove the remainder of the oligonucleotide hairpin;
• and
• repeating steps a-e adding during each round an oligonucleotide hairpin that encodes in the payload region a first bit or a second bit, the oligonucleotide hairpins added in an order based on the binary string, wherein there are only four species of oligonucleotide hairpins and the oligonucleotide hairpin added in each round is selected based on a bit to be encoded and the payload region of the oligonucleotide hairpin added immediately prior so that an overhang region of the added oligonucleotide hairpin is complementary to the payload region of the oligonucleotide hairpin added immediately prior.