DNA Anchor/Terminator

Anchoring System

The current method for Byte production (ie: USER^TM) necessitated a particular anchoring system. Longer sticky ends were also desired to increase the efficiency of recircularization. These factors led to the development of a USER^TM-based anchoring system. An anchoring piece, constructed of two annealed oligomers, is bound to the streptavidin-coated bead via a 5' biotin modification and provides a sticky 3' overhang complementary to an A end. Once the desired number of Bytes is added, a terminator (again, two annealed oligomers) is annealed and ligated to the available end of the final brick (in this case, a B end). The entire construct is then treated with USER^TM enzyme mix. The resulting end product from the digestion of uracil contained within the anchor, anneals to the terminator overhang and can be ligated to form a circular product. The ligation also yields a complete SceI site that can be used to linearize the construct for recombination into the E. coli genome. See Figure 1.

A vital component of the BioBytes method is the use of a biotinylated DNA anchor in order to allow unidirectional assembly of the Bytes on paramagnetic beads by sequestering the 5' ends of Bytes, leaving only the 3' ends available to bind incomding Bytes. The anchor itself has three vital components: A 5’ biotinylation, a double stranded DNA (dsDNA) portion that incorporates a release mechanism in order to liberate the construct from the beads, and A or B overhangs to allow Bytes to bind to the anchor. Our team has considered a number anchor systems, each with their own set of advantages and disadvantages. The three main anchor systems we investigated are summarized below. See Table 1.

Table 1: Overview of three different anchoring systems that were considered for BioBytes. 5' Biotin (BTN), single stranded DNA (ssDNA), nucleotide (nt), double stranded DNA (dsDNA).

The current BioBytes anchor system utilizes a 5’-biotin which anchors the construct to beads by binding non-covalently, but with great strength, to the covalently linked streptavidin on the surface of the paramagnetic beads. There is also a 5’-15 nucleotide spacer region of ssDNA which facilitates more efficient binding of the anchor to the bead as the binding pocket of streptavidin is deep and thus a highly flexible ssDNA linker is recommended to allow the biotin to effectively bind into this deep pocket. A 21 bp double stranded portion of the anchor contains the I-SceI recognition sequence, which when digested with I-SceI produces 4 base overhangs, but also includes four deoxyuracil residues. These uracils are excised by New England Biolab’s USER^TM system to generate single nucleotide gaps in the top strand. USER^TM digestion thus effectively destroys the anchor and produces an 18 base 3’ overhang which becomes important for recircularization of the construct. Finally the anchor contains the A or B 3’ overhangs complementary to those of the Bytes, allowing their binding to the anchor. . See Figure 1.

Figure 1: pAB and pBA multiple cloning sites with highlighted primers prA1/B1u and prA2/B2u annealing regions.