The disclosure provides novel topoisomerases which exhibit weakened ability to form a covalent bond to the 5-(C/T)CCTT-3recognition site in the presence of increased NaCl concentrations relative to wild-type, together with novel methods for synthesizing DNA in the 3 to 5 direction using the novel topoisomerase; and other compounds, compositions, methods and devices comprising or utilizing topoisomerases which exhibit reduced covalent bond formation to the 5-(C/T)CCTT-3 recognition site in the presence of increased NaCl concentrations, relative to wild-type.

Current minicircle production methods are slow, expensive, and difficult to perform under GMP conditions because, in most cases, the product is derived from bacteria. In contrast, HTLA-and CHTLA-based synthetic circular supercoiled DNA production can be done completely in a test tube, using chemically or enzymatically synthesized oligonucleotides, long single stranded DNA, and/or double stranded DNA.