Topologically Constrained Formation of Stable Z-DNA from Normal Sequence under Physiological Conditions.

Z-DNA, a left-handed duplex, has been shown to form in vivo and regulate expression of the corresponding gene. However, its biological roles have not been satisfactorily understood, mainly because Z-DNA is easily converted to the thermodynamically favorable B-DNA. Here we present a new idea to form stable Z-DNA under normal physiological conditions and achieve detailed analysis on its fundamental features. Simply by mixing two complementary minicircles of single-stranded DNA with no chemical modification, the hybridization spontaneously induces topological constraint which twines one-half of the double-stranded DNA into stable Z-DNA. The formation of Z-conformation with high stability has been proved by using circular dichroism spectroscopy, Z-DNA-specific antibody binding assay, nuclease digestion, etc. Even at a concentration of MgCl2 as low as 0.5 mM, Z-DNA was successfully obtained, avoiding the use of high salt conditions, limited sequences, ancillary additives, or chemical modifications, criteria which have hampered Z-DNA research. The resultant Z-DNA has the potential to be used as a canonical standard sample in Z-DNA research. By using this approach, further developments of Z-DNA science and its applications become highly promising.

Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration.

Preparation of large amount of single-stranded circular DNA in high selectivity is crucial for further developments of nanotechnology and other DNA sciences. Herein, a simple but practically useful methodology to prepare DNA rings has been presented. One of the essential factors is to use highly diluted T4 ligase buffer for ligase reactions. This strategy is based on our unexpected finding that, in diluted T4 buffers, intermolecular polymerization of DNA fragments is greatly suppressed with respect to their intramolecular cyclization. This promotion of cyclization is attributable to abnormally low concentration of Mg2+ ion (0.5-1.0 mM) but not ATP in the media for T4 ligase reactions. The second essential factor is to add DNA substrate intermittently to the mixture and maintain its temporal concentration low. By combining these two factors, single-stranded DNA rings of various sizes (31-74 nt) were obtained in high selectivity (89 mol% for 66-nt DNA) and in satisfactorily high productivity (∼0.2 mg/ml). A linear 72-nt DNA was converted to the corresponding DNA ring in nearly 100% selectivity. The superiority of this new method was further substantiated by the fact that small-sized DNA rings (31-42 nt), which were otherwise hardly obtainable, were successfully prepared in reasonable yields.