DNA Borromean Ring Synthesis

The Borromean rings were synthesized according to the following protocol: 300 pmoles each of strands 1a, 2a and 3a (containing proto-Z sequences) were annealed to form junction in a buffer containing 0.9M NaCl, 117 mM sodium citrate, 10 mM sodium phosphate, 1 mM EDTA, 35% formamide, pH 7.4, at 65 degrees for 60 hours. The junction was purified on a 5% nondenaturing gel, and was eluted in a buffer containing 300 mM NaCl, 20 mM Tris.HCl, 2 mM EDTA, pH 7.6. The junction was ethanol-precipitated, centrifuged 30 min at 4 degrees C, and washed with 75% ethanol. It was dissolved in a solution containing 66 mM Tris.HCl, 10 mM MgCl2, pH 7.6, and labeled with radioactive phosphate using T4 DNA kinase; the labeled material was passed over a ProbeQuant G-50 Micro Column (Pharmacia) to remove excess DTT and ATP. The same protocol was used with strands 1b, 2b and 3b to form the B-DNA junction, except that initial annealing was done in a solution containing 40 mM Tris acetate (pH 8.1), 20 mM sodium acetate, 2mM EDTA and 12.5 mM MgCl2. The two junctions were mixed to a concentration of 30 nM and the solution brought to 1 mM in Co(NH3)6Cl3 and ATP. 20 units of T4 DNA ligase were added (final volume 100 microliters) and the reaction was incubated at 16 degrees C overnight. The products were ethanol-precipitated 3 times and purified on gels.

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