Synthesis of a DNA Knot Containing Both Positive and Negative Nodes

A figure-eight (4₁) knot, containing two positive and two negative nodes, has been synthesized by ligating a single-stranded DNA molecule into a closed cycle. The molecule consists of two single-turn double-helical domains connected by four dT₁₄ or dT₁₅ linkers. One of the domains contains the sequence (dCpdGp)₆, which is capable of forming left-handed Z-DNA. Ligation yields a trefoil (3₁) knot or a 4₁ knot, depending upon the conditions in solution. A 4₁ knot results if Co(NH₃)₆³⁺ is present in the solution to promote the formation of Z-DNA; a 3₁ knot is the product in the absence of Co(NH₃)₆³⁺. Both domains of the 3₁ knot are susceptible to restriction endonuclease digestion, but only the domain of the 4₁ knot expected to be right-handed can be digested by a restriction endonuclease. The 4₁ knot has both a higher gel-electrophoretic mobility at low polyacrylamide concentrations and a higher friction constant than the 3₁ knot, when analyzed by Ferguson plots under denaturing conditions.