An RNA single strand (blue) is shown at the top of this diagram. Its Watson-Crick pairing regions, X, Y, X' and Y' are illustrated at bumps on the square, and the spacers, denoted by S are shown as the corners of the square. The arrowhead denotes the 3' end of the strand. The pathway to the left illustrates formation of the RNA circle: A red 40 nucleotide DNA linker (incompatible with knot formation) is annealed to the molecule, and it is ligated together to form an RNA circle, which survives treatment with DNase. In the other pathway, a red 16 nucleotide DNA linker is used in the same protocol to produce the RNA trefoil knot, whose three negative nodes are indicated. The interconversion of the two species by E. coli DNA Topoisomerase III (Topo III) is shown at the bottom of the figure. The 40-mer RNA strand promotes somewhat the formation of the circle from the knot.