Figure 1: Strategies of constructing ssRNA topological structures.

(a,b) Seeman’s method of using A-form RNA helix to generate ssRNA topological structures²¹. In a, one turn of an A-form RNA helix is shown with the helical and schematic representations. The two component strands form two negative nodes within this one-turn helix. In b, a strand of ssRNA (orange) is designed to contain alternating complementary pairing segments to form two one-turn A-form helices, and a trefoil knot is formed after enzymatic ligation aided by a DNA splint (green). However, topological structures constructed in this way contain very strong intrastrand base pairings. (c,d) Junction-based method to generate ssRNA topological structures. In c, a 4WJ is formed with two RNA strands (blue) as the helical strands and two DNA strands (grey) as the crossover strands, and is shown with the helical and schematic representations. The two RNA helical strands generate a node. In d, the assembly complex for the trefoil knot is formed, where the RNA scaffolds (blue) are threaded into the targeted topology by DNA staples (grey) and linked end to end by DNA splints (green). After ligation and subsequent removal of the DNA staples and splints, a ssRNA knot free of strong intrastrand base pairings is generated. (e) The ssDNA trefoil knot can be used as a template for the construction of ssRNA trefoil knot. The ssDNA knot template (red) is pre-prepared and annealed with the complementary RNA strands (blue). After ligation, the DNA–RNA hybrid knot is formed. The ds DNA–RNA hybrid is more rigid than single-stranded structure, the careful design of curvature (by adding bulges) and torsion (by adjusting the length of hybrid helix) is necessary. The hybrid knot can then be subjected to DNase I digestion to obtain the ssRNA knot.