The elusive quest for RNA knots

Burton, Aaron S.; Di Stefano, Marco; Lehman, Niles; Orland, Henri; Micheletti, Cristian

To guide the search for knotted RNA, we carried out specifically for this Point of View article a bioinformatics survey aimed at identifying candidate RNA sequences that could be susceptible to knotting. As the Seeman laboratory elegantly demonstrated, 35 nucleic acid sequences designed to contain two self-complementary 11-nucleotide sequences (X, Y, X', Y' in which X and X' pair and Y and Y' pair) can spontaneously form knots. We have diagrammed this scenario in an example RNA, and show pathways through which knotted structures could form, at least in some sub-population of folded structures (Fig. 3) . We note that in addition to forming physical knots, these molecules are also pseudoknotted, in the sense that the strands involved in the two helices are interleaved. Thus, pseudoknotted RNA sequences with relatively long base-pairing Table 1 . Knotted RNAs in the survey data set. The knots are likely artifactual results of cryo-em reconstruction. The entries are the same as those shown in Table 1 of ref. 20, though some of the PDB codes differ because the PDB introduced a new archiving system of large structures (with no change of atomic coordinates). The overall knot type was established by using the minimally-invasive closing procedure of ref. 46 . The # sign in the knot label of the last entry denotes the composition (concatenation) of various prime knots. stretches appear to be the most likely candidates to form knots. The minimal number of basepairs needed to form a knot can be estimated from a theoretical standpoint, based on the requirement of a minimum of three strand-crossings for the simplest knot. Because »10 basepairs are necessary for two strand crossings (a full helical turn), pseudoknotted structures containing multiple helices of at least ten basepairs would be prime candidates for knotting. A review of the RNA pseudoknot database reveals two candidates that approach or meet these criteria (Table 2) . Specifically, the plasmid Colib-P9 mRNA (with helices of 18 and 10 basepairs), and Homo sapiens CCR5 PRF (with helices of 25 and 13 basepairs) and telomerase RNA (helices of 22 and 9 basepairs) appear very likely to have at least a slip-knotted structure (in which a free-end has not been completely threaded through a loop). Incidentally we note that such structures have also been observed in proteins. [41] [42] [43] These candidate sequences are found within larger RNA mole-cules, which might seem to preclude the threading events necessary for knotting. In proteins, however, it has been demonstrated in vitro that addition of large structured domains to the N-and C-termini of a knotted protein does not prevent it from adopting its native, knotted conformation so it should not be assumed a priori that longer RNAs cannot form knots. 44 In addition, we have also identified a number of pseudoknotted RNAs with one helix of 10 or more basepairs and a second helix of seven or more basepairs. Although the lowest energy conformations of these molecules would likely be only pseudoknotted, the potential would seemingly exist for knotting to occur to at least a small extent as an alternative or misfolded conformation ( Table 2) . As a matter of fact, their secondary structures, as predicted by McGenus, 27, 28 are not a priori incompatible with proper knots. The table of RNA sequences we have identified, while not exhausting the repertoire of possible knotted candidates, should nevertheless provide a starting point for a targeted search