Unusual knot-like structures recently discovered in viral exoribonuclease-resistant RNAs (xrRNAs) prevent digestion by host RNases to create subgenomic RNAs enhancing infection and pathogenicity. xrRNAs are proposed to prevent digestion through mechanical resistance to unfolding. However, their unfolding force has not been measured, and the factors determining RNase resistance are unclear. Furthermore, how these knots fold remains unknown. Unfolding a Zika virus xrRNA with optical tweezers revealed that it was the most mechanically stable RNA yet observed. The knot formed by threading the 5′ end into a three-helix junction before pseudoknot interactions closed a ring around it. The pseudoknot and tertiary contacts stabilizing the threaded 5′ end were both required to generate extreme force resistance, whereas removing a 5′-end contact produced a low-force knot lacking RNase resistance. These results indicate mechanical resistance plays a central functional role, with the fraction of molecules forming extremely high-force knots determining the RNase resistance level.
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We thank J. Kieft for helpful discussions about xrRNAs and D. Ritchie for assistance in designing the optical tweezers assay. This work was supported by the Canadian Institutes of Health Research grant reference number MOP–142449 (to M.T.W), Alberta Innovates iCORE Strategic Chair (to M.T.W.) and the National Research Council Canada (to M.T.W.).
The authors declare no competing interests.
Peer review information Nature Chemical Biology thanks Pan T. X. Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Most unfolding FECs (90 ± 2%) show the extreme mechanical stability of Ir but no preceding ~20-pN unfolding event, confirming that P4 unfolds prior to Ir. Dashed lines show WLC fits to each state, cyan boxes show overstretching region, inset shows cartoon of structure before unfolding. b, A very few FECs (~0.3%) show unfolding forces in the range ~25–50 pN indicative of tertiary structure, and ΔLc consistent with state I5b, where the 5′ end is threaded but the ring is not closed. c, Some FECs (10% ± 2%) show unfolding of secondary structure only, consistent with state I1b. d, Proposed structures of Ir, I5b and I1b with oligo 1 bound. Contour length changes expected from unfolding each structure are listed in Supplementary Table 3. e, Most refolding FECs show the formation of I1b; in some cases, transitions into Ir and/or I5b are seen directly (for example in Fig. 2b), in other cases, they occur at sufficiently low force to remain undetected. f, Unfolding (black) and refolding (red) pathways in the presence of oligo 1. Arrow thicknesses indicate pathway probabilities. For comparison, the states and transitions prevented by binding of oligo 1 are shown in grey.
a, Under half of FECs (41 ± 5%) show unfolding from I5b, with the 5′ end threaded but PK prevented by oligo binding. Dashed lines show WLC fits to different states, inset and cartoon show structure before unfolding with oligo 2 bound. Contour length changes expected from unfolding each structure are listed in Supplementary Table 3. b, Over half of FECs (59 ± 5%) show unfolding from I1b. c, States I1b and I5b can be distinguished by both their lengths (Supplementary Table 3) and unfolding forces. d, Most refolding FECs show the formation of I1b; in some cases, transitions into I5b are seen directly (for example in Fig. 2b), in other cases, they occur at sufficiently low force to remain undetected. e, Unfolding (black) and refolding (red) pathways in the presence of oligo 2. Identification of I5b indirectly proves the existence of the putative intermediate I5 (Fig. 3 and Supplementary Fig. 2). Arrow thicknesses indicate pathway probabilities. For comparison, the states and transitions blocked by oligo 2 binding are shown in grey.
a, b, All of the unfolding (black) and refolding (red) FECs show the low-force transitions characteristic of secondary structure only, with length changes indicative of state I3. Dashed lines show WLC fits to different states, inset shows structure before unfolding with oligo 3 bound. c, Unfolding (top panel) and refolding (bottom panel) force distributions show low hysteresis characteristic of helix unfolding/refolding. d, Unfolding (black) and refolding (red) pathways in the presence of oligo 3. Arrow thicknesses indicate pathway probabilities. For comparison, the states and transitions blocked by oligo 3 binding are shown in grey.
a, Two-thirds of unfolding FECs (~67%) show low-force transitions consistent with the unfolding of I1 (Supplementary Fig. 2b). Dashed lines show WLC fits to each state, inset shows structure before unfolding. b, One-third of unfolding FECs (~33%) show high-force transitions consistent with state I0PK′ containing a non-native pseudoknot that inhibits the folding of P4. c, All refolding FECs show sequential formation of I4→I2→I1, indicating I1 is Mg2+-independent and suggesting I0PK′ is derived from I1. d, Cartoon of the I0PK′→I1PK′ transition. Expected contour length changes listed in Supplementary Table 3. e, Unfolding (Fu) and refolding (Fr) force distributions of the states seen in FECs. High unfolding forces for I1PK′ indicate the presence of tertiary interactions. f, Unfolding and refolding forces for I4, I2 and I1 show a small stabilization effect from Mg2+. Center line indicates median value, box edges indicate 25th and 75th percentiles, whiskers indicate 5th and 95th percentiles. N = 283, 287, and 285 respectively for unfolding I1, I2, and I4 without Mg2+; N = 339, 328, and 352 for unfolding with Mg2+; N = 386, 386, 369 for refolding without Mg2+; and N = 477, 475, and 476 for refolding with Mg2+. g, Unfolding (black) and refolding (red) pathways in the absence of Mg2+. Arrow thicknesses indicate pathway probabilities. For comparison, the states and transitions dependent on Mg2+ are shown in grey.
a, Roughly 15% of unfolding FECs (black) show low forces consistent with secondary structure only, in state I1′, which is akin to I1 (Supplementary Fig. 2b) but with shorter P2 and longer P3. Dashed lines show WLC fits to each state, inset shows structure before unfolding. b, Roughly 17% of unfolding FECs (black) show a moderately high-force state Im1 consistent with partial formation of 5′-TC and the non-native pseudoknot PK′ but without any 5′-end threading. Refolding FECs (red) show a pathway similar to I1 (Supplementary Fig. 3a), although with shorter P2 and longer P3, indicating Im1 derives from I1′. c, Roughly 15% of unfolding FECs (black) show the same extreme stability as for the wild-type ring-knot, indicating this mutant forms a ring-knot analogous to the wild-type knot, with full 5′-TC and extreme mechanical resistance. d, Roughly 53% of unfolding (black) and refolding FECs (red) show low forces consistent with state I1″ (inset), akin to I1, containing secondary structure only but with lengthened P2. e, Proposed structures of the states identified in (a-d). Base-pairing of G22 with C44 prevents native 5′-TC but allows I1′–I4′ and Im1 (with shorter P2 and longer P3) to form. Alternatively, base-pairing of G22 with C9 lengthens P2 but leaves C44 accessible for native 5′-TC formation, allowing formation of mutant states Nm1 and Irm1 containing a mechanically resistant ring-knot, in addition to intermediates I1″, I2″, and I4″. Contour length changes expected from unfolding each structure are listed in Supplementary Table 4.
Extended Data Fig. 6 Effects of anti-sense oligo 3 on C22G mutant and evidence for G22:C9 base-pairing.
a, b, All (a) unfolding and (b) refolding FECs measured in the presence of oligo 3 show secondary structure only. c, Without oligo 3 (red), two sub-populations are seen, with different average contour length changes (top) and unfolding forces (bottom). High-force transitions have lengths inconsistent with expectations for G22:C44 pairing (blue, left) but consistent with expectations for G22:C9 pairing (blue, right). As expected, ΔLc for unfolding P1 + P3 without oligo 3 (Supplementary Table 4) is anti-correlated with the change in ΔLc for P2 between the two sub-populations, being longer for G22:C44 pairing. With oligo 3 present (black), only one population is seen, matching the results for G22:C9 pairing. Fu is expected to be higher for P2 with G22:C9 pairing because two extra base-pairs are predicted by mfold to make P2 ~5.7 kcal/mol more stable. Note that these bar graphs restate results presented in Fig. 4c and Supplementary Table 4; error bars represent standard error on the mean. d, Proposed structures for intermediate states with oligo 3 present. Contour length changes expected from unfolding each structure are listed in Supplementary Table 4.
The two possible base-pairings for the mutated base (G22), with either C9 or C44, lead to heterogeneous pathways. Superscripts ‘e’ and ‘s’ denote respectively elongated and shortened versions of a helix relative to the native ones. Arrow thicknesses indicate pathway probabilities.
a, Measurements of XR activity show U4C mutant has no resistance to digestion by Xrn1 (to within 2% detection limit), in contrast to strong resistance of WT xrRNA (~80%). Note that some of the input RNA remains undigested because of incomplete loading of Xrn1. Two replicates are shown out of 3 independent measurements yielding the same result of ~0% resistance for U4C. b, Just over half (52%) of FECs show folding (red) into and unfolding (black) out of a native-like ring-knot. c, Most of the remaining FECs (44%) show folding (red) into and unfolding (black) out of intermediate I1. d, A very few FECs (1%) show folding (red) into and unfolding (black) out of an intermediate with the 5′ end natively threaded but the ring still open. e, A very few FECs (~3%) for U4C show folding (red) into and unfolding (black) out of a non-native state (I6). A speculative model of I6 involving non-native 5′-TC is illustrated (inset, dashed black line: G3:C22), but further study is needed. f, With oligo 2 blocking PK, most FECs (73 ± 7%) unfolded from the threaded intermediate I5bm2, whose unfolding force was lower than for the analogous state in the WT xrRNA (Supplementary Fig. 6a) because of disrupted 5′-TC. g, A minority of FECs (27 ± 7%) with oligo 2 unfolded from I1. h, Proposed structures of states identified in (b–g). Contour length changes expected from unfolding each structure are listed in Supplementary Table 5. i, Pathways for unfolding (black) and refolding (red). Arrow thicknesses indicate pathway probabilities.
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Zhao, M., Woodside, M.T. Mechanical strength of RNA knot in Zika virus protects against cellular defenses. Nat Chem Biol 17, 975–981 (2021). https://doi.org/10.1038/s41589-021-00829-z