Fig 1.
RNA dual graph motifs and nomenclature with two vertices.
Vertices represent stems. Edges represent junction of stems, or bulge/internal loops with more than one residue on each strand. Self-edges represent hairpin loops. Dual graphs are referred to by two numbers, listed below each respective graph. The first number indicates the number of vertices, the second number specifies the topology, e.g., 2_1 is the dual graph secondary structure motif with two vertices, specifically the first possible topology. For additional details refer to the RNA-As-Graphs database [68]. Dot bracket example structures for each respective motif shown below number labels. An open parenthesis/bracket shows the opening base on the 5′ side of the base pair, a closed parenthesis/bracket represents the closing base on the 3′ side of the base pair. Each period “.” represents an unpaired base.
Fig 2.
Dominant SARS-CoV-2 pseudoknot motif predictions via Shapify.
SARS-CoV-2 frameshift element sequence shown as a horizontal line from 5′ (left) to 3′ (right). Arcs represent predicted base pairs. Top arc diagram includes 3_6 motif components (see Table 2 for dot-bracket format) of the fifth most stable structure predicted via Shapify (see Sections 2.4–2.6) for the 144 nt sequence (free energy −29.45 kcal/mol). Initial stem 5 base pairs in red (free energy −4.22 kcal/mol). Downstream pseudoknot target sequence highlighted in red. Bottom arc diagram includes 3_3 motif component of the MFE structure predicted via Shapify for the 144 nt sequence (free energy −30.93 kcal/mol). Initial stem 2 base pairs in light blue (free energy −6.1 kcal/mol). Initial stem 1 base pairs in dark blue (free energy −11.48 kcal/mol).
Table 1.
Shortest and longest window sizes used for SARS-CoV-2 structure predictions via Shapify.
Fig 3.
Coronavirus frameshift element covariation.
Base pairs in the top arc diagram have strong covariation among the multiple sequence alignment identified by KnotAli. Bottom arc diagram displays the SARS-CoV-2 native 3_6 pseudoknot with the downstream target sequence in red. SARS-CoV-2 attenuator hairpin sequence highlighted in fuchsia and the slippery sequence in green.
Fig 4.
SARS-CoV-2 secondary structure predictions via KnotAli.
Top arc diagram: free energy −36.47 kcal/mol, EPI_ISL_426088, includes 3_6 motif. Bottom arc diagram: free energy −40.65 kcal/mol, EPI_ISL_426905, includes 3_3 motif. Mutations are indicated with red ovals. A △ symbol represents the mutation from the nucleotide on the left to the nucleotide on the right. Attenuator hairpin sequence is highlighted in fuchsia, slippery sequence in green, and the downstream native pseudoknot target sequence in red.
Fig 5.
Bat coronaviruses secondary structure predictions via KnotAli.
Top arc diagram: BtRf-BetaCov, free energy −43.24 kcal/mol, KJ473811, includes 3_6 motif. Bottom arc diagram: SARS-like WIV1-CoV, free energy −39.72 kcal/mol, KU444582, includes 3_3 motif. Mutations are indicated with red ovals. A △ symbol represents the mutation from the nucleotide on the left to the nucleotide on the right. Slippery sequence is highlighted in green and the downstream native pseudoknot target sequence in red.
Fig 6.
SARS-CoV-2 secondary structure motifs free energy per nt.
Each dot represents a Shapify predicted secondary structure for the SARS-CoV-2 frameshift sequence (independent from KnotAli predictions, see Section 2.6, Table 1). X-axis represents window size, y-axis represents free energy per nt. Dots are colored based on the four listed dual-graph motifs (legend in top-right) detected at or directly 3′ of the slippery sequence (see Table 2), or grey for others. Noteworthy structures not matched with a motif (grey dots here) are visualized in Figs 8–10.
Table 2.
Secondary structure pseudoknot motifs in dot bracket notation.
Note that motif classification allows minimal modification to structures, e.g., in the size of loops. Sequence ID: NC_045512.2 [75], indices 13467−13565. Open parentheses/brackets show the base on the 5′ side of the sequence, closed parentheses/brackets represent the base on the 3′ side of the sequence that are binding together. Each period “.” represents an unpaired base.
Fig 7.
Convergence to the most stable structures that contain the 3_3 motif.
144 nt window Shapify predictions with initial stems 1 and 2 as constraint result in the MFE and most stable structures that contain the 3_3 motif. Initial stems are labeled on the left (e.g., S1 for initial stem 1). Darker grey path indicates the structure predicted with a specific initial stem was the same for two SHAPE datasets. Light grey path indicates the structure predicted with an initial stem was specific to one SHAPE dataset. Structures on the right are labeled by free energy proximity to the MFE structure. For example, MFE-1 is the lowest free energy structure after the MFE structure. Differences from the MFE structure are marked in bold, with parentheses representing changes in paired bases, and asterisks representing predicted unpaired bases that were paired in the MFE structure. The 3_3 motif pseudoknotted base pairs are shown in light blue.
Fig 8.
Structural regions involving pseudoknots in SARS-CoV-2, 144 nt window via Shapify.
Top arc diagram: MFE-20, free energy −24.29 kcal/mol, initial stem 18 in black (free energy −0.35 kcal/mol). Bottom arc diagram: MFE-12, free energy −25.76 kcal/mol, initial stem 11 in black (free energy −1.44 kcal/mol). Attenuator hairpin sequence in fuchsia, slippery sequence in green, downstream native pseudoknot pairing region in red.
Fig 9.
SARS-CoV-2 pseudoknot predictions overlap, 222 nt window via Shapify.
Top arc diagram: MFE-5, free energy −45.04 kcal/mol, initial stem 15 in black (free energy −2.74 kcal/mol). Note that the MFE-5 pseudoknot was also detected within the 144 nt window (see MFE-19) and the 68 nt window [25]. Bottom arc diagram: MFE-29, free energy −39.34 kcal/mol, initial stem 12 in black (free energy −3.41 kcal/mol). Attenuator hairpin sequence in fuchsia, slippery sequence in green, downstream native pseudoknot pairing region in red.
Fig 10.
SARS-CoV-2 long range pseudoknot predictions, 222 nt window via Shapify.
Top arc diagram: MFE-58, free energy −34.16 kcal/mol, initial stem 16 in black (free energy −1.98 kcal/mol). Bottom arc diagram: MFE-10, free energy −42.74 kcal/mol, initial stem 2 in black (free energy −10.08 kcal/mol). Attenuator hairpin sequence in fuchsia, slippery sequence in green, downstream native pseudoknot pairing region in red.