An Orthogonal Computational Framework Identifies a Conserved RNA Motif in the SARS-CoV-2 Spike Gene

Background: Structured RNA elements in viral genomes are underexplored therapeutic targets, yet coding regions have not been systematically screened for conserved, structurally defined motifs. Methods: We screened the SARS-CoV-2 spike coding sequence across eight major variants using a staged pipeline combining thermodynamic scanning, secondary-structure cross-validation between physics-based and deep-learning predictors, tertiary prediction across seven tools with decoy-controlled analogy searches, comparison with genome-wide chemical-probing data from three laboratories, and molecular dynamics simulation. Results: The screen identified three conserved, window-robust loci at functionally important spike regions: near the N343 glycosylation site (Region A), the E484 receptor-binding motif (Region B), and the S2′ cleavage site (Region C). Only Region A satisfied all thermodynamic and structural criteria, with a dinucleotide-shuffle z-score of −3.39 (p = 0.020), near-identical secondary structure between RNAfold and MXfold2 (F1 = 0.99), and a best-supported tertiary fold from convergence of Boltz-2 and AlphaFold3 (TM-score_RNA = 0.53). In-cell chemical-probing supported Region A (F1 = 0.83–0.85; AUROC = 0.663–0.892), while Regions B and C failed. Probing data refined the predicted fold to a two-helix architecture with a flexible middle segment, and molecular dynamics of the 101 nt window revealed two individually stable domains (3′ stem-loop RMSD = 2.58 ± 0.53 Å; 5′ helix RMSD = 4.65 ± 1.30 Å) joined by a mobile hinge. Conclusions: Region A is a conserved, structurally defined RNA element within the spike coding sequence, supported by several lines of evidence of differing independence. Its biological function and target-ligand compatibility remain unproven, and it warrants functional investigation as a previously underexplored RNA feature of a gene studied mainly at the protein level. The staged framework should be applicable to coding regions beyond SARS-CoV-2.