Cytosine-Rich DNA Aptamers Reveal Recognition Principles for Structurally Diverse Lipopolysaccharides

Lipopolysaccharides (LPS) are structurally heterogeneous glycolipids whose variable composition and supramolecular organization challenge the design of analytical recognition elements. Here, independent magnetic bead-based SELEX against four LPS preparations yielded three cytosine-rich DNA aptamers that bound five structurally distinct LPS preparations, including both O-antigen-containing and O-antigen-deficient forms, with nanomolar apparent dissociation constants. Mutational analysis showed that disruption of the native C-rich sequence architecture strongly impaired binding, and the repeated emergence of such motifs across independent selections points to an underexplored sequence solution for LPS recognition. UV melting and native PAGE showed acidic pH-induced i-motif-like transitions, whereas binding was maximal at neutral pH, indicating that the acidic folded state is not the functional recognition conformation. Comparisons across immobilization formats, component competition assays with isolated lipid A and free Kdo, and biolayer interferometry with isolated lipid A indicate that recognition is not explained by isolated lipid A or free Kdo alone but is instead consistent with intact LPS presentation in a core-proximal context under the assay conditions. Together, these results reveal recognition principles that link cytosine-rich sequence motifs, pH-dependent folding, target presentation, and core-proximal LPS features in the analytical molecular recognition of heterogeneous LPS.