An aptamer highly specific to cellulose enables the analysis of the association of cellulose with matrix cell wall polymers in vitro and in muro

The current toolbox of cell wall-directed molecular probes has been pivotal for advancing basic and application-oriented plant carbohydrate research; however, it still exhibits limitations regarding target diversity and specificity. Scarcity of probes targeting intramolecular associations between cell wall polymers particularly hinders our understanding of the cell wall microstructure and affects the development of effective means for its efficient deconstruction for bioconversion. Here we report a detailed characterization of a cellulose-binding DNA aptamer CELAPT MINI using a combination of various in vitro biochemical, biophysical, and molecular biology techniques. Our results show evidence for its high specificity towards long non-substituted beta-(1-4)-glucan chains in both crystalline and amorphous forms. Fluorescent conjugates of CELAPT MINI are applicable as in situ cellulose probes and are well suited for various microscopy techniques, including super-resolution imaging. Compatibility of fluorescent CELAPT MINI variants with immunodetection of cell wall matrix polymers enabled them simultaneously to resolve the fibrillar organization of complex cellulose-enriched pulp material and to quantify the level of cellulose masking by xyloglucan and xylan. Using enzymatically, chemically, or genetically modulated Brachypodium internode sections we showed the diversity in cell wall packing among various cell types and even cell wall microdomains. We showed that xylan is the most prominent, but not the only, cellulose-masking agent in Brachypodium internode tissues. These results collectively highlight the hitherto unexplored potential to expand the cell wall probing toolbox with highly specific and versatile in vitro generated polynucleotide probes.