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

Abstract

SUMMARY 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 β‐(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.

Engineering and characterization of carbohydrate‐binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

“…Hence, the presence of calcofluor in the cell wall regeneration media could potentially be toxic for plant cells and preclude its use in monitoring cell wall regeneration in live protoplasts. Some DNA aptamers have also been recently developed to visualize cellulose in plants (Głazowska & Mravec, 2021). However, proteinaceous probes offer a slight advantage since they could be readily engineered and reliably produced in diverse research labs.…”

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots

“…There, an individual aptamer that efficiently recognizes cancerous liver tissues by binding to intracellular components in the nucleus was identified [ 18 ]. However, to the best of our knowledge, aptamers directed against plant-derived targets are relatively rare, with the recent exception of aptamers developed against celluloses, which are intended as molecular tools for the analysis of cell wall components [ 30 , 37 ]. However, no intact cell walls/plant tissues have been used previously for the in vitro selection of aptamers specific to cellulose, only the free-standing purified target molecule itself has been used [ 37 ].…”

“…Thus, these binding molecules cannot only be used in various fields, such as biomarker discovery [ 20 ], electronic biosensing [ 21 ], diagnostics [ 22 ], and drug delivery [ 23 ], and they cannot be used as pharmaceutical compounds in molecular therapy [ 24 , 25 ]; however, they are also attractive molecules for deciphering cellular processes via molecular imaging and labeling applications [ 26 ]. Even though aptamers are commonly used in biomedical research and environmental monitoring [ 27 , 28 ], their daily use has not yet been found in the field of cell wall research [ 29 , 30 ]. Therefore, to specifically label and distinguish between the growth zones of the roots of the model organism Arabidopsis thaliana , we developed a selection strategy to evolve aptamer libraries using Fluorescence Root-SELEX (“FluRoot-SELEX”) ( Scheme 1 ).…”