In this study, two respective groups of RNA aptamers have been selected against two main classes ofglycosaminoglycans (GAGs), heparosan, and chondroitin, as they have proven difficult to specifically detect in biologicalsamples. GAGs are linear, anionic, polydisperse polysaccharides found ubiquitously in nature, yet their detection remainsproblematic. GAGs comprised repeating disaccharide units, consisting of uronic acid and hexosamine residues that are oftenalso sulfated at various positions. Monoclonal antibodies are frequently used in biology and medicine to recognize variousbiological analytes with high affinity and specificity. However, GAGs are conserved across the whole animal phylogenic tree andare noni... More
In this study, two respective groups of RNA aptamers have been selected against two main classes ofglycosaminoglycans (GAGs), heparosan, and chondroitin, as they have proven difficult to specifically detect in biologicalsamples. GAGs are linear, anionic, polydisperse polysaccharides found ubiquitously in nature, yet their detection remainsproblematic. GAGs comprised repeating disaccharide units, consisting of uronic acid and hexosamine residues that are oftenalso sulfated at various positions. Monoclonal antibodies are frequently used in biology and medicine to recognize variousbiological analytes with high affinity and specificity. However, GAGs are conserved across the whole animal phylogenic tree andare nonimmunogenic in hosts traditionally used for natural antibody generation. Thus, it has been challenging to obtain highaffinity, selective antibodies that recognize various GAGs. In the absence of anti-GAG antibodies, glycobiologists have relied onthe use of specific enzymes to convert GAGs to oligosaccharides for analysis by mass spectrometry. Unfortunately, while thesemethods are sensitive, they can be labor-intensive and cannot be used for in situ detection of intact GAGs in cells and tissues.Aptamers are single-stranded oligonucleotide (DNA or RNA) ligands capable of high selectivity and high affinity detection ofbiological analytes. Aptamers can be developed in vitro by the systematic evolution of ligands by exponential enrichment(SELEX) to recognize nonimmunogenic targets, including neutral carbohydrates. This study utilizes the SELEX method togenerate RNA aptamers, which specifically bind to the unmodified GAGs, heparosan, and chondroitin. Binding confirmationand cross-screening with other GAGs were performed using confocal microscopy to afford three specific GAGs to each target.Affinity constant of each RNA aptamer was obtained byfluorescent output after interaction with the respective GAG targetimmobilized on plates; theKDvalues were determined to be 0.71−1.0μM for all aptamers. Upon the success of chemicalmodification (to stabilize RNA aptamers in actual biological systems) andfluorescent tagging (to only visualize RNA aptamers)of these aptamers, they would be able to serve as a specific detection reagent of these important GAGs in biological samples
