Screening Of SsDNA Aptamers Specific To Catalytic Region Of Glucosyltransferase By Liquid-SELEX

Glucosyltransferase (GTF) has been implicated as an important cariogenic factor of streptococcus mutans by catalyting synthesis of extracellular polysaccharide. GTF has two functional regions, a catalytic region (CAT) and a glucan binding region (GLU). The catalytic region (CAT) plays an important role in caries.In this study, the catalytic region (CAT) of GTF with N terminal fused to NusA (rNusA-CAT) was expressed in prokaryotic expression system. The NusA protein was also expressed in a same prokaryotic expression system as a control. Both the recombinant NusA-CAT protein and NusA control was soluble expressed and purified by chelating chromatography using a C terminal His tag of the vector. The catalytic activity of recombinant protein was tested and confirmed using anthrone-sulphuric acid method.The traditional SELEX requires a purified target and a selection medium that solidify the target to facilitate the separation of bound oligonucleotides with free ones. Therefore, the selection to a non-purified target, importantly, a target that function relys on native conformation is hindered. To solve this problem, we introduce the principles of EMSA to traditional SELEX process. The established liquid SELEX can be processed in a liquid incubation system and do not require target purification through a counter selection. Then the selection against non-purified rNusA-CAT expression supernatant with non-purified NusA expression supernatant as a counter target was done using a 78 base ssDNA library containing 45 bases of random sequence. After ten rounds of liquid SELEX selection, the enrichment of each round was tested by isotope labeled ssDNA binding assay. Results showed that the tenth round pool was of the best enrichment and showed specificity to CAT region. The results of our work laid the foundation for the founctional GTF inhibitor and provided information for the prevention and treatment of dental caries.The Liquid SELEX technology makes selection to biomarkers in serum, saliva or body fluids possible. This method has potential prospect in the identification of biomarkers and gain of molecular probes rapidly and effecitively, which bring out a new idea in the diagnosis and treatment of disease in the future.