Selection And Identification Of Different SsDNA Aptamers Specific To Streptococcus Mutans Isolated From Caries-free And Caries-active Individuals

Streptococcus mutans is generally considered to be the principal aetiological agent for dental caries, which is the most prevalent bacterial infectious disease in oral cavity. Varieties of risk factors interact in this disease, not only including the cariogenic agents that have been known, but also including the unknown ones. In this regard, it is necessary to study on Streptococcus mutans as a whole to reveal the cariogenic mechanism of it. And SELEX can be a useful tool for this purpose.SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a combinatorial chemistry technology for studying structure, function and molecular evolution of nucleic acid. The aptamers evolved from this in vitro selection process have a promising application in basic research, chlinical diagnosis and therapy. The recently establishment of subtractive SELEX method, which targets the whole intact cells, can even generate aptamers that differentiate target cells from counter-selected partners without the prior knowledge of the molecular differences.The objective of this research is to select and identify different ssDNA aptamers specific to Streptococcus mutans isolated from caries-free and caries-active individuals by the subtractive SELEX.Before the selection, we identified and obtained 5 clinical isolates of Streptococcus mutans from dental plaques of 11 subjects by morphology, biochemical events, automated microbiology system, and molecular biological methods. Among the isolates, there were 3 ones from caries-active individuals, while 2 from caries-free individuals. Then, we detected the cariogenic virulence characteristics of the isolates above by four classical in vitro cariogenic experiments, which were adherence analysis, extracellular polysaccharide synthesis assay, aciduricity and acidogenicity assays. Combination with statistics analysis, the results revealed that No.5 isolate was the high virulent strain, while No.4 isolate was the low one.The subtractive SELEX selection against No.5 Streptococcus mutans isolate with an 88 nucleotides ssDNA library containing 45 random nucleotides flanked by invariant primer was done, followed by counter selection to No.4 Streptococcus mutans isolate. After 9 rounds selection, that targeted the whole intact cells, one aptamer named H19, which showed high specificity and affinity to No.5 Streptococcus mutans isolate, was identified with a Kd of 69.45±38.53nM.Overall, it was possible that the ssDNA aptamers specific to Streptococcus mutans isolated from caries-free and caries-active individuals of our work might be used for clinical prediction to the susceptibility of dental caries, and might be a new idea for the prevention of dental caries.