Production and characterization of nuclease-resistant RNA Aptamers specific to Streptococcus suis

Abstract

Streptococcus suis (S. suis) is a gram-positive capsular bacteria known as a zoonotic pathogen in humans that can cause meningitis, endocarditis, and septic shock. Most cases of S. suis infection in humans, especially serotype 2, were found in the northern region of Thailand. The conventional method for S. suis identification is culture and biochemical tests, which are time-consuming and sometimes show the controversy results leading to the mis- or delay diagnostic. Nowadays, the rapid with the highly accuracy methods are required. Recently, there are studies of the molecule named “aptamer”, a short oligonucleotide (single-stranded DNA or RNA) that can specifically bind to the target cell through their conformational structure. The conventional method for producing aptamer is Systematic Evolution of Ligands by EXponential enrichment (SELEX). In this study, the monoclonal nuclease resistant RNA aptamer, R8-su12, against S. suis SS 2, strain P1/7 was established using the cell-SELEX technique. In the cell-SELEX process, firstly, the nuclease-resistant RNA library; containing 40-nt random region to yield a diversity of approximately 1014 molecules was produced. Then, it was mixed with 107 CFU/mL of target cells. The increasing of stringency in each round of the cell-SELEX resulted in high affinity and specificity of achieved aptamers. The round eighth (R8) RNA aptamer pool was selected for specificity testing. No cross-reaction was observed when tested with S. pneumoniae ATCC 49619 and S. pyogenes ATCC 19615 (95% CI, p < 0.05). After cloning and sequencing, four groups of RNA aptamer were classified according to the homology of nucleotide sequences. Group 1 showed the highest frequency in the number of homologous sequences (7/26). The monoclonal R8-su12 RNA aptamer was then selected for further study. This monoclonal RNA aptamer was 84-nt in length and the secondary structure showed an external 4-base loop with one closing helices and stems-loops structure. No cross-reaction was observed when tested with S. pneumoniae ATCC 49619, S. aureus ATCC 25923, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 (95% CI, p < 0.05). Moreover, the monoclonal R8-su12 RNA aptamer can also bind to S. suis SS 1/2, 1, 9, and 14. Importantly, this aptamer clone revealed the ability to inhibit the biofilm formation of S. suis SS 2, strain P1/7. The monoclonal R8-su12 RNA aptamer specific to S. suis SS 2, strain P1/7 is one of the candidate RNA aptamers that can be used in various diagnostic tools such as biosensors or the ELONA assay to detect S. suis in clinical isolates. Additionally, our selected RNA aptamer can be developed for use in research field as a ligand to find a new molecule involved in S. suis biofilm formation, and may also help to investigate the mechanism of biofilm formation. Furthermore, this monoclonal R8-su12 RNA aptamer can be developed as candidate ligands for drug delivery. Eventually, the RNA aptamer specific to S. suis constructed in this study was the first step of the research that could be applied in several scenarios involving in the mechanism of infection, diagnostic, and treatment of S. suis infection. At last, it could lead to the control, an early and efficient treatment of S. suis infections in humans and pigs in the near future.