PEGylation reaction of aptamers for the treatment of infections caused by multidrug-resistant bacteria (MDR)

Introduction:The emergence of infections caused by MDR bacteria and use of antibiotics has increased worldwide, which makes it necessary to develop new therapies.Aptamers are oligonucleotides with therapeutic potential that bind to a specific target, such as surface proteins of MDR bacteria.Blocking these proteins by aptamers can confer protection in animal models, but they undergo rapid renal metabolism.Conjugation with polyethylene glycol (PEG) increases its plasmatic half-life and improves its clinical efficacy.Objectives: This study standardized the PEGylation reaction of a DNA aptamer that binds to surface proteins of MDR bacteria. Methodology:The aptamer (15kDa), synthesized by the SELEX method, reacted for 1h with methyl-PEG-succinimidyl carbonate (mPEG-SCC; 12kDa) in a molar ratio of 1:20, using sodium tetraborate and dimethyl sulfoxide (DMSO).Temperature, pH and agitation were evaluated for reaction optimization.The resulting 1mL of reaction mixture was analyzed by ion exchange chromatography (IEX; Source 15Q) and DNA polyacrylamide gel electrophoresis (DNA-PAGE) with zinc sulfate/imidazole staining.The reaction was scaled up to 5 mL and purified by IEX by collecting the chromatographic peaks, whose identities were confirmed by DNA-PAGE. Results:The reactions were optimized at 43.5C, under shaking and pH 8.The DNA-PAGE method detected a band with lower migration compared to the free aptamer band; however, the staining system used did not reveal the band corresponding to mPEG-SCC.The IEX technique was effective in separating the PEGylated aptamer, free aptamer, and mPEG-SCC, and the chromatographic profile was maintained after scaled-up.DNA-PAGE analyses of the collected peaks confirmed the success of conjugation purification process. Conclusion:The results obtained so far indicate success in the conjugation, purification and analysis of the conjugate.Product characterization by mass spectroscopy and affinity tests with the target protein using Nano ITC will evaluate its efficacy.Standardized methodologies could be used to obtain PEGylated aptamers for the treatment of other MDR bacteria of epidemiological interest in the country.