Phosphoramidation反應最適化與其應用於更有效合成核酸-胜肽鍵結產物(POCs)之核酸藥物研發探討

Abstract

Extensive research endeavors have been initiated to explore the potential of nucleic acids, including siRNA, miRNA, catalytic RNA (ribozymes), aptamer RNA (RNA with exquisite roles similar to protein receptors) and antisense oligonucleotides, as therapeutic agents. Some antisense oligonucleotides and aptamer RNA have reached clinical applications, while significant amounts of clinical trials for siRNA are underway. Direct use of nucleic acids in medicine, however, faces serious hurdles such as poor cell specificity and uptake of nucleic acids, and inaccessibility of nucleic acids to cell nuclei. These uptake problems of nucleic acids are mainly ascribed to inefficiency of nucleic acids to permeate biological barriers after administration. Using peptides as vectors (ligands) to assist nucleic acid trafficking across the plasma membrane are rational strategies to develop nucleic acid-based therapeutic reagents. In the current study, we optimized previously developed aqueous phase two-step nucleic acid phosphoramidation to facilitate synthesis of peptide-oligonucleotide conjugates (POCs). We improved POC yields by exploiting 4(5)-methylimidazole, specific surfactants with defined concentrations and diamine derivatized peptides. Optimized aqueous phase two-step nucleic acid phosphoramidation was applied to efficiently conjugate peptides with oligonucleotides, with or without a disulfide-containing linker, to generate corresponding POCs with yields of 47-75%. Moreover, we exploited the optimized two-step nucleic acid phosphoramidation to developing a novel, simpler and more effective labeling method for conjugating nucleic acids with fluorophores. Studied by flow cytometry and laser scanning confocal microscope, we unequivocally demonstrated FITC-labeled POCs were taken up by A549 cells effectively. However, when employing the methods to synthesize POCs for developing nucleic acid drugs to inhibit the growth of E. coli, our results indicated that the tailored-made POCs did not meet our expectation of conferring antibacterial and antibiotic effects. In summary, this study successfully optimized previously developed aqueous phase two-step nucleic acid phosphoramidation to facilitate POC synthesis. We also demonstrated that those peptides in POCs served the purpose of Trojan horses to deliver nucleic acids into the mammalian cells. The outcomes of the research shed light on the mechanisms of aqueous-phase phosphoramidation reactions and, moreover, provide the essential supports for POCs to develop effective therapeutic reagents in clinics to treat human diseases.