Chemical synthesis and biochemical characterization of cyclic oligonucleotides containing acyl groups at both 5′- and 3′-terminal positions

Abstract

Modified oligonucleotides, whose ON-OFF switch of hybridization can be controlled by an external stimulus, are important to understanding life phenomena and efficient treatment of diseases. The ON-OFF switch can be completely controlled by chemical modification of the oligonucleotide such as cyclization. However, their chemical modifications of the previous cyclic oligonucleotides remain after the addition of an external stimulus. To overcome this problem, we carried out the first synthesis of cyclic oligonucleotides containing acyl groups at both 5′- and 3′-terminal positions, which can be hydrolyzed by intracellular esterase. The cyclic oligonucleotides were successfully synthesized via disulfide bond formation and the phosphoramidite method without base protection on polymer supports containing a silyl linker. Subsequently, we were able to introduce a functional group into the cyclic oligonucleotide using the corresponding isothiocyanate reagent. Additionally, a cyclic oligonucleotide with acyl groups was found to have a much lower binding ability than the corresponding linear oligonucleotide. Moreover, we demonstrated its structural conversion to the corresponding linear oligonucleotide with two thiol groups under reducing conditions using dithiothreitol. It was also confirmed that the two terminal acyl groups of the linear oligonucleotide were hydrolyzed by pig liver esterase. These results indicate that hybridization of cyclic acylated nucleic acid drugs with high nuclease resistance is regulated by intracellular esterase under the reducing conditions in the cell cytoplasm.