| Summary: | Previously reported (<i>S</i>)-5′-<i>C</i>-aminopropyl-2′-arabinofluoro-thymidine (<b>5ara-T</b>) and newly synthesized (<i>S</i>)-5′-<i>C</i>-aminopropyl-2′-arabinofluoro-5-methyl-cytidine (<b>5ara-<sup>Me</sup>C</b>) analogs were incorporated into a series of antisense gapmers containing multiple phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in their wing regions. The functional properties of the gapmers were further evaluated in vitro. Compared with the positive control, for the LNA-wing full PS gapmer without <b>5ara</b> modification, it was revealed that each gapmer could have a high affinity and be thermally stable under biological conditions. Although the cleavage pattern was obviously changed; gapmers with <b>5ara</b> modification could still efficiently activate <i>E. coli</i> RNase H1. In addition, incorporating one <b>5ara</b> modification into the two phosphodiester linkages could reverse the destabilization in enzymatic hydrolysis caused by fewer PS linkages. In vitro cellular experiments were also performed, and the Lipofectamine<sup>®</sup> 2000 (LFA)+ group showed relatively higher antisense activity than the LFA-free group. KN5ara-10, which contains fewer PS linkages, showed similar or slightly better antisense activity than the corresponding full PS-modified KN5ara-3. Hence, KN5ara-10 may be the most promising candidate for <i>KNTC2</i>-targeted cancer therapy.
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