Synthesis and evaluation of stability of m3G-CAP analogues in serum-supplemented medium and cytosolic extract.

Abstract Increased efficiency in splice-correction (splice-switching) has been shown by use of a synthetic RNA 5′-end nuclear localization signal composed of an m 3 G-CAP. Use of the m 3 G-CAP as an NLS signal for therapeutic compounds in vivo is likely to require additional stability towards enzymatic degradation. For this reason introduction of stabilizing modifications into the triphosphate bridge may be beneficial. Here we report on synthesis of three m 3 G-CAP derivatives with a ‘native’ (m 3 GpppA OMe ) as well as with a methylenephosphonate stabilized triphosphate bridge (m 3 GpCH 2 ppA OMe , m 3 GppCH 2 pA OMe ) and the investigation of the enzymatic stability of these compounds in 10% (v/v) fetal bovine serum (FBS) and cytosolic extract from HeLa cells, thus mimicking in vivo conditions. Our results indicate that introduction of methylene group between the β and γ phosphates in m 3 GpCH 2 ppA OMe improves to some extent stability of this analogue in 10% serum but does not prolong life of this compound in the cytosolic extract. In contrast the stabilization introduced between α and β phosphates in m 3 GppCH 2 pA OMe offers threefold longer life in 10% serum and almost complete protection in cytosolic extract.