A complete set of new photolabile nucleoside phosphoramidites were synthesized and then site-specifically incorporated in a siRNA strands for phosphate caging.Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined.Several key phosphate positions were then identified.Further multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity,suggesting a synergistic effect.The caging group on both the terminally phosphate-caged siRNA and the single-stranded caged RNA has comparatively high stability,while the hydrolysis of the caged group from the internally caged siRNA was observed,independent of the presence of Mg2+.Further molecular dynamic simulation demonstrated that the enhanced hydrolysis of caging group on internally phosphate-caged siRNAs was due to easy fragmentation of caging group upon the formation of pentavalent intermediate of phosphotriester with water attack.While the caging group in the terminally phosphate caged siRNA or single-stranded caged RNA prefers to form π-π stack with nearby nucleobases.