A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

Liu, Hong Yan; Gao, Xiaohu

The long ssRNA composed of PSMA aptamer and siRNA antisense strand ( Figure 1 ) was prepared by in vitro transcription with the presence of 29 fluoro-modified pyrimidies for improved resistance to ribonucleases. It has been shown previously that 29-F modification is compatible with dsRBD binding unlike 29-H or 29-OCH 3 substitutes 26, 37 . The transcript was annealed to chemically synthesized siRNA sense strand. Before combining the chimera with our small protein tag, we first tested the activities of the chimera. To test the targeting function of the aptamer block, PSMA-positive LNCaP and PSMA-negative PC3 prostate tumor cells were incubated with dye-labeled chimera. As shown in Figure 2c , the chimera selectively binds and enters LNCaP cells indicating targeting specificity. To test the silencing effect separately, the chimera was transfected into GFP-expressing C4-2 prostate tumor cells (a derivative of LNCaP) using conventional transfection agents, Lipofectamine. As shown in Figure 2d , the silencing effect is indistinguishable with the positive control using siRNA only, proving that chimera can be enzymatically processed intracellularly to generate functional siRNA.