A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

Liu, Hong Yan; Gao, Xiaohu

Quantitative flow cytometry studies further confirm this result (Figure 3g-l) . At the current gate value set for GFP fluorescence intensity, the original untreated cells showed a GFP-negative population of 17.4%. Treating the cells with a random sequenced siRNA with protein tag (His 18 ) shows virtually no change in this population (difference: 5.4% of total cell population, within error range) proving sequence-specific silencing of RNAi. For cells treated with GFP siRNA and chimera, the GFP negative cells only increase by 7.6% and 12.2% of the total cell population respectively. Even by increasing the chimera concentration by ten times (1 mM), the total GFPnegative cell population only increase by ,20% (Supplementary Figure S1) , strongly suggesting the need of carrier materials. Direct comparison of the chimera tagged by dsRBD-His 6 and dsRBD-His 18 shows major difference in silencing efficiency, too (14.6% and 59.6% change). Taken together, these results clearly indicate that (1) chimera alone at concentration commonly used in RNAi experiments does not lead to effective silencing, and (2) His 18 is remarkably more effective than His 6 in endosomal destabilization since the dsRBD block is identical in structure and function. To put the silencing efficiency of dsRBD-His 18 in the context of those of conventional RNA delivery vehicles such as Lipofectamine, quantitative flow cytometry was also conducted. In agreement with the microscopy results shown in Figure 2d , Lipofectamine reduces GFP-negative cells from the original 17.4% to 91.6% (74.2% change, Supplementary Figure S2 ), which is slightly more efficient than the protein tag. However, it is important to note that Lipofectamine delivers chimera into cells mainly via electrostatic interactions (positively charged Lipofectamine and negatively charged cell surface, non-targeted delivery), whereas our protein tag delivers chimera by cell type-specific molecular recognition (targeted delivery). It is also worth mentioning that the molar ratio of mixing chimera with protein tag is 152 because the siRNA block can bind up to 2 copies of dsRBD, although the second copy has very weak binding affinity. Indeed, changing the binding ratio to 1 or 4 does not affect the RNAi efficiency (Supplementary Figure S3) .