A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

Liu, Hong Yan; Gao, Xiaohu

Post expression and purification, the resulted protein tags were analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Figure 2a ). The sizes of four protein tags show in excellent agreement with theoretical values (Figure 1 ). To assess their dsRNA binding activity, siRNA-aptamer chimera labeled with fluorophore FAM were incubated with the protein tags and probed with gel electrophoresis (1% agarose). As shown in Figure 2b , the dsRNA binding capability of dsRBD with His 12 at the C terminus (total His 18 ) is well preserved compared with dsRBD without a Cterminus histag insertion. The minimum RNA length for high affinity binding with dsRBD has been determined to be 16 base-pairs 26 . At the current RNA length, the siRNA segment and the adjacent short stem in the aptamer structure can bind with 1-2 copies of dsRBD. However, it has been well documented that only the first dsRBD binds to RNA stably, while, at high dsRBD/RNA ratio, a second copy of dsRBD can bind, but at significantly lower affinity 26, 33 . Using unmodified dsRBD and siRNA alone, similar dsRBD-siRNA binding profiles have been observed previously by Kim and coworkers, who also show that the enzymatic stability of siRNA is significantly enhanced upon binding with dsRBD 34 .