Cell-SELEX Identifies a “Sticky” RNA Aptamer Sequence

Figure 3

Northern blot analysis of the aptamer clones: (a) 5′-[γ-³²P]-radiolabeled probe BW28, complementary to the 3′ constant region (CR) of the aptamer library, was used for the Northern blot analysis. Variable region (VR). (b) Total RNA (3 μg/lane), isolated from the MiaPaCa-2 cells after incubating them with the aptamers, were subjected to Northern blot analysis using the 5′-[γ-³²P]-BW28 (upper panel). For loading control, the nylon membrane was reprobed with the 5′-[γ-³²P]-U6 snoRNA probe (lower panel). Purified aptamers (0.5 μg/lane) and the starting library (Sel 3) were used as loading controls. Compared to the control (Sel 3 library), P7-14, P7-1, and M7-14 displayed 20, 17, and 24 times more binding to the MiaPaCa-2 cells, respectively. (c) To quantify the percentage of the aptamers internalized in the cells, the total RNA isolated form the MiaPaCa-2 cells after incubating them with the aptamers (P7-1 and M7-14) and with (+) or without (−) the RiboShredder (RS) treatment was subjected to the same Northern blot analysis as described above. 10.5% and 27% of the bound M7-14 and P7-1 aptamers were protected from the RS treatment, respectively, due to their internalization in the MiaPaCa-2 cells.

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