Characterization of the shRNA-intron fused to a theophylline-binding aptamer. The theophylline-binding aptamer is shown on the left. A schematic representation of the shRNA1-intron construct is shown on the right. The shRNA1-intron-Apt was constructed by substituting the region of the P6 stem, highlighted by a gray-shaded ellipse with a broken line, with the theophylline-binding aptamer in an analogous fashion to the construct "Th2P6" of Thompson, et al. (2002). In the diagram, straight lines are used to connect the three structural domains (labeled P4-P6, P1-P2 and P3-P9). P stands for the paired region. The arrowheads on the lines indicate 5' to 3' polarity. The dotted lines indicate tertiary interactions to help in correct folding. (B) Cotransfection of the shRNA1-intron-Apt into Cos cells along with vectors expressing firefly luciferase and Renilla luciferase was performed. The medium was removed and exchanged for fresh medium with or without 10 mM theophylline 24 h after transfection. Thereafter, the plates were incubated for 6 h and medium was replaced with normal, fresh medium. Luciferase activity was analyzed 36 h after transfection. (C) Caffeine treatment at a concentration of 10 mM was performed in the same way as theophylline. The results are expressed as the mean ± S.E. of the percentage of the value for the control vector, with significance determined by t tests shown by *P < 0.05.