Figure 4

Thermodynamic stability of lower theophylline aptamer stem affects splicing inhibition. (A) Schematic diagram of AdBPT pre-mRNAs exhibiting varying length of lower aptamer stem. The encircled adenosine residue represents branch nucleotide. The distorted hairpin loop structure is the simplified secondary structure of theophylline aptamer. The horizontal lines in aptamer lower stem represent hydrogen bonds between complementary bases. (B) The splicing of AdBPT15AG pre-mRNA with lower aptamer stem of 4 bp (shown in Fig. 1A) was compared with substrates in which the stem was either decreased to a single bp (AdBPT15AG-1S) or increased to eight bp (AdBPT15AG-8S). ³²P-labeled pre-mRNAs were subjected to in vitro splicing for 2 h in the absence (lanes 1, 3 and 5) or presence of theophylline (lanes 2, 4 and 6) as described in Fig. 1. The extracted RNAs were fractionated on a 13% denaturing polyacrylamide gel. The % splicing calculated as in Fig. 1 and values are expressed as mean ± SEM.