FIG. 1.

Experimental procedure for functional SELEX. The structure of the IgM M1-M2 minigene pre-mRNA is shown. The characterized natural ESE (73 nt) was replaced by 20 nt of randomized sequence by overlap-extension PCR (20). A T7 promoter (black box) was built into the PCR product. In vitro-transcribed RNA was then incubated under splicing conditions. Any spliced mRNA molecules must contain a functional ESE or winner sequence, designated by the white W in a black box. The spliced mRNA molecules were purified from a denaturing polyacrylamide gel, reamplified by RT-PCR, and cloned. Individual clones were sequenced and analyzed by a sampling algorithm to define a common motif, and a subset was rebuilt into minigene templates, transcribed, and assayed for splicing in vitro.