Figure 1. In vitro selection of RNA elements that mediate cap-independent (CI) translation.

(a) A library of human genomic DNA fragments was inserted into a DNA cassette containing all of the sequence information necessary for mRNA display. For each selection round, the dsDNA pool was in vitro transcribed into ssRNA, conjugated to a DNA-puromycin linker, and translated in vitro. Uncapped mRNA sequences that initiate translation of an intact ORF become covalently linked to a His-6 protein affinity tag encoded in the RNA message. Functional molecules are recovered, reverse transcribed, and amplified by PCR to generate the DNA for the next selection cycle. (b) RNA-protein fusion molecules are generated via the natural peptidyl transferase activity of the ribosome. (c) Selection progress was monitored by measuring the fraction of S³⁵-labeled fusion molecules recovered from the oligo-dT and Ni-NTA affinity columns.