Fending off decay: A combinatorial approach in intact cells for identifying mRNA stability elements

The strategy of systematic evolution, whereby nucleic acid sequences or con formers can be selected and amplified from a randomized population, has bee n exploited by many research groups for numerous purposes. It is, however, a technique largely performed in vitro, under nonphysiological conditions. We have now modified this in vitro approach to accomplish selection in grow ing cells, Here, we report that this new methodology has been used in vivo to select RNA elements that confer increased transcript stability. A random ized cassette was embedded in a 3 ' -untranslated region (UTR), downstream from the luciferase reporter open reading frame. A heterogeneous population of capped luciferase mRNA was then generated by in vitro transcription. Hu man liver Hep G2 cells were electroporated with this population of lucifera se mRNA and total cytoplasmic RNA was isolated after varying lengths of inc ubation. Following RT-PCR, the 3 ' UTR was used to reconstruct a new popula tion of luciferase templates, permitting subsequent cycles of in vitro tran scription, electroporation, RNA isolation, and RT-PCR. Increasing the incub ation time at each cycle before RNA isolation imposed selection for stable transcripts. The functional half-life of the luciferase mRNA population inc reased from 55 to 140 min after four cycles. Subsequent sequencing of the s elected 3 ' UTRs revealed G-U rich elements in clones with extended chemica l and functional half-lives.