The RNA world hypothesis presumes that RNA will be competent for varied essential cellular functions. One such indispensable cell function is regulation of membrane permeability. Though this was not a known RNA activity, selection-amplification yielded RNAs that bound phosphatidylcholine:cholesterol liposomes. At least eight distinct, Ϸ95-mer sequences bind well to the outside of the lipid bilayer, though randomized sequences had no such activity. No distinct sequence motif for lipid binding was found. However, truncation of one such RNA shows that a smaller, 44-nucleotide irregular RNA hairpin is an active membrane binding domain. Bound RNA increases the permeability of liposomes to 22 Na ؉ . In addition, using voltage clamp technique, four individual RNAs increase the ion permeability of the plasma membrane of cultured human cells. The existence of multiple sequences that bind membranes and provoke permeability changes suggests that these may be elementary RNA functions that could be selected in vivo.Cells communicate with their environments across phospholipid bilayer membranes. However, the permeability of such bilayers (relatively permeable to water, but virtually impermeable to polar molecules, even to small ions) is dramatically mismatched to cellular needs. Cellular life therefore absolutely requires facilitation of transport through phospholipid boundaries. The RNA-world hypothesis (1) posits ancestral cells in which RNA plays many of the roles taken by modern proteins. Might RNAs serve membrane functions?We have approached this question, first, by isolating RNAs that bind to pure phospholipid membranes by using selectionamplification (2, 3). In this technique, novel RNA activities are isolated by selecting infrequent, perhaps unique active molecules from large pools of transcripts (Ϸ10 14 different molecules) with randomized sequences. Repetition of the selection (purification) is made possible by nucleic acid amplification (replication) applied to the partially purified pools, so that large cumulative purifications are possible after multiple cycles of selection-amplification. Ultimately, cDNA cloning and in vitro transcription make available single pure active RNAs for study. Second, we have reintroduced purified membranebinding RNAs from selection-amplification into several membrane systems to measure their effects on permeability, as has long been done for channel proteins (4).To act in membranes, RNA must interact with membrane constituents. Phospholipids, the main components of the biological membrane, are chemically tripartite. They consist of a polar head group, glycerol phosphate, and fatty acids. RNAs should easily interact with polar head groups, particularly cationic ones. Glycerol phosphate (which resembles the RNA backbone) also presents easily used hydrogen-bonding opportunities to an RNA. In contrast, fatty acids might be thought of as improbable RNA ligands. However, it has previously been shown that RNAs fold to form specifically shaped, hydrophobic sites that interact favorab...

We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides. This UAUU motif (16 highly conserved positions; 27 total), is also the most abundant site in successful selections on short random tracts. The UAUU site, now isolated independently at least 63 times, is a small asymmetric internal loop. Conserved loop sequences include isoleucine codon and anticodon triplets, whose nucleotides are required for amino acid binding. This reproducible association between isoleucine and its coding sequences supports the idea that the genetic code is, at least in part, a stereochemical residue of the most easily isolated RNA-amino acid binding structures.

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We have isolated an RNA with specific affinity for the L-valine side chain, using selection-amplification. The active RNA secondary structure, identified by repeated selection, is a highly conserved asymmetric (4:10) internal loop adjacent to required G-U pairs. The binding free-energy per methylene is up to 1.5 kcal mol-1, and very dependent on group position. Amino acid binding is L-stereoselective and distinguishes aliphatic sidechains by size and, given the same total size, by configuration. Though aliphatic-RNA interactions have frequently been neglected, their avidity and specificity seem sufficient for a biological role.