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.

Simple nucleotide templating activities are of interest as potential primordial reactions. Here we describe the acceleration of 5 ′ -5 ′ AppA synthesis by 3 ′ -5 ′ poly(U) under normal solution conditions. This reaction is apparently templated via complementary U:A base-pairing, despite the involvement of two different RNA backbones, because poly(U), unlike other polymers, significantly stimulates AppA synthesis. These interactions occur in moderate (K + ) and (Mg 2+ ) and are temperature sensitive, being more efficient at 10°C than at 4°C, but absent at 20°C. The reaction is only slightly pH sensitive, despite potentially relevant substrate pK a 's. Kinetic data explicitly support production of AppA by interaction of stacked 2MeImpA and pA nucleotides paired with a single molecule of U template. At a lower rate, AppA can also be produced by a chemical reaction between 2MeImpA and pA, without participation of poly(U). Molecular modeling suggests that 5 ′ -5 ′ joining between stacked or concurrently paired A's can occur without major departures from normal U-A helical coordinates. So, coenzyme-like 5 ′ -5 ′ purine dinucleotides might be readily synthesized from 3 ′ -5 ′ RNAs with complementary sequences.

Selection for affinity for free histidine yields a single RNA aptamer, which was isolated 54 times independently. This RNA is highly specific for the side chain and binds protonated L-histidine with 10(2)-10(3)-fold stereoselectivity and a dissociation constant (K(D)) of 8-54 microM in different isolates. These histidine-binding RNAs have a common internal loop-hairpin loop structure, based on a conserved RAAGUGGGKKN(0-36) AUGUN(0-2)AGKAACAG sequence. Notably, the repetitively isolated sequence contains two histidine anticodons, both implicated by conservation and chemical data in amino acid affinity. This site is probably the simplest structure that can meet our histidine affinity selection, which strengthens experimental support for a "stereochemical" origin of the genetic code.

Selection for amino acid affinity by elution of RNAs from tryptophan–Sepharose using free L-tryptophan evokes one sequence predominantly (KD = 12 µM), a symmetrical internal loop of 3 nt per side. Though we have also isolated larger sequences with affinity for tryptophan, successively squeezed selection in randomized tracts of 70, 60, 40, 20 and 17 nt show that this internal loop is the simplest sequence that can meet the column affinity selection. From sequence variation in ∼50 independent isolates, only 26 bits of information are required to describe this loop (equivalent to only 13 fully conserved nucleotides). Thus, it is among the simplest amino acid binding sites known, as well as selective among hydrophobic side chains. Among site sequences defined as essential to affinity by conservation, protection and modification-interference, there is a recurring CCA sequence (a tryptophan anticodon triplet) which apparently forms one side of the binding site. Such conserved juxtaposition of tryptophan with a cognate coding triplet supports a stereochemical origin for the genetic code.

′ G synthesis from pG and chemically activated 2MeImpG is accelerated by the addition of complementary poly(C), but affected only slightly by poly(G) and not at all by poly(U) and poly(A). This suggests that 3 ′ -5 ′ poly(C) is a template for uncatalyzed synthesis of 5 ′ -5 ′ GppG, as was poly(U) for AppA synthesis, previously. The reaction occurs at 50 mM monoand divalent ion concentrations, at moderate temperatures, and near pH 7. The reactive complex at the site of enhanced synthesis of 5 ′ -5 ′ GppG seems to contain a single pG, a single phosphate-activated nucleotide 2MeImpG, and a single strand of poly(C). Most likely this structure is base-paired, as the poly(C)-enhanced reaction is completely disrupted between 30 and 37°C, whereas slower, untemplated synthesis of GppG accelerates. More specifically, the reactive center acts as would be expected for short, isolated G nucleotide stacks expanded and ordered by added poly(C). For example, poly(C)-mediated GppG production is very nonlinear in overall nucleotide concentration. Uncatalyzed NppN synthesis is now known for two polymers and their complementary free nucleotides. These data suggest that varied, simple, primordial 3 ′ -5 ′ RNA sequences could express a specific chemical phenotype by encoding synthesis of complementary, reactive, coenzyme-like 5 ′ -5 ′ ribodinucleotides.