SELEX is a technology for the identification of high affinity oligonucleotide ligands. Large libraries of random sequence single-stranded oligonucleotides, whether RNA or DNA, can be thought of conformationally not as short strings but rather as sequence dependent folded structures with high degrees of molecular rigidity in solution. This conformational complexity means that such a library is a source of high affinity ligands for a surprising variety of molecular targets, including nucleic acid binding proteins such as polymerases and transcription factors, non-nucleic acid binding proteins such as cytokins and growth factors, as well as small organic molecules such as ATP and theophylline. The range of applications of this technology for new discovery extends from basic research reagents to the identification of novel diagnostic and therapeutic reagents. Examples of these applications are described along with a discussion of underlying principles and future developments expected to further the utility of SELEX.

By combining crystallographic and NMR structural data for RNA-bound amino acids within riboswitches, aptamers, and RNPs, chemical principles governing specific RNA interaction with amino acids can be deduced. Such principles, which we summarize in a ''polar profile'', are useful in explaining newly selected specific RNA binding sites for free amino acids bearing varied side chains charged, neutral polar, aliphatic, and aromatic. Such amino acid sites can be queried for parallels to the genetic code. Using recent sequences for 337 independent binding sites directed to 8 amino acids and containing 18,551 nucleotides in all, we show a highly robust connection between amino acids and cognate coding triplets within their RNA binding sites. The apparent probability (P) that cognate triplets around these sites are unrelated to binding sites is %5.3 9 10 -45 for codons overall, and P % 2.1 9 10 -46 for cognate anticodons. Therefore, some triplets are unequivocally localized near their present amino acids. Accordingly, there was likely a stereochemical era during evolution of the genetic code, relying on chemical interactions between amino acids and the tertiary structures of RNA binding sites. Use of cognate coding triplets in RNA binding sites is nevertheless sparse, with only 21% of possible triplets appearing. Reasoning from such broad recurrent trends in our results, a majority (approximately 75%) of modern amino acids entered the code in this stereochemical era; nevertheless, a minority (approximately 21%) of modern codons and anticodons were assigned via RNA binding sites.A Direct RNA Template scheme embodying a credible early history for coded peptide synthesis is readily constructed based on these observations.

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An RNA has been selected that rapidly aminoacylates its 2'(3') terminus when provided with phenylalanyl-adenosine monophosphate. That is, the RNA accelerates the same aminoacyl group transfer catalyzed by protein aminoacyl-transfer RNA synthetases. The best characterized RNA reaction requires both Mg2+ and Ca2+. These results confirm a necessary prediction of the RNA world hypothesis and represent efficient RNA reaction (> or = 10(5) times accelerated) at a carbonyl carbon, exemplifying a little explored type of RNA catalysis.

A specific, reversible binding site for a free amino acid is detectable on the intron of the Tetrahymena self-splicing ribosomal precursor RNA. The site selects arginine among the natural amino acids, and prefers the L- to the D-amino acid. The dissociation constant is in the millimolar range, and amino acid binding is at or in the catalytic rG splicing substrate site. Occupation of the G site by L-arginine therefore inhibits splicing by inhibiting the binding of rG, without inhibition of later reactions in the splicing reaction sequence. Arginine binding specificity seems to be directed at the side chain and the guanidino radical, and the alpha-amino and carboxyl groups are dispensable for binding. The arginine site can be placed within the G site by structural homology, with consequent implications for RNA-amino acid interaction, for the origin of the genetic code, for control of RNA activities, and for further catalytic capabilities for RNA.

There is very significant evidence that cognate codons and/or anticodons are unexpectedly frequent in RNA-binding sites for seven of eight biological amino acids that have been tested. This suggests that a substantial fraction of the genetic code has a stereochemical basis, the triplets having escaped from their original function in amino acid-binding sites to become modern codons and anticodons. We explicitly show that this stereochemical basis is consistent with subsequent optimization of the code to minimize the effect of coding mistakes on protein structure. These data also strengthen the argument for invention of the genetic code in an RNA world and for the RNA world itself.

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.

An indispensable step in protein biosynthesis is the 2 0 ð3 0 Þ aminoacylation of tRNA by aminoacyl-tRNA synthetases. Here we show that a similar activity exists in a tiny, 5-nt-long RNA enzyme with a 3-nt active center. The small ribozyme initially trans-phenylalanylates a partially complementary 4-nt RNA selectively at its terminal 2 0 -ribose hydroxyl using PheAMP, the natural form for activated amino acid. The initial 2 0 Phe-RNA product can be elaborated into multiple peptidyl-RNAs. Reactions do not require divalent cations, and have limited dependence on monovalent cations. Small size and minimal requirements for regiospecific translational activity strongly support the hypothesis that minuscule RNA enzymes participated in early forms of translation.aminoacyl-RNA | enzyme | evolution | peptidyl-RNA | RNA A mino acids enter modern translation via attachment to a 2 0 ð3 0 Þ tRNA terminus, a reaction catalyzed by a protein aminoacyl-tRNA synthetase. Because it is implausible that primitive peptides were synthesized using already-formed protein catalysts, the RNA world hypothesis (1, 2) requires peptide synthetic reactions performed by RNA enzymes (3, 4). Indeed, a number of RNAs have been isolated which accelerate related translational reactions (5).Several ribozymes capable of catalyzing the same chemical group transfer that is today carried out by aminoacyl-tRNA synthetases (6-9) have been isolated using Systematic Evolution of Ligands by Exponential Enrichment (10, 11). However, none possess all desirable characteristics. First, an RNA world enzyme should be small, accessible after rudimentary RNA synthesis. In addition, it should act in trans, and should use universal biological, water-soluble substrates. Previously isolated ribozymes employ appropriate substrates [amino acids activated as acyladenylates (6, 12)], but are not true enzymes, as they are self-aminoacylators modified by their own reaction. Other ribozymes aminoacylate RNA with turnover; however, the amino acids must be activated as cyanomethyl, 3,5-dinitrobenzyl, or p-chlorobenzyl esters (8, 13), and hence they do not facilitate the biological reaction.The family of self-aminoacylating ribozymes exemplified by truncate C3 RNA (Fig. 1A) presented the intriguing possibility of very simple aminoacyl transfer (6). Mutational analyses as well as molecular dynamics and energy minimization of the reactants suggested a tiny active center consisting of only three essential nucleotides-a 3 0 -terminal U, and a 5 0 -GU-3 0 sequence in a loop apposed to the unpaired 3 0 -terminal U. Although the C3 RNA family possessed helical elements, adjacent helices appeared nonspecific in sequence, perhaps required only for assembly of the active center (6).Here we present a radically modified version of C3 ribozyme, unique in three ways: It functions in trans; it has been minimized to a tiny, five-nucleotide ribozyme; and it also supports peptidyl-RNA synthesis. The result is the smallest trans-aminoacylator, and arguably the smallest true ribozyme, ever observed (...