Adenine-dependent hairpin ribozymes were isolated by in vitro selection from a degenerated hairpin ribozyme population. Two new adenine-dependent ribozymes catalyze their own reversible cleavage in the presence of free adenine. Both aptamers have Mg 2؉ requirements for adenine-assisted cleavage similar to the wild-type hairpin ribozyme. Cleavage kinetics studies in the presence of various other small molecules were compared. The data suggest that adenine does not induce RNA self-cleavage in the same manner for both aptamers. In addition, investigations of pH effects on catalytic rates show that both adenine-dependent aptamers are more active in basic conditions, suggesting that they use new acid/base catalytic strategies in which adenine could be involved directly. The discovery of hairpin ribozymes dependent on adenine for their reversible selfcleavage presents considerable biochemical and evolutionary interests because we show that RNA is able to use exogenous reactive molecules to enhance its own catalytic activity. Such a mechanism may have been a means by which the ribozymes of the RNA world enlarged their chemical repertoire.The RNA world theory assumes that modern life arose from molecular ancestors in which RNA molecules both stored genetic information and catalyzed chemical reactions (1-4). According to this scenario, ribozymes of the RNA world would have been able to self-replicate (5) and to control complex metabolisms with an expanded chemical repertoire (6, 7). Until recently, RNA catalysis was believed to be restricted to phosphate chemistry, but in vitro selection experiments and recent discoveries concerning natural ribozymes have demonstrated that the catalytic capacities of RNA are far more promising and exciting than previously anticipated (8 -13). However, in comparison with proteins, the chemical spectrum of ribozymes remains limited because of the limited chemical diversity of RNA, which is composed of only four different building blocks.Yet RNA could increase its range of functionalities by incorporating catalytic building blocks such as imidazole, thiol, and functional amino and carboxylate groups (14, 15). Moreover, primeval nucleotides were not necessarily restricted to standard nucleotides; modified nucleotides may have played a role in catalysis in the RNA world (16,17).Another way for RNA to increase its chemical diversity would consist in the binding of exogenous molecules carrying reactive groups and handling them as catalytic cofactors. We recently reported the isolation of new RNA aptamers able to bind adenine in a novel mode of purine recognition (18). Adenine is a likely prebiotic analog of histidine. Its catalytic capabilities are equivalent to histidine because of the presence of a free imidazole moiety (19 -21). It was previously shown that when adenine is placed in a favorable microenvironment, its catalytic efficiency is strongly enhanced (22-24). Such favorable microenvironments could result from adenine binding to RNA and thereby providing catalytic sites. In this perspe...

RNA aptamers that are able to complex free adenine have been isolated by a SELEX (systematic evolution of ligands by exponential enrichment) procedure. The adenine binding site was revealed by sequence alignment for a prevalent cluster of aptamers, and its structure and interactions with adenine were probed by RNase digestion studies, lead cleavage, boundary determination experiments, and truncated sequences studies. A new purine binding motif was functionally and structurally characterized and compared with other RNAs specific to purine or adenylated compounds. The affinity for adenine and the specificity for other related targets were quantified. This work suggests that the adenine binding site is composed of two independent secondary structure elements forming a bipartite binding site that interacts with adenine in a new mode of purine recognition. Such binding is of great interest because the imidazole moiety is not trapped in the binding site, and would easily be available for catalytic activity.

It is assumed that modern life forms arose from a molecular ancestor in which RNA molecules both stored genetic information and catalyzed biochemical reactions. In modern cells, these functions are carried out, respectively, by DNA and proteins, but diverse cellular RNAs are also involved in key cellular functions. In this paper, we review the cellular RNAs that are ubiquitous and/or that perform essential biological functions, and we discuss the evolutionary relationships of such RNAs with a prebiotic RNA world. This unexpected biological diversity of cellular RNAs and the crucial functions they perform in cellular metabolism demonstrate the complexity of an RNA-driven metabolism in an ancient RNA world and in modern life. Cellular RNAs are involved in translation (tRNA and rRNA) but also in ribosome maturation (snoRNA) and more generally in RNA processing (snRNA and snoRNA), replication (telomerase RNA), editing, protein translocation (SRP RNA), cellular transport (vRNA) and translation quality control (tmRNA). In addition, the function of many other cellular RNAs has not yet been determined. Future investigations of RNA function will allow us to better understand not only early evolutionary biological processes but also the central metabolism of modern cells.

Molecular biology techniques have enabled us to prepare and select RNA aptamers that can bind specifically to small targets. RNA oligonucleotides can also be used as fluorescent probes. We have combined the two approaches to obtain Aptamer Beacons, in which molecular recognition is linked to the emission of an optical signal. These RNA biosensors could be used to detect directly the signatures of life in samples of mineral and extra-terrestrial material.