In Vitro Selection of Adenine-dependent Hairpin Ribozymes

Abstract

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...

In vitro RNA random pools are not structurally diverse: A computational analysis

“…We quantify the distribution of associated secondary-structural motifs using tree graphs and graph theory measures of size and complexity (particularly, tree diameter and second smallest Laplacian eigenvalue, which are defined in the Materials and Methods section). Our results show that such random RNA pools heavily favor (>90%) simple topological structures like linear stem-loop and low-branching motifs, as confirmed by many known aptamers (Bae et al 2002;Fukusho et al 2002;Khoo et al 2002;Komatsu et al 2002;Ulrich et al 2002;Zinnen et al 2002;Meli et al 2003;Laserson et al 2004). For example, the 25-nt and 40-nt pools are almost completely populated by hairpin and stem-bulge-stem-loop structures.…”

“…Indeed, in vitro selection experiments have revealed novel functional RNAs from random sequence pools, including a wide range of RNA aptamers that bind to particular compounds, such as ATP, antibiotics or proteins, and catalytic RNAs (Wilson and Szostak 1999;Hodgson and Suga 2004); see the collection of several hundred aptamers in the Aptamer Database (http:// aptamer.icmb.utexas.edu). However, an analysis of existing aptamers has shown that RNA sequences isolated from selection experiments tend to have simple topologies (Bae et al 2002;Fukusho et al 2002;Khoo et al 2002;Komatsu et al 2002;Ulrich et al 2002;Zinnen et al 2002;Meli et al 2003;Laserson et al 2004). That is, linear or slightly branched structures occur far more often than highly compact structures.…”

Competitive regulation of modular allosteric aptazymes by a small molecule and oligonucleotide effector

“…This result is encouraging, the more so as these constructs represent the first examples of hairpin ribozymes that are allosterically responsive to a small exogenous cofactor. Only one study reported an in vitro selected ATP-dependent hairpin ribozyme (Meli et al 2003) which, however, is very different from our hairpin ribozyme constructs in the sense that ATP does not act as an allosteric effector molecule but is rather required as a catalytic cofactor, similar to guanosine in group I introns. On the other hand, examples of allosteric aptazymes in which small cofactorbinding aptamers were linked to the hammerhead ribozyme via communication modules have shown that small molecules can lead to between 10-and 300-fold induced rate enhancements (Soukup and Breaker 1999b).…”

5. Prebiotic Chemistry – Biochemistry – Emergence of Life (4.4–2 Ga)

“…One of them is also active with imidazole alone. This result leads to very important perspectives (Meli et al, 2003).…”