Selection of ribozymes that catalyse multiple-turnover Diels–Alder cycloadditions by using in vitro compartmentalization

Abstract

In vitro compartmentalization (IVC) has previously been used to evolve protein enzymes. Here, we demonstrate how IVC can be applied to select RNA enzymes (ribozymes) for a property that has previously been unselectable: true intermolecular catalysis. Libraries containing 10 11 ribozyme genes are compartmentalized in the aqueous droplets of a water-in-oil emulsion, such that most droplets contain no more than one gene, and transcribed in situ. By coencapsulating the gene, RNA, and the substrates͞products of the catalyzed reaction, ribozymes can be selected for all enzymatic properties: substrate recognition, product formation, rate acceleration, and turnover. Here we exploit the complementarity of IVC with systematic evolution of ligands by exponential enrichment (SELEX), which allows selection of larger libraries (>10 15 ) and for very small rate accelerations (kcat͞kuncat) but only selects for intramolecular single-turnover reactions. We selected Ϸ10 14 random RNAs for Diels-Alderase activity with five rounds of SELEX, then six to nine rounds with IVC. All selected ribozymes catalyzed the Diels-Alder reaction in a truly bimolecular fashion and with multiple turnover. Nearly all ribozymes selected by using eleven rounds of SELEX alone contain a common catalytic motif. Selecting with SELEX then IVC gave ribozymes with significant sequence variations in this catalytic motif and ribozymes with completely novel motifs. Interestingly, the catalytic properties of all of the selected ribozymes were quite similar. The ribozymes are strongly product inhibited, consistent with the Diels-Alder transition state closely resembling the product. More efficient Diels-Alderases may need to catalyze a second reaction that transforms the product and prevents product inhibition.emulsion ͉ RNA ͉ intermolecular catalysis T he Diels-Alder [4 ϩ 2] cycloaddition reaction (1) between a 1,3 diene and an alkene dienophile is one of the most useful and important in synthetic chemistry, because it allows the formation of six-membered rings by making two simultaneous C-C bonds and at the same time generates up to four chiral centers (2). However, nature rarely seems to use this mechanism for C-C bond formation and instead prefers other mechanisms such as the aldol condensation reaction. Although Ͼ100 natural products have been proposed to be Diels-Alder cycloadducts (3), direct evidence from biosynthetic studies with purified or partially purified enzymes is available for only three naturally occurring Diels-Alder reactions (4).However, several antibodies and RNAs that catalyze Diels-Alder reactions have been generated in the laboratory. The DielsAlderase antibodies were raised against a hapten that mimicked either the Diels-Alder adduct or the transition state of the desired reactants (reviewed in ref. 5). Diels-Alderase ribozymes with both pyridyl-modified (6, 7) and unmodified RNA (8) were generated by using a variation on systematic evolution of ligands by exponential enrichment (SELEX) (9, 10). One substrate is physically tethered to the...

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“…Inspired by the natural model of cellular compartmentalisation, Agresti et al (2005) developed a system termed in vitro compartmentalization (IVC). IVC uses compartmentalization to link genotype (a nucleic acid that can be replicated) and phenotype (a functional trait such as a binding or catalytic activity).…”

Thermodynamics of Nucleic Acid Structural Modifications for Biotechnology Applications

“…In fact the Diels-Alder reaction catalyzed by DNA, which may further indicate the general nature of the requirement for hydrophobic surfaces to bring the diene and dienophile together [46]. However, in the case of the RNA J49, the specificity of binding by the hydrophobic effect reveals a second major issue in the development of artificial ribozymes, namely product inhibition [70].…”

“…While this is an excellent example of the power of in vitro selection, the tight binding of the product, rather than the transition state, shows that product inhibition is an inherent problem for the tethered substrate selection strategy [72]. It was later shown that product inhibition can be potentially overcome with appropriate selection design using an in vitro compartmentalization strategy [70]. In this strategy the reaction takes place under multiple turnover conditions in a single droplet so that selection of a true enzyme can be accomplished.…”