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 substrates/products 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 (k(cat)/k(uncat)) but only selects for intramolecular single-turnover reactions. We selected approximately 10(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.

Fig. 1.

Selection of Diels–Alderase ribozymes by using IVC. (A) Schematic diagram of the selection procedure. A repertoire of genes (DNA) encoding ribozymes, each coupled to anthracene through a PEG linker, is created (1). Genes are compartmentalized within the aqueous droplets of a water-in-oil emulsion to give, on average, less than one gene per compartment (2). Genes are transcribed, giving ≈60 RNA molecules per gene (3). Mg²⁺ and biotin-maleimide are allowed to diffuse into the compartments (4). In compartments containing active Diels–Alderase ribozymes, the formation of the cycloadduct by reaction of biotin-maleimide is catalyzed, thereby biotinylating genes encoding active ribozymes (5). The emulsion is broken (6), and active genes are enriched by binding to streptavidin-coated magnetic beads (7) and are amplified by PCR to allow further rounds of selection. For multiple-turnover selections, free anthracene is emulsified with the gene repertoire. (B) The Diels–Alder cycloaddition of biotin-maleimide (1) and AHEG (2) covalently coupled to the gene to generate the adduct (3), thereby biotinylating the gene.

Fig. 2.

Model selection of the 49mer ribozyme by IVC. Genes from before and after selection were PCR amplified and analyzed by agarose gel electrophoresis. Selections were performed starting from 1:1 or 1:5 ratios of 49mer:Δ49mer genes either emulsified or unemulsified. The upper band is a heteroduplex between the 49mer and Δ49mer DNA.

Fig. 3.

Selection of 157mer libraries by IVC. (A) Selection progress under single- and multiple-turnover conditions. The pool of selected DNA from each round was transcribed and assayed for Diels–Alderase activity by following the reduction in absorbance at 365 nm. The dotted line indicates the rate of the wild-type ribozyme (0.4 μM/min). (B) Schematic showing the different classes of ribozymes isolated from the selections. The primer binding sites and, for the ribozymes possessing the same structural motif as the SELEX ribozymes (8), helices I, II, and III (hI, hII, and hIII) and the two halves of the internal loop (IL1 and IL2) of the ribozyme are labeled and shown as colored bars. Regions with no homology to the SELEX ribozymes are shown with a black line. SELEX groups corresponding to IVC classes are indicated. An additional 13 groups, comprising 24 clones, which do not correspond to any of the IVC classes, were identified by using SELEX (8). See also Fig. 9, which is published as supporting information on the PNAS web site. (C) Comparison of the internal loop sequence of ribozymes selected by SELEX and IVC displayed in format similar to a sequence logo (34). Residues are numbered as in ref. .

Fig. 4.

Secondary structure of ribozymes. Lowest energy secondary structures as predicted by mfold (36, 37) of the 49mer ribozyme (8), and two ribozymes from the IVC selections: F12 (class 1), which has the same structural motif and internal loop sequence as the 49mer ribozymes, and E03 (class 8), which has a novel secondary structure, not found in ribozymes from SELEX (8). The predicted secondary structures were confirmed by inline attack (31) and nuclease digestion (32) (see Figs. 10 and 11). Helices I, II, and III and the internal loop of the 49mer and F12 ribozymes are labeled.

Fig. 5.

Kinetic parameters of natural and artificial Diels–Alderases. The k_cat, k_cat/K_m(diene), k_cat/K_{m(dienophile)}, and k_cat/K_m(diene)K_{m(dienophile)} for four ribozymes selected by IVC (red circles), the 49mer ribozyme derived by SELEX (SELEX¹) (33) (black circle), nine ribozymes isolated from a different SELEX experiment (SELEX²) (6, 42) (blue circles), six ribozymes derived by mutation and SELEX of a ribozyme from the previous study (SELEX³) (7) (cyan circles), six catalytic antibodies, 1E9, 39A11, 13G5, 4D5, 22C8, and 7D4 (data summarized in ref. 5) (green circles), and macrophomate synthase (41) (orange circle). Data for the SELEX² and SELEX³ ribozymes was determined under single-turnover conditions, and it was not possible to determine the K_m(diene) because these ribozymes were only active when the diene was tethered to the RNA. Circles for enzymes with very similar kinetic parameters are overlayed.