A role for hydrophobicity in a Diels-Alder reaction catalyzed by pyridyl-modified RNA

Abstract

New classes of RNA enzymes or ribozymes have been obtained by in vitro evolution and selection of RNA molecules. Incorporation of modified nucleotides into the RNA sequence has been proposed to enhance function. DA22 is a modified RNA containing 5-(4-pyridylmethyl) carboxamide uridines, which has been selected for its ability to promote a Diels-Alder cycloaddition reaction. Here, we show that DA_TR96, the most active member of the DA22 RNA sequence family, which was selected with pyridyl-modified nucleotides, accelerates a cycloaddition reaction between anthracene and maleimide derivatives with high turnover. These widely used reactants were not used in the original selection for DA22 and yet here they provide the first demonstration of DA_TR96 as a true multiple-turnover catalyst. In addition, the absence of a structural or essential kinetic role for Cu(2+), as initially postulated, and nonsequence-specific hydrophobic interactions with the anthracene substrate have led to a reevaluation of the pyridine modification's role. These findings broaden the catalytic repertoire of the DA22 family of pyridyl-modified RNAs and suggest a key role for the hydrophobic effect in the catalytic mechanism.

Figure 1.

The pyridyl-modified DA_TR96 RNA and the Diels–Alder reactions catalyzed by DA_TR96. (A) Primary sequence and predicted secondary structure (44) of the DA_TR96 RNA (28). Inset: chemical structure of pyridyl-modified UTP (pyr-UTP) (21). OPPP; triphosphate moiety; J1, junction 1; B1, bulge 1; SL1, stem–loop 1; J2, junction 2; SL2, stem–loop 2; J3, junction 3. (B) Schematic of the DA22 Diels–Alderase selection reaction (21). PEG, polyethylene glycol linker; BMCC, 1-biotinamido-4-[4′-(maleimidomethyl)cyclohexane-carboxamido]butane. (C) Primary sequence and predicted secondary structure (44) of the 39M49 RNA (6,29). (D) Schematic of the in-solution Diels–Alderase activity assay used here utilizing AHEG and MCA Diels–Alder substrates (29).

Figure 2.

DA_TR96 RNA accelerates the Diels–Alder reaction between AHEG and MCA free in solution and interacts in a nonsequence-specific manner with AHEG. (A) Plot of percent substrate conversion (product formation) and enzymatic turnover versus time with and without 20 μM CuCl₂. Error bars are standard error of the mean for three replicate measurements. The uncatalyzed reaction values were subtracted as background. (B) Plot of the change in absorbance at 365 nm over time for reactions containing 0.1 mM AHEG with various RNAs (at 7 μM) and other components under Diels–Alderase activity assay conditions (no MCA present). N-sR8, native unmodified RNA; sR8, pyridyl-modified RNA. PVHP (1 mg/ml); pyr-UTP, pyridyl-modified UTP (1 mM). (C) Plot of the change in absorbance at 365 nm over time for reactions containing 0.1 mM AHEG and increasing concentrations of DA_TR96 RNA up to 7 μM under Diels–Alderase activity assay conditions (no MCA present). Curves shown in the figure are a guide to the eye. Curve fitting to a pseudo-first-order rate constant was performed using nonlinear least squares fit with an exponential association equation in the program Prism (GraphPad).

Figure 3.

The secondary structures of pyridyl-modified and unmodified DA_TR96 RNAs are similar but the pyridyl modification reduces RNA stability. The secondary structure of (A) DA_TR96 and (B) N-DA_TR96 (native, unmodified RNA) was mapped using mung bean (MB) nuclease. Increasing concentrations of MB nuclease were titrated with for each 5′-radiolabeled RNA and reactions resolved on a denaturing polyacrylamide gel. Structural elements corresponding to the predicted secondary structure in Figure 1B are boxed and indicated. A T1 RNase digestion ladder is shown in lane 1 and an alkaline hydrolysis ladder in lane 2 to identify RNA primary sequence. (C) Thermal denaturation of modified and unmodified DA_TR96 in the absence or presence of 0.1 mM AHEG.

Figure 4.

The structure of pyridyl-modified DA_TR96 RNA does not change in the presence of CuCl₂ and Diels–Alder substrates. Mung bean (MB) nuclease or lead(II) acetate (Pb²⁺) was incubated with 5′-radiolabeled RNA and specified reaction components, as indicated above the gel, and reactions resolved on a denaturing polyacrylamide gel. Structural elements corresponding to the predicted secondary structure in Figure 1B are boxed and indicated. A T1 RNase digestion ladder is shown in lane 1 and an alkaline hydrolysis ladder in lane 2 to identify the RNA primary sequence.