Single-Molecule FRET Studies of RNA Folding: A Diels–Alderase

Article pubs.acs.org/JPCB

Single-Molecule FRET Studies of RNA Folding: A Diels−Alderase Ribozyme with Photolabile Nucleotide Modifications Andrei Yu. Kobitski,† Stefan Schaf̈ er,† Alexander Nierth,‡,§ Marco Singer,‡ Andres Jas̈ chke,‡ and G. Ulrich Nienhaus*,†,# †

Institute of Applied Physics, Institute of Toxicology and Genetics and Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany ‡ Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany # Department of Physics, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States ABSTRACT: Enzymology at the single-molecule level by using fluorescence resonance energy transfer (smFRET) offers unprecedented insight into mechanistic aspects of catalytic reactions. Implementing spatiotemporal control of the reaction by using an external trigger is highly valuable in these challenging experiments. Here, we have incorporated a light-cleavable caging moiety into specific nucleotides of the Diels−Alderase (DAse) ribozyme. In this way, the folding energy landscape was significantly perturbed, and the catalytic activity was essentially suppressed. A careful smFRET efficiency histogram analysis at various Mg2+ ion concentrations revealed an additional intermediate state that is not observed for the unmodified DAse ribozyme. We also observed that only a fraction of DAse molecules returns to the native state upon cleavage of the caged group by UV light. These constructs are attractive model RNA systems for further real-time single-molecule observation of the coupling between conformational changes and catalytic activity.

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INTRODUCTION

Enzymatic activity is not specific to proteins but can also be realized with RNA. Ribozymes have been identified in nature, and artificial ribozymes have also been generated. The mechanisms through which the RNA active sites accelerate chemical reactions are not yet well understood.8,9 Consequently, we have engaged in a research project in recent years aimed at elucidating the enzymatic action of a particular ribozyme, the Diels−Alderase (DAse) ribozyme. This small RNA consists of only 49 nucleotides and catalyzes a bimolecular cycloaddition reaction between an anthracene diene and a maleimide dienophile in a multiple turnover fashion. Its X-ray structure (Figure 1A),10 which is a key prerequisite for the development of mechanistic models of ribozyme function, shows a λ-shaped three-dimensional fold, formed by helix II and III (Figure 1B) stacking coaxially; helix I abuts the active site. The 5′-terminal GGAG sequence extends toward helical stem III; it is stabilized by Watson−Crick base pairing with residues of the asymmetric bubble consisting of UGCCA and AAUACU sequences (Figure 1A,B). Thereby, a perfectly nested pseudoknot topology is generated that encloses the catalytic pocket.

Biopolymers, proteins and nucleic acids alike, are complex physical systems that can assume a vast number of different conformations represented by minima in a multidimensional free-energy landscape.1,2 Energy landscape theory,3,4 a field pioneered and greatly shaped by P. G. Wolynes, provides a general conceptual framework for understanding biomolecular self-organization, folding, and function. Through a myriad of conformational changes, biomolecules typically fold into welldefined three-dimensional architectures. Still, even in their folded states, biomolecules display considerable structural and dynamic heterogeneity. To reveal these properties, single-molecule studies with optical detection offer key advantages over experiments performed on large ensembles.5−7 They provide access to distributions of molecular properties, whereas experiments carried out on bulk samples often yield only averages. Importantly, subspecies can be distinguished and characterized individually. Furthermore, time trajectories taken on individual molecules allow us to directly observe rate processes in real time and to resolve rarely populated and short-lived intermediate states. The observation of enzymatic reactions at the level of individual molecules is particularly intriguing. Many enzymes display highly heterogeneous reaction behavior. Both static and dynamic heterogeneity have been inferred from the experiments. © 2013 American Chemical Society

Special Issue: Peter G. Wolynes Festschrift Received: February 26, 2013 Revised: April 25, 2013 Published: April 26, 2013 12800

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Figure 1. (A) Cartoon depiction of the crystal structure of the DAse ribozyme (PDB: 1YKV)10 with marked caging sites (blue arrows), the fluorophores Cy3 (green arrow) and Cy5 (red arrow), and the 3′-biotin (gray arrow). (B) Secondary structure of the DAse ribozyme with the arrows colored as in panel A. (C) Chemical structure of cytidine caged with NPE. (D) Cartoon depiction of a single-molecule multicolor FRET experiment on immobilized ribozymes, which includes observation of conformational dynamics of the caged construct, uncaging and folding into the native state, binding of the diene and dienophile substrates, and, finally, the Diels−Alder reaction. Parts of the figure are adapted with permission from refs 12 and 13. Copyright 2007−2012 by Oxford University Press.

To observe the Diels−Alder reaction at the single-molecule level in real time, we have synthesized novel fluorogenic sensor substrates for this ribozyme,11 which report on the progress of the reaction by changing their fluorescence emission. In these substrates, anthracene is fused to a BODIPY fluorophore via a phenylacetylenyl bridge. BODIPY is significantly quenched by the anthracene aromatic ring system, most likely by photoinduced electron transfer. In the Diels−Alder reaction, the anthracene aromatic ring system is destroyed, and the BODIPY fluorescence is strongly enhanced. Moreover, the fluorescence is higher when the substrate is bound to the DAse than that in solution. Therefore, use of these substrates allows us to distinguish free substrate, bound substrate, bound product, and, finally, released product by their fluorescence intensity. Moreover, we have synthesized dye-labeled DAse variants, which were derivatized with Cy3 and Cy5 in specific locations to observe folding and function via Förster resonance energy transfer (FRET). A large number of derivatives was screened to find variants with optimal sensitivity to (Mg2+-dependent) conformational changes, and the variants with Cy5 attached to U33 and Cy3 at U6 or 5′-end (Figure 1A,B) were thoroughly characterized by single-molecule FRET (smFRET).12,13

We have further explored the feasibility of photoactivation of the ribozyme by using caging groups attached to the nucleotide bases. These groups prevent proper folding into the functionally competent form by steric interference. They can be cleaved off by light, however, so that the DAse ribozyme subsequently folds into its catalytically active form and the Diels−Alder reaction proceeds. Several DAse variants were designed and synthesized, which had (S)-1-(2-nitrophenyl)ethyl (NPE) moieties (Figure 1C) as caging groups introduced at locations that are known to be crucial for the catalytic activity.13 Two variants, NPEC25 and NPEU42, which displayed significantly suppressed catalytic activity with the NPE moiety present but complete recovery of their function in the uncaged form, have been reported earlier.14 In this work, we present singlemolecule studies of two other caged DAse constructs, NPEC21 and NPEC46, revealing significant perturbations of the conformational energy landscape by the introduced caged groups. In the future, these constructs will be employed for studying the interrelation between conformational dynamics and reaction kinetics of the ribozyme. A scheme of the single-molecule enzymatic experiment is depicted in Figure 1D. 12801

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widths at half-maxima (FWHM) as a function of the Mg2+ concentration.

METHODS Synthesis and Catalytic Activity of DAse Mutants. DAse ribozymes with and without caging groups were synthesized by a chemo-enzymatic approach utilizing the respective phosphoramidites and splinted ligation.12 The NPE-derivatized nucleotides NPEC21 and NPEC46 were introduced at positions 21 and 46 as described.14 The catalytic activity of the synthesized construct was determined by monitoring the appearance of BODIPY fluorescence by using the anthracene−BODIPY fluorescence sensor.11 For smFRET measurements, all DAse variants were fluorescently labeled at the 5′-end and the 33 position with the Cy3 and Cy5 dyes, respectively (Figure 1A,B). Furthermore, a biotin moiety was attached to the 3′-terminus for surface immobilization. Single-Molecule FRET Experiments. smFRET experiments were carried out with freely diffusing DAse solutions (∼100 pM) and also with surface-immobilized DAse, essentially as described before.12,13,15,16 Experimental data were taken on a homemade confocal microscope with multichannel detection, based on a Zeiss Axiovert 135.17 The buffer solution containing 50 mM Tris and 300 mM NaCl was supplemented with the appropriate amount of Mg2+ cations to achieve the desired concentrations in Mg2+ titration experiments. For smFRET analysis of the photon bursts emitted by freely diffusing DAse ribozymes during their brief sojourn in the focal spot, droplets (∼30 μL) of the probe solution were deposited on microscope coverslips and kept under a water-saturated nitrogen atmosphere during the measurement. Ascorbic acid (1 mM) and methyl viologen (1 mM) were added to the sample solution so as to suppress fluorophore blinking and bleaching.18 The samples were studied with the laser irradiation alternating between donor (532 nm, “green”) and acceptor (640 nm, “red”) excitation. Only those bursts indicating a functional acceptor and exceeding 50 counts under green excitation in both donor and acceptor detection channels were analyzed. For imaging immobilized RNA molecules,19 biotinylated ribozymes were sparsely attached to a glass coverslip coated with physisorbed, biotinylated bovine serum albumin (BSA), to which streptavidin was bound. By using a piezoelectric stage, identical areas were scanned with green and, subsequently, with red laser excitation to ensure that only RNA molecules with both functional donor and acceptor dyes were included in the FRET analysis. Data analysis was performed using our own software written in Matlab 2010 (MathWorks, Natick, MA). Histograms of FRET efficiency values, E, were compiled from donor and acceptor intensities, either from photon bursts for measurements on freely diffusing molecules or from spots for immobilized molecules, according to E = Ia/(Ia + γId), where Ia and Id are the intensities of the acceptor and donor emission, respectively. The parameter γ accounts for the differences in the fluorophore quantum yields and detector efficiencies of the two channels. In our smFRET experiments with the Cy3/Cy5 pair, γ was between 1.0 and 1.2 depending on the microscope adjustment and focal depth, in accordance with a reference sample, so that the FRET efficiency values from measurements on freely diffusing molecules matched those obtained on immobilized ones. FRET efficiency histograms were globally fitted (i.e., multiple data sets with varying Mg2+ concentration were simultaneously fitted with constrained parameters) by sums of two or three Gaussian distributions to obtain their fractional areas (relative populations), center positions, and full

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RESULTS AND DISCUSSION Due to the polyanionic nature of RNA, the screening of the charge by counter-cations, especially divalent ones, is crucial for their structural stability. Therefore, by measuring smFRET histograms at 0 and 80 mM Mg2+ concentrations, we screened a number of DAse variants with NPE caging groups to identify those that exhibit pronounced conformational changes upon nucleotide modification. Among these, the FRET distributions of the two constructs denoted as NPEC21 and NPEC46 showed the largest differences with respect to the unmodified (wt) DAse molecule (Figure 2). Caging of C21 at its Watson−Crick face

Figure 2. Histograms of FRET efficiency values of unmodified (wt) DAse ribozyme and two constructs with photolabile NPE groups (NPEC21 and NPEC46), measured on single molecules freely diffusing in buffer solution containing either 0 or 80 mM Mg2+.

should interfere with the formation of helix II (yellow, Figure 2), while caging C46 is expected to disturb an essential pseudoknot interaction. In earlier work,12,13 we thoroughly investigated the unmodified DAse and observed that the change of the smFRET histograms of wt DAse with the Mg2+ concentration can be described by a transition from an intermediate to a folded state, represented by two Gaussian distributions in the FRET histograms. As shown in Figure 2, the FRET distribution of wt DAse peaks at 0.47 in the presence of monovalent (50 mM Tris and 300 mM NaCl) and absence of divalent ions. This intermediate FRET value is indicative of the intermediate state. Upon addition of Mg2+ ions, the intermediate state distribution shifts to higher FRET values; the ensemble becomes more compact (peak position ⟨EI⟩ ≈ 0.52). In addition, the folding equilibrium strongly shifts toward the more compact, functionally active folded state (⟨EF⟩ ≈ 0.73). By comparing the NPEC21 and NPEC46 constructs with wt DAse (Figure 2), one can clearly observe pronounced differences in 12802

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Figure 3. FRET efficiency histograms of (A) wt, (B) NPEC21, and (C) NPEC46 DAse ribozymes exposed to buffer solution at various Mg2+ ion concentrations. The histograms were fitted with a sum of two Gaussians to obtain the populations associated with the intermediate (I, green) and folded (F, red) conformations; the black line shows the sum of the two populations.

the FRET efficiency histograms at 0 mM Mg2+ ions. For the caged variants, the maximum of the distribution appears at a FRET efficiency of 0.6, in between the values found for the intermediate (I) and folded (F) states of wt DAse, which suggests the presence of another intermediate state. At 80 mM Mg2+, the wt DAse FRET efficiency histogram displays a sharply peaked distribution at the high-FRET, folded state position, yet a significant fraction of intermediate state is also visible. For the two caged variants, the FRET distributions have broad tails toward lower FRET values, indicating that larger populations of not properly folded RNAs exist in these samples. To further explore the possible presence of a second intermediate state, we performed FRET measurements at 10 different Mg2+ concentrations for the wt DAse ribozyme and the two caged constructs. The FRET histograms were quantitatively analyzed by fitting with a sum of two Gaussian distributions assigned to an intermediate state I and the folded state F. The histograms and results of the fit at five selected Mg2+ concentrations, 0, 1.6, 6.25, 25, and 100 mM, are depicted in Figure 3. In Figure 4, the fractional population of the F state and the mean FRET efficiency, ⟨E⟩, of the I and F states are plotted as a function of Mg2+ ion concentration. For the wt DAse construct (red circles in Figure 4), the folding behavior is essentially identical to the one reported earlier.12,13 In particular, the midpoint of the I to F transition occurs at about 5.5 ± 0.8 mM. For the caged variants NPEC21 (cyan squares) and, especially, NPEC46 (brown diamonds), the iondependent interconversion between the I and F states is markedly different compared to that for the unmodified RNA. For NPEC21, the I to F transition is less steep, and the midpoint is located at about 20 mM Mg2+ concentration. Remarkably, for NPE C46, the population of the F species decreases with increasing Mg2+ concentration, and the position of its distribution in the FRET histogram, ⟨EF⟩, shifts markedly from 0.6 to 0.75 in the transition. Such a behavior does not appear reasonable for a true, structurally well-defined folded state of RNA (or proteins)12,15,20 but rather suggests that the two-state analysis employed here is inappropriate. Therefore, we introduced an additional intermediate state, I′, in the fit of

Figure 4. Mg2+ concentration dependence of parameters obtained from fitting FRET histograms of wt (circles), NPEC21 (squares), and NPE C46 (diamonds) DAse ribozymes with a two-state model. (A) Folded fraction; lines are shown to guide the eye; (B) mean FRET efficiency of the folded (⟨EF⟩, upper panel) and the intermediate (⟨EI⟩, lower panel) states.

the FRET efficiency histograms of the NPEC46 construct. Due to the large overlap of the distributions of the three species I (green), I′ (orange), and F (red), we restricted the position and FWHM parameters of the F state to values obtained from fitting wt DAse ribozyme data, that is, 0.763 and 0.17, respectively. The fit results are shown together with the FRET histograms in Figure 5A; the populations of the three species are plotted in Figure 5B as a function of the Mg2+ concentration. Obviously, the three-state model fits the FRET histograms as well as the two-state model or even better. The FRET distributions associated with the I and I′ states were found at 0.46 and 0.63, respectively (Figure 5C); they were 12803

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Figure 5. (A) FRET efficiency histograms of the NPEC46 DAse ribozyme measured in buffer solution with varying Mg2+ concentration. The histograms were fitted with a sum of three Gaussians to obtain the populations associated with two intermediate states, I (green) and I′ (orange), and the folded state F (red); the black line shows the sum of the three populations. (B) Mg2+ concentration dependence of the fractional populations of I (squares), I′ (diamonds), and F (circles). Lines are shown to guide the eye. (C) Mg2+ dependence of the mean FRET efficiency, ⟨E⟩, of the I, I′, and F populations.

Figure 6. (A) FRET efficiency histograms (at 0 mM Mg2+) of wt (upper panel) and NPEC46 DAse molecules before (middle panel) and after (lower panel) irradiation with a 405 nm laser, which photodissociates the NPE groups. Green and orange dashed lines mark the respective positions of the I and I′ states from the fit with a three-state model. (B) Cumulative FRET efficiency trace of 20 NPEC46 DAse molecules (at 0 mM Mg2+). The molecules were exposed to a 10 ms pulse of 405 nm light 200 ms after the start of recording, as indicated by the purple bar. The red dashed line represents a constant FRET efficiency level prior to illumination and an exponential decay with a characteristic time of 100 ms afterward. (C) Ensemble measurements of the relative catalytic activity of the caged DAse molecules before and after illumination with UV light, referenced to the wt DAse construct. The inset shows the progress of the catalytic reaction of the reference wt and caged NPEC21 and NPEC46 DAse ribozymes before (dashed lines) and after (solid lines) illumination with UV light. Catalytic activity was measured in the presence of 80 mM Mg2+.

observed to shift slightly (∼0.01) with increasing Mg2+ ion concentration. Interestingly, while the average FRET efficiency, ⟨EI⟩, of the I state is similar to the value observed for the wt DAse ribozyme, the average FRET efficiency, ⟨EI′⟩, of the I′ state is similar to the one of the I state of the NPEC21 construct. Also, in contrast to the fit with the two-state model, the threestate model fit revealed a pronounced increase of the F state

population upon addition of Mg2+ ions at the expense of the I′ state, while the I state population remained almost unchanged. In order to interpret the observed structural perturbations, we examined the rich tertiary architecture of the DAse ribozyme thoroughly discussed in ref 13. Thus, the caging group of the NPEC46 construct is placed exactly at the sharp turn that aligns helices I and II at the bottom of the catalytic pocket. 12804

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Moreover, there are two Mg2+ ions, Mg1 and Mg2, that screen the high local charge density and are crucial for the proper folding into the functional conformation. Therefore, on the one side, C46 base-pairs with G4 on the 5′-terminal sequence that closes the catalytic pocket, and on the other side, it directly coordinates with Mg2. C21 is also located near the sharp turn but on the other side of the Mg2 ion. It is, therefore, reasonable to assume that formation of the sharp turn can be affected by a caging group at low Mg2+ concentration, leading to the displacement of helix I and, concomitantly, to changes in the interdye distance. At high Mg2+ concentration, these conformational perturbations are compensated for in part by additionally bound cations. In addition, we have also examined the ability to photodissociate the caging group by laser irradiation at 405 nm. To this end, we measured image-based FRET efficiency distributions of NPEC46 DAse ribozyme molecules immobilized on a surface before and after applying 405 nm light (Figure 1D, “Uncaging”). These experiments were carried out at 0 mM Mg2+ concentration. In Figure 6A, the FRET efficiency histograms are shown together with the one of the unmodified wt DAse ribozyme. Prior to irradiation, the FRET histogram of the immobilized NPEC46 molecules is essentially the same as that for the freely diffusing ones (Figure 2). After irradiation, a new subpopulation at lower FRET efficiency emerged. Its FRET distribution is similar to the one of the I state of the wt construct. However, the I′ subpopulation remained significantly present, which may either result from incomplete uncaging or from incomplete relaxation into the folded form during image acquisition, that is, on the time scale of seconds. To observe conformational changes upon uncaging in real time, we have also recorded FRET efficiency time traces from individual molecules before and after illumination with 405 nm laser light. In Figure 6B, a cumulative trace obtained from 20 molecules with 50 ms dwell time binning displays an apparent change in the FRET efficiency from 0.6 prior to 405 nm illumination to about 0.55 for times >1 s after illumination. Fitting the decay with an exponential yielded a characteristic time of about 100 ms, which is similar to the one observed earlier for conformational changes between intermediate and folded states.12 Although the small change in the FRET efficiencies between the caged and uncaged constructs has so far precluded the analysis of transition pathways for individual molecules, we are confident that we will achieve this ultimate goal, for example, by using a higher photon flux and more sophisticated data analysis methods.21 Furthermore, we have also measured the enzymatic activities of the caged and uncaged constructs by monitoring the fluorescence signal of product formation in bulk samples as described earlier.14 In these experiments, we utilized the fluorogenic sensor substrate 1-AB, in which anthracene is fused to BODIPY.11 The fluorescence increase, which indicates destruction of the anthracene aromatic system so that BODIPY is unquenched, is plotted as a function of time for the NPEC21 and NPEC46 DAse ribozyme constructs before and after photolytic cleavage in the inset of Figure 6C. The relative catalytic activities with respect to the wt DAse are shown in Figure 6C. In contrast to the NPEC25 DAse ribozyme construct studied before,14 the recovery of the catalytic activity is incomplete. This observation is consistent with the observed presence of misfolded species after uncaging (Figure 6A).

Article

CONCLUSIONS Nucleotide modification is an efficient way to enrich the chemical diversity of the limited repertoire of RNA and DNA bases. Thus, post-transcriptional modifications of tRNAs are employed by nature for the fine-tuning of tertiary fold and/or contacts with proteins.22 Moreover, in some cases, for example, for the m 1 A modification in human mitochondrial tRNALys,16,23,24 localized nucleotide modifications were observed to strongly affect the RNA folding energy landscape, shifting the thermodynamic equilibrium between differently folded conformations. Artificial modifications of amino acids and nucleic acids with light-cleavable protecting groups were shown to be extremely useful for gaining spatiotemporal control of biomolecular folding and function.25 In the context of RNA, modified bases with caged groups have been successfully utilized to study conformational states of RNA molecules before and after photolysis at the ensemble level.26−32 It is worth noting that small caging groups are unlikely to alter the conformation of large RNAs in a major way. With small RNAs such as the DAse ribozyme studied here, however, incorporation of modified nucleotides is more likely to produce significant structural perturbations. Thus, in the absence of divalent ions, every construct with caged nucleotides differed more or less from the unmodified DAse ribozyme (data not shown). However, upon adding 80 mM Mg2+ to the buffer solvent to initiate folding, most constructs adopted predominantly the F conformation of the wt DAse, except those containing caging groups at C21 and C46. From a careful global analysis of FRET efficiency histograms taken at different concentrations of divalent ions, we showed that NPEC46 folds into a new intermediate conformation that might result from the disturbed formation of the sharp turn element of the DAse ribozyme. A similar scenario but with smaller consequenes may also be considered for the folding behavior of the NPEC21 construct. Our single-molecule uncaging experiments gave support for the notion that this intermediate state may act as a kinetic trap, at least on the second time scale. In the future, we will study the kinetics of DAse ribozyme conformational changes upon removal of photocleavable protection groups. In multicolor FRET experiments, we will employ dye-labeled caged DAse constructs and fluorogenic substrates to disentangle the effect of ribozyme conformational dynamics on its catalytic activity at the single-molecule level.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +49 (0)721 608 43410. Present Address §

A.N.: The Scripps Research Institute (TSRI), Department of Chemistry, Beckman Center for Chemical Sciences, La Jolla, CA 92037, U.S.A. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the Deutsche Forschungsgemeinschaft (CFN and NI 291/9) and the Karlsruhe Heidelberg Research Partnership (HEiKA) is gratefully acknowledged.

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