Noncovalent Spin Labeling of Riboswitch RNAs To Obtain LongRange Structural NMR Restraints Christina Helmling, Irene Bessi, Anna Wacker, Kai A. Schnorr, Hendrik R. A. Jonker, Christian Richter, Dominic Wagner, Michael Kreibich,† and Harald Schwalbe* Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt, Germany S Supporting Information *

ABSTRACT: Paramagnetic relaxation enhancement (PRE) NMR is a powerful method to study structure, dynamics and function of proteins. Up to now, the application of PRE NMR on RNAs is a significant challenge due to the limited size of chemically synthesized RNA. Here, we present a noncovalent spin labeling strategy to spin label RNAs in high yields required for NMR studies. The approach requires the presence of a helix segment composed of about 10 nucleotides (nt) but is not restricted by the size of the RNA. We show successful application of this strategy on the 2′dG sensing aptamer domain of Mesoplasma f lorum (78 nt). The aptamer domain was prepared in two fragments. A larger fragment was obtained by biochemical means, while a short spin labeled fragment was prepared by chemical solid-phase synthesis. The two fragments were annealed noncovalently by hybridization. We performed NMR, cw-EPR experiments and gel shift assays to investigate the stability of the two-fragment complex. NMR analysis in 15N-TROSY and 1H,1H-NOESY spectra of both unmodified and spin labeled aptamer domain show that the fragmented system forms a stable hybridization product, is in structural agreement with the full length aptamer domain and maintains its function. Together with structure modeling, experimentally determined 1H-Γ2 rates are in agreement with reported crystal structure data and show that distance restraints up to 25 Å can be obtained from NMR PRE data of RNA.


complexes such as ribosomes, and regulation of gene expression by riboswitches. These functional RNAs exhibit a large degree of structural diversity and therefore represent a very interesting target for structural biology studies. The formation of RNA complexes, stimulated by interactions with proteins, ions, or small molecules, is often associated with substantial, long-range conformational changes. In this context, PRE NMR could represent a powerful tool to augment traditional NOE-based NMR methods that record short-range distances and to characterize transient encounter complexes occurring during molecular recognition processes. PREs provide long-range structural restraints, which are particularly valuable in longhelical segments of RNAs, where the number of NOE contacts are limited and NOEs tend to be insensitive to helix−helix reorientation as part of the conformational changes. Site-specific spin labeling of RNA has been previously accomplished by conjugation of a spin label to chemically synthesized RNA fragments or by incorporation of a spin labeled phosphoramidite into the oligonucleotide during chemical synthesis.22 Spin labels have been attached at the 5′end,23 at the 3′-end of RNA fragments,24 or at internal sites of

n NMR and EPR studies of protein structure and dynamics, paramagnetic tags serve as marker spins.1,2 The presence of a paramagnetic center induces a distance-dependent relaxation enhancement (PRE) of nearby nuclear spins through dipolar interactions. Since the magnetic moment of paramagnetic centers is about 103 times larger than the magnetic moment of nuclear spins, PREs are particularly suited to obtain long-range structural restraints3,4 and serve as a powerful NMR parameter to characterize transient, lowly populated states.5 Paramagnetic NMR represents a unique method to detect transient states and has been applied to investigate encounter interactions,6−8 motions of lowly sampled states,9 and probing of conformational space.10 The requirement to integrate a spin label within the biopolymer constitutes the main practical challenge of applying PRE NMR for studies on proteins and oligonucleotides. While several strategies based on nitroxide tags or chemical11−13 and encodable metal binding tags14,15 have been established for proteins,16,17 spin labeling of RNA is less common. Up to now, only few PRE studies on RNA have been published despite renewed interest in NMR studies of sizable RNAs.18,19 In the past decade, for example, the discovery of a variety of noncoding RNAs led to increased interest in the structure of such functional RNAs.20,21 The function of noncoding RNAs covers catalysis by ribozymes, involvement in RNA-protein © 2014 American Chemical Society

Received: January 23, 2014 Accepted: March 27, 2014 Published: March 27, 2014 1330 | ACS Chem. Biol. 2014, 9, 1330−1339

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Figure 1. (a) Elongated 2′dG sensing I−B aptamer used as model system to optimize the noncovalent spin labeling approach. Nucleotides enclosing the ligand to form the binding pocket of the aptamer domain are marked in gray. (b) Schematic representation of spin labeling in trans by hybridizing a spin labeled oligomer fragment to a helical section of the RNA of interest.

the RNA, at the phosphodiester backbone,25 at the sugar ring,26 or at the nucleobase.27−29 In particular, spin label coupling chemistry has been successful on pyrimidine residues, modified either at the 5- or 2′-position of the nucleotide with a functional group.26,28 As site-directed spin labeling of RNA relies on chemical synthesis, these techniques are limited to RNAs up to about 40 nucleotides, while spin labeling of larger RNAs still remains challenging. Therefore, RNA riboswitches with a length of up to 150 nt require an alternative spin labeling approach. Recently, spin labeling of such a large RNA has been facilitated by DNA-catalyzed ligation of smaller fragments to obtain a spin labeled SAM riboswitch (118 nt).30 However, ligation procedures are accompanied by a significant loss of RNA and are hardly cost-effective for the preparation of milligram quantities of isotope labeled RNA as required for NMR. One approach of noncovalent spin labeling has been proposed by Sigurdsson. In their approach, a spin labeled nucleobase analogue was hybridized to the nucleobase guanine in an abasic site of a double-stranded RNA.31,32 Later on, the pRNA (packaging RNA) has been spin labeled by assembling RNA fragments to form a slightly modified pRNA.33 All strategies mentioned above have been optimized for EPR experiments. The requirements for NMR analysis differ regarding the amount and homogeneity of sample needed. To our knowledge, a protocol to produce spin labeled RNAs larger than 40 nucleotides in high yields for NMR studies has not yet been reported. Here, we report a noncovalent spin labeling approach for large RNAs, which avoids chemical ligation and is accessible to most biochemical laboratories. We show the general applicability of this method using the aptamer domain of the 2′dG riboswitch from Mesoplasma f lorum (78 nt).34 The approach relies on hybridization of a short, chemically synthesized oligomer fragment containing a nitroxide label, to a large RNA fragment transcribed in vitro. Since RNAs commonly contain a significant amount of helical regions, this method can be generalized to a variety of RNA systems. The spin labeling method provides sufficient amounts of RNA for NMR measurements, allows segmental isotope labeling, and does not require advanced chemistry. We characterized the system by gel shift assays, cw-EPR, and NMR spectroscopy to prove near-complete hybridization and a high stability of the complex. In addition, we show that experimentally determined PREs obtained from such a system yield valuable structural restraints.

(Figure 1a). Compared to the native aptamer domain (70mer),35 four base pairs were inserted into stem P1, while nucleotides inserted at the 3′-end represent the native sequence of the full length riboswitch. Spin labeling in trans was accomplished by cutting the RNA of interest into two pieces and hybridizing the fragments in a later step. Stem P1 of the 2′dG aptamer constitutes a helical region of appropriate length for hybridization of the nitroxide labeled oligomer fragment (Figure 1b). The four base pairs adjoining the binding pocket in stem P1 were not included to ensure stability of the binding pocket. The resulting aptamer domain consists of a large 68 nt RNA fragment and a short spin labeled 10 nt RNA fragment. The 68 nt fragment (68mer) was synthesized biochemically as a fusion construct with a 100 nt long hepatitis delta virus (HDV) ribozyme at the 3′-end by in vitro transcription with T7-polymerase.36 Introduction of a ribozyme to generate a defined 3′-end is crucial for successful application of this method as additional nucleotides randomly added by T7polymerase37 may interfere in the hybridization procedure. In case the oligomer fragment has to be attached to the 5′-end, it is recommended to ensure 5′-end homogeneity by transcribing the RNA with a 5′-hammerhead ribozyme. The shorter oligomer fragment (10mer) was spin labeled in a condensation reaction of 4-isocyanato-TEMPO with a 2′-amino modified uracil residue (U81) post solid-phase synthesis as previously described in the protocol by Edwards et al.38 This step is technically feasible for standard biochemical laboratories as both compounds required for the coupling reaction are commercially available. Hybridization conditions to form the complex between transcribed and spin labeled RNA fragments were optimized by adapting the Mg2+ concentration and temperature. The nitroxide was reduced by addition of a small excess of ascorbic acid to obtain a diamagnetic reference system required to determine PRE rates. Reduction of the spin label is the most reliable approach to generate a reference system, since the spin label may cause small structural alterations and concentration determination errors prevent reliable reproducibility. The redox state of the nitroxide radical was analyzed by cw-EPR spectroscopy prior to performing PRE measurements (Supplementary Figure S1). Hybridization of the Oligomer Fragment. We performed gel shift assays of the 68mer in the absence and presence of the TEMPO-labeled 10mer fragment and the cognate ligand 2′dG to analyze whether the two fragments form a tight complex (Figure 2). To investigate potential destabilizations caused by the presence of the spin label, the 68mer RNA fragment was also loaded in combination with unmodified 10mer. The native full length I−B aptamer domain (70mer) was loaded as reference, as free form and in the ligand bound state.

RESULTS AND DISCUSSION Spin Labeling Strategy and Construct Design. The noncovalent spin labeling strategy reported here was applied to the study of a 78 nt elongated version of the 2′dG responsive I−B aptamer domain of Mesoplasma f lorum34 as model system 1331 | ACS Chem. Biol. 2014, 9, 1330−1339

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unmodified 10mer, 2′dG, and Mg2+. Structural changes upon addition of a binding partner were monitored via changes in the imino proton resonance pattern in 1D NMR spectra. As imino proton signals can be observed only if they are protected from solvent exchange, they serve as reporters for RNA secondary structure. Preliminary assignments of all imino resonances of the native full length I−B aptamer domain in the ligand-bound form were obtained on the basis of the assignment of the structurally related I−A aptamer domain of M. f lorum by Wacker et al.35 Optimal hybridization conditions were determined by analyzing imino proton signals corresponding to the 10mer fragment, which can be detected only in the hybridized state. While the 68mer was prepared in 15N isotope labeled form, the 10mer obtained from solid-phase synthesis contains the natural abundance nitrogen isotopes (15N, 0.3%). This feature allows signal filtering41 of the two fragments and simplifies the detection of particular resonances in crowded regions of NMR spectra. Upon hybridization of the 10mer, five distinct imino proton signals are expected in 14N selected spectra. On the basis of these experiments, we determined that high excess of Mg2+ (>32 equiv) destabilizes the complex, whereas a minimum of 8 equiv of Mg2+ is necessary for ligand binding by the aptamer domain (Supplementary Figure S2). Full secondary structure characterization of the system was accomplished by sequential assignment of imino proton signals in a NOESY spectrum (Figure 3). The NOESY spectrum shows the formation of a stable and functional aptamer domain as all three helical segments P1, P2, and P3, as well as expected tertiary contacts of the ligand-bound state,35 could be assigned. In addition, we analyzed the thermal stability of the complex by monitoring imino proton resonances upon increasing the sample temperature. 1D spectra recorded at different temperatures showed that in the presence of 12 equiv of Mg2+ the 10mer does not dissociate from the 68mer below 40 °C and melting occurs cooperatively and together with the overall complex at approximately 50 °C (Supplementary Figure S3). During the melting procedure, U83 and U84 dissociate last and

Figure 2. Native gel electrophoresis analysis of the complex stability. 1: full length I−B aptamer (70mer); 2: full length I−B aptamer (70mer) + 2′dG; 3: 68mer; 4: 68mer + 10mer; 5: 68mer + 10mer + 2′dG; 6: 68mer + 10merTEMPO; 7: 68mer + 10merTEMPO + 2′dG; 8: 68mer; 9: 68mer + 10mer; 10: 68mer + 10mer + 2′dG; 11: I−B 68mer + 10merTEMPO; 12: I−B 68mer + 10merTEMPO + 2′dG. 2′dG and oligomer were added in 2-fold excess. Lanes 1−7 contain 8 equiv of Mg2+, and lanes 8−12 contain 32 equiv of Mg2+. Gels were run at RT.

According to analysis by native gel electrophoresis, the full length 2′dG aptamer does not show any difference in migration in the free and the ligand-bound state. The presence of 8 equiv of Mg 2+ presumably already stabilizes an RNA apoconformation with migration properties similar to those of the ligand-bound state.39,40 At 8 equiv of Mg2+, the migration speed of the 68mer in the presence of both unmodified and TEMPO-labeled 10mer is reduced compared to the 68mer alone. This reduction in speed indicates the formation of a stable duplex between the two fragments. Furthermore, we detect a decrease in hybridization stability for the TEMPOlabeled 10mer at higher Mg2+ concentrations underlining the significance of optimizing the Mg2+ concentration to obtain a stable RNA duplex. Rotational correlation time analysis of the TEMPO-label in cw-EPR spectra of the 10mer fragment and the 68 + 10mer complex also indicate successful hybridization (Supplementary Figure S1). NMR Characterization of the Unmodified Complex. Hybridization was first characterized by NMR with unmodified 10mer to ensure that the fragmented 68 + 10mer system forms a functional aptamer domain. The purified 68mer fragment obtained from in vitro transcription was titrated with

Figure 3. NOESY spectrum of the unmodified 68 + 10mer system in the ligand-bound form. The sample contained 450 μM 68mer, 2 equiv of 10mer, 2 equiv of 2′dG, and 16 equiv of Mg2+ in NMR buffer. The spectrum was recorded at 700 MHz and 283 K with a mixing time of 200 ms. 1332 | ACS Chem. Biol. 2014, 9, 1330−1339

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Figure 4. Overlay of 15N-HSQC (full length 70mer, black) and 15N-TROSY (68 + 10mer in the diamagnetic state, red). Green labels indicate residues that are fundamentally shifted due to sequential differences in the two structures. Residues that cannot be detected as they contain the 15N isotope only in natural abundance are labeled in blue.

NMR Characterization of the Nitroxide Labeled Complex. Hybridization of the TEMPO-labeled 10mer was optimized in accordance to the unmodified complex after reduction with ascorbic acid in the diamagnetic state. To avoid additional relaxation effects from intermolecular PREs in the paramagnetic state, we chose a sample concentration of 0.3 mM and kept the amount of oligomer to a minimum of 1.2 equiv. Slight excess was added to compensate for errors occurring during concentration determination by UV. The spin labeled 68 + 10mer system was characterized in both dia- and paramagnetic states in NOESY and 15N-TROSY spectra. The NOESY spectrum illustrates that both diagonal and cross peaks for imino protons of residues U17, U18, G79, G80, and U81 are missing in the paramagnetic state (Figure 5a). In addition, the 15N-TROSY spectrum (Figure 5b) shows significant attenuation of signals for U17, U18, and G14 in the paramagnetic state, indicating considerably enhanced proton relaxation induced by the TEMPO label. Since the chemically synthesized RNA is not 15N-labeled, the 15N-TROSY spectrum shows only resonances of the 68mer obtained from in vitro transcription, while G79 and G80 and U81 are not detectable. According to the published crystal structure of the I−A aptamer domain,43 particularly residues U17, U18, and G14 exhibit the closest distance toward the free electron of the spin label (13.2, 16.2, and 12.7 Å, respectively). In the diamagnetic state, diagonal peaks of residues G79 and U81 and the strong cross peak between G79 and U17 reappear in the NOESY spectrum (Figure 5a). Diagonal peaks for residues U17, U18, and G80 cannot be explicitly confirmed in the NOESY spectrum due to spectral overlap. The absence of NOE cross peaks and the weak intensity of diagonal peaks for residues in close proximity of the spin label in the diamagnetic state suggest slight weakening of the base pairing by the spin label, leading to enhanced solvent exchange of these imino protons. Since the detection of imino proton signals is highly dependent on their exchange rate with water, these perturbations can be considered minor. Consistent with findings for the unmodified 68 + 10mer system, residues U83, U84, G9, and G10 give rise to strong imino signals, indicating that the 3′-end of the 10mer is strongly hybridized to the 68mer. NMR analysis of the melting behavior is in line with these results as melting occurs cooperatively with the 68mer RNA, while the 3′-end of the oligomer fragment dissociates last.

therefore show stronger binding in comparison with guanine and uracil residues at the 5′-end of the oligomer fragment. Structural Integrity Fragmented vs Full Length Riboswitch. Regarding noncovalent attachment of spin labels, it is a central aspect whether the fragmented RNA construct is structurally in line with the full length construct, and most importantly, if it still represents a functional system. To address this question we have analyzed structural features and the ligand binding competence of the 68 + 10mer system and compared it to that of the native full length I−B aptamer domain (70mer). According to theoretical folding predictions,42 the truncated form of the aptamer domain (68mer) still folds helices P2 and P3, while stem P1 is absent. Prior to hybridizing the 10mer fragment, we analyzed if the cognate ligand 2′dG binds to the 68mer to induce folding of P1 by four base pairs in the absence of 10mer. This kind of prefolding would release the 5′ strand of P1 and therefore support hybridization of the 10mer in the ligand-bound state. Our results show that the 68mer binds 2′dG only at high excess of Mg2+ (32 equiv) and therefore requires strong Mg2+-induced prefolding of tertiary contacts (Supplementary Figure S4). On the contrary, the presence of the 10mer allows 2′dG binding of the aptamer domain at relatively low Mg2+ concentration (

Noncovalent spin labeling of riboswitch RNAs to obtain long-range structural NMR restraints.

Paramagnetic relaxation enhancement (PRE) NMR is a powerful method to study structure, dynamics and function of proteins. Up to now, the application o...
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