High yield spin labeling of long RNAs for EPR spectroscopy

ABSTRACT: Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we int...
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High yield spin labeling of long RNAs for EPR spectroscopy Mark Kerzhner, Hideto Matsuoka, Christine Wuebben, Michael Famulok, and Olav Schiemann Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00040 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Biochemistry

High yield spin labeling of long RNAs for EPR spectroscopy Mark Kerzhner1, Hideto Matsuoka†, 2, Christine Wuebben2, Michael Famulok*,1,3 and Olav Schiemann*,2 1

Life & Medical Sciences Institute Chemical Biology & Medicinal Chemistry Unit c/o Kekulé-Institut für Organische Chemie und Biochemie University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany 2 Institute of Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115 Bonn, Germany 3

Max Planck Fellowship Chemical Biology Group, Stiftung caesar , Ludwig-Erhard-Allee 2, 53175 Bonn, Germany

ABSTRACT: Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we introduce a spin labeling strategy based on click-chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex and long RNAs with short reaction times and suppressed RNA degradation. With this approach a 34 nucleotides long aptamer domain of the preQ1 riboswitch and a 81 nucleotides long TPP riboswitch aptamer could be labeled with two labels in several positions. We then show that conformations of the preQ1 aptamer and its dynamics can be monitored in the absence and presence of Mg2+ and preQ1 ligand by continuous wave (cw) Electron Paramagnetic Resonance (EPR) spectroscopy at room temperature and pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) in the frozen state.

INTRODUCTION RNA molecules are essential for many biological processes. Metabolite-sensing regulatory RNAs, or riboswitches, found in the non-coding regions of bacterial mRNAs control a broad range of genes in bacteria. In response to specific binding by cognate metabolites, riboswitches regulate protein-free gene expression. Due to their conformational variability, structural studies towards the dynamics of riboswitches are of great interest. Nuclear magnetic resonance (NMR)1-4 and Fluorescence Resonance Energy Transfer (FRET)5 are most commonly used to elucidate structural dynamics of riboswitches. Complementary to these methods, Electron Paramagnetic Resonance (EPR) spectroscopy in combination with site-directed spin labeling has also been employed to investigate local dynamics and conformational changes in riboswitches.6,7 Especially, pulsed EPR methods such as Pulsed Electron-Electron Double Resonance (PELDOR or DEER)8,9 enables one to measure distances in the range of 1.5 to 16 nm between sitespecifically incorporated spin labels in biomacromolecules10 and to follow their conformational dynamics.11,12 Two main strategies for site-directed spin labeling of RNA exist. One is the phosphoramidite method, in which spinlabeled phosphoramidites are incorporated at specific positions during the RNA solid phase synthesis.13,14,18 The other is postsynthetic spin labeling of reactive pre-functionalized sites in the RNA.15-18 The latter avoids exposing spin labels to the reducing environments during the oligonucleotide synthesis. This important advantage led to the development of various post-synthetic spin labeling approaches including the incorporation of paramagnetic nitroxides to all three sub-units of RNA: phosphate backbone,19,20 nucleobase21,22 and ribose sugar.23,24 For example, Grant et. al. used a deoxyribophosphorothiolate linkage in RNA as a reactive site for spin labeling with 1-oxyl-2,2,5,5-tetramethylpyrroline (R5).19 Another strategy, introduced by Ramos and Varani, attaches 3-(2iodoacetamido)-PROXYL (IAP) to 4-thiouridine modified

RNA.25 However, both spin labeling approaches are rather time consuming with reaction times of 12 – 20 h and have only limited yields. To improve the efficiency, a new approach was proposed on the basis of click chemistry on solid support and successfully applied to a 16 nucleotides long RNA in our previous work.26 The advantage of using click chemistry is that the reaction can be carried out in a straightforward way, provides high selectivity, specificity, high yields and convenient product purification. However, there are several disadvantages in spin labeling based on the solid support approach. Requiring 24 h of reaction time and two deprotection steps this method still remains rather tedious. In addition, all of the mentioned labeling methods are restricted to RNAs of lengths around 50 nucleotides (nt) that are amenable to chemical synthesis. This limit was overcome in some cases by spin labeling of two or more smaller RNA strands during solid phase synthesis followed by subsequent enzymatic ligation of the spin labeled RNA fragments. For example Duss et. al assembled a 70 kDa proteinRNA complex by spin labeling two 4-thiouridine modified RNA fragments and ligating them to each other with 20% – 40% efficiency using T4 DNA ligase.27 Thereby, a DTT-free ligation protocol was applied to prevent the reduction of the spin labels. Although this approach provides access to spin labeled RNA sequences of 70 nt or larger, the IAP spin label attached to the 4’-thio position drastically changes the hydrogen bonding pattern of the 4'-thiouridine base and changes the bonding situation within its ring system. Alternatively, Hoebartner and co-workers synthesized a 118 nt long SAM-I riboswitch containing a single spin-label, by site specific labeling of an 18 nt long RNA fragment at the exocyclic amino group of cytidine with 4-amino-TEMPO and subsequent ligation to an in vitro transcribed unmodified RNA using DNAzyme-catalysed ligation.7 The ligation yield of isolated spin labeled product amounted to 30% only and this approach

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is restricted by the sequence requirements at the ends of RNA sites. Here, we employ click chemistry (Scheme 1a) in solution combined with a ligation strategy to provide access to long spin labeled RNAs in high yields. The effectiveness of the insolution click-labeling procedure is shown on the aptamer domain of a preQ1 riboswitch from Fusobacterium Nucleatum consisting of 34 nt (Scheme 1b) and its applicability for labeling long RNAs on the 81 nt long TPP riboswitch aptamer domain (Scheme 1c). The remarkably high yields that this approach achieves enabled not only cw EPR but also PELDOR studies on these functional RNAs. RESULTS AND DISCUSSION Spin labeling by click chemistry in solution Click-labeling of DNA has been reported previously28,29 and we showed recently that it can be extended to RNA with the oligonucleotide still on solid support and fully protected.26 Especially the protection of the chemically unstable 2’hydroxy group was crucial. However, click labeling would be easier and more straightforward to use if it were possible to perform on the fully deprotected RNA in solution. In a first step, this was tested on the aptamer domain of a class I preQ1 riboswitch, which specifically binds 7-aminomethyl-7deazaguanine (preQ1). The preQ1 riboswitch contains only 34 nt and thus can be fully synthesized by conventional solid phase RNA synthesis. As shown in scheme 1, the click reaction was carried out with the RNA functionalized with the alkyne group and the nitroxide label functionalized with the azide group. The reason for using the functionalities in this order is that the 5-ethynyl2’-deoxyuridine is commercially available and that the alkyne group is chemically more stable under the reducing conditions of automated RNA synthesis than the azide. In addition, the N3-functionalized label 2 can be easily synthesized.30 Furthermore, due to the previously reported partial oxidation of ethynyl moieties during alkaline nucleobase deprotection31, commercially available triisopropylsilyl (TIPS)-protected 5ethynyl-2’-dU phosphoramidites were used for RNA synthesis. The reaction was performed in the following way: First the catalytic Cu(I)/THPTA complex was prepared by mixing Cu(I) with an excess of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) in a solution of DMSO. The resulting mixture was incubated for five minutes at room temperature. Using an excess of THPTA ensured that no free Cu(I) was present, which could damage the RNA. This solution was then added to a DMSO/water solution of alkynylated RNA and spin label 2, and the resulting mixture was incubated for 30 min at 37 °C. Using the solvent mixture DMSO/water ensured that all reactants were soluble. In contrast to previous reports on clicklabeling of DNAs,32 degassing of the solvent was not necessary (Fig. S1). Using these reaction conditions, the singly spin labeled preQ1 constructs dU4, dU23 and dU32 as well as the doubly labeled ones dU4-dU32, dU4-dU23 and dU4-dU32(G11A) were prepared for cw EPR and PELDOR measurements. Mutant dU4-dU32(G11A) was chosen as a control because the point mutation G11A leads to a drastically reduced affinity of the riboswitch to the ligand preQ1.33 LCMS monitoring of the reactions revealed that all RNAs were fully spin-labeled after 30 minutes. This reaction time is considerably shorter then reported previously for click-labeling of DNA.29,32 Quantita-

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tive conversion of the starting material was achieved even for the doubly labeled constructs and RNA degradation was almost completely suppressed, as shown exemplarily for preQ1 dŲ4-dŲ32 in figure 1 (see Fig. S2 for the others).

Scheme 1 a) Spin labeling of 5-ethynyl-2’-dU modified RNA 1 with the azide-functionalized nitroxide 2 in solution using click chemistry. b) Secondary structure of the preQ1 aptamer in the folded state. Spin labeled and mutated positions are indicated as X1, X2, X3 and X4. c) Secondary structure of the TPP aptamer domain in the metabolite-bound state. Spin labeled positions are indicated as dŲ. The black arrow indicates the ligation site.

According to ESI mass spectrometric analysis only small traces of attached but reduced nitroxide could be observed. Pure spin labeled oligos where obtained after one HPLC run. Starting with 2.5 nmol RNA, 1.8 nmol of labeled RNA were obtained on average, which corresponds to a yield of about 72%. The identity of the modified oligos was confirmed by ESI mass analysis (Fig. 1 and Table S1). For the sake of comparison, the single mutant preQ1 dŲ32 and the double mutant preQ1 dŲ4-dŲ32 were also labeled by click reaction on solid support.26 For this approach, the RNA on solid support was incubated with the label and the catalyst for a total duration of 24 hours, during which the solid support was washed, dried and treated with freshly prepared reaction mixture three times. Even after these three successive spin labeling procedures, considerably lower conversion of the starting material (36%) was observed for both RNAs (Figure S3). This observation clearly demonstrates that not only the reaction time is shorter for the in-solution approach but also the handling is easier and the yields are twice as large. The in-solution labeling strategy was then applied to the 81 nt long TPP riboswitch (Scheme 1c). Due to the large RNA size two singly ethynyl pre-functionalized RNA fragments of the TPP aptamer, consisting of 45 nt (TPP-dŲ20) and 36 nt (TPP-dŲ68), respectively, were spin labeled quantitatively using click chemistry in solution (Fig. 2). After labeling the two fragments, they were assembled by splinted enzymatic T4

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Biochemistry nor effect of the dŲ label on the secondary structures of the preQ1 and TPP riboswitches (Fig. S4). In addition, only small decreases in Tm were observed, where preQ1 dŲ4 exhibits the largest decrease at 8 °C (Tab. 1), supporting the finding that the dŲ label has only a minor and likely just local effect on the preQ1 and TPP structures. For dŲ32, the Tm decrease by 5 °C correlates with the observation, that this label site is fairly occluded according to the mtsslWizard results (SI page S4).

Figure 1 HPLC and ESI mass analysis of preQ1 dŲ4-dŲ32 after 30 min of reaction. a) HPLC profile with the UV peaks recorded at 260 nm identified as follows: I = two reduced nitroxides, II = one reduced nitroxide, III = double spin labeled RNA. The correlation of UV signals is based on LCMS analysis. b) ESI mass spectrometric analysis of the UV peak III (collected from 9.710.1min). Mcalcd(preQ1 dŲ4-dŲ32) = 11376.78 m/z and Mfound(preQ1 dŲ4-dŲ32) = 11376.71 m/z.

DNA ligation analogously to previously published protocols.34,35 Afterwards, the doubly spin labeled full-length TPP aptamer was separated from the unreacted RNA fragments at 80 °C on an anion-exchange HPLC. Performing the ligation on a 5 nmol scale, 2.5 nmol of doubly labeled full-length TPP aptamer were obtained after purification (yield 50%). The purity of the doubly labeled product was verified by HPLC and ESI spectroscopic analysis (Fig. 2). The presence of dithiothreitol (DTT) in the ligation buffer did not affect the spin labels demonstrating the high stability of the dŲ label towards reduction.

Figure 2 HPLC profiles of a) TPP dŲ68 and of b) TPP dŲ20 after 30 min of reaction. c) HPLC profile of TPP dŲ20-dŲ68 after purification. The major UV signal in all HPLC chromatograms corresponds to the spin labeled RNA. d) ESI mass spectrometric analysis of TPP dŲ20-dŲ68. Mcalcd(TPP dŲ20-dŲ68) = 26596.17 m/z and Mfound(TPP dŲ20-dŲ68) = 26595.14 m/z.

Tm and CD control In a previous study reporting the influence of the dŲ label on the structure of a RNA duplex26 only minor differences were found between the unlabeled and labeled construct with respect to their Tm values and CD spectra. Also here, the CD spectra of the labeled riboswitches revealed only a slight decrease of the extrema at 210 nm and 260 nm indicating a mi-

Table 1. Differences in Tm-values for the spin labeled riboswitches as compared to the unlabeled ones.a

Construct preQ1 dŲ4 preQ1 dŲ23 preQ1 dŲ32 preQ1 dŲ4-dŲ23 preQ1 dŲ4-dŲ32 preQ1 dŲ4-dŲ32 (G11A) TPP dŲ20-dŲ68

∆Tm [°C]b w/o Mg2+

∆Tm [°C]b,c with Mg2+

-8 -3 -5 -2 -5 -5 -2

-4 +3 +1 -2 -6 -2 ±0

a

The absolute values are collected in the SI. Values are given per label. Melting curves of the constructs with the preQ1 ligand bound could not be recorded due to the UV absorbance of the ligand at 260 nm. b c

pre Q1 riboswitch: CW-EPR All labeled preQ1 riboswitch constructs display cw X-band EPR spectra at room temperature that are typical for partially immobilized nitroxide spectra and show no evidence for the presence of free label. Spin counting reveals quantitative spin labeling (Table S4). As an example, the cw EPR spectra of three samples of preQ1 dŲ32 (preQ1 dŲ32 alone, with Mg2+, and with Mg2+ plus the preQ1 ligand) are shown in Figure 3a (for the others see SI). Each of the three spectra is a superposition of two spectra, one with broad spectral width yielding the broad shoulder on the low field site and the other with narrower spectral width contributing mainly to the three intense lines. Only changing the rotational correlation time τc and the ratio of both spectra but using the same parameters otherwise can simulate both spectra. Figure 3 shows the best-fit simulated EPR spectra overlaid with the experimental ones. The narrower spectrum corresponds to a more mobile nitroxide label with a τc of 0.7 ns (Fig. 3, blue line) and the broader one to a more immobile one (Fig. 3, green line) with a τc of 6.3 ns. In the absence of Mg2+ and ligand, the fraction of the mobile species is 71% and that of the more immobile one 29% (Tab. 2). Upon addition of Mg2+ the fraction of the mobile species is reduced to 37% and further to 19% after adding the ligand. Simultaneously, the fraction of the immobile species increases to 63% and 81% after adding Mg2+ and then the ligand, respectively. This observation can be explained in accordance with the model of Breaker39 that was later confirmed by Micura using NMR36 in which the preQ1 riboswitch adopts a stem-loop structure with a single stranded 3’ overhang (Scheme 2, conformation I) in the absence of Mg2+.36 Upon addition of Mg2+, the riboswitch pre-organizes into a pseudoknot-like structure (conformation II). Subsequent addition of the preQ1 ligand leads only to small structural changes (conformation III).37

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Figure 3 Room temperature cw X-band EPR spectra of a) preQ1 dŲ32 and b) preQ1 dŲ4-dŲ32(G11A) in the absence of preQ1 and Mg2+ (red), after addition of Mg2+ (black) and after addition of Mg2+ and preQ1 (blue). The signal intensity was normalized to the central peak height. The peak marked by an asterisk is due an impurity in the glass of the tube. Experimental (red) and calculated (black) CW-EPR spectra of c) preQ1 dŲ32 and d) preQ1 dŲ4-dŲ32(G11A) in the absence of preQ1 and Mg2+, after addition of Mg2+ and after addition of preQ1 at room temperature. The calculated spectra composed of two contributions with different mobility: more mobile (blue) and less mobile (green) spin labels. Conditions: cpreQ1 dŲ32 = 100 µM, cpreQ1 dŲ4-dŲ32(G11A) = 50 µM, 10 mM cacodylate buffer; cMg(II) = 2 mM, cpreQ1 = 1.2 mM.

In conformation I, the spin label at position U32 is located in a mobile, single stranded region. This conformation yields the narrow/mobile cw EPR spectrum, which is the dominating fraction in the absence of Mg2+. Adding Mg2+, this fraction reduces and the broader, immobile fraction increases in weight, which is assigned to conformation II. In conformation II, the single stranded overhang is bound to the loop and thus mobility at position U32 is greatly reduced giving rise to the EPR spectrum corresponding to the more immobile conformation. Obviously, this motional model assumes that pseudoknot formation leads to a spectrum that is dominated by slower global tumbling and that the local faster motion can be Table 2. Quantifications of mobile and immobile components of cw EPR spectra for the preQ1 constructs. w/o [%]a

Mg2+ [%]a

I

II+III

I

II+III

I

II+III

71 12 71 71 12 59

29 88 29 29 88 41

37 10 37 37 10 29

63 90 63 63 90 71

19 10 19 19 10 17

81 90 81 81 90 83

24

76

20

80

15

85

neglected.The small structural changes after addition of the ligand are not mirrored in the rotational correlation time. Instead, the ligand leads to a larger population of the two more immobile conformations II and III. Interestingly, the pseudoknot-like conformations are already populated in the absence of Mg2+. This has not been seen by room temperature NMR36 but maybe a consequence of the lower sensitivity of NMR with respect to EPR and the small extent to which these conformations are populated in the absence of Mg2+. The cw EPR spectra of preQ1 dŲ4 (Fig. S8) are very similar to that of preQ1 dŲ32 and the slow motion fraction also increases upon addition of Mg2+ and the ligand indicating that also position U4 can sense the structural rearrangement of the preQ1 riboswitch (Tab. 2).

preQ1 [%]a

preQ constructs dŲ4 dŲ23 dŲ32 dŲ4-dŲ23b

dŲ4-dŲ32c dŲ4-dŲ32 (G11A)d a

The number in front of the back slash denotes the fraction of the mobile, the number behind the slash, the fraction of the immobile component. b Simulations were done by assuming four components, fast and slow for each label site. The weight ratio was set to 50:50 for the two sites. c In these two cases only one mobile and one immobile component was sufficient to simulate the spectra, because preQ1 dŲ4 as well as preQ1 dŲ32 have identical τc values. d Simulations were done by assuming only two components, fast and slow.

Scheme 2 Schematic view of the conformational exchange proposed for the preQ1 riboswitch aptamer.36,38, 39 Spin labeled positions are depicted by dŲ.

This result is somewhat unexpected because position U4 locates within the stem region for which the movement of the label should be more restricted. However, the Tm studies revealed the largest drop of -8 °C in Tm for dŲ4, which may indicate that the stem is disrupted at this site leading to a higher mobility than expected. Although the U4 site is not involved in the loop/overhang interaction the sensing of the conformational rearrangement is traced back to the 3D structure (Fig. 4), where the folding in conformations II and III restricts the mobility of the label. Also for preQ1 dŲ23, the cw EPR spectra are a superposition of two spectra (Tab. 2 and

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Biochemistry

Figure 4 Top: The derived distance distributions (red lines) for doubly labeled preQ1 dŲ4-dŲ32 a) without Mg2+ and preQ1, b) in presence of Mg2+ and c) after addition of preQ1. Each distance distribution was deconvoluted by two Gaussian components (gray and green dashed lines). Bottom: d) MtsslWizard distance distributions for preQ1-bound states of preQ1 dŲ4-dŲ23 (black) and preQ1 dŲ4dŲ32(blue). Next are shown the crystal structures of the preQ1 riboswitch for (e) preQ1 dŲ4-dŲ32 and (f) preQ1 dŲ4-dŲ23 in the preQ1bound state (PDB 3Q50), where the spin labels have been attached by means of the program mtsslWizard, are drawn as blue sticks with a red-coloured oxygen atom. Conditions: 20% deuterated ethylene glycol/buffer (10 mM cacodylate buffer), cRNA = 50 µM, cMg(II) = 2 mM, cpreQ1 = 1.2 mM.

Fig. S10). But the mobile and immobile species have slower rotational correlation times than found for dŲ32 and dŲ4. In addition, the 88% level of the fraction of the more immobile species is fairly high in the absence of Mg2+ and increases only marginally to 90% upon adding Mg2+ and preQ1. This steady level indicates that the label at position U23 is located within a rather rigid region that is not much influenced by the overall conformational change. Also these observations fit to the Micura model, where U23 locates right next to the stem and is not involved in the loop/single strand interaction. CW EPR measurements were also performed on the doubly labeled constructs preQ1 dŲ4-dŲ23 and preQ1 dŲ4-dŲ32 (Fig. S12 and S13). The spectra are a superposition of the spectra of the respective singly labeled constructs with slight differences in the fractions. To confirm that the observed spectral change after addition of Mg2+ and the ligand is due to the conformational change, cw EPR measurements were also performed on the mutant preQ1 dŲ4-dŲ32(G11A). Interestingly, its cw EPR spectrum contains a large fraction of immobile component already without Mg2+ and ligand, which only increases marginally upon addition of Mg2+ or Mg2+ plus preQ1 (Fig. 3b). This result is in nice agreement with a previous ligand binding study, which reported that the binding of the ligand was severely reduced because the loop overhang interaction is hindered.33 This supports the interpretation that the spectral change observed for the unmutated constructs is correlated with the conformational change. However, the reason why the immobile component dominates the spectra of preQ1 dŲ4-dŲ32(G11A) still remains unclear since the structure of this mutant is unknown.

preQ1 riboswitch: PELDOR If Mg2+ binding leads to a conformational change in the riboswitch, it should also be possible to observe this in PELDOR experiments. PELDOR measurements between spin labels yield label-to-label distance distributions, are wellestablished and have been used frequently on proteins11,40 but are rarely applied to riboswitches.6 In contrast to the cw EPR data, PELDOR data are collected in the frozen state. Previous PELDOR studies of a dŲ modified RNA26 duplex revealed orientation selection, however in the case of preQ1 orientation selectivity was found to be negligible most likely due to the higher conformational flexibility of preQ1 as compared to a duplex (Fig. S15). Figure 4 shows the PELDOR derived distance distributions for preQ1 dŲ4-dŲ32. The distance distribution of dŲ4-dŲ32 in the absence of Mg2+ can be deconvoluted with two Gaussians that peak at 1.9 nm and 2.8 nm with weights of 59% and 41%, respectively. In order to determine the appropriate conformation for the two Gaussian components, the X-ray structures of conformation II and III were labeled in silico with dŲ at positions U4 and U32 within the program mtsslWizard41-43 and the distance distributions were generated (Fig. 4). In the preQ1-bound state, conformation III, a mean distance of 2.7 nm was obtained, which fits nicely to the experimental distance at 2.8 nm, as shown in Figure 4e. For conformation II, the in silico labeling in mtsslWizard reveals that this site in conformation II is fairly occluded. This might indicate, that the introduction of the label at this site comes along with local structural changes. However, conformation II yields within mtsslWizard under these conditions a mean distance of 1.7 nm (Fig. S25), which fits nicely to the experimental distance of 1.9 nm. The secondary structure of

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conformation I suggests a longer distance than for conformations II or III, but such longer distance was not observed in the PELDOR measurements. NMR showed that the pseudoknot formation occurs at lower temperature even in the absence of Mg2+,36 indicating that conformation I is mostly changed to conformation II at 50 K. After addition of preQ1, the weights of the two components at 1.9 nm and 2.8 nm change to 21% and 79%, respectively. The result indicates that only the fraction of conformation III increases after addition of preQ1, which is in accordance with the NMR data. As described above, the cw EPR measurements allowed us to discriminate between conformation I and II/III. On the other hand, the PELDOR measurements enabled to investigate the difference of conformation II and III. Table 3. Deconvolutions of the distance distributions for doubly labeled preQ1 mutants w/o Mg2+, with Mg2+ only and with Mg2+ and preQ1. w/o

preQ constructs dŲ4-dŲ23 dŲ4-dŲ32 dŲ4-dŲ32 (G11A)

Mg

preQ1

nm

%

nm

%

nm

%

II

1.9

100

1.9

100

1.9

100

II III II III

1.9 2.7 1.9 3

59 41 61 39

1.9 2.7 1.9 3

53 47 70 30

1.9 2.7 1.9 3

21 79 89 11

To verify the assignment of the two distance components, PELDOR measurements were also performed on the mutant preQ1 dŲ4-dŲ32(G11A). As shown in Fig. 5a, the resulting distance distribution does not change after adding Mg2+ and preQ1 ligand. This finding strongly supports the assignment made here because the G11A mutation inhibits ligand binding and pseudoknot-formation.33 After addition of Mg2+ and preQ1 a small distance shift of 0.2 nm is observed, which may be connected to a small structural change upon ligand binding but the change is too small as to be regarded as reliable. For conformation I, the result indicates that the distance between the spin labels might be outside the range of the current PELDOR measurements, or that the interspin distance is widely distributed due to the very high conformational flexibility. This speculation would be supported by the shallow modulation depth shown in Fig. S18b. PELDOR measurements were also performed for the preQ1 dŲ4-dŲ23 construct, which should be insensitive to the pseudoknot formation according to the cw EPR data and the model. Indeed, independent of the presence or absence of Mg2+ and preQ1, the peaks of the distance distributions were only observed around 1.8 nm, as shown in the Fig.4d, e and 5b.

Figure 5 Distance distributions derived from the PELDOR time traces for a) preQ1 dŲ4-dŲ32(G11A) and b) preQ1 dŲ4-dŲ23

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in the absence of preQ1 and Mg2+ (red), after addition of Mg2+ (black) and after addition of preQ1 (blue). Conditions: 20% deuterated ethylene glycol/buffer (10 mM cacodylate buffer), cRNA = 50 µM, cMg(II) = 2 mM, cpreQ1 = 1.2 mM.

TPP riboswitch For the TPP riboswitch, the cw EPR and PELDOR data are only shown as proof-of-principle and only for the riboswitch without Mg2+ and TPP ligand. The cw EPR spectrum of TPP dŲ20-dŲ68 in the absence of Mg2+ and TPP ligand is shown in Fig. 6a. The spectrum is typical for a partially immobilized nitroxide demonstrating the successful incorporation of the spin labels into the TPP riboswitch. Computer simulations revealed again two a more mobile and a more immobile fraction, where the fraction of the more mobile species has a τc of 5.0 ns and amounts to 63.5% while the fraction of the less mobile species has a τc of 101.9 ns and a weight of 36.5% (see Table S13 for parameters). The PELDOR measurement on the same construct yields the time trace in Figure 6b. The time trace reveals one long oscillation period with a modulation depth of 20%. The time window of 12 µs is long enough to yield reliable distances of up to 8 nm. In the obtained distance distribution, two broad peaks are observed at 5.1 nm and 7.2 nm, indicating the presence of at least two conformations in the absence of both, Mg2+ and ligand (Fig. 6b, inset).

Figure 6 a) Experimental (red) and simulated (black) cw X-band EPR spectra of TPP dŲ20-dŲ68 in the absence of both TPP and Mg2+ at room temperature. The calculated spectra are composed of a more mobile (blue) and a less mobile (green) spin label. b) The PELDOR derived distance distribution for the same construct. Conditions: 20% deuterated ethylene glycol/buffer (50 mM HEPES buffer), cRNA = 50 µM.

CONCLUSIONS The spin labeling of a preQ1 and a TPP riboswitch was accomplished with unprecedented efficiency by click chemistry in solution. The new spin labeling strategy enabled a significantly reduced reaction time, a high labeling efficiency, and a significant suppression of RNA degradation during the spin labeling procedure. For the preQ1 riboswitch, the conformational equilibrium and pseudoknot formation could be monitored by cw EPR and PELDOR measurements. The correlation between the conformational change of the preQ1 riboswitch and EPR/PELDOR results were confirmed through the EPR and PELDOR measurements of the preQ1 dŲ4-dŲ32(G11A) mutant. The application of the spin labeling strategy was extended to the large TPP riboswitch that cannot be synthesized on solid support in sufficiently high quantity. The distance measurement showed that two stable conformations for the TPP riboswitch exist

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Biochemistry even in the free state. Thus, cw EPR and PELDOR in combination with site directed spin labeling of RNA via clickchemistry is well suited for studying the conformational dynamics of large functional RNAs.

ASSOCIATED CONTENT Supporting Information Original PELDOR time traces, CD-spectra, Tm-values etc. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected] Present Addresses † Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan

Author Contributions All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT Funding from the DFG via the SFB813 “Chemistry at Spin Centers” project A6, and by the Max-Planck Society is gratefully acknowledged.

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SYNOPSIS TOC Click chemistry in combination with ligation is applied for site-selective and high yield spin labeling of large riboswitches. The incorporated nitroxide dŲ allows monitoring the conformational change within the preQ1 riboswitch using cw EPR and PELDOR spectroscopy. TOC

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