Alkyne-Functionalized Coumarin Compound for Analytic and

Subscriber access provided by University of Newcastle, Australia

Article

Alkyne-functionalized coumarin compound for analytic and preparative 4-thiouridine labeling Katharina Schmid, Maria Adobes-Vidal, and Mark Helm Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00035 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Alkyne-functionalized coumarin compound for analytic and preparative 4-thiouridine labeling Katharina Schmid†, Maria Adobes-Vidal‡ and Mark Helm†*

†

Institute of Pharmacy and Biochemistry, Staudingerweg 5, Johannes-Gutenberg University Mainz, D55128 Mainz, Germany ‡

Electrochemistry & Interfaces Group, Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Bioconjugation of RNA is a dynamic field recently reinvigorated by a surge in research on posttranscriptional modification. This work focusses on the bioconjugation of 4-thiouridine, a nucleoside that occurs as a post-transcriptional modification in bacterial RNA, and is used as a metabolic label and for crosslinking purposes in eukaryotic RNA. A newly designed coumarin compound, named 4-bromomethyl-7-propargyloxycoumarin (PBC) is introduced, which exhibits remarkable selectivity for 4-thiouridine. Bearing a terminal alkyne group, it is conductive to secondary bioconjugation via “click chemistry”, thereby offering a wide range of preparative and analytical options. We applied PBC to quantitatively monitor the metabolic incorporation of s4U as a label into RNA, and for site specific introduction of a fluorophore into bacterial tRNA at position 8, allowing the determination of its binding constant to an RNA modification enzyme.

Introduction Bioconjugate functionalization is a useful and powerful approach that has advanced RNA research throughout the last decades. RNA, in its versatile functions, is a highly dynamic biomolecule which is continuously transcribed and degraded, making it an interesting target for various avenues of research, and therefore, for functionalization and labeling.1 For many analytical strategies, effective functionalization of RNA is a prerequisite. One may roughly divide strategies for bioconjugation and functionalization in co-synthetic versus post-synthetic as one aspect, and distinguish in vitro versus in vivo labeling as another aspect. A further important aspect is site-specificity versus random labeling. Relevant examples for site-specific co-synthetic labeling are typically found in applications of solidphase synthesis2 with modified building blocks.3 This strategy has enabled the site-specific incorporation of dyes,4 non-natural nucleosides,5,6 or bioidentical RNA modifications.3 In contrast, cotranscriptional functionalization of RNA with chemically modified nucleosides is distributive thus leading to statistical incorporation of the label. It can either be conducted in vitro or in vivo, but is restricted by the substrate spectrum of the RNA polymerase. Typical in vitro transcription experiments use T7-RNA polymerase and may incorporate a single site specific label at the 5’-end by an appropriate initiator nucleotide.7,8 All subsequent incorporation of non-standard ribonucleotidetriphosphates typically involves application of the latter in a mixture with the corresponding unmodified ribonucleotide-triphosphates, e.g. Ψ or s4U.9–11 Incorporation of modified nucleobases or nucleosides in vivo suffer from additional restrictions with respect delivery, e.g. membrane permeability or transporter mediated uptake in tissue culture. Here, several synthetic uridine derivatives including 5-ethinyluridine,12 5-bromouridine13 and 4-thiouridine (s4U)14 have successfully been established. Post-synthetic derivatization approaches are frequently distributive, if the target RNA is only composed of canonical building blocks. Attachment of fluorescent dyes or other labels has been accomplished with different highly reactive electrophiles,15–18 including also electron poor bromomethylcoumarin derivatives.19 A particularly interesting approach involves the derivatization of RNA containing non-standard nucleosides. In these cases, site specificity is not governed by the bioconjugate reagent, but determined by the initial site of modification. A point in case is 4-thiouridine (s4U), the focus in this study. This thiolated uridine is popular in the context of its application as metabolic label in eukaryotes, were it is randomly distributed during transcription. In addition, it is a naturally occurring RNA modification in bacteria. It is post-transcriptionally and site-specifically introduced by the s4U synthethase ThiI in the tRNAs of eubacteria,20–22 mainly at position 8 of tRNAs, and infrequently at position 9. The level of thiolation varies with the growth rate of the bacteria. Data on its importance for translation regulation are sparse,23 but it has been reported to be responsible for photoprotection and growth-delay upon near UV irradiation.24,25 Due to the s4U’s dual relevance as natural occurring modification and metabolic label, several analytical strategies have been described for this nucleoside. 2 ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


This includes methods which rely on organomercurial affinity matrices,14,23 methods based on the afore mentioned UV-crosslinking26,27 as well as the recently developed, methylthiosulfonate-activatedbiotin (MTS-biotin) which depends on formation of reversible disulfide bonds between the biotinreagent and s4U.28 Even though there is a variety of established methods for studies on s4U, desirable properties of a bioconjugate reagent include facile detection by (i) UV absorption and/or by (ii) fluorescence. Furthermore, when PAGE and chromatography techniques are used for workup, multicore heterocyclic aromats tend to offer new possibilities for efficient separation and purification, e.g. by (iii) increasing the overall lipophilicity of the conjugate. Also desirable in the context of the purification of conjugates is a so-called (iv) click-handle, i.e. a functional group that allows even further derivatization via so called click chemistry, which has been shown to open numerous routes to new applications in labeling and isolation of thus tagged RNAs.29 A cardinal criterion is obviously (v) high selectivity for s4U in the face of a large population of structurally similar uridine residues. Our starting point for the design for a new selective s4U label with a maximum number of the above mentioned properties (i) – (v), was the 4-bromomethylcoumarin scaffold, which had been established as an RNA label already.19,30,31 4-bromomethylcoumarin derivatives can alkylate nucleobases in a nucleophilic substitution reaction with the bromine as leaving group.

Results and Discussion Design and synthesis of a selective bionconjugation reagent for s4U The design of a selective and multifunctional bioconjugation reagent for thiouridines was based on the considerations already outlined above, namely (i-ii) analytics, (iii-iv) purification of conjugates, including facilities for tagging, labeling or other bioconjugation concepts and, last but not least (v) selectivity. Based on previous results, we continued work on a coumarin-scaffold, which offered numerous substitution sites for fine-tuning of reactivity and selectivity via electron-density of the aromatic system, as previously reported.32 As an inherent benefit, the coumarin moiety shows UVabsorption around 320 nm, allowing side-by-side detection with nucleic acids. An additional feature facilitating analytics is the blue fluorescence of coumarins. While we had found an electron-poor 7azido-4-bromomethlycoumarin to be quite reactive and rather little selective19, various electron-rich bromomethylcoumarins showed a strongly increased selectivity in comparison. Among the latter, subtle differences in selectivity caused by variegated substitution patterns were relatively minor when compared to effects caused by variation of external parameters such as pH and solvent content.32 In general, the nucleophilicity in such reactions can be influenced by buffer pH and amount of aprotic solvents, such as dimethyl sulfoxide (DMSO) in the reaction mixture.31 Looking at the reaction of nucleosides with coumarins, which is likely to be an SN2 reaction, it was shown that high amounts of DMSO promote nucleophilicity, which is expected to increase reactivity, and, thereby, reduce selectivity. Slightly alkaline pH led to less substrate deprotonation and hereby to an overall increased reactivity.32 The above suggested that the introduction of a “clickable” moiety (such as a terminal alkyne) to an electron-rich bromomethylcoumarin derivative might lead to a compound displaying the combined requirements (i)-(v), as outlined above. Given our previous work with commercial 4-bromomethyl-7methoxycoumarin (BMB), we reasoned that replacement of the 7-methoxy group with a 7propargyloxy group would minimally affect electron density (and hence, selectivity) and simultaneously allow to conduct further bioconjugation of derivatized RNA via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).33–36 We hence pursued the synthesis and characterization of 4bromomethyl-7-propargyloxycoumarin (PBC) (Figure 1). 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1: Concept of PBC as a bioconjugate reagent with selectivity for s4U over U. The selectivity arises from different nucleophilic moieties attacking the bromomethylgroup of 4-bromomethyl-7propargyloxycoumarin, namely sulphur (from s4U) being largely more nucleophilic than oxygen (from U). This nucleophilic substitution is likely to be an SN2 reaction. The propargyloxy-substituent supplies the possibility for secondary conjugation via CuAAC chemistry (red).

4-bromomethyl-7-propargyloxycoumarin (PBC) (1) was obtained in a total yield of 3.6 % in a fivestep synthesis (Fig. 2). 7-hydroxy-4-methylcoumarin (3) was readily accessible via a Pechmann condensation37 using 1,3-dihydroxybenzene (2) and ethyl acetoacetate. To prevent bromination in position 7, the hydroxy-group was protected via acetylation with acetic anhydride leading to 7acetoxy-4-methylcoumarin (4) in a very good yield of 99%.38 Subsequent radical bromination provided 4-bromomethyl-7-acetoxy-coumarin (5).39 The low yield of 11% compared to 60%, described in literature, might be related to the simultaneous formation of the 4,4-dibromo-product. Deprotection was carried out according to Furusho et al., yielding 98 % of 4-bromomethyl-7hydroxycoumarin (6).40 The ultimate reaction step was terminal alkylation41 of the coumarin compound at the hydroxy group at position 7, finally providing PBC (1). The pale-yellow solid was analyzed by 1H NMR and 13C NMR (Figure S1) spectroscopy as well as FD-mass spectrometry and IR-spectroscopy (shown in Figure S2).

Figure 2: Synthesis scheme of PBC. A: ethyl acetoacetate, 14 h, room temperature. B: acetic anhydride, reflux for 3 h. C: N-bromosuccinimide in carbon tetrachloride and catalytic amount of benzoyl peroxide, reflux overnight. D: HClaq in tetrahydrofuran, 48 h. E: Potassium carbonate and propargyl bromide in acetone, reflux for 12 h. Percentages show obtained yields. The numbers in parentheses show the yields given in literature.


Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Evaluation of reaction conditions to enhance 4-thiouridine selectivity

To assess the selectivity of PBC for s4U, native tRNATyr from Escherichia coli and a corresponding in vitro transcribed tRNA (IVT) were derivatized with PBC and subsequently “clicked” to a fluorescent dye by CuAAC. Selectivity would then be determined as the ratio of fluorescently-labeled thiouridine to fluorescently-labeled uridine residues (Figure 3A). The native tRNA, which contains s4U residues at positions 8 and 9, thus served as positive control, while the IVT constituted the negative control since it did not contain any modification. Given that selectivity of coumarin compounds significantly depends on the composition of the reaction mixture regarding buffer pH, and amount of DMSO, we intended optimization of these parameters. Per previously conducted experiments with other coumarin derivatives a starting condition set I was defined as pH 8, 70 % (v/v) DMSO, 3 h and at 37 °C. Because preliminary experiments had indicated a very low IVT signal as a consequence of the reagent’s selectivity for s4U, the reaction mixture of the negative control contained 10 mM PBC, rather than 1 mM as in the native control. Furthermore, the RNA concentration was increased from 0.1 µM in the native RNA to 1 µM in the negative control. Two other conditions designed to attenuate nucleophilicity included a decrease to pH 7.0, one of which (condition set II) maintained DMSO content at 70% as in condition set I, and in the other one (condition set III), the DMSO content was lowered to 50% (conditions set III). RNA samples treated under these conditions were precipitated by ethanol, conjugated to ATTO 488 azide by CuAAC, and again precipitated. Equal fractions of the reactions, (i.e. 10-fold more RNA for the IVT samples) were then run on a 10% urea-PAGE (Figure 3 B). The intensity of bands emitting fluorescence at 520 nm (bandpass 40 nm) was scanned upon excitation at 488 nm (for scanning details see supplement S1). Upon decreasing nucleophilicity from condition set I to II, and further to III, visual inspection of the gel in Figure 3B shows a strong decline of the IVT signal, and a moderated decline in the native tRNA signal, both observations indicating a significant drop in reactivity. An interesting side observation concerns the different running speeds of IVT tRNA and native tRNA in the gel. For verification, the identity of both samples was confirmed by Deep Sequencing (data not shown), the results suggesting that the differential migration might be assigned to the influence of the nucleoside modifications in the native tRNA. A numeric value for selectivity was approximated from the fluorescence signal obtained for native tRNA and that from the IVT. The intensities were measured by ImageJ42 software and corrected for the gel’s background (Figure 3B). Figure 3C shows the mean signals and standard deviation from three independent derivatizations. The background-corrected signal from native tRNA was corrected for the number of s4U residues, and correspondingly the IVT signal was corrected for the number of uridine residues. The latter also included corrections for the increased concentrations of IVT and reagent used (vide supra) in comparison to the reaction of the native tRNA. The contribution of unmodified uridines in the native tRNA was considered negligible, since it amounted to less than 1% for selectivity values above 100. The corresponding selectivity values for condition sets I, II and III are shown in Figure 3D, including error bars which depict the standard deviation of the selectivity factors of three independent derivatizations. Of note, selectivity factors for condition sets I and III were verified by LC-MS/MS to be in the correct order of magnitude (see Fig. S3). Very clearly, the reduction of nucleophilicity due to lower pH and less organic solvent led to decreased reactivity, and a correspondingly increased selectivity.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3: Experimental determination of selectivity factors. A: Structures of PBC-dye conjugates. U-PBCdye conjugate appeared as sole product in reactions with in vitro transcribed tRNA, while s4U-PBC-dye conjugate is the predominant product from native tRNA samples. Sulphur and C4-oxygen in the figure are not to scale. Blue stars represent ATTO 488 labels introduced by CuAAC. B:10% urea-PAGE showing one example of each tested reaction condition. The red arrow indicates the band for IVT under most selective reaction conditions (middle grey bar in 3C) which is barely detectable. Settings for fluorescence scan are detailed in Table S1. C: Mean pixel-intensities from tRNATyr, E. coli and IVT samples, run on a 10% denaturing PAGE. Signal intensity of ATTO 488 was measured with ImageJ software and corrected to the gels background. D: selectivity factors calculated from mean-pixel intensities, revealing buffer pH of 7, 50% (v/v) DMSO and reaction time of 2 h as the most selective conditions. Error bars are standard deviations from selectivity factors of three independent derivatizations.

To assess a different target RNA sequence, we switched further investigations to native tRNAVal, which, in contrast to the previously used tRNATyr, contains only a single s4U residue. For further characterization of condition set III, the PBC concentration was varied between 0 and 10 mM at equal concentrations of native tRNAVal and IVT, and with otherwise unchanged parameters. As shown in Figure 4A, the signal for in vitro transcribed tRNA evolves from barely detectable at 1 mM PBC to reasonably apparent at 10 mM PPC, whereas native tRNA is well visible at 1 mM and strongly visible at 10 mM. As another negative control, previous treatment with a concentration of H2O2 that oxidizes s4U to U 43 (Fig. S4) leads to a decrease of the native tRNA signal to near-IVT level, confirming that the observed effect is dependent on the presence of s4U. Aliquots of the above-mentioned samples had been set aside before click derivatization for LCMS/MS analysis. Figure 4B shows analyses of the U-PBC and s4U -PBC conjugates for IVT and native tRNAVal at 1 mM and 5 mM PBC (see Table S2 for the monitored mass transitions). While no 6 ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


signal for U-PBC was detectable from native tRNA at 1 mM PBC, a weak signal appears at 5 mM which is just under the LOD, i.e. the signal-to-noise ratio (S/N) was 2.6, while limit-of-detection is defined as S/N >3. In contrast, signals from equivalent amounts of IVT were well above LOD at 1 mM PBC, and yet more pronounced at 5 mM PBC. Furthermore, analysis of the s4U-PBC conjugate showed no signal from IVT samples, and a strong signal for native tRNA that increased from 1 mM to 5 mM PBC. At the latter concentrations, detection of residual s4U confirmed, that the conjugation reaction was incomplete up to 5 mM PBC, whereas the absence of detected s4U at 10 mM PBC indicated complete turnover under these conditions, however at the expense of significant levels of unspecific U-PBC conjugate (data not shown). Of note, the intensity of peaks from s4U-PBC conjugate and the U-PBC conjugate (Figure 4B) cannot be compared without normalization as they underlie different ionization efficiencies. The reactivity of PBC towards pseudouridine (Ψ) was also investigated via LC-MS/MS. Two different tRNAs of E. coli were treated with varying concentrations of the coumarin, digested and analyzed. For both no substantial product peaks for Ψ-PBC were recorded, up to a PBC concentration of 10 mM (Figure S5). After optimization of the reaction conditions and characterization of the nucleoside-PBC conjugates we sought for further applications of our labeling reagent, starting with evaluation of several dyes for applicability in gel experiments together with PBC. Dyes from green to red emission, namely ATTO 488, TAMRA, ATTO 590, Cy5 and Cy5.5, were tested. The labeling and CuAAC was easily adapted to all of them. Even the attachment of a trifunctional TAMRA-azide-biotin reagent was easy and uncomplicated. From here we expanded the experiments to biological applications. We conclude that – in keeping with expectations - reaction conditions known to diminish nucleophilic reactivity (such as low pH and content of organic solvent) concomitantly increase selectivity for derivatization of sulphur nucleophiles over all other nucleophiles in RNA. In summary, we have so far established a reagent and a corresponding set of conditions that allow flexible derivatization and labeling of s4U in RNA with a selectivity >1000 fold.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4: Characterization of reactivity by LC-MS/MS and urea-PAGE: A: Fluorescence scans of 10% denaturing PAGE showing tRNAs treated with increasing PBC amount. Samples labeled “H2O2 ctrl” and “tRNA” denote E. coli tRNAVal, IVT denotes in vitro transcribed tRNA. The upper panel displays the Cy5.5 label, the lower panel shows the loading control after GelRedTM staining. B: Detection of U-PBC and s4U-PBC conjugates via LC-MS/MS for tRNAVal, E. coli (indicated as tRNA in figure) and in vitro transcribed tRNA (indicated as IVT). MS chromatograms were normalized to injected RNA amount.

Monitoring of metabolic s4U labeling with PBC

As an application, we envisaged PBC as a selective reagent for tracing metabolic incorporation of s4U into eukaryotic RNA, where it is typically used in UV-crosslinking-immunoprecipitation studies (CLIP)26,27. We decided to determine a saturation kinetic of incorporation using HEK293 cells, which were s4U labeled using supplemented growth medium according to the protocol by Hafner et al.44. Cells were grown to 80% confluency in the absence of s4U before exchange with medium containing s4U and continued growth for another 24 hours. During this period, cells were harvested at time points of 3, 6, 12, 18 and 24 h after the addition of s4U label. RNA extracted from these cells was analyzed for its s4U content by two methods, one newly established based on our PBC-labeling approach and, for comparison, by LC-MS/MS. Total RNA, i.e. preparations including all cellular RNAs, were derivatized with PBC, applying condition set III. The conjugated total RNA was further functionalized with Cy5.5-azide to enable fluorescence detection in denaturing PAGE. Band intensities were assessed by imageJ software and normalized to the GelRedTM loading control. The intensity values were used to calculate relative s4U enrichment factors, as shown in Figure 5A. The PAGE experiments revealed that s4U was, after incubation of no more than 24 h, best detectable in the total tRNA faction with this method, which was therefore used for PAGE-based quantification. Fig. S6 shows an exemplary PAGE scan, used for 8 ACS Paragon Plus Environment

Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


quantification. Fitting the results to a saturation curve (Fig. 5A), indicated that s4U integration into tRNA has a ceiling effect. This observation was confirmed in the LC-MS/MS experiments, as shown in Figure 5B. The fold-change of s4U amount started at 1.7 between 3 h and 6 h of incubation, then declined to 1.1-fold from hour 12 to 18 and stayed level from 18 h to 24 h of s4U treatment. Figure 5C compares the fold-changes between various time points, as determined by our fluorescence-PAGE method versus LC-MS/MS and clearly shows that the bona vide LC-MS/MS quantification validates the relative quantification by the PBC based method. In order to develop this into a method for absolute quantification, a calibration with commercially available native tRNAVal from E. coli was added. This commercial tRNA was derivatized with PBC and conjugated to Cy5.5. This sample was titrated to calibrate the fluorescent signal in tRNA context on a denaturing polyacrylamide gel (Fig. 6A). Hereby we achieved an absolute quantification of s4U incorporation of ~1 pmol s4U/pmol tRNA in a total tRNA sample from HEK cells that had undergone 16 h of labeling with s4U. This was confirmed by quantitative LC-MS/MS measurements (Fig. 6B, Fig. S7 for LC-MS/MS calibration curves of s4U). In conclusion, we showed that PBC can be used to monitor metabolic s4U labeling via a technically simple biomolecular approach in a relative quantification, and after proper calibration, also for absolute quantification.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5: Relative quantification of s4U levels after metabolic labeling via LC-MS/MS and urea-PAGE. A: Relative quantification of s4U in cellular RNA from HEK cells after metabolic labeling, as analyzed after PBC treatment, followed by CuAAC conjugation with Cy5.5 azide. Fluorescence from the subsequent 10% PAGE gel was scanned and quantified by the area under the curve (AUC) using ImageJ software. B: Relative quantification of the same samples as in A, using LC-MS/MS, values are given as s4U in % (s4U/A). C: Comparison of the fold-changes of incorporated s4U, calculated from LC-MS/MS results and PAGE experiments. Error bars represent standard deviation of biological triplicates.


Page 10 of 24

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6: Absolute quantification of s4U in total tRNA after metabolic labeling. A: Example of a calibration of the Cy5.5 fluorescence signal as a function of labeled tRNAVal in the range of 11-23 pmol. B: Results of absolute quantification in total tRNA extracts after 16 h of metabolic s4U labeling. LC-MS/MS measurements (grey bar) yielded 0.93 pmol s4U/pmol tRNA. For quantification by the PBC-based PAGE assay 12-16 pmol total tRNA were derivatized, labeled, and run on a 10% PAGE followed by fluorescence scan. An average value of 1.2 pmol s4U/pmol tRNA was obtained from four replicates (error bar shows standard deviation) by comparison to a calibration with a tRNA of known s4U content.

Synthesis of tRNA-PBC-Cy5 conjugate for Kd measurements

As a preparative application for s4U labeling in biophysical approaches by PBC, tRNAVal from E. coli was used because of its commercial availability and the presence of a single s4U at position 8. The tRNA was derivatized using 5 mM PBC in condition set III, click-conjugated with Cy5-azide, and purified by ion-pair chromatography on an HPLC equipped with a diode array detector (DAD) and a fluorescence detector. This setup allowed, as shown in Figure 7, to monitor elution of nucleic acids via absorption at 254 nm, of coumarins via absorption at 320 nm, and of the Cy5-dye at 646 nm as well as the dye’s fluorescence at λEm: 662 nm. Recording all four signals combined with PAGE analysis of the LC eluted product allowed the explicit identification of the product by spectroscopic properties and RNA size. The LC traces in figure 6A-D reveal four peaks. Free tRNA and tRNA-PBC conjugate elute from 10.2-12.5 (peak a: 10.2-12.5). The second one, eluting from 14.8 to 15.7 minutes, corresponded to intact tRNA-PBC-Cy5 conjugate which was confirmed by gel electrophoresis (product b, Fig. 7E). The denaturing PAGE revealed a defined degradation product of the labeled tRNA in the second peak (product c: 16.0-17.9 minutes). Comparison of fluorescence intensity of the two products (b and c) to the same RNA amount of commercially Cy5-labeled oligonucleotides in the denaturing PAGE (Fig. 7E) reveals that product c shows relatively more intense fluorescence, supporting the hypothesis that it carries multiple labels. The third fluorescent product peak (d: 17.0-18.9 minutes) was not further analyzed, as reproducibly no RNA could be precipitated. Throughout three separate preparations, the desired tRNA-PBC-Cy5 conjugate was obtained in final yields of 0.5-3%, relative to tRNA input. Various other azide dyes and other s4U containing tRNAs were successfully conjugated incurring slight adjustment of the LC gradient (data not shown).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7: HPLC chromatograms and urea-PAGE of PBC and Cy5 labeled tRNA A-D: HPLC traces of one run with PBC-Cy5 labeled tRNAVal, E. coli, detected by A: Absorbance at 254 nm (free tRNA), B: Absorbance at 320 nm (coumarin absorption) C: Absorbance at 646 nm (Cy5 label) and D: Emission at 662 nm (Cy5 fluorescence). Throughout panels A-E, label “a” denotes free and PBC-labeled tRNA, b corresponds to tRNAPBC-Cy5 product while c and d are multiply labeled side products. E shows a 10% denaturing PAGE of the LC purified fractions. Two Cy5 labeled oligonucleotides were loaded as fluorophore control, denoted “Cy5 ctrl”.


Page 12 of 24

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The functionality of the tRNA-PBC-Cy5 conjugate in biophysical assays, its binding constant towards a tRNA binding protein was determined by means of microscale thermophoresis (MST) 45,46. Based on our experience of MST-based characterization of the tRNA binding properties, we used the tRNA modification enzymes TruB for this purpose.47–49 This Ψ-synthase is monomeric and soluble at concentrations required for this type of assay.50 A dilution series of a fix amount of Cy5 tagged tRNAVal from E. coli, against decreasing concentrations of TruB was submitted to MST measurements. NT analysis software was applied to evaluate the obtained data: The measured raw fluorescence data was normalized (Figure 8A) and fluorescence ratios plotted in a one-to-one binding curve (Figure 8B), from which Kd values were determined by the quadratic solution of the binding reaction equilibrium, deduced from the law of mass action, where Kd was considered the only free parameter.51 The Kd-values, calculated by the Nanotemper software, from two independent experiments (Figure 8C) were in good agreement with previously published data on other dye-labeled tRNA conjugates.50 In summary, the PBC-based preparative labeling approach opened an avenue to labeling of s4U containing tRNAs with a fluorescent dye and subsequent measurement of the binding constant to a tRNA binding protein with very short turnaround time.

Figure 8: Results from microscale thermophoresis. A: Typical MST time traces of a protein binding experiment. Regions a (dark grey) and b (light grey) denote time points for calculation of differential normalized fluorescence ratios. Thermophoresis starts (region a, dark grey) after the initial fast decline of fluorescence, the so-called T-Jump (dashed line). Brownian motion balances the thermophoretic movement, leading to a steady state of fluorescence (region b, light grey) B: Normalized fluorescence ratios (b/c) fitted to a one-to-one binding model with Nanotemper analysis software (NT analysis). C: Kd s were calculated from one-to-one binding models by NT analysis applying an equation derived from the law of mass action with Kd as single free parameter. Error bars are standard deviations from technical triplicate measurements.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Conclusion & Outlook We here describe the synthesis of 4-bromomethyl-7-propargyloxycoumarin (PBC) and its characterization as a highly selective electrophilic reagent for covalent labeling and bioconjugate chemistry of the RNA modification s4U. Other reagents have been reported,23,28,52 which exploit different reactivties typically associated with sulphur-organic compounds. For example, early methods for s4U detection and enrichment rely on coordinative binding of organomercurial affinity cellulose matrices.14 This has been further developed to an affinity electrophoresis approach, applying PAGE which is cast with (N-acryloylamino) phenyl mercuric chloride (APM).23 Regents such as HPDPbiotin (a 2-pyridylthio-activated disulfide derivative of biotin),53,54 and MTS-biotin28 exploit the sulphur’s propensity to selectively form covalent disulfide bonds. Conjugation of such reagents to biotin has been exploited for enrichment of the s4U labeled RNA species via application of a streptavidin matrix. In combination with NGS, this allowed turnover and decay studies of e.g. miRNA species.28,52 Similar applications may be envisaged for the PBC reagent. Beyond such analytical approaches, PBC was also found useful in preparative labeling of bacterial tRNAs that naturally contain s4U at positions 8 or 9. We are aware that exploitation of the sulphur selectivity of the PBC is reagent is restricted to the in vitro derivatization of pure, i.e. protein-free RNA preparations, since proteins carry cysteines that are likely to rapidly quench the PBC reactivity. The isolated yield of these labeling reactions was rather low and varied between 0.5-3% with respect to the input RNA amount. Both, the low yield and its variability are not uncommon when working with low amounts of RNA in complex purification procedures. In the case at hand, the purification included two steps of ethanol precipitation and HPLC purification, followed by yet another ethanol precipitation. The subsequent determination of a binding constant in the triple digit nanomolar range (Figure 8) can be taken as a strong indication that the tRNA did not suffer substantial structural changes to its overall structure. Some advantages of the coumarin system that surface during the preparation procedure include facile detection of the heterocyclic system by absorption at 320 nm, or detection by fluorescence of either the coumarin,19 or other dyes conjugated via click chemistry. We furthermore applied PBC for quantitative assessment of metabolically-labeled RNA. These results were validated by quantitative LC-MS/MS, confirming reproducibility of the PAGE quantification method. The lack of absolute quantitative conversion accounts for the limitation, that reliable detection is dependent on a certain minimal abundance of s4U in the target RNA. In addition, the limited yields of preparative RNA-PBC functionalization represents a certain restriction for downstream investigations of such conjugates. On the other hand, the high selectivity and especially the versatility of PBC make it applicable in a wide range of settings, e.g. with different fluorophores that allow multiplexed quantification at different wavelengths. Such approaches would be extremely laborious and costly with dyes already attached to the PBC before the reaction with RNA. Including such possibilities as attaching a purification tag like biotin via CuAAC, one might think of PBC as a modular tagging reagent, paving the way to secondary derivatization in a variety of applications. Sulphur as an excellent nucleophile has been vastly exploited in protein bioconjugation chemistry55, where electrophiles ranging from “soft”, (such as maleimides56,57, including coumarin maleimides58) to relatively “hard”, (such as acetyliodide) compounds have been applied to label cysteins,59,60. Reagents of the latter class have also been used in RNA bioconjugation strategies.61,62 In contrast to such highly reactive electrophilic reagents as acetyliodides, PBC is rather moderate in its reactivity, which in turn can be fine-tuned via substituents on the aromatic coumarin scaffold.19,32 The data presented in the present paper revealed dramatic influence on reactivity and selectivity by parameters known to affect nucleophilicity, namely content of protic vs. non-protic solvent, and pH. Such optimization has, to our knowledge, not been conducted for other sulphur-reactive nucleic acid bioconjugate reagents, and it is consequently difficult to truly compare selectivity of electrophilic bromomethylcoumarins to the 14 ACS Paragon Plus Environment

Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


abovementioned reagents which are based on a different chemical reactivity. However, until proven otherwise, we maintain that a selectivity above 1000 is remarkable and should allow efficient separation of sulphur-modified RNAs from others, e.g. for identification by deep sequencing, as has been implemented with other reagents to determine metabolic stability of RNA species.63 In a slightly different scenario outside metabolic labeling, PBC might be used for isolation of RNA that naturally contains s4U, potentially followed by identification of conjugation sites by e.g. reverse transcription and deep sequencing.64,65 Efforts directed this way are indeed underway in our laboratory. So far, we cannot exclude that cross-reactivity with other thiolated modifications, such as s2U or 5methoxycarbonylmethyl-2-thiouridine, as well as other s2-substituted, so-called wobble U modifications,66 might occur when investigating other RNA preparations than those which have been discussed in the above. This must be addressed in further studies.

Experimental Procedures PBC synthesis

If not otherwise declared the required chemicals were purchased from Sigma-Aldrich (Steinheim, Germany). The numbers in parentheses in this paragraph refer to Figure 2. 7-hydroxy-4methylcoumarin (3) was synthesized according to Pechmann et al..37 Briefly, 1,3-dihydroxybenzene (17.9 g, 0.162 mol, 1 eq.) was dissolved in 60 mL conc. sulphuric acid at -5 °C, and ethyl acetoacetate (21.1 g, 0.162 mol, 1 eq.) was added. The reaction allowed to take place at room temperature for 14 h before it was quenched with ice. 7-hydroxy-4-methylcoumarin (3) (17.1 g, 0.097 mol, 60%) was purified via recrystallization from ethanol. Sm = 186 °C, TLC (silica gel, petrol ether:ethyl acetate 1:3) Rf = 0.33. 1H NMR (Bruker AC-300, 300 MHz, DMSO-d6, 25°C): δ = ppm 2.37 (3H, s), 6.13 (1H, s), 6.70 (1H, d, 4J= 2.3 Hz), 6.80 (1H, dd, 3J= 8.7 Hz, 4J= 2.4 Hz), 7.68 (1H, d, 3J= 8.7 Hz), 10.5 (1H, s). The spectrum is listed in Figure S7. 7-acetoxy-4-methylcoumarin (4) was synthesized according to Wollowitz et al..67 Briefly, 1.21 g of (3) (6.86 mmol) were refluxed in 3.6 mL acetic anhydride for 3 h. The product (4) was precipitated by cooling to room temperature, and remaining anhydride and formed acid were removed under reduced pressure. TLC revealed complete consumption of (3), and (4) (1.49 g, 6.84 mmol, 99%) was used without further work-up. TLC (silica gel, cyclohexane:ethyl acetate =7:3): Rf= 0.26; m.p. 149°C; 1H NMR (Bruker AC-300, 300 MHz, CDCl3, 25°C): δ = ppm 2.35 (3H, s), 2.43 (3H, s), 6.27 (1H, s), 7.09 (2H, m), 7.61 (1H, pd, 3J= 8.6 Hz). The corresponding

spectrum can be found in Figure S8. 4-bromomethyl-7-acetoxycoumarin (5) was synthesized according to a protocol derived from Belluti et al..39 Compound (4) (900 mg 4.1 mmol) was refluxed with N-bromosuccinimide (807 mg, 4.5 mmol, 1.1 eq) (Acros Organics, Geel, Belgium) with a catalytic amount of benzoyl peroxide in carbon tetrachloride. After 1 h, another catalytic amount of benzoyl peroxide was added, followed by refluxing for another 3 h. 1 eq. N-bromosuccinimide and a third catalytic amount of benzoyl peroxide were added and further reaction was allowed for 12 h. The product (5) was extracted with dichloromethane and purified via flash column chromatography (FCC) (cyclohexane:ethyl acetate =7:3). Pure product was obtained (137 mg, 0.4 mmol) with a yield of 11%. TLC (silica gel, cyclohexane:ethyl acetate =7:3): Rf= 0.33; m.p. 175°C (dichloromethane); 1H NMR (Brucker AC-300, 300 MHz, CDCl3, 25°C): δ = ppm 2.36 (3H, s), 4.48 (2H, s), 6.51 (1H, s), 7.14 (2H, m), 7.75 (1H, d, 3J= 8.6 Hz). See Figure S9 for the corresponding NMR spectrum. Deprotection of compound (5) was achieved dissolving 331 mg (1.11 mmol), pooled from three batches of (5), in 11.1 mL tetrahydrofuran and 5 mL 37% hydrochloric acid. The mixture was stirred for 48 h and subsequently extracted with dichloromethane and water. After drying over Na2SO4 and removal of solvent under reduced pressure, (6) (277 mg, 1.08 mmol, 97%) was obtained as a white solid. 1H NMR (Brucker AC-300, 300 MHz, DMSO-d6, 25°C): δ = ppm 4.96 (2H, s), 6.42 (1H, s), 6.75 (1H, pd, 4J= 2.2 Hz), 6.84 (1H, dd, 3J= 8.6 Hz, 4J= 2.2 Hz), 7.68 (1H, d, 3J= 8.6 Hz), 10.68 (1H, s). The NMR spectrum is listed in Figure S10. Finally, 4-bromomethyl-7-propargyloxycoumarin (1) was obtained by alkylation as described by Kosiova et al..41 Compound (6) (174 mg, 0.7 mmol, 1 eq.) and 103 mg 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

K2CO3 (0.75 mmol, 1.1 eq.) were dissolved in 1.4 mL dried acetone under argon atmosphere. After 0.5 h, 80 µL (0.75 mmol 1.1 eq.) of propargyl bromide in 2 mL acetone were added. This mixture was refluxed for 12 h at 56 °C. (1) was extracted with ethyl acetate, dried with Na2SO4 and the solvent removed under reduced pressure. FCC (cyclohexane:ethyl acetate = 4:1) yielded the pure product (119 mg, 0.4 mmol, 57%, total yield 3.6%). TLC (silica gel, cyclohexane:EtOAc=4:1): Rf= 0.22; m.p.150°C (ethyl acetate); 1H NMR (DRX-400, 400 MHz, CDCl3, 25°C): δ = ppm 2.59 (1H, t, 4J= 2.5 Hz), 4.63 (2H, s), 4.78 (2H, d, 4J= 2.5 Hz), 6.43 (1H, s), 6.97 (2H, m), 7.60 (1H, m); 13C NMR (DRX-400, 100 MHz CDCl3, 25°C): δ= ppm 41.2, 56.2, 76.6, 77.2, 102.5, 111.4, 113.1, 125.2, 149.4, 155.4, 160.5, 160.6; NMR Spectra are listed in Figure S1. FD-MS (Thermo Finnigan MAT95, Thermo Fischer Scientific, Dreieich, Germany) : 292.2, 294.2 [M+H]+ ; IR (AVATAR 330FT-IR Thermo Nicolet, Thermo Fisher Scientific, Dreieich, Germany) ν cm-1: 3268.1 (ν (C-H) alkyne) 3080.0 (ν (CH) aromatic), 2127.7 (C≡C), 1713.7 ((C=O) lactone), 1603.4 (ν (C=C)). See Figure S2 for FD-MS and IR spectra.

Cell culture of HEK293 cells and isolation of total RNA and total tRNA

HEK293 cells, kindly provided by the group of Prof. A. Dalpke (Heidelberg, Germany), were grown to 80% confluency in growth medium consisting of 90% (v/v) D-MEM supplemented with 10% (v/v) fetal bovine serum and 100 µg/mL penicillin/streptomycin (all Thermo Fisher Scientific, Dreieich, Germany). The growth medium was removed and exchanged by D-MEM supplemented as above and additionally 100 µM s4U (Sigma Aldrich, Steinheim, Germany). Cells were further incubated for 3 h, 6 h, 12 h, 16 h, 18 or 24 h. For 3 h and 24 h a control without s4U was grown. After incubation, the medium was removed and cells were lysed by TriReagent (Sigma Aldrich, Steinheim, Germany). RNA extraction was performed per the manufacturer’s instruction. RNA was dissolved in MilliQ water and stored at -20 °C until further use. To isolate the total tRNA from the total RNA extract, 50 µg total RNA were loaded to a 10% denaturing PAGE and run for 1.5 h at 8 W. The band corresponding to the tRNA was detected via 15 min staining in 1 x GelRedTM, (Biotium, Fremont, CA, USA) and scanning with a Typhoon 9400 variable mode imager (GE Healthcare, Freiburg, Germany). The scanning set-up is specified in Table S1. The RNA band which corresponded to the total tRNA fraction was excised, shredded and shaken at 850 rpm in 0.5 M ammonium acetate overnight. Subsequent filtration (NanoSep 0.45 µm, PALL, Dreieich, Germany) and ethanol precipitation of the flow-through provided total tRNA which was also dissolved in MilliQ water and stored at -20 °C.

H2O2 treatment of tRNAs

Native tRNA, valine specific from E. coli (Sigma Aldrich, Steinheim, Germany), was incubated for 2 h in a 0.35% (w/v) H2O2 solution, buffered to pH 8, at 20 °C. RNA was recovered by ethanol precipitation and used for continuative experiments without further purification.

Derivatization of RNA with PBC and fluorescent dye

The required RNA, respectively native tRNATyr or tRNAVal, E. coli (Sigma Aldrich, Steinheim, Germany), optionally H2O2 treated; total tRNA E. coli (Roche, Basel, Suisse); HEK293 total RNA; HEK293 total tRNA or in-house in vitro transcribed tRNAs68, was charged to a 1.5 mL reaction tube. Phosphate buffer of pH 7 or 8, dimethyl sulfoxide to a final (v/v) % of 50 or 70 were added. Finally, the derivatization was started by PBC of a final concentration of 1 mM, 5 mM or 10 mM. RNA concentrations were 0.05-0.1 µg/µL. Samples were incubated 2 or 3 h at 37 °C. The reaction was 16 ACS Paragon Plus Environment

Page 16 of 24

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


quenched by ethanol precipitation of the RNA. The pellet was dissolved in MilliQ water and either frozen on -20 °C or directly further derivatized by CuAAC. Required reagents were added to the RNA in the following direction: 5 mM Tris(3-hydroxypropyltriazolmethyl) amine (in-house synthesized), 5 mM sodium ascorbate, 0.5 mM copper(II)sulfate, 50% (v/v) dimethyl sulfoxide (all Sigma Aldrich, Steinheim, Germany) and 0.05 mM fluorescent dye-azide (all concentrations are final concentrations). This reaction mixture was kept at 25 °C for 2 h. Used dye-azides were ATTO 488 (ATTO-TEC, Siegen, Germany), Cy5, Cy5.5 or TAMRA-azide-biotin (all Jena Bioscience, Jena, Germany). Clicked RNA was again ethanol precipitated and submitted to PAGE, LC-MS/MS or HPLC analysis.

HPLC purification of tRNA constructs

Separation of labeled and unlabeled tRNAs was carried out on an Agilent 1100 series HPLC, equipped with a DAD, as well as an FLD detector (Agilent Technologies, Waldbronn, Germany). Eluent A was a 0.1 M triethylammonium acetate buffer, pH adjusted to 7, eluent B consisted of HPLC-grade acetonitrile (all reagents Sigma Aldrich, Steinheim, Germany). To enhance separation power, eluents were applied in a gradient (Table S3) on a YMC Triart C18 (particle size 3 µM, pore size 120 Å, column size 150x3.0 mm I.D., YMC Europe, Dinslaken, Germany) column at 35 °C. To avoid overloading a maximum amount of 1 nmol PBC-treated and Cy5-conjugated tRNA was injected. The DAD detector was set to 254 nm to monitor the tRNA, 320 nm for coumarin detection and 646 nm to monitor the Cy5’s λAbsorption. For fluorescence analysis of Cy5 the FLD detector was set to λExcitation: 646 nm and λEmission: 662 nm. Fractions were collected from the FLD, according to Figure 7. Products were precipitated with ethanol directly out of the HPLC eluate, supplemented with 10% (v/v) 5 M ammonium acetate. Quantification of tRNA conjugates was conducted on a NanoDrop 2000 UV-Vis spectrophotometer (NanoDrop products, Wilmington, DE, USA) in RNA mode.

Qualitative and quantitative LC-MS/MS analyses of derivatized and underivatized RNA

All LC-MS/MS measurements were conducted on an Agilent 1200 series HPLC, equipped with a DAD detector and an Agilent 6460 QQQ mass spectrometer (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved by a Phenomenex Synergi Fusion RP18 column (particle size 4 µM, pore size 80 Å, column size 250x2 mm, Phenomenex, Aschaffenburg, Germany) at 35 °C, using a 5 mM ammonium acetate buffer (pH 5.3) as eluent A and LC-MS grade acetonitrile (Sigma Aldrich, Steinheim, Germany) as eluent B. Methods, including s4U quantification, have been adapted according to Thüring et al.69 Briefly, all RNAs which had to be analyzed were hydrolyzed to nucleosides by nuclease P1 (Sigma Aldrich, Steinheim, Germany), snake venom phosphodiesterase (Worthington, Lakewood, NJ, USA) in 25 mM ammonium acetate buffer containing 0.2 mM zinc chloride and FastAP (Thermo Fischer Scientific, Dreieich, Germany) in manufacturer supplied buffer. If sample was prepared for quantification, it was spiked with 100 ng digested all 13C total RNA extract from E. coli per injection, as internal standard. Main nucleosides were monitored via UV detection, modifications and PBC-conjugates were observed by MS detection and characteristic fragmentation of the glycosidic bond (see Table S2 for the monitored mass transitions). All data extraction was performed by Mass Hunter software. Data was then further analyzed in Excel. Analysis of PBC conjugates of derivatized and digested in vitro transcribed and native tRNAs, as well as digested total tRNA from E. coli, were analyzed by UV detection at 254 nm for the nucleoside and 320 nm for the coumarin and additionally by MS/MS in dynamic multiple reaction mode. See Table S4 for method details. Table S2 shows monitored mass transitions and corresponding retention times. The MS peaks of the conjugates have been related to the UV signal of cytidine for normalization to the injected RNA amount to allow inter-sample comparability. The normalized data was plotted again, 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA). Injection amounts varied from 0.5 pmol derivatized tRNA to 10 pmol derivatized tRNA. Data for quantification of underivatized s4U via internal calibration, applying stable isotope 13C internal standard from E. coli culture, was acquired in dynamic multiple reaction monitoring mode, while data for A quantification was recorded by UV-absorption at 254 nm, which enabled external UV-calibration of adenosine nucleoside standard (Sigma Aldrich, Steinheim, Germany). See Figure S11 for the calibration curves. Method details such as gradient and source parameters can be found in Table S5, monitored mass transitions are recorded in Table S2. Calculation of absolute adenosine amounts, by UV calibration, and absolute s4U amounts, quantified by internal standard, allowed specification of %(s4U/A) in total RNA of HEK293 cells, which was plotted against the time of s4U incubation. For total tRNA the absolute amount of pmol s4U/pmol tRNA could be calculated. Therefore, the amount of tRNA was approached by the approximation that 1 µg tRNA are ~40 pmol. Absolute amounts of s4U in the injected sample were again calculated via internal standard. The injection amount of total HEK293 RNA was usually 500 ng and ~750 fmol for total tRNA of HEK293.

Qualitative and quantitative PAGE analysis of labeled RNA species

For all polyacrylamide gel electrophoresis experiments a 10% denaturing polyacrylamide gel was casted. RNA was loaded in amounts from 2 to 23 pmol. For electrophoresis, demand was kept constantly at 8 W for 45 minutes. Bands of labeled tRNAs were detected by fluorescence with a Typhoon 9400 variable mode imager (see Table S1 for laser and filter settings for different dyes).Post staining by 1x GelRedTM for 15 minutes and a second fluorescence scan (scanner set-up as describen in Table S1) was applied as a loading control. Ultra Low Range DNA ladder (Thermo Fisher Scientific, Dreieich, Germany), as well as untreated tRNA were loaded to each gel for size control. Obtained data was further analyzed using ImageJ software. For evaluation of selectivity factors: Mean pixel-intensity of the bands was measured and background intensity normalized. These background-corrected signals were once more normalized to the number of s4U residues (in case of the native tRNA) and to the number of uridines (in the case of IVT). Bands of IVT were further normalized by loaded sample amount (2 pmol for native tRNA, 20 pmol for IVT) and higher reaction rate due to higher PBC concentration (10 mM for IVT and 1 mM for native tRNA). Conservative estimation of increase of reaction rate by a factor of 10 was proven by measuring mean pixel-intensities of bands in Figure 4A where different PBC concentrations were applied to the same RNA. Further, background normalized pixel-intensity for native tRNA was divided by normalized mean-pixel intensity of IVT. For relative quantification: Bands were plotted as profiles and integrated. Calculation of the ratio of the areas under the curve (AUC) of the Cy5.5 signal to GelRedTM signal, which was evaluated in the same manner, allowed normalization to loaded RNA amount. Obtained values were plotted against the time of incubation with s4U. For absolute quantification: A calibration curve was obtained from titration of tRNAVal, E. coli-PBCCy5.5 conjugate from 11 to 23 pmol. Bands of the Cy5.5 signal have been extracted as profiles and integrated. The AUCs were then plotted against the amount in a linear fit. The resulting equation was used to calculate s4U amount in total tRNA extracts from metabolic labeling of HEK293 cells.

MST analyses


Page 18 of 24

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To be able to compare the results with already published data, MST analyses have exactly been conducted as described previously.50 Briefly, aliquots of labeled RNA were mixed with 1:1 dilutions of TruB in MST buffer (20 mM Tris-HCl, pH 7.5, 60 mM KCl, 0.02% (v/v) Tween-20 (Sigma Aldrich, Steinheim, Germany). In total 16 samples, containing 50 nM fluorescently-labeled tRNA and increasing amounts of TruB were analyzed, including a negative control without the enzyme. Analysis was run on a Nanotemper Monolith NT115 (Nanotemper, Munich, Germany). The obtained data was analyzed by manufacturer supplied software (NT analysis 1.4.27) and further plotted in GraphPad Prism 7.

Abbreviations 4-bromomethyl-7-methoxycoumarin (BMB), 4-bromomethyl-7-propargyloxycoumarin (PBC), area under the curve (AUC), copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), diode array detector (DAD), in vitro transcribed tRNA (IVT), microscale thermophoresis (MST), Next Generation Sequencing (NGS),

Acknowledgements Dr. Felix Spenkuch is gratefully acknowledged for kindly providing TruB enzyme, as well as Dr. Thomas Fritz for substantial help with the cell culture experiments and Dr. Stefanie Kellner for extensive scientific advice. The authors appreciate the support of all members of the Helm lab in terms of scientific discussion. HEK293 cells were a generous gift from the group of Prof. A. Dalpke (Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital, Heidelberg, Germany).

Supporting Information Analytical data of PBC, Selectivity factors, derived from LC-MS/MS, LC-MS/MS chromatogram of H2O2 treated tRNA, chromatograms of Ψ-PBC conjugates, Gel scan of relative quantification, LC-UVMS/MS calibration of adenosine and s4U, mass transitions and retention times, HPLC gradient, method details for LC-MS/MS (gradients and source parameters), Settings for Typhoon variable imager. (PDF)

Funding This work was supported by a DFG grant to M.H. (Helm, HE3397/9-1).

Author Information *

To whom correspondence should be addressed. Phone : +49-6131-3925731 3920373 Email : [email protected] Note: The authors declare no competing financial interest.


Fax : +49-6131-


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References (1) Paredes, E., Evans, M., and Das, S. R. (2011) RNA labeling, conjugation and ligation. Methods 54, 251–9. (2) Slim, G., and Gait, M. J. (1991) Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 19, 1183–1188. (3) Riml, C., and Micura, R. (2016) Synthesis of 5-Hydroxymethylcytidine- and 5Hydroxymethyl-uridine-Modified RNA. Synthesis (Stuttg). 48, 1108–1116. (4) Gait, M. J. (1991) DNA/RNA synthesis and labelling. Curr. Opin. Biotechnol. 2, 61–8. (5) Seidu-Larry, S., Krieg, B., Hirsch, M., Helm, M., and Domingo, O. (2012) A modified guanosine phosphoramidite for click functionalization of RNA on the sugar edge. Chem. Commun. (Camb). 48, 11014–6. (6) Domingo, O., Hellmuth, I., Jäschke, A., Kreutz, C., and Helm, M. (2015) Intermolecular “crosstorque”: The N4-cytosine propargyl residue is rotated to the “CH”-edge as a result of Watson-Crick interaction. Nucleic Acids Res. 43, 5275–5283. (7) Seelig, B., and Jäschke, A. (1997) Site-specific modification of enzymatically synthesized RNA: Transcription initiation and Diels-Alder reaction. Tetrahedron Lett. 38, 7729–7732. (8) Wolf, J., Dombos, V., Appel, B., and Müller, S. (2008) Synthesis of guanosine 5’-conjugates and their use as initiator molecules for transcription priming. Org. Biomol. Chem. 6, 899–907. (9) Kato, Y., Minakawa, N., Komatsu, Y., Kamiya, H., Ogawa, N., Harashima, H., and Matsuda, A. (2005) New NTP analogs: The synthesis of 4-thioUTP and 4-thioCTP and their utility for SELEX. Nucleic Acids Res. 33, 2942–2951. (10) Minakawa, N., Sanji, M., Kato, Y., and Matsuda, A. (2008) Investigations toward the selection of fully-modified 4-thioRNA aptamers: Optimization of in vitro transcription steps in the presence of 4’thioNTPs. Bioorganic Med. Chem. 16, 9450–9456. (11) Karikó, K., Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., and Weissman, D. (2008) Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–40. (12) Jao, C. Y., and Salic, A. (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. U. S. A. 105, 15779–84. (13) Tani, H., Mizutani, R., Salam, K. A., Tano, K., Ijiri, K., Wakamatsu, A., Isogai, T., Suzuki, Y., and Akimitsu, N. (2012) Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Res. 22, 947–56. (14) Melvin, W. T., Milne, H. B., Slater, A. A., Allen, H. J., and Keir, H. M. (1978) Incorporation of 6-thioguanosine and 4-thiouridine into RNA. Application to isolation of newly synthesised RNA by affinity chromatography. Eur. J. Biochem. 92, 373–9. (15) Bakin, A., and Ofengand, J. (1993) Four newly located pseudouridylate residues in Escherichia 20 ACS Paragon Plus Environment

Page 20 of 24

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. Biochemistry 32, 9754–9762. (16) Gupta, V., Cherkassky, A., Chatis, P., Joseph, R., Johnson, A. L., Broadbent, J., Erickson, T., and DiMeo, J. (2003) Directly labeled mRNA produces highly precise and unbiased differential gene expression data. Nucleic Acids Res. 31, e13. (17) Mortimer, S. A., Johnson, J. S., and Weeks, K. M. (2009) Quantitative analysis of RNA solvent accessibility by N-silylation of guanosine. Biochemistry 48, 2109–2114. (18) Weeks, K. M., and Mauger, D. M. (2011) Exploring RNA structural codes with SHAPE chemistry. Acc. Chem. Res. 44, 1280–91. (19) Kellner, S., Seidu-Larry, S., Burhenne, J., Motorin, Y., and Helm, M. (2011) A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA. Nucleic Acids Res. 39, 7348–60. (20) Mueller, E. G., Palenchar, P. M., and Buck, C. J. (2001) The Role of the Cysteine Residues of ThiI in the Generation of 4-Thiouridine in tRNA. J. Biol. Chem. 276, 33588–33595. (21) Mueller, E. G., and Palenchar, P. M. (1999) Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis. Protein Sci. 8, 2424–7. (22) Neumann, P., Lakomek, K., Naumann, P.-T., Erwin, W. M., Lauhon, C. T., and Ficner, R. (2014) Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification. Nucleic Acids Res. 42, 6673–85. (23) Emilsson, V., Näslund, A. K., and Kurland, C. G. (1992) Thiolation of transfer RNA in Escherichia coli varies with growth rate. Nucleic Acids Res. 20, 4499–505. (24) Thomas, G., and Favre, A. (1980) 4-Thiouridine triggers both growth delay induced by nearultraviolet light and photoprotection. Eur. J. Biochem. 113, 67–74. (25) Ramabhadran, T. V., Fossum, T., and Jagger, J. (1976) In vivo induction of 4-thiouridine-cytidine adducts in tRNA of E. coli B/r by near-ultraviolet radiation. Photochem. Photobiol. 23, 315–21. (26) Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano, M., Jungkamp, A.-C., Munschauer, M., et al. (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–41. (27) Garzia, A., Meyer, C., Morozov, P., Sajek, M., and Tuschl, T. (2016) Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins. Methods. (28) Duffy, E. E., Rutenberg-Schoenberg, M., Stark, C. D., Kitchen, R. R., Gerstein, M. B., and Simon, M. D. (2015) Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry. Mol. Cell 59, 858–66. (29) Li, X., Zhu, P., Ma, S., Song, J., Bai, J., Sun, F., and Yi, C. (2015) Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome. Nat. Chem. Biol. 11, 592–7. (30) Secrist, J. A., Barrio, J. R., and Leonard, N. J. (1971) Attachment of a fluorescent label to 4thiouracil and 4-thiouridine. Biochem. Biophys. Res. Commun. 45, 1262–1270. (31) Yang, C., and Söll, D. (1974) Covalent attachment of fluorescent groups to transfer ribonucleic acid. Reactions with 4-bromomethyl-7-methoxy-2-oxo-2H-benzopyran. Biochemistry 13, 3615–21. (32) Kellner, S., Kollar, L. B., Ochel, A., Ghate, M., and Helm, M. (2013) Structure-function relationship of substituted bromomethylcoumarins in nucleoside specificity of RNA alkylation. PLoS One 8, e67945. (33) Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click Chemistry: Diverse Chemical 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Function from a Few Good Reactions. Angew. Chem. Int. Ed. Engl. 40, 2004–2021. (34) Paredes, E., and Das, S. R. (2011) Click chemistry for rapid labeling and ligation of RNA. ChemBioChem 12, 125–131. (35) Rao, H., Tanpure, A. A., Sawant, A. A., and Srivatsan, S. G. (2012) Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization. Nat. Protoc. 7, 1097–112. (36) Schulz, D., and Rentmeister, A. (2014) Current approaches for RNA labeling in vitro and in cells based on click reactions. Chembiochem 15, 2342–7. (37) Von Pechmann, H., and Duisberg, C. (1883) Ueber die Verbindungen der Phenole mit Acetessigäther. Berichte der Dtsch. Chem. Gesellschaft 16, 2119–2128. (38) Wollowitz, S., and Nerio, A. Psoralens for pathogen inactivation, U.S. Patent 6,455,286. Sep 24, 2002. (39) Belluti, F., Fontana, G., Bo, L. D., Carenini, N., Giommarelli, C., and Zunino, F. (2010) Design, synthesis and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel proapoptotic agents. Bioorganic Med. Chem. 18, 3543–3550. (40) Furusho, H., Oishi, M., Kishi, T., Yasumori, A., and Nagasaki, Y. (2010) Preparation and molecular recognition of SERS probe based on gold nanoparticles constructed from PEG-oligoamine copolymer possessing a coumarin group between PEG and oligoamine. Chem. Lett. 39, 52–53. (41) Kosiova, I., Kovackova, S., and Kois, P. (2007) Synthesis of coumarin-nucleoside conjugates via Huisgen 1,3-dipolar cycloaddition. Tetrahedron 63, 312–320. (42) Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012) Fiji: an open source platform for biological image analysis. Nat. Methods 9, 676–682. (43) Watanabe, K. (1980) Reactions of 2-thioribothymidine ad 4-thiouridine with hydrogen peroxide in transfer ribonucleic acids from Thermus thermophilus and Escherichia coli as studied by circular dichroism. Biochemistry 19, 5542–9. (44) Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano, M., Jungkamp, A.-C., Munschauer, M., et al. (2010) PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins. J. Vis. Exp. 2400, 2–6. (45) Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P., and Duhr, S. (2011) Molecular interaction studies using microscale thermophoresis. Assay Drug Dev. Technol. 9, 342–53. (46) Seidel, S. A. I., Markwardt, N. A., Lanzmich, S. A., and Braun, D. (2014) Thermophoresis in nanoliter droplets to quantify aptamer binding. Angew. Chemie - Int. Ed. 53, 7948–7951. (47) Nurse, K., Wrzesinski, J., Bakin, A., Lane, B. G., and Ofengand, J. (1995) Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli. RNA. (48) Gutgsell, N., Englund, N., Niu, L., Kaya, Y., Lane, B. G., and Ofengand, J. (2000) Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells. RNA 6, 1870–1881. (49) Friedt, J., Leavens, F. M. V, Mercier, E., Wieden, H. J., and Kothe, U. (2014) An arginineaspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation. Nucleic Acids Res. 42, 3857–3870. (50) Spenkuch, F., Hinze, G., Kellner, S., Kreutz, C., Micura, R., Basché, T., and Helm, M. (2014) Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor. Nucleic Acids Res. 42, 12735–45. 22 ACS Paragon Plus Environment

Page 22 of 24

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(51) Baaske, P., Wienken, C. J., Reineck, P., Duhr, S., and Braun, D. (2010) Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew. Chemie - Int. Ed. 49, 2238–2241. (52) Dölken, L., Ruzsics, Z., Rädle, B., Friedel, C. C., Zimmer, R., Mages, J., Hoffmann, R., Dickinson, P., Forster, T., Ghazal, P., et al. (2008) High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14, 1959–72. (53) Cleary, M. D., Meiering, C. D., Jan, E., Guymon, R., and Boothroyd, J. C. (2005) Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay. Nat. Biotechnol. 23, 232–7. (54) Miller, M. R., Robinson, K. J., Cleary, M. D., and Doe, C. Q. (2009) TU-tagging: cell typespecific RNA isolation from intact complex tissues. Nat. Methods 6, 439–41. (55) Wall, S. B., Smith, M. R., Ricart, K., Zhou, F., Vayalil, P. K., Oh, J. Y., and Landar, A. (2014) Detection of electrophile-sensitive proteins. Biochim. Biophys. Acta - Gen. Subj. Elsevier B.V. (56) Chen, Z., Sun, Q., Yao, Y., Fan, X., Zhang, W., and Qian, J. (2017) Biosensors and Bioelectronics Highly sensitive detection of cysteine over glutathione and homo-cysteine : New insight into the Michael addition of mercapto group to maleimide. Biosens. Bioelectron. 91, 553–559. (57) Henkel, M., Roeckendorf, N., and Frey, A. (2016) Selective and Efficient Cysteine Conjugation by Maleimides in the Presence of Phosphine Reductants. Bioconjug. Chem. 27, 2260–2265. (58) Farmer, W. H., and Yuan, Z. (1991) A continuous fluorescent assay for measuring protease activity using natural protein substrate. Anal. Biochem. 197, 347–352. (59) Kouyama, T., and Mihashi, K. (1981) Fluorimetry Study of N1-(-Pyrenyl)iodoacetamideLabelled F-Actin: Local Structural Change of Actin Protomer both on Polymerization and on Binding of Heavy Meromyosin. Eur. J. Biochem. 114, 33–38. (60) Hill, B. G., Reily, C., Oh, J. Y., Johnson, M. S., and Landar, A. (2009) Methods for the determination and quantification of the reactive thiol proteome. Free Radic. Biol. Med. 47, 675–683. (61) Johnson, A. E., Adkins, H. J., Matthews, E. A., and Cantor, C. R. (1982) Distance moved by transfer RNA during translocation from the A site to the P site on the ribosome. J. Mol. Biol. 156, 113–140. (62) Watson, B. S., Hazlett, T. L., Eccleston, J. F., Davis, C., Jameson, D. M., and Johnson, A. E. (1995) Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.GTP ternary complex. A fluorescence energy transfer study. Biochemistry 34, 7904–12. (63) Neymotin, B., Athanasiadou, R., and Gresham, D. (2014) Determination of in vivo RNA kinetics using RATE-seq. RNA 20, 1645–52. (64) Hauenschild, R., Tserovski, L., Schmid, K., Thüring, K., Winz, M.-L., Sharma, S., Entian, K.-D., Wacheul, L., Lafontaine, D. L. J., Anderson, J., et al. (2015) The reverse transcription signature of N1-methyladenosine in RNA-Seq is sequence dependent. Nucleic Acids Res. 43, 9950–64. (65) Hauenschild, R., Werner, S., Tserovski, L., Hildebrandt, A., Motorin, Y., and Helm, M. (2016) CoverageAnalyzer (CAn): A Tool for Inspection of Modification Signatures in RNA Sequencing Profiles. Biomolecules 6, 1–7. (66) Machnicka, M. A., Milanowska, K., Osman Oglou, O., Purta, E., Kurkowska, M., Olchowik, A., Januszewski, W., Kalinowski, S., Dunin-Horkawicz, S., Rother, K. M., et al. (2013) MODOMICS: a database of RNA modification pathways--2013 update. Nucleic Acids Res. 41, D262-7. (67) Wollowitz, S., and Nerio, A. (2002) Psoralens for pathogen inactivation. Google Patents. (68) Hayrapetyan, A., Grosjean, H., and Helm, M. (2009) Effect of a quaternary pentamine on RNA stabilization and enzymatic methylation. Biol. Chem. 390, 851–61. 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(69) Thüring, K., Schmid, K., Keller, P., and Helm, M. (2016) Analysis of RNA modifications by liquid chromatography-tandem mass spectrometry. Methods 107, 48–56.

TOC Graphic


Page 24 of 24

Alkyne-Functionalized Coumarin Compound for Analytic and

Recommend Documents