For pairs of RNA MedChemExpress CB-5083 modifications and corresponding labeling agents [7], we found only one example for a coalescence of the reactive electrophile and a fluorescent dye into a single scaffold, as opposed to just linking the two moieties via a series of covalent bonds. Most interestingly, this example concerned bromomethylcoumarines, in particular 4-bromomethyl-7-methoxycoumarin (BMB) (see Figure 1 first panel). BMB was reported to selectively alkylate the uridine derivatives pseudouridine () and 4-thiouridine in reactions with native tRNA [36]. Furthermore, the reaction conditions were reported to influence the selectivity to a significant extend, including selective alkylation of pseudouridine. Compared to various aforementioned, relatively small alkylating agents, the coumarin scaffold has the advantage of being directly detectable due to its fluorescent properties, and to allow incorporation of additional functionalReaction of coumarins with RNARNA used in this study. Total tRNA E. coli was prepared by gel-purification of total RNA from E. coli (Roche, Mannheim, Germany) In-vitro-transcript of Mus musculus (M. m.) tRNAAsp was prepared by in vitro transcription as described by Jurkowski et al. [42] and gel-purified. The quality of the used tRNA was analyzed on a 10 denaturing urea polyacrylamide gel. The tRNA bands were stained with GelRed (Biotium, Hayward, CA, USA) and imaged on a Typhoon (GE Healthcare) with excitation at 532 nm and emission filter 610 nm. For IVT, only one major band can be seen. The native tRNA shows two tRNA bands (70 and 85 nt), as expected (see Figure S1 in File S1).Specific Alkylation of Modified NucleosidesFigure 1. Coumarins used in this study. The coumarins are 1315463 isomeric pairs, substituted with 3 different The assays. Thus, patients in the MC and NE groups had substituents (color code: methoxy-red, methyl-blue, phenyl-annulated green).doi: 10.1371/journal.pone.0067945.gDerivatization conditions 1. 0.5 / tRNA (16.67 of 3 / tRNA stock solution) was incubated with 8.8 mM coumarin (44.13 of stock at 20 mM dissolved in pure DMSO), 62.5 mM phosphate buffer (6.25 of 1 M stock pH 6.5) and 75 DMSO (30.88 pure DMSO and 2.1 water) at 37 for 300 minutes under light protection. After thereaction, 10 volumes of a 2 LiClO4 in aceton solution were added and RNA was precipitated by centrifugation at 15000 g at room temperature for 30 minutes and the pellet washed with pure aceton. The RNA was redissolved in pure water to yield a 0.5 / solution.Specific Alkylation of Modified NucleosidesDerivatization conditions 2. 0.5 / tRNA (16.67 of 3 / tRNA stock solution) was incubated with 10 mM coumarin (50 of stock at 20 mM dissolved in pure DMSO), 100 mM phosphate buffer (10 of 1 M stock pH 8.25) and 70 DMSO (20 pure DMSO and 3.23 water) at 37 for 180 minutes under light protection. The tRNA workup was the same as described for conditions 1. PAGE analysis of coumarin treated tRNA. The concentration of coumarin-conjugated tRNA was determined using a Nanodrop-ND-2000 (Peqlab, Erlangen, Germany) and 50 were analyzed on a 10 urea gel. Blue fluorescence of coumarin labeled tRNA was observed upon radiation with UV light (365 nm) and imaged with a Geldoc (Peqlab, Erlangen, Germany). Afterwards, the tRNA was stained for 10 minutes with GelRed (Biotium, Hayward, CA, USA).LC-MS method and analysisSample preparation. 10 of coumarin treated tRNA (final concentration 1 / ) was digested with Nuclease P1, Snake Venom Phosphodiesterase and Shrimp Alkaline Phospha.For pairs of RNA modifications and corresponding labeling agents [7], we found only one example for a coalescence of the reactive electrophile and a fluorescent dye into a single scaffold, as opposed to just linking the two moieties via a series of covalent bonds. Most interestingly, this example concerned bromomethylcoumarines, in particular 4-bromomethyl-7-methoxycoumarin (BMB) (see Figure 1 first panel). BMB was reported to selectively alkylate the uridine derivatives pseudouridine () and 4-thiouridine in reactions with native tRNA [36]. Furthermore, the reaction conditions were reported to influence the selectivity to a significant extend, including selective alkylation of pseudouridine. Compared to various aforementioned, relatively small alkylating agents, the coumarin scaffold has the advantage of being directly detectable due to its fluorescent properties, and to allow incorporation of additional functionalReaction of coumarins with RNARNA used in this study. Total tRNA E. coli was prepared by gel-purification of total RNA from E. coli (Roche, Mannheim, Germany) In-vitro-transcript of Mus musculus (M. m.) tRNAAsp was prepared by in vitro transcription as described by Jurkowski et al. [42] and gel-purified. The quality of the used tRNA was analyzed on a 10 denaturing urea polyacrylamide gel. The tRNA bands were stained with GelRed (Biotium, Hayward, CA, USA) and imaged on a Typhoon (GE Healthcare) with excitation at 532 nm and emission filter 610 nm. For IVT, only one major band can be seen. The native tRNA shows two tRNA bands (70 and 85 nt), as expected (see Figure S1 in File S1).Specific Alkylation of Modified NucleosidesFigure 1. Coumarins used in this study. The coumarins are 1315463 isomeric pairs, substituted with 3 different substituents (color code: methoxy-red, methyl-blue, phenyl-annulated green).doi: 10.1371/journal.pone.0067945.gDerivatization conditions 1. 0.5 / tRNA (16.67 of 3 / tRNA stock solution) was incubated with 8.8 mM coumarin (44.13 of stock at 20 mM dissolved in pure DMSO), 62.5 mM phosphate buffer (6.25 of 1 M stock pH 6.5) and 75 DMSO (30.88 pure DMSO and 2.1 water) at 37 for 300 minutes under light protection. After thereaction, 10 volumes of a 2 LiClO4 in aceton solution were added and RNA was precipitated by centrifugation at 15000 g at room temperature for 30 minutes and the pellet washed with pure aceton. The RNA was redissolved in pure water to yield a 0.5 / solution.Specific Alkylation of Modified NucleosidesDerivatization conditions 2. 0.5 / tRNA (16.67 of 3 / tRNA stock solution) was incubated with 10 mM coumarin (50 of stock at 20 mM dissolved in pure DMSO), 100 mM phosphate buffer (10 of 1 M stock pH 8.25) and 70 DMSO (20 pure DMSO and 3.23 water) at 37 for 180 minutes under light protection. The tRNA workup was the same as described for conditions 1. PAGE analysis of coumarin treated tRNA. The concentration of coumarin-conjugated tRNA was determined using a Nanodrop-ND-2000 (Peqlab, Erlangen, Germany) and 50 were analyzed on a 10 urea gel. Blue fluorescence of coumarin labeled tRNA was observed upon radiation with UV light (365 nm) and imaged with a Geldoc (Peqlab, Erlangen, Germany). Afterwards, the tRNA was stained for 10 minutes with GelRed (Biotium, Hayward, CA, USA).LC-MS method and analysisSample preparation. 10 of coumarin treated tRNA (final concentration 1 / ) was digested with Nuclease P1, Snake Venom Phosphodiesterase and Shrimp Alkaline Phospha.
