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Electronic Supplementary Information

Electronic Supplementary Information. Synthesis and evaluation of fluorescent cap analogues for mRNA labelling Marcin Ziemniak a , Mariusz Szabelski b , Maciej Lukaszewicz a , Anna Nowicka a , Edward Darzynkiewicz a , Robert E. Rhoads c , Zbigniew Wieczorek b , Jacek Jemielity a , d *.

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Electronic Supplementary Information

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  1. Electronic SupplementaryInformation Synthesis and evaluation of fluorescent cap analogues for mRNA labelling Marcin Ziemniaka, Mariusz Szabelskib, Maciej Lukaszewicza, Anna Nowickaa, Edward Darzynkiewicza, Robert E. Rhoadsc, Zbigniew Wieczorekb, Jacek Jemielitya,d*. a Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, ZwirkiiWigury 93, 02-089 Warsaw, Poland b Department of Physics and Biophysics, University of Warmia and Mazury in Olsztyn, Oczapowskiego 4, 10-719 Olsztyn, Poland c Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA d Centre of New Technologies, University of Warsaw, ZwirkiiWigury 93, 02-089 Warsaw, Poland

  2. Data presented in Table S1 illustrates effect of polarity of different solvents on the spectroscopic properties of AntOMe and MantOMe. In this study MeOH, EtOH and iPrOH were chosen as appropriate organic solvents since theirs protic properties and dipole moments provide a close resemblance to water. Despite that absorption maxima and absorption coefficients displays only a slightly variation upon a change of solvent in case of AntOMe a 9 nm shift on changing from PBS to organic solvents was observed. Interestingly, fluorescence of AntOMe and MAntOMe decreases in intensity and is red shifted with increasing solvent polarity. Quantum yields calculated for alcohol solutions are almost five times higher than in phosphate buffer solutions. Time-resolved experiments revealed a tendency to increase fluorescence lifetime if solvent polarity is decreased. The most significant changes were observed for AntOMe as its lifetime in aqueous medium (PBS, pH 5.175) was equal 1.81 ns, however, in ethanol it was prolonged to 8.25 ns (Table S1.).

  3. Table S1. Spectroscopic properties of methyl anthranilate and methyl N-methylanthranilate in phosphate buffer and organic solvents.

  4. Fig. S1. Absorbance and emission spectra of AntOMe Fig. S2. Absorbance and emission spectra of MantOMe

  5. Fig. S4. Changes of fluorescenceintensity of Mant-m7GpppGand Trpresidues in eIF4E upon complexformation. The excitationwavelengthused in the experiment was 280 nm (maximum absorbtion for Trpresidues). eIF4Econcentration was 100 nM and Mant-m7GpppG concentration was 200 nM. • Fig. S3. MantOMe fluorescence intensity observed upon addition of increasingamounts of eIF4E protein. The concentration of MantOMe was 200 nM and the eIF4E concentrations were in range 50-1000 nM.

  6. Table S2. 1H NMR signals of 2' and 3' regioisomers of cap analogues 1-2

  7. Table S3. Conformational and regioisomericequilibria for some (M)Ant labelled nucleotides

  8. Ant-m7GpppG isomer C2’ 29% isomer C3’ 64% 7% HPLC profile

  9. Ant-m7GpppG λ (nm) Emission spectrum

  10. Ant-m7GpppG λ (nm) Absorption spectrum

  11. Ant-m7GpppG ESI (-) MS

  12. Ant-m7GpppG 1H NMR

  13. Ant-m7GpppG 31P NMR

  14. Ant-m7GpppG

  15. Mant-m7GpppG isomer C3’ 60% isomer C2’ 40% HPLC profile

  16. Mant-m7GpppG λ (nm) Emission spectrum

  17. Mant-m7GpppG λ (nm) Absorption spectrum

  18. Mant-m7GpppG ESI (-) MS

  19. Mant-m7GpppG 1H NMR

  20. Mant-m7GpppG 31P NMR

  21. Mant-m7GpppG

  22. Mant-m7GpCH2ppG isomer C2’ 34% isomer C3’ 50% 4% 7% 5% HPLC profile

  23. Mant-m7GpCH2ppG λ (nm) Emission spectrum

  24. Mant-m7GpCH2ppG λ (nm) Absorption spectrum

  25. Mant-m7GpCH2ppG ESI (-) MS

  26. Mant-m7GpCH2ppG 1H NMR

  27. Mant-m7GpCH2ppG 31P NMR

  28. Mant-m7GppCH2pG isomer C3’ 63% isomer C2 31% 6% HPLC profile

  29. Mant-m7GppCH2pG λ (nm) Emission spectrum

  30. Mant-m7GppCH2pG λ (nm) Absorption spectrum

  31. Mant-m7GppCH2pG ESI (-) MS

  32. Mant-m7GppCH2pG 1H NMR

  33. Mant-m7GppCH2pG 31P NMR

  34. Ant-m27,2’-OGpppG 100% HPLC profile

  35. Ant-m27,2’-OGpppG λ (nm) Emission spectrum

  36. Ant-m27,2’-OGpppG λ (nm) Absorption spectrum

  37. Ant-m27,2’-OGpppG ESI (-) MS

  38. Ant-m27,2’-OGpppG 1H NMR

  39. Ant-m27,2’-OGpppG 31P NMR

  40. Ant-m27,2’-OGpppG

  41. Ant-m7GDP isomer C3’ isomer C2’ HPLC profile

  42. Ant-m7GDP λ (nm) Emission spectrum

  43. Ant-m7GDP λ (nm) Absorption spectrum

  44. Ant-m7GDP ESI (-) MS

  45. Ant-m7GDP 1H NMR

  46. Ant-m7GDP 31P NMR

  47. Mant-m7GDP isomer C3’ isomer C2’ HPLC profile

  48. Mant-m7GDP ESI (-) MS

  49. Mant-m7GDP λ (nm) Emission spectrum

  50. Mant-m7GDP λ (nm) Absorption spectrum

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