Supplementary Data Set 1

Transcription

1 Supplementary Data Set 1 Contents: Supplementary text: Synthesis of m 6 A phosphoramidite, pages 2-4. Uncropped images, pages 5-8. ature Structural & olecular Biology: doi:.38/nsmb.3462

2 Supplementray ote 1: Synthesis of m 6 A phosphoramidite. Synthesis of m 6 A phosphoramidite. H H H 1) tbu 2 Si(Tf) 2 PacCl, DBU 2) TBDSCl, HBt 4-DAP, DBU CH 2 Cl 2, pyridine 22 h, RT g 80 C Si Si H H 49% TBDS 75% TBDS over 2 steps ) HF pyridine 2) DTCl, pyridine DT CEDCl, ipr 2 Et, CH 2 Cl 2 5 h, RT DT 58% over 2 steps H 4 TBDS 99% P TBDS C 5 6 -methyladenosine (1) was synthesized according to published procedures methyladenosine (1.50 g, 5.33 mmol, 1.00 eq.) was dissolved in DF (4 ml) and the solution cooled to 0 C. Di-tert-butyl-silyl bis(trifluoromethanesulfonate) (1.91 ml, 5.87 mmol, 1. eq.) was added dropwise and the reaction stirred for 75 min, after which more di-tert-butyl-silyl bis(trifluoromethanesulfonate) (0.3 ml, 921 µmol 0.17 eq.) was added. After stirring at 0 C for another 3 h, the reaction was ended by adding a solution of ahc 3 (aq. sat., 15 ml). The suspension was diluted with EtAc ( ml), the layers separated and the aqueous phase extracted with EtAc (2 20 ml). The combined organic layers were dried over a 2 S 4 and evaporated. The resulting sticky yellow foam was dissolved in ec (15 ml) and 1,8-Diazabicyclo[5.4.0]undec-7-ene (2.79 ml, mmol 3.50 eq.), TBDSCl (2.01 g, mmol, 2.50 eq.) and 4-(Dimethylamino)pyridine (65 mg, 530 µmol, 0. eq.) were added. After stirring at room temperature for 15 h, methanol (20 ml) was added and the reaction stirred for further 30 min. The solvents were removed in vacuo, the residue taken up in EtAc ( ml) and the solution washed with a semi-saturated solution of ahc 3 (aq., 4 50 ml), acl (aq. sat., 50 ml). It was dried over a 2 S 4 and the solvent evaporated to give a yellow foam. Purification by silica flash chromatography (column packed and crude loaded with CH 2 Cl % pyridine, elution with hexanes/acetone 5: % pyridineg 4: % pyridine) gave pro-tected nucleoside 2 as a white foam (1.41 g, 2.63 mmol, 49%). ature Structural & olecular Biology: doi:.38/nsmb.3462

3 1 H R (800 Hz, CDCl 3 ) δ = 8. (s, 1H), 7.75 (s, 1H), 6.28 (bs, 1H), 5.89 (s, 1H), 4.60 (d, J = 4.8 Hz, 1H), 4.53 (dd, J = 9.6, 4.8 Hz, 1H), 4.46 (dd, J = 9.3, 5.2 Hz, 1H), 4.19 (ddd, J =.4, 5.6, 4.8 Hz, 1H), 4.01 (dd, J =.4, 9.4 Hz, 1H), 3.15 (s, 3H), 1.06 (s, 9H), 1.02 (s, 9H), 0.91 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H). 13 C R (201 Hz, CDCl 3 ) δ = 1.6, 153.5, 138.0, 92.5, 75.9, 75.6, 74.8, 67.9, 27.6, 27.1, 26.0, 22.8, 20.5, 18.4, -4.2, HRS-ESI (m/z): calcd for C H Si 2 + [+H] + : Found: IR (ATR, neat): ν = 3284 (s), 3234 (s), 2931 (s), 2895 (s), 2859 (s), 2360 (s), 2341 (s), 1637 (m), 1583 (s), 1541 (s), 1472 (m), 1413 (s), 1388 (s), 1363 (s), 1346 (s), 1330 (s), 4 (s), 12 (m), 1226 (s), 1203 (s), 1163 (m), 1131 (l), 15 (m), 76 (m), 50 (l), 22 (s), 1 (l), 938 (m), 907 (s), 889 (m), 833 (l), 826 (l), 793 (m), 783 (l), 751 (m), 740 (s), 720 (s), 683 (s), 668 (s). Synthesis of m 6 A probes. The synthesis of the RA oligonucleotides was performed on an ABI 394 DA/RA Synthesizer (Applied Biosystems) using typical reagent concentrations (activator: 0.3 benzylthiotetrazole in ec (Link Technologies), detritylation: 3% dichloroacetic acid in CH 2 Cl 2, oxidation: m I 2 in ec/h 2 /2,6-lutidine (65/30/6), capping: Ac 2 /2,6-lutidine/eC (30 ppm H 2 ) (20/30/50) and 20% -methylmidazole in ec ( ppm H 2 ). The oligonucleotide syntheses were performed on 1 µmol scale, 0 Å CPG carriers using 0.1 RA CE-TBDS-phosphoramidites: A (Bz- A), C (Ac-C), G (dmf-g), U, 5 -Biotin, obtained from Link Technologies. m 6 A phosphoramidite was synthesized according to the included procedure and incorporated into RA using the standard protocol. The coupling times for m 6 A and biotin were increased to 20 min to ensure maximum coupling efficiency. Partially degenerate sequences were prepared using premixed equimolar mixtures of the intended building blocks for the individual positions and also coupled for 20 min at the randomized positions. After removing the resin from the cartridges, all strands were treated with 1 ml AA (28% H 4 H in H 2 /40% eh 2 in H 2, 1:1) at room temperature for 5 minutes and at 65 C for 5 minutes to ensure complete cleavage from the carrier and base deprotection. The supernatant was dried in vacuo and the residue taken up in DS ( µl) and Et 3 3HF (1 µl) and heated to 65 C for another 90 minutes. After addition of a solution of aac (3, µl) the oligoribonucleotides were precipitated by addition of n-buh (1 ml) and cooling to -80 C for 12h. Centrifugation at 4 C/21 g/60 min afforded the crude products. Analysis and purification was performed on a Waters HPLC system (Waters Alliance 2695 with PDA 2996, preparative HPLC: 15 with 2487 UV detector) with VP / ucleosil -7 C 18 columns from acherey agel using a gradient of 0.1 triethylamine/acetic acid in water and 80% acetonitrile. After purification, the products were desalted using Sep-Pak C18 Classic Cartridges (Waters) according to the manufacturers protocol. The identity of the strands was confirmed by ALDI-ToF-S and the purity determined by analytical HPLC. ature Structural & olecular Biology: doi:.38/nsmb.3462

4 1. Hobartner, C. et al. The synthesis of 2 '--[(triisopropylsilyl)oxy] methyl (T) phosphoramidites of methylated ribonucleosides (m(1)g, m(2)g, m(2)(2)g, m(1)i, m(3)u, m(4)c, m(6)a, m(2)(6)a) for use in automated RA solid-phase synthesis. onatshefte Fur Chemie 134, (2003). ature Structural & olecular Biology: doi:.38/nsmb.3462

5 Supplementary ote 1: Uncropped images Supplementary Fig. 1d Control m6a before after before after Figure 2b GGACU input control m6a GAACU control m6a ature Structural & olecular Biology: doi:.38/nsmb.3462

6 Figure. 3f G3BP1 E G3BP1 KD sicon sig3bp1 Con F-G3BP1 Figure. 3h G3BP1 Ponceau loading control ETTL3 ature Structural & olecular Biology: doi:.38/nsmb.3462 G3BP1 ETTL3

7 Figure. 4f IP FT Input IP con m6a FT input IP input con m6a FT FH-FR1iso1 FH-FR1iso1I304 FH Ctrl FR1 I304 Input FR1 Input Figure 4a Figure 4j FR1 E o Dox Dox FR1 I304 E Ponceau o Dox Dox Ponceau o Dox Dox o Dox Dox ETTL3 KD Ponceau Control KD Control KD ature Structural & olecular Biology: doi:.38/nsmb.3462

8 Supplementary Fig. 4b FR1 Con1 Con2 Con3 Con4 Con5 Supplementary Fig. 4c FH Ctrl FH FR1iso1 FH FR1iso1I ature Structural & olecular Biology: doi:.38/nsmb.3462