Mean and Low Frequency Wave Forces on Semi Subs - JA Pinkster NSMB
Supplementary Data Set 1 95 55 36 28 17 10 130 250 FH Ctrl FH FMR1iso1 FH FMR1iso1I304N...
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Supplementary Data Set 1 Contents: Supplementary text: Synthesis of m 6 A phosphoramidite, pages 2-4. Uncropped images, pages 5-8. Nature Structural & Molecular Biology: doi:10.1038/nsmb.3462
Transcript of Supplementary Data Set 1 95 55 36 28 17 10 130 250 FH Ctrl FH FMR1iso1 FH FMR1iso1I304N...
Supplementary Data Set 1
Contents: Supplementary text: Synthesis of m6A phosphoramidite, pages 2-4. Uncropped images, pages 5-8.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.3462
SupplementrayNote1:Synthesisofm6Aphosphoramidite.
Synthesisofm6Aphosphoramidite.
N6-methyladenosine(1)wassynthesizedaccordingtopublishedprocedures1.
N6-methyladenosine(1.50g,5.33mmol,1.00eq.)wasdissolvedinDMF(4mL)andthesolutioncooledto 0 °C. Di-tert-butyl-silyl bis(trifluoromethanesulfonate) (1.91 mL, 5.87 mmol, 1.10 eq.) was addeddropwise and the reaction stirred for 75 min, after which more di-tert-butyl-silylbis(trifluoromethanesulfonate)(0.3mL,921µmol0.17eq.)wasadded.Afterstirringat0°Cforanother3 h, the reaction was ended by adding a solution of NaHCO3 (aq. sat., 15 mL). The suspension wasdilutedwithEtOAc(250mL),thelayersseparatedandtheaqueousphaseextractedwithEtOAc(2×20mL).ThecombinedorganiclayersweredriedoverNa2SO4andevaporated.
TheresultingstickyyellowfoamwasdissolvedinMeCN(15mL)and1,8-Diazabicyclo[5.4.0]undec-7-ene(2.79mL,18.67mmol3.50eq.),TBDMSCl(2.01g,13.33mmol,2.50eq.)and4-(Dimethylamino)pyridine(65mg,530µmol,0.10eq.)wereadded.Afterstirringatroomtemperaturefor15h,methanol(20mL)wasaddedandthereactionstirredforfurther30min.Thesolventswereremovedinvacuo,theresiduetakenupinEtOAc(250mL)andthesolutionwashedwithasemi-saturatedsolutionofNaHCO3(aq.,4×50mL),NaCl (aq. sat., 50mL). Itwas driedoverNa2SO4 and the solvent evaporated to give a yellowfoam.Purificationbysilicaflashchromatography(columnpackedandcrudeloadedwithCH2Cl2+0.5%pyridine, elution with hexanes/acetone 5:1 + 0.5% pyridineg4:1 + 0.5% pyridine) gave pro-tectednucleoside2asawhitefoam(1.41g,2.63mmol,49%).
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Nature Structural & Molecular Biology: doi:10.1038/nsmb.3462
1HNMR(800MHz,CDCl3)δ=8.35(s,1H),7.75(s,1H),6.28(bs,1H),5.89(s,1H),4.60(d,J=4.8Hz,1H),4.53(dd,J=9.6,4.8Hz,1H),4.46(dd,J=9.3,5.2Hz,1H),4.19(ddd,J=10.4,5.6,4.8Hz,1H),4.01(dd,J=10.4,9.4Hz,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).
13CNMR (201MHz,CDCl3)δ=155.6,153.5,138.0,92.5,75.9,75.6,74.8,67.9,27.6,27.1,26.0,22.8,20.5,18.4,-4.2,-4.9.
HRMS-ESI(m/z):calcdforC25H46N5O428Si2+[M+H]+:536.3083.Found:536.3083.
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),1304(s),1252(m),1226(s),1203(s),1163(m),1131(l),1105(m),1076(m),1050(l),1022(s),1001(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 m6A probes. The synthesis of the RNA oligonucleotides was performed on an ABI 394DNA/RNA Synthesizer (Applied Biosystems) using typical reagent concentrations (activator: 0.3Mbenzylthiotetrazole in MeCN (Link Technologies), detritylation: 3% dichloroacetic acid in CH2Cl2,oxidation: 25mM I2 in MeCN/H2O/2,6-lutidine (65/30/6), capping: Ac2O/2,6-lutidine/MeCN (30 ppmH2O)(20/30/50)and20%N-methylmidazoleinMeCN(10ppmH2O).Theoligonucleotidesyntheseswereperformedon1µmolscale,1000ÅCPGcarriersusing0.1MRNACE-TBDMS-phosphoramidites:A(Bz-A), C (Ac-C), G (dmf-G), U, 5’-Biotin, obtained from Link Technologies. m6A phosphoramidite wassynthesized according to the included procedure and incorporated into RNA using the standardprotocol.Thecouplingtimesform6Aandbiotinwereincreasedto20mintoensuremaximumcouplingefficiency. Partially degenerate sequences were prepared using premixed equimolar mixtures of theintended building blocks for the individual positions and also coupled for 20min at the randomizedpositions.
Afterremovingtheresinfromthecartridges,allstrandsweretreatedwith1mLAMA(28%NH4OHinH2O/40%MeNH2inH2O,1:1)atroomtemperaturefor5minutesandat65°Cfor5minutestoensurecompletecleavagefromthecarrierandbasedeprotection.ThesupernatantwasdriedinvacuoandtheresiduetakenupinDMSO(100µL)andEt3N·3HF(125µL)andheatedto65°Cforanother90minutes.AfteradditionofasolutionofNaOAc(3M,25µL)theoligoribonucleotideswereprecipitatedbyadditionofn-BuOH(1mL)andcoolingto-80°Cfor12h.Centrifugationat4°C/21130g/60minaffordedthecrudeproducts.AnalysisandpurificationwasperformedonaWatersHPLCsystem(WatersAlliance2695withPDA2996,preparativeHPLC:1525with2487UVdetector)withVP250/10Nucleosil100-7C18columnsfromMachereyNagelusingagradientof0.1Mtriethylamine/aceticacidinwaterand80%acetonitrile.Afterpurification,theproductsweredesaltedusingSep-PakC18ClassicCartridges(Waters)accordingtothemanufacturersprotocol.TheidentityofthestrandswasconfirmedbyMALDI-ToF-MSandthepuritydeterminedbyanalyticalHPLC.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.3462
1. Hobartner,C.etal.Thesynthesisof2'-O-[(triisopropylsilyl)oxy]methyl(TOM)phosphoramiditesofmethylatedribonucleosides(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)foruseinautomatedRNAsolid-phasesynthesis.MonatshefteFurChemie134,851-873(2003).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.3462
Supplementary Fig. 1dControl m6A
before after before after
input control m6A control m6A GGACU GAACU
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Supplementary Note 1: Uncropped images
Nature Structural & Molecular Biology: doi:10.1038/nsmb.3462
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Nature Structural & Molecular Biology: doi:10.1038/nsmb.3462
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Nature Structural & Molecular Biology: doi:10.1038/nsmb.3462
