新規がん抑制遺伝子型microRNAの探索と創薬への展開
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(2) ưʨ OƇéˬ¹ĺĦ microRNA :ƙɡ6 ïʖ>:Œ˻. 2017 Ţ. ŕ˾ijļijļ̂ˢČ˖ļɁɋɆ ȣȘˇȇɆļŋƨ (ŕ˾ijļ) ¥dž ʌ³.
(3) ưʨ OƇéˬ¹ĺĦ microRNA :ƙɡ6 ïʖ>:Œ˻. ¥dž ʌ³.
(4) Ȼ Ǟ ȫʺʠ. ɑ 1 Ɏ Ŧʾ. 1̤1 RNA Šȁ. 1. 1̤2 miRNA 6Ȳø. 2. 1̤3 miRNA 9GJǘȵ mRNA :ʸˁ. 3. 1̤4 amNȥ.ǘȵ mRNA :©Ȇ. 4. 1̤5 ǘȵ mRNA N©Ȇ)Jȑ:Ł̠ȵ8ƅǯ. 5. 1̤6 ׹Źźǡĩ. 8. 1̤7 tXofQf. 8. ɑ 2 Ɏ ׹ŹźǡĩNōÙ'.ưʨ RNA :ČƁ6,: RNA ƕȞʀ. 2̤1 ׹Źźx]XeuQv^Ą=,:QnSt½:ČƁ. 11. 2̤2 x]XeuQv^ 1 G= 2 NōÙ'. RNA :ČƁ 17 2̤3 Qv^ 1 G= 2 NďCªdž˸:ȖȵĽŀź. 18. 2̤4 RNA : RNA ƕȞʀ. 22. 2̤5 Qv^ 2 NōÙ'. miRNA 9GJˬ¹ĺȳȠƇéʀ:ǔʴ 25 ɑ 3 Ɏ ׹Źź miRNA 9GJǘȵ mRNA :ƕȞ. 3̤1 miRNA :ʲʯ. 27. 3̤2 miRNA :˯æ. 28. 3̤3 Qv^ 2 NōÙ'. miR-145 9GJǘȵˬ¹ĺ: Ƈéʀǔʴ. 30. 3̤4 Qv^ 2 NōÙ'. miR-145 9GJǘȵ mRNA :ƕȞ 33.
(5) ɑ 4 Ɏ ׹ŹźǡĩĄ=VowĩNōÙ'.ưʨ RNA :ČƁ6,: RNA ƕȞʀ. 4̤1 jȢ9fQfwĩĄ=VowĩNǁ)JQaʹō ½:ČƁ. 36. 4̤2 x]XeuQv^ 25 Ą=,:QnSt½:ČƁ. 37. 4̤3 Qv^ 25 NōÙ'. RNA :ČƁ. 38. 4̤4 RNA : RNA ƕȞʀ. 38. 4̤5 Qv^ 25 NōÙ'. miRNA 9GJˬ¹ĺȳȠƇéʀ:ǔ 39. ʴ ɑ 5 Ɏ cRGD af`t:ČƁ6,:ˬ¹ĺȳȠƇéʀ 5̤1 miRNA ùʖĖ:˻ȳ. 42. 5̤2 ǐ˲ùʖĖ:s|. 43. 5̤3 džɁɋ:Ȼȵ. 45. 5̤4 cRGD-siRNA af`t:ʲʯ. 46. 5̤5 QzÊ̞QnStĄ=ġȽü½:ČƁ. 47. 5̤6 gcQnSt:ČƁ. 47. 5̤7 QzÊ̞ RNA :ČƁ. 48. 5̤8 RNA54 :ČƁ. 49. 5̤9 RNA55 :ČƁ. 51. 5̤10 RNA56 :ČƁ. 53. 5̤11 cRGD-siRNA af`t:ɣɿʆ˞˦ź. 55. 5̤12 cRGD-siRNA af`t:ˬ¹ĺȳȠƇéʀ. 57. 5̤13 QÊ̞ cRGD-siRNA af`t:ČƁ6ˬ¹ĺȳȠƇ éʀ 5̤14 x]Qjɸź miRNA :ČƁ6ǜʀʵÄ. 59 62. ɑ 6 Ɏ ɧʺĄ=²Ű:Œǂ. 41. 6̤1 ɫƏ. 66. 6̤2 ²Ű:Œǂ. 68.
(6) ɑ 7 Ɏ Ł̠:ʷɣ. 7̤1 ÀȥǜĜĄ=Àȥʶʖ. 69. 7̤2 x]XeuQv^:ČƁ. 72. 7̤3 Xbx]Xou:ČƁ6ɚʤ. 85. 7̤4 Ȗİźǯ9GJ 50 %ʙʬȅŧ (Tm) :Ȇŀ. 86. 7̤5 Dual luciferase reporter assay. 87. 7̤6 RNA ƕȞʀ:ʵÄ. 88. 7̤7 TUgmprR^9GJˬ¹ĺƇéʀ:ʵÄ. 88. 7̤8 miRNA 9GJǘȵ mRNA :ƕȞ. 89. 7̤9 cRGD af`tXbx]Xou:ČƁ. 89. 7̤10 ɣɿʆ˞˦ź:ǔʴ. 90. 7̤11 ˬ¹ĺȳȠƇéʀʶ̠. 90. 7̤12 ƉʄȰǵźʵÄ. 90. 7̤13 x]Qjɸźʶ̠. 91. ăɶƭȝ. 92. ˀ˕. 99.
(7) ȫʺʠ. džɁɋÚʍ9ȥ.ȫʺ G=ȫʱĊ;·9Ʉ)˟I5J Ago. Argonaute. APS. ammonium peroxodisulfate. CPG. controlled pore glass. DIPEA. N, N-diisopropylethylamine. DMAP. 4-dimethylaminopyrideine. DMF. dimethylformamide. DMSO. dimethylsulfoxide. DMTr. 4,4-dimethoxy trityl. ds. double strand. EDTA. ethylenediamine-N, N, N’, N’-tetraacetic acid. MALDI-TOF. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. MASS. MASS spectroscopy. MeCN. Acetonitrile. MEM. minimum essential medium. mRNA. messenger ribonucleic acid. n-BuLi. n-butyllithium. NMR. nuclear magnetic resonance. OD. optical density. PAGE. Poly-Acrylamide Gel Electrophoresis. PCR. polymerase chain reaction. RNA. ribonucleic acid. ss. single strand. TBAF. tributylammoniumfluoride. TBDMSCl. tert-buthyldimethylsilyl chloride. TBE. tris-bornic acid-EDTA. TEAA. triethylamine-acetic acid. TEMED. N,N,N’,N’-tetramethyl-ethylenediamine. THF. tetrahydrofuran.
(8) TLC. thin-layer chromatography. Tm. melting temperature. TMSOTf. Trimethylsilyl Trifluoromethanesulfonate. Tris. tris(hydroxymethyl)aminomethane. TsCl. p-toluensulfonyl chloride. UV. ultraviolet.
(9) ɑ 1 Ɏ Ŧʾ 1-1 RNA Šȁ. RNA Šȁ (RNAi; RNA interference) 6;ɣɿÜ>ōÙ&K.ªdž˸ RNA ˊȐ681 4˯æșȭȵ9ǘȵ mRNA äʬ&KJȠ˃5J$:Ƞ˃ ǀç9ĪĒ&K.:; 1998 Ţ5IFire 6 Mello H;ɬʗNȥ.Ɂɋ H$:Ƞ˃Nʧã'. 1,:ŰƷ ʗʒ̚ʦ̚ɣɿäʡ˱ǣvQ875EčǗ:Ƞ˃ ʪŇ&KRNA Šȁ Ť!ɀHKJG981.$: RNA Šȁ:ȳʧ9GIRNA ;DNA Hˬ¹żĪ Nm}]ˈ9ɵʳ)JA5:³ģȘ6'4:ǜʀ/"5;8!RNA Ȝʊ:Ǘ8ǜʀ NƐ2$6 ʬƹ&K;(D.. ds RNA Cytoplasm Nucleus DNA. Dicer. siRNA RISC. pri-miRNA. target mRNA. Drosha pre-miRNA. degradation Exportin-5. mature miRNA. Dicer. RISC. target mRNA repression of gene expression. Figure 1. RNA n. 1.
(10) RNA Šȁ;ȣ½Ü9ōÙ&K.ªdž˸ RNA (dsRNA) ũ. ˷68Iũ ˊ$&KJ. (Figure 1) ɣɿÜ9ōÙ&K. dsRNA ;RNase III P9œ)J Dicer 9G14 2 Ĭĩ:X|{^ˮ»NƐ2 21~25 Ĭĩʼnɉŧ:¼äĺ RNA (siRNA; small interfering RNA) >6åƯ&KJɨ4ªdž˸ siRNA ; Argonaute2 m}]ˈ9ćI˗AK4 ´:Ğĺ66E9 RNA-induced silencing complex (RISC) NŬƁ)J,:Űªdž˸ siRNA ;Ʊ:˸ Aronaute2 :Vux]jǵź9GJåƯNĈ"4dž˸ø'ƁȕĦ RISC 68J$:̅Argonaute2 9ǡJ˸;ZSu˸äʬ&KJ˸;}pif˸ 6ē<K4JRISC ;ZSu˸6Ƚʣȵ8˯æNďCǘȵ mRNA Nʸˁ'ZSu˸: ¥ĵˮä5ǘȵ mRNA NåƯ)J$65ˬ¹ĺȳȠNƇé)J 2. ƱmicroRNA (miRNA) ; 20~24 Ĭĩɉŧ:ÜĞźªdž˸ RNA 5ɵʳ 5m }]ˈ:ȳȠʽɖNʜɣɿÜʽɖĞĺ5JmiRNA ǀç:ĪĒ;1993 ŢLee H 9G148&K. 3 ŮH;ɬʗ:Ɍȓİȭ½Nȥ.Ł̠ Hɾȳȣ:ƻ˼éų9 miRNA 5J lin-4 ˽¡'4J$6NƹH 9'.˙Ţ5;ʦöȘș9~t9 4EIJ!: miRNA ʧ2 14 IȠģA59ɝ 1900 Ɋ̚ Ƀʸ&K4JmiRNA ˬ¹ĺ;çD9 RNA j9G14primary miRNA (pri-miRNA) 6ē<KJQ Ț: RNA >ːÞ&KJɨ4ǐÜ5Drosha 6ē<KJ RNase III uS˱ɠ9 G14åƯ&K4grǖˡNǁ)J precursor miRNA (pre-miRNA) NȣƁ)J pre-miRNA ; Exportin-5 9G14ɣɿˈ9˔˜&K.ŰDicer 9G14åƯ&K4ƁȕĦ miRNA 68J$:ªdž˸ miRNA ;,:ŰArgonaute NďCˢ:m}]ˈ66E9 RISC NŬƁ'ªdž˸ Hdž˸>:ʬ̉J;åƯNĈ"4dž˸68IRISC 9ǡ 1.˸NZSu˸6'4ǘȵ mRNA N˯æșȭȵ9ʸˁ)J$65m}]ˈ>:ɵʳN Ƈé)J 4miRNA ;ˮ9gpoĬĩʼnNďB8 H mRNA 9ɧČ)J$6 H 2: miRNA ʥƫ:ǘȵ mRNA ɧČ'ɵʳNƇé'4J$:.Dċ miRNA 9 ʼn)Jǘȵ mRNA :ʬNJ;ğ̊5IIJ!;DŽčŀ:AA5J. 1-2 miRNA 6Ȳø. ˙ŢIJ!:Ȯȯ9 4miRNA :ȳȠȭŞ ,:āĞ5J$6 ƹH 9814 ș9,:ȳȠ:ĭòĄ=ȳȠ:¼ ɣɿ:Ȳø:ȼƚȵ8āĞ68IŲJ miRNA J 5 ;oncomiR ē<K̎Ş9ʎ!Ɂɋ&K4J 6Ł̅9!2 : miRNA ;ǠŞɣɿ NȲɣɿ9ʹō)J$6F (oncomiR)Ȳɣɿ9QtegNʹō)J$6 ɀHK4 I (Table 1)oncomiR :˿Ńë (antagomir) F miRNA ʊ½ ư.8Ɖ Oë6'4DzȻN ̈D4JA.$KHȲ˽ˢ miRNA :ȳȠ˶NʪŇ)J$65ưʨ|SXY. 2.
(11) 6'4:èȥEǃů5. J. Table 1. \
(12) ±L miRNA. miRNA. ʄȰŬſ. ǜʀ. Reference. miR-122. ɹʈȲ. C ĦɹȎTRg:ʥʤNÇˣ. 7. miR-10b. ɻȲ. Ȳɣɿ:ːɈNÇˣ. 8. miR-221. ɹɣɿȲ. ʛɕŬƁNÇˣ. 9. miR-380-5p. Ʌɦʐɣɿʄ. ʄȰƇéĞĺ p53 :ȳȠNƇé. 10. miR-296. Ʌɦʇʄ. ʛɕŬƁNÇˣ. 11. let-7. IJ!:Ȳɣɿ. KRAS,MYC,HMG2 ɓ:Ȳˬ¹ĺNƇé. 12. miR-34. IJ!:Ȳɣɿ. QtegNʹō. 13. miR-143/145. ijʅȲɣɿ. MYC :ȳȠNƇé. 14. miR-26a. ɹɣɿȲ. cS] D2E2 :ȳȠNƇé. 15. ·:G9miRNA ;Ǘ8ȣȘļȵig9˽¡'4 Iǘȵ mRNA :čŀ; ɣɿÜʽɖĞĺ6'4: miRNA :Ó. Nȡʬ)J/"58!miRNA Nèȥ'.ư'. Ɖ OëF|SXY:˻ȳ61.ùȱä˵>:Œ˻Eǃů5 J'. 14 miRNA :ǘȵ mRNA :čŀ;˙Ţ˴ʦ8Ɂɋʼ̘:26814J. 1-3 miRNA 9GJǘȵ mRNA :ʸˁ. ê˛'.G9Argonaute 6ʥČ½NŬƁ'. miRNA ;Ś ƃ'NĈ"4dž˸9 ʬ̉)J$69GIƁȕĦ miRNA 68JArgonaute 9ǡ1.ZSu˸:0 5´Džɏ H 2-7 ȬȻ:Ĭĩ;eu̕ħ6ē<K$:̕ħ6ǘȵ mRNA : 3´UTR ˼5tk] p]Ĭĩʼn G!ÈĻ&K4J 16$:$6 HmiRNA 9GJǘȵ mRNA :ʸˁ9 ;eu̕ħ6ǘȵ mRNA :Ƚʣź ˴ʦ5J6ɶ HK4.Ł̅9 X ɬɧ ƾǖˡʬNJ HEArgonaute :ɧČ9G14eu̕ħ ǘȵ mRNA 6:ª˴ʚƳ ǖˡN"pre-organize"'4J. Figure 2. 6/ Argonaute2 0° RNA ¬. 3.
(13) $6 Ʉ&Keu̕ħ9 "Jǘȵ mRNA :ÕÖȵ8ĬĩʼnŬƁ;Vtȵ9 ǁè5J.D/6ʴƹ&K.(Figure 2) 17 A.ʍȵ9 miRNA :ǘȵ mRNA ȳȠƇ éʀ;ZSu˸ 5´Džɏ:ȽʣĬĩ (t1) Qsze:̅9Ď)JÒĎ9JIJ!: ʦ̚9 4t1 »9;Qsze G!ÈĻ&K4J$6 ɀHK4 I 18X ɬɧ ƾǖˡʬNJ9GI~t: Argonaute2 9 4 t1 Nʸˁ'4J MID uS9;Qsw ˮ»6șȭȵ9Ƚ«¾ȥ)JɧČ_pt Ļģ'4J$6 ƹH 981. (Figure 3) 19. A. B. 2Ia= -/ (B) t1
(14) edmW Figure 3. (A) MID 0AI
(15) hc.2 ° Argonaute2 a%. . 1-4 amNȥ.ǘȵ mRNA :©Ȇ. $KA5˛?.G8 miRNA 6ǘȵ mRNA ˼:ɧČǗŨNĩ9'4ǘȵ mRNA :© ȆQbh ƫIJ!˻ȳ&K4J (Table 2) TargetScanPicTarEMBLEMMO ; eu̕ħ:ȽʣźNÕÖȵ9ɶƀ'.^5I©Ȇ&KJǘȵ mRNA ;G! º4J' 'Ú4čǗ:ɧNj98Jʳ5;8!,K-Kʣˍȵ8˫ƋĩȈ Q bh9ɥB˗AK4JÁ < TargetScan ;eu̕ħ:Ƚʣź9ò t1 » Qsze5JG8 mRNA N»Ëʣ9ƒ#J $:»ɲ9ĬĩʼnNŬƁ)J mRNA N»Ëʣ9ƒ#JQbhEĻģ)Jű14ZSu˸: 5´Džɏ Te :īČ9;4 Qbh5čǗ:ɧNj ŲHKJ Te·ı:īČ5;ɧNj ȭ814!J´9Eȭ8J˫ƋĩȈ ċ^9ɥB˗AK4J rXk ʬNJ6ǥˑNʜ1.̅9ǀEʎƁɯNĆD.:; TargetScan 51. 20. 4.
(16) Table 2. ǘȵ mRNA N©Ȇ)J.D:am^ ^. ʼn˃Ɋ. . a. TargetScan. m,w,f. ǘȵˬ¹ĺ:˫ãĩȈ. URL. Reference. eu̕ħ:Ƚʣź. http://www.targetscan.org. 21. http://russell.embl-heidelberg.de. 22. http://pictar.mdc-berlin.de. 23. http://www.mirz.unibas.ch/ElMMo2. 24. http://www.microrna.org. 25. http://microrna.sanger.ac.uk. 26. Jɉŧ:eu̕ħ. http://genie.weizmann.ac.il/pubs/. 27. :ȽʣźɧČ̕ħ:. mir07/mir07_data.html. ɧČ̕ħ:ƫǘȵ 1 :˯æ EMBL. f. eu̕ħ:Ƚʣź ɧČ̕ħ:ƫǘȵ6 :ªdž˸:ȖȵĽŀź. PicTar. m,f,w. eu̕ħ:Ƚʣź ɧČ̕ħ:ƫǘȵ6 :ªdž˸:ȖȵĽŀź. EIMMo. m,f,w. Jɉŧ:eu̕ħ :ȽʣźɧČ̕ħ: ƫ. Miranda. m,f,w,+. Jɉŧ:eu̕ħ :Ƚʣźeu̕ħ :ƫ. MirBase. m,f,w,+. Targets. Jɉŧ:eu̕ħ :Ƚʣźeu̕ħ :ƫªdž˸Ú½:Ƚ ʣź. PITA Top. m,f,w. ƫǘȵ6:ªdž˸: ȖȵĽŀź RNA22. m,f,w. Jɉŧ:eu̕ħ. http://cbcsrv.watson.ibm.com/. :ȽʣźɧČ̕ħ:. rna22.html. ƫªdž˸Ú½:Ƚʣ źĄ=ȖȵĽŀź a. m = mammalian/vertebrate, f = fly, w= worm, + = additional clades. 5. 28.
(17) 1-5 ǘȵ mRNA N©Ȇ)J.D:Ł̠ȵ8ƅǯ. am^Nȥ.ǘȵ mRNA :©Ȇ;miRNA :ǜʀʬNJ9 4̎Ş 9Ūð5J' '©Ȇ&KJǘȵ mRNA ;ʍȵ8 miRNA :ɧČǗŨ9ÃĻ'. E: IJ!Áıȵ8ɧČǗŨNƐ2ǘȵ mRNA :©Ȇ;ğ̊5JA.Ł̅9;ǘ ȵ5;8 mRNA ǘȵ6'4©Ȇ&KJÐ̄źE'<'<Ě̘6814JJiang H; ǘȵ mRNA ©Ȇ^5J miRanda Nȥ4 CyclinD1 Nǘȵ6)J miRNA Nƙɡ 'eUjmQpiSNèȥ'.Ł̠9GIdž^:É̗źN ǔʴ'. 29,:ɧNjmiRanda 9G14©Ȇ&K. 45 Ɋ̚:Ëʣ miRNA :0Ł̅9 CyclinD1 : 3´UTR 9ɧČ'4ȳȠNƇé'.E:; 7 Ɋ̚ (16%) /"51.$:G 9am^9GJǘȵ mRNA :©Ȇ;ɪɳźFǠɃź9ǝ!.DG IɃŁ8Ł̠ȵƅǯ:˻ȳ ǪDHK4J. 1-5-1 tg]tʬNJ9GJǘȵ mRNA :čŀ miRNA Nɣɿ>ōÙ)J6ˮ:ǘȵ mRNA 9 4ȳȠ˶:¼ Ƀʸ&KJ 30 ű14 miRNA N˦íȳȠ'.Ű:Ú mRNA ˶ (tg]t) NʬNJ)J$65 ǘȵ mRNA Nčŀ5 J6ɶ HKJLim H;$:ȳŽ9ĩ3. miRNA N Hela ɣɿ. >ōÙ'4 12 ƻ˼Ű:Ú mRNA ˶NS]QS5ʬNJ'.,:ɧNjmiRNA :˦ íȳȠ9GIɝ 1000 Ɋ̚: mRNA 9 4ȳȠ˶:¼ Ƀʸ&K.A.ȳȠ˶:¼ '. mRNA :IJ! ,: 3´UTR 9ōÙ'. miRNA :eu̕ħ6Ƚʣ8˯æNƐ1 4 ImiRNA :ȼƚȵ8ǘȵ5J$6 Ʉę&K. 31S]QSʬNJ9G14 ŤɗĠ9ȃJǘȵ mRNA ƛŀ&K.$6 H,:Ű´:^9G14EčǗ: Ł̠ ƫIJ!ʜMK4J 32-34miRNA :Ƚʣ˯æNɣɿÜ>˦íȳȠ'.īČmiRNA :ǜʀ ˿Ń&Kǘȵ mRNA :ȳȠ˶;ĭò)J6ɶ HKJű14 miRNA :ǜʀ˿ ŃN'.īČEčǗ9S]QSʬNJ9GJǘȵ mRNA :ƛŀ ĉʀ5J 35& H9miRNA :˦íȳȠ6ǜʀ˿Ń:£ƱNʜ6ȳȠ ĭò)J mRNA 6¼)J mRNA : 2 }m:ʬNJɧNj ŲHKJ 70H:}m9EÛ˟)J mRNA ; ǘȵ mRNA 6'4:ĉʀź ǕD4̢!8J$6 Nicolas H:Ɂɋ9G14Ʉ&K. 36 ·:G9tg]tʬNJNȥJ$65ɪɳȵ8ǘȵ mRNA :čŀ ĉ ʀ68J' '©Ȇ5. Jǘȵ mRNA miRNA :ōÙ9G14ȳȠ˶ İø)JE. :)8M0 miRNA 9G14äʬ&KJE:9́ŀ&KJ$6F˼ƚȵ8ȳȠ˶:İø. 6.
(18) 9G14ȣ(JÐ̄ź87 Ě̘Ȑ6'4ƒ#HKJ. 1-5-2 HITS-CLIP. HITS-CLIP ; UV cross-linking immunoprecipitation (CLIP) ǯ6 high-throughput sequencing (HITS) NɥBČM+.˘ˠ5ɪɳȵ8ǘȵ mRNA :ʬNJǯ5J 37×]g]ʀ NƐ2 4-oXTfNɣɿ>Ǿò'4 UV NȔŌ)J64-oXTfNćI˗O/ RNA RISC ʥČ½9 4 Argonaute 6×njǛ&KJ Ɖ Argonaute Ɖ½5 RNA-Argonaute SDS-PAGE 5ɚʤ)J$65 mRNA-Argonaute ʥČ½Nɚʤ'.Ű RNA ˮäN 32P 5ǘˁ ʥČ½N˫Ƌȵ9ŲJ$6 5. J,KHNȥ4{Sgpte_e^N. ʜ$69GIArgonaute 9ďAK4J (miRNA 9ǘȵ6&K4J) mRNA Nɪɳȵ 9äNJ)J$6 ĉʀ68J (Figure 4) ' 'džƅǯ H miRNA ɧČ)J mRNA Nȼƚčŀ)J$6;5. *|SXSWrR]g:ƅǯ6ɥBČM+Jŷʦ . J UV cross-linking 4S 4S. U. U immunoprecipitation. miRNA. Cell lysis. target mRNA. AA A. Alkaline phosphatase. RNase A. AA A. 5´OH. 3´P. 5´OH. 3´OH. RNA ligase Deep Sequencing. AGO+mRNA AGO+miRNA. SDS-PAGE ќ32 P. Free RNA. polynucleotide kinase 5´OH 3´RNA adapter.
(19) . 1-5-3 miR-CLIP. microRNA crosslinking and immunoprecipitation (miR-CLIP) ;׹Źźäĺ5Jk 6Xo5øļÊ̞'. miRNA 9GJǘȵ mRNA :čŀǯ5J 39k;T e6˫Ƌȵ9×njǛ)J.DÊ̞ miRNA ; RISC NŬƁŰUV ȔŌ9G14ǘȵ mRNA 6ȼƚÛǁɧČNŬƁ)JƉ Argonaute Ɖ½5 RISC Nɚʤ'.ŰgttQ f6Xo:Ƚ«¾ȥNèȥ'4 miRNA 6ǘȵ mRNA :ʥČ½Nü̉'{Sg pte_e^Nʜ$69GImiRNA ȼƚɧČ)Jǘȵ mRNA Nɪɳȵ. 7.
(20) 9ʬNJ)J$6 ĉʀ5J (Figure 5) Jochen H;$:ƅǯNȥ4 miR-106a G= miR-17-5p :ǘȵ mRNA Nƙɡ'am^ H;©Ȇ&K8Áıȵ 8ɧČ}mNƐ2ǘȵ mRNA :čŀ9Ɓñ'4J 39 UV cross-linking . immunoprecipitation. . . . . . . miRNA mimic. . . target mRNA. . . . AA A. RNA extraction. . . . . . AA A. AA A streptavidin -biotin affinity isolation. Deep Sequencing. . . . AA A. Figure 5. miR-CLIP. 1-6 ׹Źźäĺ. ¶ʠȵ8ǘȵ mRNA :ʬNJǯ5J HITS-CLIP F miR-CLIP ;70HEÛ˟'4׹ ŹźäĺNèȥ'4J׹Źźäĺ6;×Vy\5ńƺ9äʬ)JøļĿʀĩ NƐ2äĺ5Iɢı̕ħ:×ȔŌ9GIǀŅI:äĺ6ÛǁɧČNŬƁ)J$6 ĉ ʀ5Jșŀ:Zu9H (D׹ŹźǡĩNōÙ'˩å8mS^5×Ȕ Ō)J$65DŽɀ:im9ÛǁɧČNŬƁ)J$6 5. JA.×ȔŌ9GJ. ɣɿǤź;ŏ8!ȣȡǵźNÈ1.AAÛǁɧČ:ŬƁ ĉʀ5J.Dȣ½̢äĺ :ʬNJ9˩'4JmiR-CLIP ;miRNA 9ōÙ&K.käĺ9G14ǘȵ mRNA :BN˫Ƌȵ9ƕȞ5 ȵ8ąŹź:.DƕȞ5. JŪð8ƅǯ5;JE::ōÙ'.k:Te˫Ƌ Jǘȵ mRNA 9;é́ Iɪɳȵ8ʬNJǯ6;ʮ 8. DŽɀ:ǘȵ mRNA Nɪɳȵ9ƕȞʬNJ)J.D9;ȽʣĬĩ9ÃĻ'8ąŹźNƐ 2ư':˻ȳ ŷʦ5J. 1-7 tXofQf. ׹Źźǡĩ5JtXofQf;365 nm µ˙:˺DZ˺Ï: UV Ȕ Ō9GIY Nȣ(J (Scheme 1) 40Y ;̎Ş9ąŹź ̢!C-HO-HN-H C-C ɧČNďCIJ!:Ŀʀĩ6ąŹ)J$6 ĉʀ5J3 »:Uwĩ6tX oĩ;ČƁLJ¸9 "JfQfȢ:ȖȵĽŀźN̢DJ6čƻ9TːɈ. 8.
(21) 6,K9ɨ!Ǫ̌ĺɲƜąŹ9G14ȣ(JfQlȭź½:ȣƁNƇé'4J (Scheme 2) fQlȭź½; UV ȔŌ9G14Y NȣƁ5 :Ǫǐë6ąŹ'4̎șȭȵ8ǘˁNũ. J ąŹˠŧ ˥.D´. ˊ$)ĉʀź J,$5ɲƜĩ6'4t. XoĩNȥJ$65fQl½:òǨäʬNƇé'4J N N. hν. CF3. CF3. X Y. X Y CF3. Scheme 1. $$EI ^rA2&@ N N. hν. hν. hν N N. slow. diazo. Scheme 2. UVl
(22) $)sQ t. ȣ½̢äĺ:ǘˁø9fQfNȥJèȐ6'4ɣɿǤź:¼˺DZ˺:UVȔŌ 9G14ǵźø&KJ$6UVȔŌ9G14ȣ(JY :ąŹź ǕD4̢$6 ƒ#HKJUVȔŌ9G14ȣ(.Y ;:Țſ5;Ǩ6ˠF 9ąŹ'4 ]Vo&KJ.DǘȵNʻǘˁ)Jĉʀź;ǕD4¼˙ŢfQfǡĩNȥ .ȣ½̢äĺ:ʬNJ ȹO9ʜMK I41,42ūɁɋ5EZSu˸:3´Džɏ9fQf ǡĩNōÙ'.siRNANČƁ'RISC:ǖƁĞĺ5JArgonaute2:ƕȞ9Ɓñ' 4J (Figure 6) 43. Figure 6. V^rssiRNA:HJ9
(23) Argonaute2 y. 9.
(24) 2Ɏ ׹ŹźǡĩNōÙ'.ưʨRNA:ČƁ6,:RNAƕȞʀ ê˛'.Ǘ9׹ŹźǡĩtXofQf;˺DZ˺:UVȔŌ9G14 Y Nȣ(˙Ñ:Ǘ8øČȘ6ÛǁɧČNŬƁ)J$6 ĉʀ5Jű14 fQfǡĩNōÙ'.miRNANɣɿ>ōÙ'UVNȔŌ)J$65ǘȵ mRNANɪɳȵ9ǘˁ5. J6ɶ . (Figure 7) . UV cross-linking . . . . label molecule. miRNA mimic. target mRNA. AA A. AA A. Figure 7. miRNA:HJ9
(25) mRNA §\. fQfǡĩNōÙ'.miRNANČƁ)J.Dư.9x]XeuQv ^1 G=2Nʲʯ'. (Figure 8) 1 G=2;Ĵȓ:RNA6čǗ:ɛ-˲̡ǒNǁ) J$6 H$KHNōÙ'.miRNA;Ĵȓ:miRNA6čɓ:ȣȡǵźNɄ)$ 6 ǃů5 JA.UVȔŌ9G14ȣ(JY ;ǕD4ąŹź ̢.DȽʣ ˯æ9ÃĻ+*ǘȵmRNANɪɳȵ9ƕȞ5. J6ɶ HKJdžɁɋ5;1 G=2. NōÙ'.RNANČƁ',:RNAƕȞʀNǔʴ'.. F 3C. CF3 N N HO O OH. HO. O. O. OH. OH. 1. N N. O. OH 2. Figure 8. $$EId3 G#01H . 10.
(26) 2-1 ׹Źźx]XeuQv^Ą=,:QnSt½:ČƁ. x]XeuQv^1 G=2;tXofQf:Qaʹō½. 6β-D-zgN^aeø)J$65ŲHKJ6ɶ . (Scheme 3) 8 t XofQf:Qaʹō½;Ƶɀ:ĪĒ9ű14ČƁ)J$66 '.41. CF3 N N. CF3 N N. O BzO +. O. O BzO O. OBz OBz. HO. CF3 N N HO. O. O. OBz OBz. OH. O. OH 1. Scheme 3. V^rs3 G#01H atu. $:^aeøąŹ5;2´-»Ǩ˲ĩ9QeɜÈ˂ĩNǁ)Jβ-D-zg NãȳāƮ6'4˫Ƌ)J$65ōÙ'.Qeĩ:̆ƚĩõNj9GIβ-»˫Ƌȵ9Q [ĩNōÙ)J$6 ĉʀ5J (Figure 9) . R OH. H O BzO O. O. BzO. BzO O. BzO O. O O. OBz OBz. OBz O. O. OBz. O. O. O. OBz. BzO O. O. OBz OBz. Figure 9. #FT¨d ²zd[. 11. R.
(27) 2-1-1 jȢm»9fQfwĩNǁ)JQaʹō½:ČƁ. m-Iodobenzyl alcoholNãȳāƮ6'4TBDMSClNąŹ&+J$69GIǨ˲ĩN TBDMSĩ5È˂'.øČȘ3NĆȟ92%5Ų.ɨ4øČȘ3NTHF9ȉʬ'-78 °C94 n-BuLiâȡ)J$69GI jȢ:m»NoXø'.ŰtX˰˲Vo NǾò)J$65øČȘ4NĆȟ95%5Ų.øČȘ49~u[eQĬ˲ĬN¾ȥ&+ X[e½6'.Ű9Ǩ˲ĩNteø'øČȘ5NĆȟ82%5Ų.ɨ4te½5 NŊɕ¥52ƶ˼QwQǨ6ąŹ&+4øČȘ6NĆȟ88%5ČƁ'.ŲHK.fQf fʹō½6NI25˲ø'4øČȘ7NĆȟ78%5Ų.ǀŰ9TBDMSĩNTBAF5âȡ) J$65̃Ă'Ȼȵ:Qaʹō½8NĆȟ86%5ČƁ'. (Scheme 4) . TBDMSCl imidazole. HO. I. n-BuLi,CF3COOEt. TBDMSO. DMF. TBDMSO. CF3. THF. I. O. 3: 92 %. 4: 95 % δ 5.07. 19FNMR:. 1. HONH 3Cl pyridine. TBDMSO. CF3. 2. TsCl, NEt3,DMAP pyridine. N. NH 3 aq.. THF. TBDMSO. HN NH. OTs. 5: 82 %. I2 NEt 3. TBDMSO. CF3. CH3OH. 6: 88 %. TBAF THF. HO. N N 7: 78 %. CF3 N N 8: 86 %. Scheme 4. A*P
(28) $$E2FdF. 12. CF3. JFQ at.
(29) 2-1-2 x]XeuQv^1Ą=,:QnSt½:ČƁ. ɨ4øČȘ861-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose:^aeøąŹNʜ 1.øČȘ861-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoseNCH2Cl2¥TMSOTfĻģ5 ąŹ&+.6$LȻȵ6)JøČȘ9NĆȟ89%5ŲJ$6 5. .ʍ9x]Xe. u:Qz»:ɍ½˯ɲ;ɛˮ:1´-»Ą=2´-»:H:˼:Yp^ŀƫ (JÍ) N ĩ9ǫŀ)J$6 5. Jþ01´-»:ɍ½˯ɲ β-»:ƻ91´-H62´-H˼5:ª̏ʫ. ɝ90°NƁ)$6 Hª̏ʫΦ6KarplusŨ:˽Æ9GIJÍ;Ŏ&!e^pt6 '4ʪŇ&KJ6ƛȆ&KJŁ̅øČȘ9:1H NMRʬNJN $81.6$L5.31ppm µ˙91´-»:H9ŝœ&KJe^pt:] ʪŇ&K.G14ȣƁȘNβ½6ǫŀ'.ɨ4øČȘ99ʭĹ˶:CH3ONaN¾ȥ&+ʂ lSø)J$65 x]XeuQv^1NĆȟ85%5ČƁ'.ƿ9øČȘ19DMTrClN1ū˶ąŹ&+ 5´-»:1ɟǨ˲ĩN˫Ƌȵ9DMTrĩ9GIÈ˂'øČȘ10NĆȟ91%5Ų.ČƁ'. DMTr½10NTBDMSCl94âȡ'ŲHK.ǽČȘNeY`Y]t^R 94ɚʤ)J$6522:øČȘNü̉'.,K-KN1H NMRĄ=1H-1H COSYNȥ4 ʬNJN $81.6$LƱ5;Ǩ˲ĩ:] 2.5ppm9npt6'4ʪŇ& K3´-»9»ɲ)JH6:Yp^ ʪȆ&K.$:$6 HǨ˲ĩ;3´-»9» ɲ'TBDMSĩ;2´-»9»ɲ'4J$6 Ƀʸ5. .A.EƱ5;Ǩ˲ĩ:. ] 2.8ppm9npt6'4ʪŇ&K2´-»9»ɲ)JH6:Yp^ ʪȆ& K.$:$6 HǨ˲ĩ;2´-»9»ɲ'TBDMSĩ;3´-»9»ɲ'4J$6 Ƀʸ 5. .G14$KH:ʪȆsmGI2´-TBDMS½5JøČȘ11Ą=3´-TBDMS½. 5JøČȘ12NĆȟ38%45%94,K-KŲHK.$6 ä 1.ǀŰ92´-TBDMS ½11NŞǯ9GI˲ø'x]XeuQv^1:QnSt½5JøČȘ13N Ćȟ62 %5ČƁ'. (Scheme 5) . 13.
(30) CF3 N N. O BzO O. O. +. HO. CF3. OBz OBz. CH3ONa. TMSOTf CH2Cl2. N N. CF3 N N. BzO O. CH3OH. O. HO. 8. OBz OBz. OH. 9: 89 %. CF3 N N Pyridine. DMTrO O OH. CF3 N N DMTrO O. OH. OH. 10: 91 %. O. P Cl DIPEA. CF3 N N +. DMF. O. O. OTBDMS 11: 38 %. DMTrO O TBDMSO. DMTrO. THF. O. NC. O. OH. O. O OTBDMS P N(i-Pr)2 13: 62 % 31P NMR: δ 149.5, 150.0. Scheme 5. 3 G#01H 1] ?+/Q at. 14. O. 12: 45 %. CF3 N N. N(i-Pr)2. OH 1: 85 %. TBDMSCl TEA. DMTrCl. NC. O. O.
(31) 2-1-3 jȢ}»9fQfwĩNǁ)JQaʹō½:ČƁ. øČȘ8:ČƁ6čǗ:ƅǯ9GI}ʹō½19NČƁ'.p-Iodobenzyl alcoholNãȳ āƮ6'4TBDMSCl6ąŹ&+J$69GIǨ˲ĩNTBDMSĩ5È˂'øČȘ14Nŀ ˶ȵ9Ų.ɨ4øČȘ14NTHF¥-78 °C5n-BuLiâȡ)J$69GI jȢ} »NoXø'.ŰtX˰˲VoNǾò)J$65øČȘ15Nŀ˶ȵ9Ų. øČȘ159~u[eQĬ˲ĬN¾ȥ&+X[e½6'.Ű9Ǩ˲ĩNte ø'øČȘ16NĆȟ90%5Ų.ɨ4te½16NŊɕ¥52ƶ˼QwQǨ6ąŹ &+4øČȘ17NĆȟ88%5ČƁ'ŲHK.fQffʹō½NI25˲ø'4øČȘ18 NĆȟ 96%5Ų.ǀŰ9TBDMSĩNTBAF5âȡ)J$65̃Ă'Ȼȵ:Qa ʹō½19NĆȟ92%5ČƁ'. (Scheme 6) . I HO. TBDMSCl imidazole. DMF. I. CF3. n-BuLi,CF3COOEt. O. THF. TBDMSO. TBDMSO. 14: quant.. 15: puant. δ 5.10. 19FNMR:. 1. HONH 3Cl pyridine. CF3. 2. TsCl, NEt3,DMAP pyridine. N. THF. TBDMSO. TBDMSO 17: 88 %. 16: 90 %. CF3 N N. I2 NEt 3. CH3OH. TBDMSO. CF3 NH N H. NH 3 aq.. OTs. CF3 N N. TBAF THF. HO. 18: 96 %. 19: 92 %. Scheme 6. 5DP
(32) $$E2FdF. 15. JF¥mQ at.
(33) 2-1-4 x]XeuQv^2Ą=,:QnSt½:ČƁ. øČȘ13:ČƁ6čǗ:ƅǯ9GIQv^2:QnSt½NČƁ'.øČȘ196. 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoseNÖ6čǗ:LJ¸5^aeø'øČȘ 20NĆȟ92%5ČƁ'.1H NMR9GJʬNJN $81.6$L5.31ppmµ˙91´-» :H9ŝœ&KJe^pt:] ʪŇ&K..DȣƁȘNβ-½6ǫŀ'.ƿ9 øČȘ209ʭĹ˶:CH3ONaNȥ4ʂ lSø)J$65x]XeuQv^2 NĆȟ95%5ČƁ'.ɨ4øČȘ29DMTrClN1ū˶ąŹ&+5´-»:1ɟǨ˲ĩN˫Ƌ ȵ9DMTrĩ9GIÈ˂'øČȘ21NĆȟ93%5Ų.øČȘ21NTBDMSCl94âȡ' ŲHK.ǽČȘNeY`Y]t^R94ɚʤ'.ü̉'.22:øČ ȘN,K-K1H NMRĄ=1H-1H COSYNȥ4ʬNJ'2´-TBDMS½5JøČȘ22Ą= 3´-TBDMS½5JøČȘ23N,K-KĆȟ30 %Ą=45 %94ŲHK.$6 ä 1.ǀ Ű92´-TBDMS½22NŞǯ9GI˲ø'x]XeuQv^2:QnSt½ 5JøČȘ13NĆȟ95%5ČƁ'.(Scheme 7) N. F 3C. F 3C. N. O BzO O. O. CF3 N N. + HO. CH3ONa. TMSOTf CH2Cl2. CH3OH. BzO. O. O. OBz OBz 19. HO. OBz OBz. OH. 20: 92 %. N. F 3C. F 3C. N. Pyridine. O OH. DMTrO. O. O. OH. OH. 21: 93 %. NC. P Cl DIPEA. O. OTBDMS. DMTrO O TBDMSO. 22: 30 %. F 3C O. F 3C. N. +. DMF. DMTrO. N N. N(i-Pr)2. DMTrO. THF. O. NC. O. O. O OTBDMS P N(i-Pr)2 24: 95 % 31P NMR: δ 149.1, 149.8. Scheme 7. 3 G#01H 2] ?+/Q at. 16. OH 2: 95 %. N. TBDMSCl TEA. DMTrCl. O. O. O. OH 23: 45 %. N N. N N.
(34) 2-2 x]XeuQv^1 G=2NōÙ'.RNA:ČƁ. 2-2-1 ġȽgQnStǯ. ǐ˲;ʍȵ9ġȽgQnStǯ9ű14øļČƁ&KJ (Figure 10) Ö* ˲âȡ5ǚʁ9ġŀø&K.x]Xou:DMTrĩNʂÈ˂' (Řɉ
(35) ) ɨ4Q nSt½Nrtlɓ:˲ʭĹ9GIǵźø'4ǚʁ9ġŀø&K.x]Xe u:5´-Ǩ˲ĩ6:Yp^ąŹNʜ (Řɉ ) DŽąŹ:5´-Ǩ˲ĩNQioĩ5 È˂'.Ű (Řɉ) ˲VgrNI25˲ø)J$651cS] ɤ¨)J (Řɉ ) $:cS]NɱI˚)$65DNA/RNANøļȵ9ČƁ)J$6 ĉʀ5J. DMTrO O. Base O. O. DMTrO O. Base. N H. O. CPG. 3% Cl3COOH/CH2Cl2.
(36) . O O P O. HO. O NC. O. Base. O. O. O O. N H. O. Base. O. CPG. N H. O. CPG. DMTrO. . 0.02M I2/THF/pyridine/H2O. O. NC. DMTrO O. Base. O O O. . Base. O. O O O. P. O. N(i-Pr) 2. O P NC. O. N H. O O. CPG AcO O. N N. Base O. O O. N H. CPG. Figure 10. b<%<H?+/. 17. Base.
(37) 2-2-2 Qv^1 G=2NōÙ'.RNA:ČƁ. ČƁ'.x]XeuQv^1 G=2:ċQnSt½Nȥ4ǐ˲ʊöČƁǜ9 4Xbx]XouNČƁ'. (Table 3) ˯æ;Ĵȓ:miRNA5Jlet-7NǙ'4 I 5´Džɏ:8-10ȬȻ9Qv^1 G=2NōÙ'. (RNA2-9) let-7;¶ʠȵ8ȲƇéˬ¹ĺ ĦmiRNA:1Ɋ5IɻȲɣɿ9 4̙ʓ8ȣɺ˿ŃõNjNɄ)$6 ĪĒ&K4J 44. A.RNAƕȞʀǔʴ:.D5´-DžɏNFITC5ǘˁ'.ǘȵ˸EčǗ9ČƁ'.˯. æ;ĴȓĦ:let-76Ƚʣ8˯æ H8JE: (RNA10) 6gpoĬĩʼnN12ďCE : (RNA11) NČƁ'.ČƁ'.ċXbx]Xou:ǖˡ;MALDI-TOF/MS9GI Ƀʸ'.. Table 3. `E!3 G,0 ®X. RNA. Sequencea. calculated. observed. RNA 1. 5'-UGA GGU AGU AGG UUG UAU AGU-3'. 6791.9. 6790.3. RNA 2. 5'-UGA GGU A1U AGG UUG UAU AGU-3'. 6856.9. 6854.6. RNA 3. 5'-UGA GGU AG1 AGG UUG UAU AGU-3'. 6895.9. 6897.7. RNA 4. 5'-UGA GGU AGU 1GG UUG UAU AGU-3'. 6872.9. 6875.9. RNA 5. 5'-UGA GGU AGU A1G UUG UAU AGU-3'. 6856.9. 6854.1. RNA 6. 5'-UGA GGU A2U AGG UUG UAU AGU-3'. 6856.9. 6855.9. RNA 7. 5'-UGA GGU AG2 AGG UUG UAU AGU-3'. 6895.9. 6895.0. RNA 8. 5'-UGA GGU AGU 2GG UUG UAU AGU-3'. 6872.9. 6871.9. RNA 9. 5'-UGA GGU AGU A2G UUG UAU AGU-3'. 6856.9. 6855.6. RNA 10. 3'-ACU CCA UCA UCC AAC AUA UCA-F-5'. 7078.1. 7078.0. RNA11. 3'-ACU CCA UGA UCC AAC AUA UCA-F-5'. 7118.1. 7118.0 a. F = FITC. 2-3 Qv^1 G=2NďCªdž˸RNA:ȖȵĽŀź. Üˮ9x]XeuQv^1 G=2NōÙ'.RNA (RNA2-9) 6ǘȵRNA (RNA10,11) NɥBČM+ªdž˸:50%ʙʬȅŧ (Tm) N100mM:NaClNďC˲|p P (pH =7.0) ¥5Ȇŀ'.Ĵȓ:x]Xeu H8JRNA16ǘȵRNA (RNA10,11) ªdž˸:TmÍ6ǥˑ)J$65Qv^:ōÙ9GJªdž˸RNA:ȖȵĽŀź>:ŭ̑ NʵÄ'.. 18.
(38) 2-3-1 Qv^1NōÙ'.ªdž˸RNA:ȖȵĽŀź. Qv^1NōÙ'.RNA (RNA2-5) 6ǘȵRNA (RNA10,11) NɥBČM+4 ªdž˸NŬƁ&+50 %ʙʬȅŧ (Tm)NȆŀ'. (Figure 11,12) . ds RNA. Tm (°C). ΔTm (°C). RNA 1 + 10. 70.7. -. RNA 2 + 10. 59.8. -10.9. RNA 3 + 10. 62.8. -7.9. RNA 4 + 10. 63.3. -7.4. RNA 5 + 10. 58.8. -11.9. ΔTm = Tm – Tm (RNA 1 + 10). Figure 11. 1H 1mWRNA:HJ9 RNA10
(39) N° jks. ds RNA. Tm (°C). ΔTm (°C). RNA 1 + 11. 63.0. -. RNA 2 + 11. 60.4. -2.6. RNA 3 + 11. 59.2. -3.8. RNA 4 + 11. 56.8. -6.2. RNA 5 + 11. 50.3. -12.7. ΔTm = Tm – Tm (RNA 1 + 11). Figure 12. 1H 1mWRNA:HJ9 RNA11
(40) N° jks. Qv^1NōÙ'.RNA5;ĴȓĦ:RNA1:īČ6ǥˑ'4ªdž˸9 " JȖȵĽŀź Ȃŏ'4J$6 Ƀʸ&K.A.1:ōÙ9GJªdž˸: Ľŀø; 1:ȽʣĬĩ CG:īČ (RNA2 + 10, RNA 2 + 11) 6ǥˑ'4ȽʣĬĩ AU:īČ (RNA 3 + 10, RNA 4 + 10) 5;Ŏ& 1.ĴȓĦ:C-GĬĩʼn;ǨɠɧČN3džŬƁ)J: 9ʼn'4A-UĬĩʼn5;2dž' ŬƁ'8ű14C-GĬĩʼn>:1:ōÙ;ǨɠɧČ. 19.
(41) N3džåƯ)J.Dªdž˸NGI Ľŀø)JɧNj681.A.1:ōÙ9GJTmÍ: ¼;ĴȓĦ:gpo˯æ (RNA 1 + 11) 6čɉŧ/1.$6 H1:ōÙ;miRNA :gpo̕ħ (eu̕ħı) 5K<miRNA:ǵź9ŭ̑N¡ 86ɶ HKJ. 2-3-2 Qv^2NōÙ'.ªdž˸RNA:ȖȵĽŀź. čǗ9Qv^2NōÙ'.RNA (RNA6-9) 6ǘȵRNA (RNA10,11) NɥBČ M+4ªdž˸NŬƁ&+50 %ʙʬȅŧ (Tm) NȆŀ'. (Figure 13,14) . ds RNA. Tm (°C). ΔTm (°C). RNA 1 + 10. 70.7. -. RNA 6 + 10. 61.0. -9.7. RNA 7 + 10. 64.1. -6.6. RNA 8 + 10. 64.4. -6.3. RNA 9 + 10. 60.1. -10.6. ΔTm = Tm – Tm (RNA 1 + 10). Figure 13. 1H 2mWRNA:HJ9 RNA10
(42) N° jks. ds RNA. Tm (°C). ΔTm (°C). RNA 1 + 11. 63.0. -. RNA 6 + 10. 61.3. -1.7. RNA 7 + 10. 59.6. -3.4. RNA 8 + 10. 57.6. -5.4. RNA 9 + 10. 52.0. -11.0. ΔTm = Tm – Tm (RNA 1 + 11). Figure14. 1H 2mWRNA:HJ9 RNA11
(43) N° jks. . 20.
(44) Qv^2NōÙ'.RNA5EĴȓĦ:RNA1:īČ6ǥˑ'4ªdž˸9 "J ȖȵĽŀź Ȃŏ'4J$6 Ƀʸ&K.A.2:ōÙ9GJªdž˸: Ľŀø; 2:ȽʣĬĩ CG:īČ (RNA2 + 10, RNA 2 + 11) 6ǥˑ'4ȽʣĬĩ AU:īČ (RNA 3 + 10, RNA 4 + 10) 5;Ŏ& 1.$K;Ö@76čǗ9C-GĬĩʼn>:2:ōÙ 9G14A-UĬĩʼnGIEIJ!:ǨɠɧČ åƯ&K..D5JA.2:ōÙ9GJ TmÍ:¼;ĴȓĦ:gpo˯æ (RNA 1 + 11) 6čɉŧ/1.$6 H2:ōÙ EA.miRNA:gpo̕ħ (eu̕ħı) 5K<miRNA:ǵź9ŭ̑;8E :6ɶ HKJ. 21.
(45) 2-4 RNA:RNAƕȞʀ. Qv^NōÙ'. RNA 6Ƚʣ˸ RNA Ȗȵ9Ľŀ8ªdž˸NŬƁ)J$6 Ƀʸ&K.ɨ4x]XeuQv^NōÙ'. RNA : RNA ƕȞʀN ǔʴ'.RNA 6Ƚʣ˸ RNA N 1:1 :îČ5ǽČ'Qw^âȡ9GIª dž˸ŬƁ&+.Ű9UV (365 nm 30 ä302 nm 10 ä) NǩǸ5ȔŌ'.7 M TQȉ ǻNò 4ąŹNÎǟ&+ŲHK.ǽČǻN 20% İźQ]Qu`̌ǧdzö (20% PAGE) 94äNJ'.. 2-4-1 Qv^ 1 NōÙ'. RNA : RNA ƕȞʀ. ds RNA. ȽʣĬĩ. Ćȟ (%). RNA 10 + 2. C. 6. RNA 10 + 3. A. 18. RNA 10 + 4. U. 13. RNA 10 + 5. C. 10. RNA 11 + 2. G. 6. RNA 11 + 3. A. 11. RNA 11 + 4. U. 7. RNA 11 + 5. C. 11. Figure 15. 1H 1 mW RNA :HJ9 RNA y. ,:ɧNjÚ4:ɥBČM+5̢äĺ˶̕ħ9ư.8|u ʪŇ&K.ˮNɚʤ '4 ESI-TOF/MS 94äNJ'.6$LRNA 6Ƚʣ˸ RNA :]g]µò½ 5J$6 Ƀʸ&K. (Figure 17) A.gpoNďCɥBČM+5;,58 E:6ǥˑ'4]g]Ćȟ;¼)JÒĎ91.$K;µ˙:ǨɠɧČ: ǺĶ9G14 1 :ġŀø úä68IȽʣ̕ħ9˙ƚ5 8!81..D/6ɶ H KJA.Ú4:ɥBČM+5]g]ąŹ ˣʜ'.$6 H1 NōÙ'. RNA 9GJȽʣ˸ RNA :ƕȞ;1 :ȽʣĬĩ:Ɋ̚9ÃĻ'8$6 ƹH 98 1.. 22.
(46) 2-4-2 Qv^ 2 NōÙ'. RNA : RNA ƕȞʀ. ds RNA. ȽʣĬĩ. Ćȟ (%). RNA 10 + 6. C. 32. RNA 10 + 7. A. 25. RNA 10 + 8. U. 13. RNA 10 + 9. C. 17. RNA 11 + 6. G. 25. RNA 11 + 7. A. 19. RNA 11 + 8. U. 9. RNA 11 + 9. C. 13. Figure 16. 1H 2 mW RNA :HJ9 RNA y. 1 NōÙ'.īČ6čǗ9Ú4:ɥBČM+5̢äĺ˶̕ħ9ư.8|u Ƀʸ&K .ˮNɚʤ'4 ESI-TOF/MS 94äNJ'.6$LRNA 6Ƚʣ˸ RNA :] g]µò½5J$6 Ƀʸ&K. (Figure 18) gpoNďCɥBČM+5Ćȟ ¼)JÒĎE1 :īČ6čǗ51.' '8 HÚ½ȵ8Ćȟ; 1 NōÙ'. RNA 6ǥˑ'4ij. !Ď'.$K;ąŹˮ» m» H}»9ɲƜ'. .$69G14Ƚʣ̕ħ9GIƚ˙'..D/6ɶ HKJA.Ú4:ɥBČM+ 5ąŹ ˣʜ'.$6 H2 NōÙ'. RNA 9GJȽʣ˸ RNA :ƕȞEȽʣ Ĭĩ:Ɋ̚9ÃĻ'8$6 ƹH 681.. 23.
(47) Relative intensity (%). RNA 2 + RNA 10 [M-H]- : 13951.6; C431H519F3N154O298P41. 13953. 100. 50. 13974. 14058. 13913. 0 13700. 13800. 14070. 14009. 13927. 13900. 14000. 14100. 14200 m/z. Figure 17. RNA 2 + RNA 10 H%EI Q >%%; /F. 13972. 100. RNA 7 + RNA 11 [M-H]- : 13991.6; C432H519F3N156O298P41. Relative intensity (%). 13953. 50. 13993. 14009 14027. 0 13800. 13850. 13900. 13950. 14000. 14059. 14050. 14079. 14100. 14150. m/z. Figure 18. RNA 7 + RNA 11 H%EI Q >%%; /F. 24.
(48) 2-5 Qv^ 2 NōÙ'. miRNA 9GJǘȵˬ¹ĺȳȠƇéʀ:ǔʴ. 2 NōÙ'. RNA õȟG!Ƚʣ˸ RNA NƕȞĉʀ5J$6 ƹH 68 1.,$5 2 NōÙ'. miRNA Nư.9ČƁ',:ˬ¹ĺȳʮƇéʀNʵÄ )J$65 2 :ōÙ miRNA ǵź9¡ Jŭ̑NʵÄ'.. 2-5-1 miRNA :ČƁ. 2 NōÙ'. miR-199a-5p (RNA12, 14, 16) G= miR-199a-3p (13, 15, 17) N,K-Kġ ȽgQnStǯ9űǐ˲ʊöČƁǜ5ČƁ'. (Table 4) A.5p 6 3p N ɥBČM+4Qw^âȡ'miR-199a ªdž˸NʽƬ'. (Table 5) (Figure 19) . Table 4. miR-199a-5p/3p ®X. RNA. Sequence. calculated. observed. RNA 12. 5'-CCC AGU GUU CAG ACU ACC UGU UC-3'. 7220.3. 7219.3. RNA 13. 5'-ACA GUA GUC UGC ACA UUG GUU A-3'. 7003.2. 7000.6. RNA 14. 5'-CCC AGU G2U CAG ACU ACC UGU UC-3'. 7321.2. 7324.5. RNA 15. 5'-ACA GUA G2C UGC ACA UUG GUU A-3'. 7104.2. 7106.2. RNA 16. 5'-CCC AGU GU2 CAG ACU ACC UGU UC-3'. 7321.2. 7322.2. RNA 17. 5'-ACA GUA GU2 UGC ACA UUG GUU A-3'. 7105.1. 7104.8. Table 5. miRNA N° a. miRNA. miR-199a-5p. miR-199a-3p. miRNA 1. RNA 12. RNA 13. miRNA 2. RNA 14. RNA 13. 5´- C. miRNA 3. RNA 16. RNA 13. 3´- A U U. miRNA 4. RNA 12. RNA 15. miRNA 5. RNA 12. RNA 17. miR-199a-5p C C A GU GU U C A GA C U A C GGU U A C A C GUC U GAU G. CU GU U C-3´ A C A -5´. miR-199a-3p. Figure 19. miR-199a N¬. 25.
(49) 2-5-2 Qv^ 2 NōÙ'. miRNA :ˬ¹ĺȳȠƇéʀ. miR-199a ;-5p ˯æ6 3p ˯æ:ªdž˸ H8J miRNA 5I70H:˸EɣɿÜ5 ǘȵ mRNA 9ɧČ',:ɵʳNƇé)J2 NōÙ'. 5p G= 3p H8J miR-199a Nȥ4,K-K:ǘȵ9ʼn)Jˬ¹ĺȳȠƇéʀNȆŀ'2 : miRNA ǵź>:ŭ̑N ʵÄ'.ʵÄǯ9; Dual-luciferase reporter assay Nȥȳ×m}]ˈ5J Renilla luciferase :Ƕ9 5p A.; 3p :ɧČ˯æNƔÙ'. ]mNʽƬ'miR-199a 6čƻ 9ɣɿ>ōÙ'.24 ƻ˼Ű: Renilla 6 Firefly luciferase ȧLj:ȳ×˶NȆŀ'miRNA 9GJ Renilla luciferase :ȳȠƇéõNjNʵÄ'. miRNA NōÙ'48īČ:ȳ×˶ Nat6'4 100 %6'. (Figure 20) . Figure 20. (A) 5p ®XxW Renilla luciferase vY[ (B) 3p ®Xx W Renilla luciferase vY[. ,:ɧNj2 NōÙ'. miR-199a (miRNA2-5) ;ĴȓĦ: miR-199a (miRNA1) 9;ó JE::Renilla luciferase :ȳȠNƇé'4J$6 Ƀʸ&K.$K;2 NōÙ' . miRNA ɣɿÜ5 RISC N³'4ǘȵ mRNA 9ɧČ'4J$6NɄ'4 I 2 NōÙ'. miRNA NȥJ$65UV ȔŌ9GJǘȵ mRNA :ƕȞ ǃů5 J. 26.
(50) 3 Ɏ ׹Źź miRNA 9GJǘȵ mRNA :ƕȞ $KA59miRNA :ǘȵ mRNA :ɪɳȵ8ʬNJǯ:˻ȳNȻƑ'4ưʨ׹Źźx ]XeuQv^ 1 G= 2 NČƁ'.1 G= 2 NōÙ'. RNA ;ȽʣĬ ĩ:Ɋ̚9˽MH*ǘȵ RNA 6×njǛ)J$6ƿ9 2 NōÙ'. miRNA ;ɣ ɿÜ5 RISC N³'ǘȵ mRNA 9ɧČ)J$6 Ƀʸ&K.,$5Ł̅9 2 G= ǘˁäĺNōÙ'. miRNA NČƁ'ɣɿÜ5ǘȵ mRNA :ƕȞNʶB.. 3-1 miRNA :ʲʯ. ²ĝ2 NōÙ)J miRNA 6'4 miR-145 N˫Ƌ'. (Figure 21) miR-145 ;Ȳɣɿ9. 4 FSCN1 mRNA ǘȵ6',:ȳȠNƇé)J$6 ĪĒ&K4J 45A.ǘˁ äĺ9;XoN˫Ƌ'.Xo;Āȴ¥9Ļģ)Jm}]ˈ5JQf6 ̎Ş9ŪȽ«¾ȥNɄ)Ŏäĺ5ImiRNA : 3´Džɏ>:Ê̞ÁEƵ9ĪĒ&K4 J 46ś˅: CPG ǚʁNȥ4ċ miRNA : 3´-Džɏ9YN³'4XoNōÙ '. (Figure 22) 2 NōÙ)J»ɲ6'4ǘȵ mRNA 9ɧČ)JZSu˸ 5´Džɏ: 1-23 ȬȻ ɶ HK.,$52 NZSu˸: 1-23 ȬȻ9 1 2*2ōÙ'. 23 Ɋ̚: miRNA NČƁ'ǘȵˬ¹ĺ5J FSCN1 :ȳȠƇéõNjNTUgmprR^ 5ʵÄ)J$652 :˩å8ōÙ»ɲNǔʰ'.. 1. U. UC. 23. C. C U-3´. guide strand 5´- G C A GU U U C C A GG A A U C C C GU C A A A GGU C CU U A GG -5´ passenger strand 3´- U U U U A Figure 21. miR-145 N° ¬. O HN. O O O O PH. NH H N. S. O. O. O. O. O OH. Figure 22. 3´-Biotin TEG. 27.
(51) 3-2 miRNA :˯æ. ċ miR-145 NġȽgQnStǯ9űČƁ'. ČƁ'. RNA :˯ æN Table6 G= 7 9Ʉ'.ċ RNA :ǖˡ; MALDI-TOF/MS GIɃʸ'.. Table6. at miR-145 0°. RNA. Sequence. calculated. observed. RNA 18. 5´-GUC CAG UUU UCC CAG GAA UCC CU-3´. 7220.30. 7220.30. RNA 19. 5´-GUC CAG UUU UCC CAG GAA UCC CU-B-3´. 7789.91. 7789.75. RNA 20. 5´-2UC CAG UUU UCC CAG GAA UCC CU-B-3´. 7854.94. 7855.91. RNA 21. 5´-G2C CAG UUU UCC CAG GAA UCC CU-B-3´. 7893.98. 7892.14. RNA 22. 5´-GU2 CAG UUU UCC CAG GAA UCC CU-B-3´. 7894.97. 7894.33. RNA 23. 5´-GUC 2AG UUU UCC CAG GAA UCC CU-B-3´. 7894.97. 7894.82. RNA 24. 5´-GUC CAG UUU UCC CAG GAA UCC CU-B-3´. 7870.94. 7870.13. RNA 25. 5´-GUC CA2 UUU UCC CAG GAA UCC CU-B-3´. 7854.94. 7854.48. RNA 26. 5´-GUC CAG 2UU UCC CAG GAA UCC CU-B-3´. 7893.98. 7893.19. RNA 27. 5´-GUC CAG U2U UCC CAG GAA UCC CU-B-3´. 7893.98. 7895.33. RNA 28. 5´-GUC CAG UU2 UCC CAG GAA UCC CU-B-3´. 7893.98. 7893.70. RNA 29. 5´-GUC CAG UUU 2CC CAG GAA UCC CU-B-3´. 7893.98. 7895.31. RNA 30. 5´-GUC CAG UUU U2C CAG GAA UCC CU-B-3´. 7894.97. 7893.84. RNA 31. 5´-GUC CAG UUU UC2 CAG GAA UCC CU-B-3´. 7894.97. 7895.40. RNA 32. 5´-GUC CAG UUU UCC 2AG GAA UCC CU-B-3´. 7894.97. 7894.11. RNA 33. 5´-GUC CAG UUU UCC C2G GAA UCC CU-B-3´. 7870.94. 7871.07. RNA 34. 5´-GUC CAG UUU UCC CA2 GAA UCC CU-B-3´. 7854.94. 7854.60. RNA 35. 5´-GUC CAG UUU UCC CAG 2AA UCC CU-B-3´. 7854.94. 7856.63. RNA 36. 5´-GUC CAG UUU UCC CAG G2A UCC CU-B-3´. 7870.94. 7876.67. RNA 37. 5´-GUC CAG UUU UCC CAG GA2 UCC CU-B-3´. 7870.94. 7871.08. RNA 38. 5´-GUC CAG UUU UCC CAG GAA 2CC CU-B-3´. 7893.98. 7894.25. RNA 39. 5´-GUC CAG UUU UCC CAG GAA U2C CU-B-3´. 7894.97. 7896.05. RNA 40. 5´-GUC CAG UUU UCC CAG GAA UC2 CU-B-3´. 7894.97. 7895.08. RNA 41. 5´-GUC CAG UUU UCC CAG GAA UCC 2U-B-3´. 7894.97. 7894.37. RNA 42. 5´-GUC CAG UUU UCC CAG GAA UCC C2-B-3´. 7893.98. 7894.16. 28.
(52) Table7. at miR-145 5-'I$BJ°. RNA. Sequence. calculated. observed. RNA 43. 5´-GGA UUC CUG GAA AUU CUG UUC U-3´. 6989.15. 6979.82. ČƁ'. miR145 :}pif˸ (RNA42) 6ċZSu˸ (RNA18-42) NɥBČM+4 25 Ɋ̚:ªdž˸ miR-145 NʽƬ'.. Table 8. ¦| miR-145 :HJ9. miRNAa. ZSu˸. }pif˸. miR-145. RNA 18. RNA 43. miR-145-B. RNA 19. RNA 43. miR-145-G1. RNA 20. RNA 43. miR-145-G2. RNA 21. RNA 43. miR-145-G3. RNA 22. RNA 43. miR-145-G4. RNA 23. RNA 43. miR-145-G5. RNA 24. RNA 43. miR-145-G6. RNA 25. RNA 43. miR-145-G7. RNA 26. RNA 43. miR-145-G8. RNA 27. RNA 43. miR-145-G9. RNA 28. RNA 43. miR-145-G10. RNA 29. RNA 43. miR-145-G11. RNA 30. RNA 43. miR-145-G12. RNA 31. RNA 43. miR-145-G13. RNA 32. RNA 43. miR-145-G14. RNA 33. RNA 43. miR-145-G15. RNA 34. RNA 43. miR-145-G16. RNA 35. RNA 43. miR-145-G17. RNA 36. RNA 43. miR-145-G18. RNA 37. RNA 43. miR-145-G19. RNA 38. RNA 43. miR-145-G20. RNA 39. RNA 43. miR-145-G21. RNA 40. RNA 43. 29.
(53) a. miR-145-G22. RNA 41. RNA 43. miR-145-G23. RNA 42. RNA 43. 3´-Džɏ9XoNōÙ'. miR-145 N miR-145-B 6XoĄ=Qv^NōÙ'.. miR-145 ;Qv^:ōÙ»ɲ (N) N miR-145-GN 6Ʉ'4J. 3-3-1 Qv^ 2 NōÙ'. miRNA 9GJˬ¹ĺȳȠƇéʀ. ʽƬ'. 25 Ɋ̚: miR-145 9GJǘȵˬ¹ĺ FSCN1 :ȳȠƇéʀNTUgmp rR^94ʵÄ'. (Figure 23) ijʅȲɣɿ DLD-1 N 6well 5 24 ƻ˼Ĩ̟Ű ċ miR-145 NǀɤȌŧ 40 nM 5tgU]e'.ƿ9 48 ƻ˼Ĩ̟Űɫm}] ˈNƌã'FSCN1 :ȳȠ˶Nǥˑ)J$65ċ miR-145 :ˬ¹ĺƇéʀNʵ Ä'.. C N B 1 2 3 4 5 6 7 8. C 9 10 11 12 13 14 15 16. FSCN1 ћDFWLQ C 17 18 19 20 21 22 23 FSCN1. & &RQWUROPL51$. % PL5%. ћDFWLQ. 1 1DWLYHPL5. 1XPEHU 1
(54) PL5*1. Figure 23. miR-145 :HJ9 OgvY£S. ĴȓĦ: miR-145 6ǥˑ'4XoNōÙ'. miR-145-B 5;ˬ¹ĺȳȠƇéʀ ¼)J$6 Ƀʸ&K.$:ɧNj;Wilson H9G14ĪĒ&K. 3´-Džɏ>:Xo Ê̞ miRNA : RISC ŬƁNķ#J6ɀʧ6ʋ)J 47A.2 N 5´-Džɏ: 1-8 ȬȻ:»ɲ9ōÙ'.īČ5;ˬ¹ĺȳȠƇé @6O7Ƀʸ&K8 1.miRNA ; ɣɿ>ōÙŰ5´-Džɏ:Ǩ˲ĩ ˲ø˱ɠ9G14˲øNĈ"JX ɬɧƾǖˡ ʬNJGI5´-Džɏ:˲ĩ; Argonaute : MID uS6Ū!Ƚ«¾ȥ'4 IRISC :ŬƁ9ŷʦ5J$6 ĪĒ&K4J 48ű141 ȬȻ>: 2 :ōÙ; 5´-Džɏ:Ǩ ˲ĩ>:˲øN˿Ń'RISC ŬƁNķ#J$6 Ʉę&K.A.eu̕ħNď C 2-8 ȬȻ;miRNA 9 4ǘȵ6ĬĩʼnNŬƁ'F)̕ħ5JŁ̅sm. 30.
(55) g H©Ž&KJ FSCN1 mRNA 6 miR-145 :ªǞǖˡ HE$:̕ħ>:ǨɠɧČ Ƀʸ&K. (Table 9) 2 ;Ĭĩʼn:ŬƁʀðNƐ.8.D$:̕ħ>:ōÙ; miR-145 :ǘȵ>:ɧČNķ#J$6 Ʉę&K.15-17 ȬȻ>: 2 :ōÙE miR-145 : ˬ¹ĺȳȠƇéʀNij. !¼&+4J $KEčǗ9ǘȵ6:ǨɠɧČNķ#4. J.D/6ɶ HKJ' '8 H,:´:̕ħ9 2 NōÙ'.īČ5;miR-145-B 6čɉŧ:ˬ¹ĺȳȠƇé Ƀʸ5 2 :ōÙ9GJŭ̑;ŏ86ɶ HK.. Table 9. FSCN1 mRNA miR-145 {kao. Position of FSCN1 3´UTR. Predicted consequential paring. Position of 116-123 FSCN1 3´UTR hsa-miR-145 Position of 377-384 FSCN1 3´UTR hsa-miR-145 Position of 729-735 FSCN1 3´UTR hsa-miR-145 Position of 1140-1247 FSCN1 3´UTR hsa-miR-145 TargetScanHuman (http://www.targetscan.org). 31.
(56) 3-3-2 miR-145-G9 9GJǘȵˬ¹ĺƇéʀ. $KA59˛?4 .Ł̠GIQv^ 2 NōÙ)J»ɲ6'4eu̕ħ9̆ƚ )J 9 ȬȻ:Ĭĩ ǀE˩å6ɶ HK.,$5$:»ɲ9Qv^ 2 NōÙ'. miR-145-G9 Nȥ4 luciferase assay NʜčǗ9ǘȵˬ¹ĺ:ƇéʀNʵÄ'.ʵÄ ǯ9; Dual-luciferase reporter assay Nȥȳ×m}]ˈ5J Firefly luciferase :Ƕ9 FSCN1 mRNA : 3´UTR NƔÙ'. ]mNʽƬ'miR-145-G9 6čƻ9ɣɿ>ōÙ' .24 ƻ˼Ű: Firefly 6 Renilla luciferase 9ȧLj)Jȳ×˶NȆŀ'miRNA 9GJ Firefly luciferase :ȳȠƇéõNjNʵÄ'.n˯æ: miRNA N'.īČ:ȳ×˶Nat. Firefly luciferase / Renilla luciferase (%). 6'4 100 %6'. (Figure 24) . 140 120 100. **. **. miR-145. 145-g9. 80 60 40 20 0 Control. Figure 24. miR-145-G9
(57) OgvY. Qv^ 2 G=XoNōÙ'. miR-145-G9 5EĴȓĦ6čǗ9 firefly luciferase :ˬ¹ĺȳȠƇé Ƀʸ&K.$6 HmiR-145-G9 ;ɣɿÜ5 RISC N³'ǘȵ mRNA 9ȼƚɧČ)J$65,:ȳȠNƇé'4J$6 Ʉ&K.. 32.
(58) 3-4 Qv^ 2 NōÙ'. miR-145 9GJǘȵˬ¹ĺ:ƕȞ. Qv^ 2 G=XoNōÙ'. miR-145-G9 ɣɿÜ5 RISC NŬƁ'FSCN1 9 ɧČ)J$65,:ȳȠNƇé'4J$6 Ƀʸ5. .:5Ł̅9džNȥ. 4 FSCN1 mRNA :ƕȞNʶB. ijʅȲɣɿ DLD-1 N 6 well 5 24 ƻ˼Ĩ̟ŰmiR-145-G9 NǀɤȌŧ 40 nM 5tgU]e'.ƿ9 24 ƻ˼ŰUV (365 nm 20 ä302 nm 10 ɇ) NȔŌ'ɫ RNA Nƌã'.miR-145-G9 9G14Xoǘˁ&K.ǘȵ mRNA NgttQf5arR^'.^ypth5ɚʤ'Ý=ɫ RNA 6' 4ƌã'.^ypth9GJɚʤêŰ:ɫ RNA ¥9ďAKJ FSCN1 mRNA ˶N QmS PCR 9GIŀ˶' miR-145-G9 9GJǘȵˬ¹ĺ:ƕȞʀNǔʴ'. (Figure 25) . 900. Relative FSCN1 mRNA level ћDFWLQ
(59). 800. EHIRUHSXULILFDWLRQ DIWHUSXULILFDWLRQ. ***. 700 600 500 400 300 200 100 0 145-B (+). 145-G9 (+). 145-G9 (-). Figure 25. 7J&¡Zq FSCN1 mRNA ¯. at6'4Qv^ 2 NōÙ'48ɜ (miR-145-B(+))6 UV NȔŌ'4 8ɜ (145-G9 (-)) E¿+4ʜ1. ,:ɧNj Qv^ 2 NōÙ'UV NȔŌ'. 145-G9 (+) 5:Bh:ɚʤŰ9 FSCN1 mRNA ij. !Ƹ'.$:ɧNj HQ. v^ 2 Ą=XoNōÙ'. miR-145-G9 ;UV ȔŌ9GIǘȵˬ¹ĺ5J FSCN1 mRNA 9×njǛNŬƁ'.$6 Ƀʸ&K.. 33.
(60) A.,:´:ǘȵˬ¹ĺ6'4miR-145 ; c-MYC mRNA 9ɧČ',:ȳȠNƇé )J$6 ɀHK4J 49DLD-1 ɣɿ9 4 miR-145 9GJ c-MYC :ȳȠƇé;Ƀ ʸ5. 8 1. (Figure 26) '. 14DLD-1 ɣɿ5; miR-145 ; c-MYC Nǘȵ6'. 8ĉʀź Ʉę&K. C. N. B. 9. c-MYC ћ-actin (48 h, 40 nM). Figure 26. miR-145 :HJ9
(61) c-MYC vY. ,$5 DLD-1 ɣɿGIE c-MYC :ȳȠ˶ IJ COLO-201SW480 ɣɿ5 miR-145 9G. J c-MYC :ȳȠƇéNɃʸ'. (Figure 27) . (a). c-MYC β-actin (b) COLO201. (c) SW480. c-MYC. c-MYC. FSCN1. FSCN1. β-actin. β-actin. Figure 27. (a) DLD-1, COLO-201, SW480
(62) c-MYC ¯ (b) COLO-201 M miR-145
(63) c-MYC vY£S (c) SW480 M miR-145
(64) . c-MYC vY£S. DLD-1 ɣɿ5:Ł̠ɧNj6čǗ9FSCN1 :ȳȠ˶;¼'4J c-MYC :ȳȠ˶9 İø;ʧHK8 1.ű14Öʜƭȝ:ɧNj6;ȭ8J ŏ8!6E²ĝƂ ø. 34.
(65) ļČƁ'. miR-145 ; c-MYC Nǘȵ6'48$6 ƹH 681. ,$5čǗ9^ypthɚʤêŰ:ɫ RNA ¥9ďAKJ c-MYC mRNA ˶N QmS PCR 94ŀ˶'. (Figure 28) ,:ɧNj^ypth9GJɚʤêŰ5 c-MYC mRNA ˶9İø;8!miR-145-G9 9×njǛ;ŬƁ&K48$6 Ʉ&K. ű14 dž miRNA ;ǘȵ mRNA :BN˫Ƌȵ9ǘˁ'4J$6 Ƀʸ&K.. Relative c-MYC mRNA level ћDFWLQ
(66). 800 700. EHIRUHSXULILFDWLRQ DIWHUSXULILFDWLRQ. 600 500 400 300 200 100 0. 145-B(+). 145-G9 (+) 145-G9 (-). Figure 28. 7J&¡Zq c-MYC mRNA ¯. 35.
(67) 4Ɏ ׹ŹźǡĩĄ=VowĩNōÙ'.ưʨRNA:ČƁ6,: RNAƕȞʀ ·˛?4Lj.Ǘ9ưʨ׹Źźx]XeuQv^2Ą=XoNōÙ'. miRNANČƁ',:ǘȵmRNANǘˁø)J$69Ɓñ'.' '8 H3´Džɏ>:XoōÙ;miRNA:RISC:ŬƁNķ#ǘȵmRNA>:ɧČN¼&+J$ 6 Ƀʸ&K.,$5GI̢ǵź8miRNA:ČƁNȻƑ'ư.9x]X euQv^25Nʲʯ'. (Figure 29) . CF3 N N HO O OH. O. OH. 25. Figure 29. 3 G#01H 25 ¬. Qv^25;űLjĦ:Qv^29ò 4ư.9VowĩN jˮ»9µò'. ǖˡNǁ'4JōÙ'.Vowĩ;ȢøµòąŹ9GIQfu6˫Ƌȵ9ąŹ& +J$6 ĉʀ5I$:$6 H×]g]ąŹ9GJǘȵmRNA:ƕȞŰ9 9XoäĺNōÙ)J$6 ĉʀ68Jű14Qv^25NȥJ$6 5RISC:ŬƁN˿Ń)J$68!ǘȵmRNA:ǘˁĄ=ƕȞ ĉʀ98J6ɶ . (Figure 30) dž̓5;Qv^25NōÙ'.RNANČƁ',:RNAƕȞʀN ǔʴ)J66E9ǘȵmRNA:ȳȠƇéʀNʵÄ'.. UV cross-linking Click reaction CF3 N N. CF3 N N. miRNA mimic. target mRNA. label molecule N3. AA A. N N N. AA A. Figure 30. }¢V^rsmiRNA:HJ9
(68) mRNA y §. 36. AA A.
(69) 4-1 jȢ9fQfwĩĄ=VowĩNǁ)JQaʹō½:ČƁ. Qv^25;øČȘ1 G=26čǗ9Qaʹō½6-D-zgN^ aeø)J$65ŲHKJ6ɶ .A.Qaʹō½;ĪĒ&K4Jƅ̔ Năɶ9ČƁ'. (Scheme 8)50 3,5-diaminobenzoic acidNãȳāƮ6'4QzĩN sandmayerąŹ5Tɠ½9İƜ'.ŰȣƁȘNCH3OH9ȉʬ'˲źLJ¸5oVg r>6İƜ)J$65øČȘ26NĆȟ51%5Ų.ƿ9NaBH4Nȥ4VgrNQ a>İƜ'ŲHK.Qa½NTBDMSCl6ąŹ&+Ǩ˲ĩNTBDMSĩ5È˂ øČȘ27NĆȟ92%5Ų.ɨ4øČȘ27NTHF9ȉʬ'-78 °CLJ¸1ɓ˶:n-BuLi 5âȡ)J$69GIƱ:TɠNoXø'.ŰtX˰˲VoNǾò) J$65øČȘ28NĆȟ85%5Ų.øČȘ289~u[eQĬ˲ĬN¾ȥ&+X[ e½6'.Ű9Ǩ˲ĩNteø'øČȘ29NĆȟ71%5Ų.ɨ4te½29 N2ƶ˼QwQǨ6ąŹ&+4øČȘ30NĆȟ92%5ČƁ'.ŲHK.fQff ʹō½30NI25˲ø'4øČȘ31NĆȟ93%5Ų.øČȘ316TIPS-QioNʕ̖Yp ^5ˢɧ'øČȘ32Nŀ˶ȵ9ČƁ'ċeĩN2ɓ˶:TBAFâȡ5ʂÈ˂ )J$65Ȼȵ:Qaʹō½33NĆȟ91%5ČƁ'.. H 2N. NH 2. O. 1. NaNO 2, urea, KI 25% H 2SO 4 aq. 2. H 2SO 4 CH3OH. OH. I. I. O. I. 1. NaBH 4 EtOH/THF (1/1). I. 2.TBDMSCl, imidazole DMF. OCH3. OTBDMS. 26: 51%. 27: 92%. O n-BuLi, CF3COOEt. I. CF3. THF OTBDMS. N. 1. HONH 2Cl pyridine. I. N N I 2, NEt 3. CF3. CF3. 29: 71%. TIPS PdCl 2(PPh) 3, CuI. TIPS. I. CF3. OTBDMS. OTBDMS. 30: 92%. N N. N N CF3. NEt 3. CH3OH. HN NH NH 3 aq. THF. 2. TsCl, NEt 3, DMAP CH2Cl2. 28: 85%. I. OTs. CF3. TBAF THF. OTBDMS. OTBDMS. 31: 93%. 32: quant.. OH 33: 91%. Scheme 8.fQfwĩĄ=VowĩNǁ)JQaʹō½:ČƁ. 37.
(70) 4-2 x]XeuQv^25Ą=,:QnSt½:ČƁ. øČȘ12:ČƁ6čǗ:ƅǯ9GIQv^25:QnSt½NČƁ'.øČȘ33 61-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoseNÖ6čǗ:LJ¸5^aeø'øČ Ș34NĆȟ89%5ČƁ'.1H NMR9GJʬNJN $81.6$L5.31ppmµ˙91´»:H9ŝœ&KJe^pt:] ʪŇ&K..DȣƁȘNβ-½6ǫŀ'.ƿ 9øČȘ349ʭĹ˶:CH3ONaNȥ4ʂ lSø)J$65x]XeuQv ^25NĆȟ86%5ČƁ'.ɨ4øČȘ29DMTrClN1ū˶ąŹ&+5´-»:1ɟǨ˲ĩN ˫Ƌȵ9DMTrĩ9GIÈ˂'.øČȘ35NĆȟ90%5Ų.øČȘ35NTBDMSCl94âȡ '.ŰŲHK.ǽČȘNeY`Y]t^R94ɚʤ'ü̉'.22: øČȘN,K-K1H NMRĄ=1H-1H COSYNȥ4ʬNJNʜ1.6$L2´-TBDMS½5 JøČȘ36Ą=3´-TBDMS½5JøČȘ37N,K-KĆȟ25 %27 %94ŲHK.$6 ä 1.ǀŰ92´-TBDMS½36NŞǯ9GI˲ø'x]XeuQv^25: QnSt½5JøČȘ38NĆȟ76%5ČƁ'.(Scheme 9) . CF3 N N. O BzO O. O. +. HO. CF3. OBz OBz. CH3ONa. TMSOTf CH2Cl2. N N. CF3 N N. BzO O. CH3OH. O. HO. 33. OBz OBz. OH. 34: 89 %. CF3 N N Pyridine. DMTrO O OH. CF3 N N DMTrO O. OH. OH. 35: 90 %. O. P Cl DIPEA. CF3 N N +. DMF. O. O. OTBDMS. DMTrO O TBDMSO. 36: 25 %. CF3 N N. N(i-Pr)2. DMTrO. THF. O. NC. O. OH 25: 86 %. TBDMSCl TEA. DMTrCl. NC. O. O. O. O OTBDMS P N(i-Pr)2 38: 76 % 31P NMR: δ 149.4, 149.8. Scheme 9. 3 G#01H 25] ?+/Q at. 38. O. OH 37: 27 %.
(71) 4-3 Qv^25NōÙ'.RNA:ČƁ. ČƁ'.x]XeuQv^25:QnSt½Nȥ4ǐ˲ʊöČƁǜ94Xb x]XouNČƁ'. (Table 10) ˯æ;Ĵȓ:miRNA5Jlet-7NǙ'4 I5´-Dž ɏ:8ȬȻ9Qv^25NōÙ'. (RNA 44) A.RNAƕȞʀǔʴ:.D 5´-DžɏNFITC 5ǘˁ'.ǘȵ˸EčǗ9ČƁ'.˯æ;let-7:Ƚʣ˸5IQv^:ȽʣˮäNİ .4Ɋ̚:RNANČƁ'. (RNA 45-48) ČƁ'.ċXbx]Xou:ǖˡ; MALDI-TOF/MS9GIɃʸ'.. Table 10. `E!3 G,0 ®X. RNA. aSequence. calculated. observed. RNA 44. 5´-UGA GGU AXU AGG UUG-3´. 4963.99. 4962.79. RNA 45. 5´-F-CAACCU AAU ACC UCA-3´. 5178.79. 5178.80. RNA 46. 5´-F-CAACCU AGU ACC UCA-3´. 5194.28. 5193.99. RNA 47. 5´-F-CAACCU ACU ACC UCA-3´. 5154.25. 5154.83. RNA 48. 5´-F-CAACCU AUU ACC UCA-3´. 5155.24. 5154.96. a. X = Qv^25 F = FITC. 4-4 RNA:RNAƕȞʀ. Qv^25NōÙ'.RNA9GJRNAƕȞʀNǔʴ'.RNA6Ƚʣ ˸RNAN1:1:îČ5ǽČ' Qw^âȡ9GIªdž˸NŬƁ&+.Ű9 UV (365 nm 30ä302 nm 10ä) NǩǸ5ȔŌ'.7MTQȉǻNò 4ąŹNÎǟ&+ŲHK .ǽČǻN20% İźPAGE94äNJ'. (Figure 31) Target RNA. ds RNA. ss RNA. RNA 44 - 45 46 47 48. ds RNA. ȽʣĬĩ. Ćȟ (%). RNA 44 + 45. A. 14. RNA 44 + 46. G. 9. RNA 44 + 47. C. 15. RNA 44 + 48. U. 13. Figure 31. 25mWRNA:HJ9 RNAy. 39.
(72) ,:ɧNjÚ4:ɥBČM+5̢äĺ˶̕ħ9ư.8|u ʪŇ&KRNA 6Ƚʣ˸RNA:]g]ąŹ:ˣʜNɃʸ'.A.Ú4:ɥBČM+5]g ]ąŹ ˣʜ'.$6 HQv^25NōÙ'.RNA9GJȽʣ˸RNA:ƕ Ȟ;ȽʣĬĩ:Ɋ̚9ÃĻ'8$6 Ƀʸ&K.. 4-5 Qv^25NōÙ'.miRNA9GJˬ¹ĺȳȠƇéʀ:ǔʴ. 25NōÙ'.RNA ȽʣĬĩ:Ɋ̚9˽MH*Ƚʣ˸RNANƕȞĉʀ5J$ 6 Ƀʸ&K..D25NōÙ'.miR-145NČƁ'ǘȵˬ¹ĺ5JFSCN1: ȳȠƇéõNjNTUgmprR^5ʵÄ'.A.25:ōÙ»ɲ;$KA5: Ł̠9Ì5´-Džɏ H9ȬȻ:Ĭĩ6'miR-145-G9:FSCN1ȳȠƇéʀ6ǥˑ'.. 4-5-1 miRNA:ČƁ. ZSu˸:9ȬȻ925NōÙ'.miR-145NġȽgQnStǯ9űČ Ɓ'.RNA:ǖˡ;MALDI-TOF/MSGIɃʸ'.. Table 11. atmiR-1450°. RNA. Sequencea. calculated. observed. RNA 49. 5´-GUC CAG UUX UCC CAG GAA UCC CU-3´. 7350.41. 7349.52. a. X = Qv^25. ZSu˸:9ȬȻ9Qv^25NōÙ'.RNA436}pif˸NɥBČM+ªdž˸ miRNA5JmiR-145-X9NʽƬ'.. Table 12. ¦|miR-145:HJ9. miRNA. ZSu˸. }pif˸. miR-145-X9. RNA 49. RNA 43. 40.
(73) 4-5-2 Qv^25NōÙ'.miRNA9GJˬ¹ĺȳȠƇéʀ. ʽƬ'.miR-145-X99GJǘȵˬ¹ĺFSCN1:ƇéʀNTUgmprR^94 ʵÄ'. (Figure 32) ijʅȲɣɿDLD-1N6well524ƻ˼Ĩ̟ŰmiR-145Nǀɤ Ȍŧ20-40 nM5tgU]e'.ƿ948ƻ˼Ĩ̟Űɫm}]ˈNƌã' FSCN1:ȳȠ˶Nǥˑ)J$65miR-145:ˬ¹ĺȳȠƇéʀNʵÄ'.A. ǥˑʼn˃6'4ĴȓĦ:miR-1453´-Džɏ9XoNōÙ'.miR-145-BXoĄ=9 ȬȻ9Qv^2NōÙ'.miR-145-G9EČM+4Ł̠'ˬ¹ĺȳȠƇéʀNǥˑ'.. N. B. G9. X9. C 20 20 20 20 FSCN1 ћDFWLQ & &RQWUROPL51$. % PL5%. 1 1DWLYHPL5. * PL5* . ; PL5; . Figure 32. miR-145-:HJ9
(74) FSCN1 vY. $KA5:Ł̠čǗĴȓĦ:miR-1456ǥˑ'4XoNōÙ'.miR-145-BF miR-145-G95;ˬ¹ĺȳȠƇéʀ ¼)J$6 Ƀʸ&K.' '8 H9ȬȻ9 25:BNōÙ'.miR-145-X9;ĴȓĦ:miR-1456čɓ:ˬ¹ĺȳȠƇéʀNɄ)$6 ƹH 981.ű1425NōÙ'.miRNANȥJ$65GIõȟȵ8ǘ ȵmRNA:ǘˁø ǃů5. J. . 41.
(75) 5 Ɏ cRGD af`t:ČƁ6,:ˬ¹ĺȳȠƇéʀ 5-1 miRNA ùʖĖ:˻ȳ. Ŧʾ5˛?.Ǘ9miRNA ;Ǘ8Ȯȯ9˽¡'4 Iș9 Oɣɿ5ȳȠ ¼'4 J miRNA ;¼äĺùʖ9İMJư.8Ɖ Oë6'4ǃů&K4JÁ <ɹɣɿ Ȳ9 4ȳȠ˶ ¼'4J miR-26a ; Oɣɿ9QtegNʹō',:ĭ ǢNƇ J$6 ɀHK4J 51-52miR-34a EčǗ9ɹɣɿȲ:ȣɺ˿ŃNɄ'öȘ s5EʄȰ:ȂŏNũ. ˊ$)6Û9ȣĻȟNijş9Ƹ&+J$6 ĪĒ&K4. J 53' '8 HȠģʉťɁɋA5ˣO5J miRNA ùʖĖ;˛'. miR-34a N ďCªĖȻ/"5I,:´ǐ˲ùʖĖ6ǥˑ'4,:˻ȳ ij. !˥K4JȚǮ. 9JÜĞź: miRNA NùʖĖ>6Źȥ)Jȑ9;·9ƒ#Jʼ̘Nʬǫ)Jŷʦ J. 1) ùʖĖËʣ68J miRNA :ƙɡ ıĞź: siRNA FQoig DNA 6;ȭ8IÜĞź: miRNA NȥJīČ;ù ʖĖËʣ68J miRNA N˫Ɗ'8"K<8H8,:ȑ9;ʼn˃:ȮȯÁ < O ɣɿ¥5ȳȠ˶ ¼'4J miRNA Nʼn˃9,KH:ǘȵ mRNA Nčŀ' Oɣ ɿÜ5:Ó NƹH 9)Jŷʦ J' '8 HmiRNA 6ǘȵ mRNA :ɧČ}m ;gpoĬĩʼnNďC$6 H̎Ş9IJǗ5I,:ʬNJ9;IJ!:ƻ˼6ˆ ȥNŷʦ6)J 2) up^s|egr:˻ȳ ǐ˲ùʖĖ:˻ȳ9Û˟)Jʼ̘6'4ǐ˲:˲fVgrȧLj:˄̌ʑ9GI ɣɿʆ˞˦ź ¼Ě̘Ȑ I$KNʬǫ)Jup^s|egr:˻ȳ ŷʦ5J. ˛'. 2 ʼ̘:0 1) 924;$KA5:Ɂɋ5׹Źź miRNA 9G Jǘȵ mRNA :ʬNJǯN˻ȳ'4 IdžƅǯNȥJ$65 miRNA :ǜʀʬNJN˘ˠ 9ʜ$6 ĉʀ681.,$5džɎ5;miRNA :ùʖĖ>:ŹȥNŽŀ'ư.8 up^s|egr:˻ȳNȻȵ6'.. 42.
(76) 5-2 ǐ˲ùʖĖ:s|. ǐ˲ùʖĖ:s|Ʊǯ;ij !ä"41) ʁˈvzəĺ (Lipid Nano Particle: LNP) N[Q6)J LNP ǯ62) ɣɿʆ:Ĉń½9ʸˁ&KJZuäĺNǐ˲9ɧČ &+JZuaf`tǯ Ļģ)J (Figure 33) 54 (A) LNP法. (B) リガンドコンジュゲート法. Figure 33. (A) LNP (B) EI0. I$C J/ ¬. 5-2-1 LNP ǯ LNP ǯ;ȠģǀEŤ!ȥHK4Jǐ˲:s|Ʊǯ5J 55-58LNP ǯ5;˄̌ ʑ:ǐ˲ YoXź:ʁˈ6vzəĺNŬƁ'VucSteg9GIõȟʎ!ɣ ɿÜ>ōÙ&KJüɞ8 LNP ;Ĩ̟ɣɿɜ5;õNjȵ5JE::in vivo 5ȥJ9 ;˩&8YoXźʁˈ;ʛ¥:m}]ˈ6Ū!Ƚ«¾ȥ',:ɧNjɣɪÜȷ ɜ:PbcSteg9G14ˠF 9ʛ¥ HƗã&K4'Aȑ5Jű14in vivo 5ȥHKJ LNP ;ʍȵ9 ʛ¥:m}]ˈ6:Ƚ«¾ȥN˭"Jȑ9¥ź: PEG äĺ5arR^&K4JA.ʍȵ8 LNP ;əĺɜ 100-200 nm 6ǥˑȵcS h:ij. 8ʥČ½5Jȑin vivo 5s|ĉʀ8ɥɰ;ʛɕ˞˦ź:̢ɹʈʃʈ. Ą=ˮ:ʄȰ9é́&KJò 4ʤëø)J̅9÷ơ)J siRNA :˶əĺ:ĥ ø87 ʼ̘6814J. 5-2-2 Zuaf`tǯ. ˙Ţư.8up^s|egr6'4ǐ˲>:Zuaf`tǯ DzȻN̈D4J. 59-62. Zuaf`tǯ; LNP ǯ6;ȭ8I̎Ş9äĺc. Sh Ŏ&!m}]ˈ>:̎șȭȵ8ĐȿNˊ$'9!ȑPbcSteg9G JƗãN˭"HKJ´LNP ǯ6ǥˑ'4Ǥź ǕD4¼6ɶ HK4JA.ä. 43.
(77) ĺcSh Ŏ&:5ʛɕ˞˦ź9EÕK4 IIJǗ8ɥɰ>:s| ĉʀ5 J6©Ž&KJ&H9Zu6Xbx]Xou HƁJe8ǖˡ5J .DǖˡʬNJ ńƺ5Iʤë:ĥø9ÕK4JƱ5LNP ǯ6ǥˑ)J6 ǐ˲:ɣɿÜ>:ōÙõȟ ¼6Ě̘ȐEĻģ)JA.LNP ǯ6ȭ8Iǐ˲ ì. ã'5Ļģ)Jȑx]Qj9GJäʬNĈ"F). 5-2-3 miRNA ùʖĖ:ʄȰ>:s|Ʊǯ. Qoig DNA FsiRNA 5;Zuaf`tǯ ȹO9ɁɋĪĒ&K4 J miRNA 5;DŽ/Zuaf`tǯ9GJs|:ĪĒÁ;8 $K;ǘȵșȭź:̢Qoig DNA F siRNA 6ǥˑ'4ʥƫ:ǘȵ mRNA N éų)J miRNA ;ƈ¡˶ IJ!8JÒĎ9IōÙõȟ:¼Zuaf`t ǯ6:Ƚź Żȑ5JƱ5˜˧ɥɰN˫<8Zuaf`tǯ;ʄ Ȱ>:s|9ǁõ5JȑGIōÙõȟ:̢ miRNA-Zuaf`te gr:˻ȳ ǪDHK4J. 5-2-4 Cyclic RGD ou. cyclic RGD (cRGD) ; RGD ˯æNďC 5 ǡĩ:ȢȚou5I (Figure 34) ɣɿʠ ̏:Ĉń½5J αVβ3 Sr^9˫Ƌȵ9ɧČ)J$6 ɀHK4J. 63. αVβ3 S. r^;ʛɕưȣ¥:ÜȷɣɿFIJ!: Oɣɿ5̢ȳȠ'4JȑcRGD ; Oǭ ȱ9 "Js|äĺ6'4Ť!ȥHK4J 64Qoig DNA F siRNA >: cRGD Ê̞ÁEƵ9ĪĒ&K4 I 65-66cRGD NʥƫƟ˒'. siRNA ;LNP 5ˬ ¹ĺȳȠƇéʀNɄ)$6EƹH 9814J 67 HN O. HS O. NH NH. NH. HN O HN H N. O O. O OH. c(RGDfC). Figure 34. cRGD ¬. 44. NH 2.
(78) A.cRGD ;ü˶½GIEª˶½˶½ GIĈń½9ʼn)Jʩĕź ̢$6 IJ !:Ɂɋ5ƹH 9814J $K; cRGD :őƄȌŧ:Ƹ6IJÄõNj9GJE: 6ɶ HK4J 69IJÄõNj6;ʥƫ:Zu čƻ9ʥƫ:Ĉń½9ɧČ)J$6 5ɧČð ̛ˏȵ9Ƹ)JȠ˃5IIJÄõNjNȳȠ&+Jȑ9;Zu˼: Y˺ ŀ·:˺&5Jŷʦ J. 5-3 džɁɋ:Ȼȵ. ·:Ǘ8ɼƽ HdžɁɋ5; miRNA 9˩ȥĉʀ8ɣɿÜōÙõȟ:̢ cRGD a f`t:˻ȳNȻȵ6'. Ɵ˒)J cRGD ;IJÄõNj9GJɧČð:ƸNț gc˺Nİ 4ǖˡǵźȽ˽Nʜ$6NʯȨ'.$K9GIōÙõȟ:̢ cRGD af`t:ǖˡNƙɡ)J²ĝcRGD Naf`t)J˯æ9; miRNA 5;8! siRNA NƘȥ'.siRNA ;°ȑȵ9ʲʯ'.ıĞź:ªdž˸ RNA 5 ImiRNA GIE¼ń˶5ˬ¹ĺȳȠƇéõNjNɄ)ȑcRGD af`t:ǖˡǵ źȽ˽Nʜ5ǀ˩5J6ɶ . A.Zuaf`tǯ5;ǐ˲äĺ x]Qj9GJäʬNĈ"F) ȑøļÊ̞Nʜ$65x]Qjɸź miRNA ˯æNƙɡ)J ǀɤȵ9;ǵźȽ˽9GIǀ˩ø&K. cRGD ǖˡ6x]Qjɸź miRNA Nȥ 4 cRGD-miRNA af`t:ČƁNʜmiRNA ùʖĖ>:ŹȥNȻƑ). 45.
(79) 5-4 cRGD-siRNA af`t:ʲʯ. cRGD : siRNA >:ōÙ9; post-synthetic modification ǯNƘȥ'.þ0ąŹź:̢ Q z ĩ 5 Ê ̞ ' . RNA N ǐ ˲ ʊ ö Č Ɓ ǜ 5 Č Ɓ ' . Ű N-ε-maleimidocaproyl-oxysuccinimide ester (EMCS) 6:ɮČąŹ5 RNA 9SuĩNō Ù)Jɨ"4cRGD :oXĩ:Suĩ>:S_µòąŹ9GIRNA 9 cRGD NōÙ)J$6NʯȨ'. (Scheme 10) . O. O N. O. O. RNA. O. N O. O. O. EMCS. NH 2. N H. OH. O. RNA. O OH. Phosphate buffer, pH 7.4. O. H N. N H. N O. O. HN O. HS O. NH NH. NH. NH 2 NH 2. HN. HN. NH. O HN H N. O O. O. O O. RNA. OH. OH. N H. O. O. H N. N O. O. c(RGDfC). O N H. S. HN. NH O. H N. HN. O. O O. OH. Scheme 10. cRGD ;:,0 RNA mW. A.RNA 9QzĩNōÙ)Jȑ9ư.9QzÊ̞QnStNʲʯ'. (Figure 35A) ò 4cRGD :Y˺Nʽɖ)J.D:gcQnStEčƻ9ʲʯ '. (Figure 35B) QzÊ̞QnSt6gcQnStNɥBČM+J$65 ʥƫ: cRGD NƟ˒'Y˺Nʽɖ'. cRGD af`t:ČƁ ĉʀ98J 6ɶ . (A). (B). O. NC. DMTrO O. P N. O. N H. H N. CF3 O. DMTrO. O 6. P N. O. CN. Figure 35. (A) ?4U´?+/ ¬ (B) %;J"J?+/ ¬. 46.
(80) 5-5 QzÊ̞QnStĄ=ġȽƍ½:ČƁ. 6-aminohexanoic acid NãȳāƮ6'QzĩNtX˰˲Vo5âȡ)J$ 69GItXQioĩ5È˂'.øČȘ 40 NĆȟ 43%5Ų.ɨ"4ø ČȘ 40 N (S)-3-amino-1-O-(4,4´-dimethoxy)trytily-2-propandiol (39)69 6:ɮČąŹ9Â'ø ČȘ 41 NĆȟ 76%5Ų.ǀŰ9øČȘ 41 NżĪ9ű˲ø)J$65Ȼȵ:Q nSt½ 42 NĆȟ 42%5ČƁ'.A.øČȘ 41 NȒǨa{]˲6ąŹ&+4g] ew½6'.ŰQzĩNǁ)J CPG (controlled pore glass) ǚʁ6ʂǨɮČ)J$69 GIġȽƍ½ 43 NōÙȟ 35.8 μmol/g 5Ų. (Scheme 11) . DMTrO. NH 2 OH 39. CF3COOEt. O. TEA. O NH 2. HO. H N. HO. MeOH. CF3. O. EDCl, DMAP, TEA DMTrO. DMF O. OH. 41: 76 % 31P. O DIPEA NC. CH2Cl2. DMTrO O. P N. O. CF3 O. 40: 43 %. iPr 2NP(Cl)O(CH 2)2CN. H N. N H. H N. N H. NMR: δ 149.0, 149.7. CF3 O. 42: 51 %. O. 1) succinic anhydride, CPG pyridine. O DMTrO OH. H N. N H. CF3. DMTrO O. 2) CPG, EDCl, DMF. O. H N. N H. CF3 O. O HN O CPG. 41. 43. Scheme 11. ?4U´?+/]bwQ at. 5-6 gcQnSt:ČƁ. hexaethylene glycol NãȳāƮ6'0.5 ɓ˶: DMTrCl NąŹ&+4̥Ʊ:Ǩ˲ĩ :BN DMTr ĩ5È˂'.øČȘ 44 NĆȟ 48%5Ų.ƿ9EƱ:Ǩ˲ĩNŞ ǯ9ű˲ø)J$65Ȼȵ:QnSt½ 45 NĆȟ 54%5Ų. (Scheme 12) iPr 2NP(Cl)O(CH 2)2CN HO. O. H 6. DMTrCl Pyridine. DMTrO. O. DIPEA. H. CH2Cl2. 6. O. DMTrO. 6. 44: 48%. P N. O. 45: 54% 31P. Scheme 12.%;J"J?+/ at. 47. NMR: δ 149.1. CN.
(81) 5-7 QzÊ̞ RNA :ČƁ. QzÊ̞QnStġȽƍ½Ą=gcQnStNȥ4ǐ˲ʊöČƁǜ 94QzÊ̞ RNA NČƁ'.˯æ;`zÊŴ9˽¡)J RecQL1 mRNA Nǘȵ6' . siRNA 5I Ƶ9 2´-O-metyl Ê̞9GIx]QjɸźNȞŲ'4J (Figure 36) QzĩĄ=gc;ǘȵ6;ɧČ'8}pif˸: 3´Džɏ9ōÙ'.A .ǘȵ mRNA 9ɧČ)JZSu˸ RNA 57,58 Eǐ˲ʊöČƁǜNȥ4ČƁ'.ČƁ '.ċ RNA :ǖˡ;MALDI-TOF/MS 9GIɃʸ'. (Table13) . Table 13. cRGD-RNA. I$C J/ ®X. RNA. Sequence. calculated. observed. RNA 50. 5´-Guu cAG ACC ACu ucA Gcu uTT-3´. 6693.19. 6693.04. RNA 51. 5´-Guu cAG ACC ACu ucA Gcu uTT XXX-3´. 7491.88. 7490.30. RNA 52. 5´-Guu cAG ACC ACu ucA Gcu uTT sXXX-3´. 7836.18. 7835.73. RNA 53. 5´-Guu cAG ACC ACu ucA Gcu uTT sXsXsX-3´. 8524.78. 8522.44. RNA 57. 5´-AAG CUG AAG UGG uCu GAA cTT-3´. 6785.18. 6784.57. RNA 58. 5´-F-AAG CUG AAG UGG uCu GAA cTT-3´. 7313.64. 7314.83. u, c = 2´-OMe RNA, F = Fluorescein, s = hexaethylene glycol, X = amine. ɨ"4QzĩNōÙ'. RNA 51-53 9 cRGD NōÙ'3 Ɋ: cRGD-RNA af `tNư.9ČƁ'.cRGD NōÙ'. RNA54-56 :ǖˡ;MALDI-TOF/MS 9GI Ƀʸ'. (Table 14) . O NH N. HO. O. O OH. OMe. Figure 36. 2´-OMe RNA ¬. 48.
(82) 5-8 RNA 54 :ČƁ. ČƁ'.QzÊ̞ RNA 51 N pH 7.4 :˲ɭʞǻ¥9ȉʬ'Qzĩ6 N-ε-maleimidocaproyl-oxysuccinimide ester (EMCS) 9GJɮČąŹ5 RNA 9SuĩN ōÙ'.Suµò½N HPLC 5ɚʤ'.ŰcRGD :oXĩ6Su: S_µòąŹ9GIRNA 54 NŲ. (Scheme13) (Figure 37) . (1) マレイミドの付加 RNA. O HO. O. O P. O. RNA NH 2. N H. O O P OH O. O. O N. O. O. P. HO. O. O EMCS. O. O. OH. O. H N. O. N O O. H N. NH 2. N H. O. O. O O P OH O. Phosphate buffer, pH 7.4. N O. N H. O O P OH O. O. H N. N H. O O P OH O. N O. NH 2. N H. OH. O. O. O. O. N H. N O. O. RNA 51 50 RNA. NH 2 HN. NH. (2) cRGDの付加. O O. O. HS O. NH NH. NH. HN O HN. NH 2. RNA HO. O. O P. S. N. H N. O HN. NH. H N. O. O N H. O. NH 2. HN. NH. O OH. c(RGDfC). O. OH. O O. O. O O P OH. H N. O O. NH. O. HN. HN. O. O. N H O O P OH. O. O. H N. N H. N O. S. NH O. O. HN HN. H N O. O. O. OH. O O HO. N H. HN H N. O O. O. N O. S O. NH. N H. O HN. NH HN RNA RNA 52 54. O O O OH. Scheme 13. RNA 54 at. 49. NH 2 NH.
(83) (A) Su:µò. RNA 51. RNA 51 + maleimide. 0. 10. 20. 30. 40. (min.). ǔã: UV 260 nm, Ƕˠ: 1.0 mL/min, ɈöȽ: (A) 5%Qitwt0.1 M TEAA (pH =7.0) (B) 50%Qitwt0.1 M TEAA (pH=7.0)^fVt: B conc. 0% (0 min) 35% (20 min) 100% (30-35 min) 0% (35-40 min). (B) cRGD :µò RNA 51 + maleimide. RNA 54. 0. 5. 10. 15. 20. (min.). ǔã: UV 260 nm, Ƕˠ: 1.0 mL/min, ɈöȽ: (A) 5%Qitwt0.1 M TEAA (pH =7.0) (B) 50%Qitwt0.1 M TEAA (pH=7.0)^fVt: B conc. 0% (0 min) 35% (2.5 min) 60% (22.5 min) 100% (25-35 min) 0% (40-45 min). Figure. 37 RNA 54 ^r«©. 50.
(84) 5-9 RNA55 :ČƁ. Č Ɓ' . Q zÊ ̞ RNA 52 N pH7.4 : ˲ ɭʞ ǻ ¥9 ȉʬ ' Q z ĩ 6 N-ε-maleimidocaproyl-oxysuccinimide ester (EMCS) 9GJɮČąŹ5 RNA 9SuĩN ōÙ'.Suµò½N HPLC 5ɚʤ'.ŰcRGD :oXĩ6Su: S_µòąŹ9GIRNA 55 NŲ. (Scheme 14) (Figure 38) . (1) マレイミドの付加. RNA. O. HO O P O. O. 6. O NH 2. N H. O O P OH O. O O. N. RNA. N O. O. O. O. HO O P O O 6. O EMCS. NH 2. N H O O P OH O. OH. O. O. O. O. O N. N H. OH. O O. H N. NH 2. N H. O. H N O. O O P OH O. O. N O. O O P OH O. N H. Phosphate buffer, pH 7.4. O. H N. N H. N O. O. RNA RNA 52 51. NH 2 HN. NH. (2) cRGDの付加. O O. O. HS O. NH NH. NH. NH 2. HN O HN RNA. H N. O O. NH. O. HO O P O O 6. NH S. N. H N. O. O HN. O. HN. HN. H N. O. O N H O O P OH. NH 2. HN. NH. O OH O. c(RGDfC). OH. O O. O. O. O. N H O O P OH. O. O. H N. N H. N O. S. NH O. O. HN HN. H N O. O. O. OH. O O HO. N H. HN H N. O O. O. N O. S O. NH. N H. O HN. NH HN 53 RNA 55. O O O OH. Scheme 14. RNA 55 at. 51. NH 2 NH.
(85) (A) Su:µò. RNA 52. RNA 52 + maleimide. 0. 10. 20 (min.). 30. 40. ǔã: UV 260 nm, Ƕˠ: 1.0 mL/min, ɈöȽ: (A) 5%Qitwt0.1 M TEAA (pH =7.0) (B) 50%Qitwt0.1 M TEAA (pH=7.0)^fVt: B conc. 0% (0 min) 35% (20 min) 100% (30-35 min) 0% (35-40 min). (B) cRGD :µò. RNA 52 + maleimide RNA 55. 0. 5. 10 (min.). 15. 20. ǔã: UV 260 nm, Ƕˠ: 1.0 mL/min, ɈöȽ: (A) 5%Qitwt0.1 M TEAA (pH =7.0) (B) 50%Qitwt0.1 M TEAA (pH=7.0)^fVt: B conc. 0% (0 min) 35% (2.5 min) 60% (22.5 min) 100% (25-35 min) 0% (40-45 min). Figure 38. RNA 55 ^r«©. 52.
(86) 5-10 RNA56 :ČƁ. Č Ɓ' . Q zÊ ̞ RNA 53 N pH7.4 : ˲ ɭʞ ǻ ¥9 ȉʬ ' Q z ĩ 6 N-ε-maleimidocaproyl-oxysuccinimide ester (EMCS) 9GJɮČąŹ5 RNA 9SuĩN ōÙ'.Suµò½N HPLC 5ɚʤ'.ŰcRGD :oXĩ6Su: S_µòąŹ9GIRNA 55 NŲ. (Scheme 15) (Figure 39) . (1) マレイミドの付加. RNA. O. O. HO O P O. O. 6. NH 2. N H. O O P OH O. O. O. O EMCS. HO O P 6 O. N O. HO O P O 6 O. O. O. O. O. N O. O. O. HO O P 6 O. O. H N. N H. OH. (2) cRGDの付加. O. O. NH 2. N H O O P OH OH. O. H N. N H O O P OH. Phosphate buffer, pH 7.4. O. O. H N. N H. O O P OH. 6. N O. O. HO O P O. O. NH 2. O O P OH O. O O. O N H. 52 RNA 53. O N. HO O P O 6 O. RNA. N O. O. NH 2 HN. NH. HN O. HS O. NH NH. NH. O NH 2 O. HN O. HN H N O RNA. OH. N H. O HN. O. O O. NH. O. HO O P O O 6. c(RGDfC). H N. OH O O. O. NH 2 HN. O. O. N H. S. N. H N. O. NH. NH. O O P OH O. O O. HO O P 6 O. O O. H N. N H O O P OH. NH HN. O N O. NH. S O. O. H N. O. HN. O. OH. O. O O HO O. P. 6. NH 2. HN. NH. O O. RNA RNA 54 56. HO. N H. H N. N H. N O. O. O. O O. S. HN. NH O. H N O. Scheme 15. RNA 56 at. 53. HN. O. O. OH.
(87) (1) Su:µò. RNA 53. RNA 53 + maleimide. 0. 10. 20 (min.). 30. 40. ǔã: UV 260 nm, Ƕˠ: 1.0 mL/min, ɈöȽ: (A) 5%Qitwt0.1 M TEAA (pH =7.0) (B) 50%Qitwt0.1 M TEAA (pH=7.0)^fVt: B conc. 0% (0 min) 35% (20 min) 100% (30-35 min) 0% (35-40 min). (2) cRGD :µò. RNA 53 + maleimide RNA 56. 0. 5. 10. 15. 20. (min.). ǔã: UV 260 nm, Ƕˠ: 1.0 mL/min, ɈöȽ: (A) 5%Qitwt0.1 M TEAA (pH =7.0) (B) 50%Qitwt0.1 M TEAA (pH=7.0)^fVt: B conc. 0% (0 min) 35% (2.5 min) 60% (22.5 min) 100% (25-35 min) 0% (40-45 min). Figure 39. RNA 56 ^r«©. 54.
(88) 5-11 cRGD-siRNA af`t:ɣɿʆ˞˦źǔʴ. ČƁ'.ċ cRGD-RNA af`tNĩ9ʘ×ǘˁ siRNA NʽƬ'ɣɿ>:ćI˗ BNʘ×̙ŵ˹5ʪŇ)J$65PEG gc ɣɿʆ˞˦ź9¡ Jŭ̑NʵÄ' .. 5-11-1 ʘ×ǘˁ cRGD-siRNA af`t:ʽƬ. ʘ×ǘˁ'. RNA 58 6 cRGD NōÙ'.Ƚʣ˸ RNA 54-56 NɥBČM+4Qw^ âȡN'4 Ɋ:ʘ×ǘǘˁ siRNA 1-4 NʽƬ'. (Table 14) . Table 14. V § cRGD-siRNA. I$C J/ ®X. siRNA. RNA. Sequece. calculated. observed. siRNA 1. RNA 58. 3´-TTc AAG uCu GGU GAA GUC GAA F-5´. -. -. RNA 50. 5´-Guu cAG ACC ACu ucA Gcu uTT-3´. -. -. RNA 58. 3´-TTc AAG uCu GGU GAA GUC GAA F-5´. -. -. RNA 54. 5´-Guu cAG ACC ACu ucA Gcu uTT XXX-3´. 9867.43. 9869.47. RNA 58. 3´-TTc AAG uCu GGU GAA GUC GAA F-5´. -. -. RNA 55. 5´-Guu cAG ACC ACu ucA Gcu uTT sXXX-3´. 10151.73. 10150.28. RNA 58. 3´-TTc AAG uCu GGU GAA GUC GAA F-5´. -. -. RNA 56. 5´-Guu cAG ACC ACu ucA Gcu uTT sXsXsX-3´. 10840.33. 10842.11. siRNA 2. siRNA 3. siRNA 4. u, c = 2´-OMe RNA, F = Fluorescein, s = hexaethylene glycol, X = cRGD. 55.
(89) 5-11-2 cRGD-siRNA af`t:ɣɿʆ˞˦źʶ̠. . ʘ×ǘˁ'. siRNA1-4 N~tzɣɿ A2058 >Ǿò',:ʘ×NʪŇ)J$. 65ɣɿʆ˞˦źNʵÄ'.A2058 N 24 ƻ˼Ĩ̟'ĨĤNʛǿ:ĨĤ9İƿ' .ŰsiRNA NɤȌŧ 250 nM 5Ǿò'.37 °C LJ¸CO2 S[ m5 18 ƻ˼ Ĩ̟'.ŰǐǍʏʶʖ6'4 Hoecst-33342 Nò 4ƿ9 10 ä˼S[ t'.Ĩ ĤNćĨ. 1PBS 5ǴǷ'.Űʘ×̙ŵ˹5ɣɿNʪŇ'. (Figure 40) . RGD. RGD. (EG)6. RGD. RGD. RGD. (EG) 6. RGD. siRNA 3. siRNA 2. (EG) 6. RGD. (EG) 6. RGD. siRNA 4. merge. Hoechst. Fluorescein. siRNA 1. RGD. 20. Figure 40. cRGD-siRNA. I$C J/ _ª¤µ. cRGD NōÙ'48 siRNA 1 5;ʘ× @6O7ʪŇ&K8 1.:9ʼn'4 cRGD NōÙ'. siRNA 2-4 5;Ú4XiSȧLj:ʘ× ʪŇ&K.A.siRNA 2 6 siRNA 3 5;@6O7ʘ×Ūŧ9ř 8 1.Ʊ53 äĺ: cRGD ˼9 PEG gc NōÙ'. siRNA 4 5ǀEŪʘ× ʪŇ&K.$K;ūç:ǃů˟IgcN ōÙ'. 3 äĺ: cRGD IJÄõNj9G14ɧČð ijş9Ƹ'.ȑ6ɶ HKJ ,$5·̀:Ł̠;siRNA 4 :}pif˸5J RNA 56 Nȥ4Ł̠Nʜ$6 6'.. 56.
(90) 5-12 cRGD-siRNA af`t:ˬ¹ĺȳȠƇéʵÄ. ɣɿÜćI˗Bʶ̠5ǀEōÙõȟ:ʎ 1. RNA 56 Nȥ4 siRNA NʽƬ' cRGD-siRNA af`t:ˬ¹ĺȳȠƇéʀNQmS PCR ǯ5ʵÄ'.yZ rRat6'4cRGD NōÙ'48 siRNA 5 Ečƻ9ʽƬ'. (Table 15). Table 15. cRGD-siRNA. I$C J/ ®X. siRNA. RNA. Sequece. siRNA 5. RNA 57. 3´-TTc AAG uCu GGU GAA GUC GAA-5´. RNA 50. 5´-Guu cAG ACC ACu ucA Gcu uTT-3´. RNA 57. 3´-TTc AAG uCu GGU GAA GUC GAA-5´. RNA 56. 5´-Guu cAG ACC ACu ucA Gcu uTT sXsXsX-3´. siRNA 6. u, c = 2´-OMe RNA, s = hexaethylene glycol, X = cRGD. 5-12-1 cRGD-siRNA af`t:ˬ¹ĺȳȠƇéʀ. cRGD :ōÙ siRNA :ǵź9¡ Jŭ̑NʵÄ)J.D9ś˅:ǐ˲ōÙʶʖ5 JU]m RNAiMAX Nȥ4 siRNA 5 Ą= 6 :ˬ¹ĺȳȠƇéʀNʵÄ'. ~tzɣɿ A2058 N 96well 5 24 ƻ˼Ĩ̟Űċ siRNA 6 RNAiMAX :ʥČ½N ǀɤȌŧ 20 nM 5tgU]e'.ƿ9 24 ƻ˼Ĩ̟Űɫ RNA Nƌã'4 ǘȵ5J RecQL1 mRNA NQmS PCR ǯ9GIŀ˶)J$65siRNA :ˬ¹ĺ ȳȠƇéʀNʵÄ'. (Figure 41A) A.cRGD af`t9GJˬ¹ĺs|õNjNǔʴ)Jȑǐ˲ōÙʶʖN ȥ*9ˬ¹ĺȳȠƇéʀ:ʵÄNʜ1.čǗ9 A2058 N 96well 5 24 ƻ˼Ĩ̟'ĨĤ Nʛǿ:ĨĤ9İƿ'.Űċ siRNA NǀɤȌŧ 250 nM 5Ǿò'.ƿ9 72 ƻ˼ Ĩ̟Űɫ RNA Nƌã'4ǘȵ5J RecQL1 mRNA NQmS PCR ǯ9GIŀ˶ )J$65siRNA :[Q5:ˬ¹ĺȳȠƇéʀNʵÄ'. (Figure 41B). 57.
(91) (B) キャリアフリー. 120. 120. 100. 100. 80. 80. % expression. % expression. (A) キャリアを使用. 60. 60. 40. 40. 20. 20. 0. 0 Control. siRNA 5. siRNA 6. Control. siRNA 5. siRNA 6. Figure 41. (A) BEROgvY¤µ, (B) BE8EJ Og vY¤µ. (A) cRGD N}pif˸: 3’Džɏ9ōÙ'. siRNA 6 ;U]mĻģ5 DŽÊ̞: siRNA 5 6čǗ8ˬ¹ĺȳȠƇéõNjNɄ'.$:ɧNjGIsiRNA }pi f˸: 3´Džɏ>: cRGD äĺ:ōÙ; RISC :ŬƁĄ=ǘȵ mRNA :ʸˁ9ŭ̑N¡ 8$6 Ƀʸ&K.. (B) Ʊ5U]m:Ł̠5;siRNA 56 Û9 250 nM :̢ȌŧLJ¸ 5Eˬ¹ĺȳȠƇéNɄ&8 1.ʘ×̙ŵ˹:Ł̠5;cRGD :ōÙ9GIʘ×Ūŧ :ĭò ʪŇ&K4.$6 H²ĝʲʯČƁ'. cRGD-siRNA af`t;V ucSteg5ɣɿÜ9ōÙ&K.ŰVuk HʂããLj*9kk¥ 5äʬ&K4Jĉʀź Ʉę&K.. 58.
(92) 5-13 QÊ̞ cRGD-siRNA af`t:ČƁ6ˬ¹ĺƇéʀʵÄ. ê˛'.Ǘ9²ĝČƁ'. cRGD-siRNA af`t;[Q5ˬ¹ĺȳ ȠƇéNɄ&8 1.$:āĞ6'4. 1) Ƙȥ'. siRNA :ǵź ¼ (IC50 ̢) 2) cRGD-siRNA af`t:Vukʂã ʜMK48. ·: 2 Ȑ ɶ HK. 1) 9˽'4af`tĦ siRNA ;ʍȵ9[QNȥ.s|6ǥˑ' 4ōÙõȟ ̎Ş9¼$6 ɀHK4JāĞ6'4siRNA :˄̌ʑ9ȧLj)Jɣ ɿʆ6:ąȳ ƒ#HKJA.[Qǯ5;ij˶: siRNA YoXźʁˈ9÷A K4ɣɿÜ>s|&K4J:9ʼn'af`tĦ siRNA ; 1 äĺ%69s |&K4 I$:ȐEōÙõȟ Żȡȧ:25J,:ȑȠģʉťɁ ɋ9ȥHK4Jaf`tĦ siRNA ;[Q5ȥ.īČ:ōÙõȟ :¼Nʧˌ'[QNÀȥ'.̅9 IC50 ƫ pM 68JG8˯æ ˫Ɗ&K4 J²ĝƂ ȥ. RecQL1 siRNA ; IC50 nM ˋ5I[Q5ˬ¹ĺȳȠ ƇéõNjNɄ)ȑ9; ˩å/1.6ƛȆ&K.' '8 HdžɁɋ:ǀɤȻǘ;a f`tĦ miRNA :˻ȳ5Iê˛'.G9 miRNA ; siRNA GIEǘȵșȭź ¼.DʖõNɄ).D9;ƿ9̢ȥ˶Nŷʦ6)Jű14siRNA ˯æ:Ƨě; džɁɋ:˧Ɓ9 "JǑdžȵ8ʬǫ6;8H8 2) 9˽'4˙Ţaf`tĦ siRNA ;ƫIJ!ĪĒ&K4J VucSt egɦˎĄ=Vukʂãɦˎ:ʷɣ;DŽ/ƹH 98148²ĝ:Ł̠ɧ Nj HcRGD-siRNA af`tEčǗ5I@6O7;Vuk HʂããLj *9kk¥5äʬ&K4J6Ʉę&K.. ,$5cRGD-siRNA af`t:s|õȟ:ƧěNȻȵ6'4ư.9 QÊ̞ cRGD-siRNA af`tNɶǓ'. (Figure 42) . 59.
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