Title
糖部開環型人工核酸の合成と遺伝子発現抑制法への応用( 本
文(Fulltext) )
Author(s)
小縣, 綾
Report No.(Doctoral
Degree)
博士(農学) 甲第669号
Issue Date
2017-03-13
Type
博士論文
Version
ETD
URL
http://hdl.handle.net/20.500.12099/56217
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。⢾
⢾㒊㛤⎔ᆺேᕤ᰾㓟ࡢྜᡂ
㑇ఏᏊⓎ⌧ᢚไἲࡢᛂ⏝
2016 ᖺ
ᒱ㜧ᏛᏛ㝔㐃ྜ㎰Ꮫ◊✲⛉
⏕≀㈨※⛉Ꮫᑓᨷ
(ᒱ㜧Ꮫ)
ᑠ⦩ ⥤
⢾
⢾㒊㛤⎔ᆺேᕤ᰾㓟ࡢྜᡂ
㑇ఏᏊⓎ⌧ᢚไἲࡢᛂ⏝
␎
␎ㄒ⾲
ᮏㄽᩥ⏝࠸ࡓ␎ㄒ࠾ࡼࡧ␎グྕࡣࠊ௨ୗ♧ࡍ㏻ࡾ࡛࠶ࡿࠋ rA = adenosine dA = deoxyadenosine rC = cytidine dC = deoxycytidineCPG = controlled pore glass
EDCl࣭HCl = 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl DMAP = 4-dimethylaminopyridine
DMF = N,N-dimethylformamide DMTr = 4,4′-dimethoxytrithyl
EDTA = ethylenediamine-N,N,N ′,N ′-tetraacetic acid rG = guanosine
dG = deoxyguanosine
MALDI-TOFMS = matrix assisted laser desorption ionization - time of flight MEM = minimum essential medium
NIS = N-iodosuccinimide
NMR = nuclear magnetic resonance dT = thymidine
TBAF = tetrabutylammonium fluoride TBDMS = tert-buthyldimethylsilyl TBDPS = tert-buthyldiphenylsilyl THF = tetrahydrofuran
Tris = tris (hydroxymethyl) aminomethane TMS = tetramethylsilane
rU = uridine
┠
┠ḟ
➨
1❶ ᗎㄽ
➨1 ⠇ ᰾㓟་⸆ရ 1-1-1. RNA ᖸ΅ 1-1-2. ࣥࢳࢭࣥࢫἲ ➨2 ⠇ ᰾㓟ࡢᏛಟ㣭 1-2-1. ࣜࣥ㓟㒊ࡢಟ㣭 1-2-2. ⢾㒊ࡢಟ㣭 1-2-3. ሷᇶ㒊ࡢಟ㣭 1-2-4. ◊✲┠ⓗ➨
2❶ ⢾㒊㛤⎔ᆺேᕤ᰾㓟ࡢྜᡂ
➨1 ⠇ ⢾㒊㛤⎔ᆺ᰾㓟 (ࢳ࣑ࢪࣥࢼࣟࢢ) ࡢྜᡂ 2-1-1. ⢾㒊ྜᡂ 2-1-2. ሷᇶ㒊ྜᡂ 2-1-3. ࢳ࣑ࢪࣥࢼࣟࢢࡢྜᡂ ➨2 ⠇ ⢾㒊㛤⎔ᆺ᰾㓟 (ࢩࢳࢪࣥࢼࣟࢢ) ࡢྜᡂ 2-2-1. ሷᇶ㒊ྜᡂ 2-2-2. ࢩࢳࢪࣥࢼࣟࢢࡢྜᡂ➨
3❶ RNAi ࡢᛂ⏝᳨ウ
➨1 ⠇ siRNA/miRNA ࡢタィ 3-1-1. ࢜ࣇࢱ࣮ࢤࢵࢺຠᯝࡢᅇ㑊 3-1-2. ᅇ᳨ウࡍࡿ siRNA/miRNA ➨2 ⠇ ࢼࣟࢢ Taࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞࢳࢻࡢྜᡂ≀⌮Ꮫⓗᛶ㉁ 3-2-1. ࢼࣟࢢ TaࢆᑟධࡋࡓRNA 㔜㙐ࡢ⇕ⓗᏳᐃᛶࡢ᳨ド 3-2-2. ࢼࣟࢢ TaࢆᑟධࡋࡓRNA ࡢ࢚࢟ࢯࢾࢡ࣮ࣞࢮ⪏ᛶࡢ᳨ド 3-2-3. ࢼࣟࢢ TaࢆᑟධࡋࡓRNA ࡢ࢚ࣥࢻࢾࢡ࣮ࣞࢮ⪏ᛶࡢ᳨ド ➨3 ⠇ ࢼࣟࢢ TaࢆᑟධࡋࡓsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド 3-3-1. ࢼࣟࢢ TaࢆᑟධࡋࡓsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ドϨ 3-3-2. ࢼࣟࢢ TaࢆᑟධࡋࡓsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ドϩ 3-3-3. ࢼࣟࢢ TaࢆᑟධࡋࡓsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ドϪ 1 7 23 11 16 14 18➨4 ⠇ ࢼࣟࢢ TTaࢆᑟධࡋࡓmiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド 3-4-1. ࢼࣟࢢ TaࢆᑟධࡋࡓmiR-199a-3p ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド 3-4-2. ࢼࣟࢢ TaࢆᑟධࡋࡓmiR-199a-5p ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
➨
4❶ ࣥࢳࢭࣥࢫἲࡢᛂ⏝᳨ウ
➨1 ⠇ ࣥࢳࢭࣥࢫ᰾㓟 4-1-1. ࣥࢳࢭࣥࢫ᰾㓟ࡢᏛಟ㣭 4-1-2. ᅇ᳨ウࡍࡿࣥࢳࢭࣥࢫ᰾㓟 ➨2 ⠇ ࢼࣟࢢࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢྜᡂ≀⌮Ꮫⓗᛶ㉁ 4-2-1. ࢼࣟࢢࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢྜᡂ 4-2-2. ࢼࣟࢢࢆᑟධࡋࡓ ON/RNA 㔜㙐ࡢ⇕ⓗᏳᐃᛶࡢ᳨ド 4-2-3. ࢼࣟࢢࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢሷᇶ≉␗ᛶࡢ᳨ド 4-2-4. ࢼࣟࢢࢆᑟධࡋࡓ ON/RNA 㔜㙐ࡢ⇕ຊᏛⓗᏳᐃᛶࡢ᳨ド 4-2-5. ࢼࣟࢢࢆᑟධࡋࡓ ON/RNA 㔜㙐ࡢ㧗ḟᵓ㐀ࡢ᳨ド ➨3 ⠇ ࢾࢡ࣮ࣞࢮ⪏ᛶࡢ᳨ド 4-3-1. ࢼࣟࢢ Taࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢྜᡂ 4-3-2. ࢚࢟ࢯࢾࢡ࣮ࣞࢮ⪏ᛶࡢ᳨ド ➨4 ⠇ ࢼࣟࢢࢆᑟධࡋࡓ ON/RNA 㔜㙐ࡢ RNase H άᛶ⬟ࡢ᳨ド 4-4-1. ࢼࣟࢢࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢྜᡂ 4-4-2. RNase H ࡼࡿ RNA ศゎ⬟ࡢ᳨ドϨ 4-4-3. RNase H ࡼࡿ RNA ศゎ⬟ࡢ᳨ドϩ⥲ᣓ
ᐇ㦂㡯
ᘬ⏝ᩥ⊩
ㅰ㎡
78 61 73 51 38 49 58 31 36
1
➨
➨
1 ❶ ᗎㄽ
➨
1 ⠇ ᰾㓟་⸆
㏆ᖺࠊsiRNA ࡸࣥࢳࢭࣥࢫ᰾㓟ࢆࡣࡌࡵࡍࡿ᰾㓟་⸆ရࡣ 1) పศᏊ་⸆ရࡸᢠ య་⸆ရ࡛ࡣᶆⓗ࡛ࡁ࡞ࡗࡓRNA ࡸ DNA ࡞ࢆ⸆ࢱ࣮ࢤࢵࢺ࡛ࡁࡿⅬࠊ2) ᇶ㉁≉␗ᛶࡀ㧗࠸ࡓࡵస⏝ࡀᑡ࡞࠸Ⅼࠊ3) 〇ရࡢつ᱁ࡀᢠయ་⸆࡞ẚ㍑ࡋ࡚ ᐜ࡛᫆࠶ࡿⅬ࡞ࡢ⌮⏤ࡽࠊḟୡ௦་⸆ရࡋ࡚ὀ┠ࢆ㞟ࡵ࡚࠾ࡾࠊࡇࢀࡲ࡛ᵝࠎ ࡞◊✲ࡀ㐍ࡵࡽࢀ࡚࠸ࡿ (Table 11) 1)ࠋ ୍⯡ྡ ศ㢮 ୖᕷᮇ ᶆⓗ 㐺ᛂ ᢞ᪉ἲ (ၟရྡ) Fomivirsen ࣥࢳࢭࣥࢫ 1998 (⡿) ࢧࢺ࣓࢞ࣟ࢘ࣝࢫ (CMV) 㑇ఏᏊIE2 mRNA CMV ᛶ⥙⭷⅖ (AIDS ᝈ⪅) ◪Ꮚయෆᒁᡤ (Vitravene) 1999 (EU) Pegaptanibࣉࢱ࣐࣮ 2004 (⡿) Vascular endothelial growth
factor (VEGF) 165 ࢱࣥࣃࢡ㉁ ຍ㱋ᛶ㯤ᩬኚᛶ ◪Ꮚయෆᒁᡤ (Macugen) 2006 (EU) Mipomersen ࣥࢳࢭࣥࢫ 2013 (⡿) ApoB 100 mRNA ࣍ࣔ᥋ྜᆺᐙ᪘ᛶ㧗 ࢥࣞࢫࢸ࣮ࣟࣝ⾑ ⓶ୗ (Kynamro) ࡇࡢ୰࡛ὀ┠ࡍࡁࡣࠊࡑࢀࡲ࡛ࡢ᰾㓟་⸆ရࡀᒁᡤᢞ࡛࠶ࡿࡢᑐࡋࠊ2013 ᖺ Kynamro ࡀ㌟ᢞࡢ᰾㓟་⸆ရࡋ࡚ࡣࡌࡵ࡚ୖᕷࡉࢀࡓࡇ࡛࠶ࡿࠋࡇࡢ Kynamro ࡣ DDS ศᏊ࡞ࡋ࡛ࡢ⓶ୗᢞࡀྍ⬟࡛࠶ࡾࠊ⫢⮚Ⓨ⌧ࡍࡿ ApoB mRNA ࡢ㑇ఏᏊⓎ⌧ࢆᢚไࡍࡿࡇ࡛ຠᯝࢆ♧ࡍࣥࢳࢭࣥࢫ᰾㓟࡛ࡍࠋࡇࢀࡣ20 ሷᇶࡽ ࡞ࡿDNA ࡛࠶ࡾࠊ࣍ࢫ࣍ࣟࢪ࢚ࢫࢸࣝ⤖ྜ௦ࢃࡾ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺ⤖ྜࢆ⏝࠸ࡿ ࡇ࡛⾑୰␃ᛶࡀྥୖࡋ⓶ୗᢞࢆྍ⬟ࡋ࡚࠸ࡿ2)ࠋ
ࡇࡢ ࠊ2016 ᖺ 9 ᭶ࡣࢹࣗࢩ࢙ࣥࢾᆺ➽ࢪࢫࢺࣟࣇ࣮⒪⸆ࡋ࡚ eteplirsen3)ࠊ12 ᭶ࡣ⬨㧊ᛶ➽ጤ⦰ (SMA) ⒪⸆ࡋ࡚ SPINRAZA (ࢾࢩࢿࣝࢭ ࣥ) ࡀ FDA (Food and Drug Administrationࠊ⡿ᅜ㣗ရ་⸆ᙜᒁ) ᢎㄆࡉࢀࡓ4)ࠋࡇ ࢀࡽࡣୖᕷࡉࢀࡓୡ⏺ึࡢࢫࣉࣛࢩࣥࢢไᚚᆺࡢࣥࢳࢭࣥࢫ᰾㓟࡛࠶ࡾࠊヲ⣽ࡣ 1-1-2. ࣥࢳࢭࣥࢫἲグ㍕ࡋࡓࠋ
ࡋࡋࠊ᰾㓟་⸆ရ㛤ⓎࡣゎỴࡍࡁၥ㢟ⅬࡀࡲࡔᩘከࡃᏑᅾࡋࠊูࡋ࡚௨ୗࡢ 3 Ⅼࡀᣲࡆࡽࢀ࡚࠸ࡿ5)ࠋ
2 1. ኳ↛ᆺࡢ᰾㓟ࡢ⏕యෆᏳᐃᛶࡢஈࡋࡉ ᰾㓟ࢆ་⸆ရᛂ⏝ࡍࡿࡣࠊ⏕యෆ࠾࠸࡚ࡶᏳᐃᏑᅾࡋࠊᶆⓗ≉␗ⓗຠᯝ ࢆ♧ࡍࡇࡀᚲ㡲᮲௳࡛࠶ࡿࠋࡋࡋࠊኳ↛ᆺࡢ᰾㓟ࡣ⏕యෆ࠾࠸࡚ᬑ㐢ⓗᏑᅾ ࡍࡿࢾࢡ࣮ࣞࢮࡼࡾᐜ᫆ศゎࡉࢀࡿࡓࡵ㠀ᖖᏳᐃ࡛࠶ࡿࠋ 2. ࢹࣜࣂ࣮ࣜࡀᅔ㞴࡛࠶ࡿ ᰾㓟ࡣࣜࣥ㓟ࢪ࢚ࢫࢸࣝ⤖ྜ⏤᮶ࡍࡿ㈇㟁Ⲵࡺ࠼⣽⬊⭷㏱㐣ᛶࡀప࠸ࠋࡲࡓࠊ Ỉ⁐ᛶࡣᴟࡵ࡚㧗ࡃࠊẟ⣽⾑⟶ࡽ₃ฟࡋࠊ⣒⌫యࢁ㐣ࢆ⤒࡚㏿ࡸᒀ୰ἥࡉ ࢀࡿࠋ 3. ࣥࢱ࣮ࣇ࢙ࣟࣥᛂ⟅ࡼࡿస⏝ ⏕యෆഛ࠼ࡘ࠸ࡓ⏕Ꮡᚲ㡲ࡢᶵᵓ࡛࠶ࡿࡀࠊ᰾㓟་⸆ရ㛤Ⓨࡗ࡚㔜⠜࡞ స⏝ࡢཎᅉࡶ࡞ࡾ࠺ࡿࠋ ࡇࢀࡽࡢၥ㢟ࢆඞ᭹ࡍࡿᡭẁࡢࡦࡘࡋ࡚ࠊ᰾㓟ࡢᏛಟ㣭యࡢᑟධࡀ┒ࢇ◊ ✲ࡉࢀ࡚࠸ࡿࠋᏛಟ㣭యࡘ࠸࡚ࡣ➨2 ⠇グ㍕ࡋࡓࠋ
3
11-1-1. RNA ᖸ΅
㏆ᖺࠊࣥࢳࢭࣥࢫἲࡸRNA ࣉࢱ࣐࣮⥆ࡃ᪂ࡓ࡞᰾㓟་⸆ᡭἲࡋ࡚ RNA ᖸ ΅ (RNA interference, RNAi) ἲࡀᮇᚅࡉࢀ࡚࠸ࡿࠋ1998 ᖺࡢ Fire Mello ࡽࡼࡿ ⥺ࡼࡿᐇ㦂࡛RNA ᖸ΅6) ࡣࡁ࡞ὀ┠ࢆ㞟ࡵࡓࠋࡇࢀࡲ࡛ࡢ◊✲ࡽࠊࡇࡢRNA ᖸ΅ࡣ⥺ࢆࡣࡌࡵࠊࢩࣙ࢘ࢪࣙ࢘ࣂ࢚ࠊ့ங㢮ࠊ᳜≀࡛ࡶಖᏑࡉࢀ࡚࠸ࡿࡇࡀ▱ࡽ ࢀ࡚࠸ࡿࠋࡑࡋ࡚ࠊ2001 ᖺࡣ Elbashir ࡽࡀᏛྜᡂࡋࡓ 21-22 ሷᇶࡢ siRNA ࡀ့ ங㢮⣽⬊࠾࠸࡚ࣥࢱ࣮ࣇ࢙ࣟࣥᛂ⏝ࢆㄏ㉳ࡏࡎ㓄ิ≉␗ⓗ࡞㑇ఏᏊⓎ⌧ᢚไࢆ♧ ࡍࡇࢆሗ࿌ࡋࡓ7)ࠋ
RNA ᖸ΅ࡢస⏝ᶵᵓࡣ௨ୗࡢ㏻ࡾ࡛࠶ࡿ (Figure 11)ࠋRNA ᖸ΅ࡣ㛗㙐㔜㙐 RNA
ࡀᘬࡁ㔠࡞ࡗ࡚ᘬࡁ㉳ࡇࡉࢀࠊࣄࢺࢆࡣࡌࡵࡍࡿ့ஙື≀࠾࠸࡚ࠊ⣽⬊ෆᑟධ ࡉࢀࡓ㛗㙐㔜㙐RNA ࡣ Dicer (RNaseϪࢱࣥࣃࢡ㉁ࣇ࣑࣮ࣜ) ࡼࡾ 25-21 ሷᇶ⛬ ᗘࡢsiRNA (small interfering RNA) ษ᩿ࡉࢀࡿࠋࡑࡋ࡚ࠊsiRNA ࡣ ATP 㠀౫Ꮡⓗ ࡞ᕳࡁᡠࡋࢆཷࡅ࡚ࣃࢵࢭࣥࢪ࣮ࣕ㙐ࡀྲྀࡾ㝖ࢀ୍㔜㙐࡞ࡾࠊArgonaute ࢱࣥࣃࢡ ㉁ (Ago) ࡞ඹ RISC (RNA-induced silencing complex) ࢆᙧᡂࡍࡿࠋࡑࡢᚋࠊ RISC ࡣ࢞ࢻ㙐┦⿵ⓗ࡞㓄ิࢆྵࡴᶆⓗ mRNA ࢆㄆ㆑ࡋࠊAgo2 ࡢ Slicer άᛶࡼ ࡾ࢞ࢻ㙐୰ኸ㒊ศ࡛ษ᩿ࡉࢀࡿࡇࡼࡾ㑇ఏᏊⓎ⌧ᢚไࢆᘬࡁ㉳ࡇࡍࡇࡀ▱ࡽ ࢀ࡚࠸ࡿ8) 9)ࠋ
4
ࡇࡢ RNA ᖸ΅ࢆᘬࡁ㉳ࡇࡍ siRNA ࡣ 3′ᮎ➃ࡀ 2 ሷᇶ✺ฟࡋࡓᵓ㐀ࢆᣢࡘ㙐㛗 21-23mer ࡢ㔜㙐 RNA ࡛࠶ࡾࠊDNA/RNA ⮬ື᰾㓟ྜᡂᶵࡼࡾᐜ᫆ྜᡂྍ⬟࡛ ࠶ࡿࠋࡑࡢࡓࡵࠊ᰾㓟་⸆ࡢၥ㢟ⅬࢆゎỴࡍࡿࡓࡵࡢᏛಟ㣭ࡢᑟධࡶᐜ࡛᫆࠶ࡾࠊᵝࠎ ࡞Ꮫಟ㣭siRNAࡢ㛤Ⓨࡀ㐍ࡵࡽࢀ࡚࠸ࡿࠋ2015 ᖺ 12 ᭶Ⅼ࡛ࠊୖᕷࡋ࡚࠸ࡿ siRNA ་⸆ࡣ࡞࠸ࡶࡢࡢࠊ᪥ᮏᅜෆ࡛ࡶ⫢◳ኚ⒪⸆ࡢND-L02-s0201 (᪥ᮾ㟁ᕤ/ᮐᖠ་⛉ /ᾏ㐨) ࠊஙࡀࢇࢆᶆⓗࡋࡓ TMD-812 (ᅜ❧ࡀࢇ◊✲ࢭࣥࢱ࣮/3DM) ࢡ࣮ࣟࣥ ࡢྜేࡋ࡚⏕ࡌࡿ⭠⟶⊃✽ࢆᑐ㇟ࡋࡓ STNM01 (᪂₲/ࢫࢸࣜࢵࢡ⏕་ ⛉Ꮫ◊✲ᡤ) ࡢ 3 ௳ࡢ⮫ᗋヨ㦂ࡀ㐍ࡵࡽࢀ࡚࠸ࡿ10)ࠋ ࡲࡓࠊsiRNA ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ RNA ᖸ΅ᑐࡋ࡚ࠊෆᅾᛶపศᏊ࡛࠶ࡿ miRNA (micro RNA) ࡼࡗ࡚ࡶ RNA ᖸ΅ࡣᘬࡁ㉳ࡇࡉࢀࡿ (Figure 11)ࠋmiRNA ࡣ ࡲࡎDNA ࡽ㛗࠸୍ḟ㌿⏘≀ (pri-miRNA) ࡋ࡚㌿ࡉࢀࠊࡑࡢ࠺ࡕ࣊ࣆࣥ≧ ࡢ㧗ḟᵓ㐀ࢆࡗ࡚࠸ࡿ㒊ศࡀษࡾฟࡉࢀ (pre-miRNA)ࠊࡇࢀࡀ Dicer ࡼࡿࣉࣟࢭ ࢩࣥࢢࢆཷࡅ࡚miRNA ࡞ࡿࠋࡑࡋ࡚ࠊsiRNA 㢮ఝࡢ⤒㊰ࢆ㎺ࡾ mRNA ⤖ྜࡋ ษ᩿ࠊ࠶ࡿ࠸ࡣmRNA ࡢ 3′-UTR ⤖ྜࡋ࡚⩻ヂࢆ㜼ᐖࡍࡿࠋ miRNA ࡣከᩘࡢᶵ⬟ࡀᮍࡔゎ᫂ࡉࢀ࡚࠸࡞࠸ࡀࠊ⏕≀ࡢⓎ⏕ࡸᙧែᙧᡂࠊ⣽⬊ቑ Ṫࡸ⒴࡞ࠊ㠀ᖖ㔜せ࡞⏕≀Ꮫⓗᶵ⬟ࢆ⦓ᐦไᚚࡋ࡚࠸ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋ ≉㏆ᖺࠊ⒴miRNA ࡢ㛵㐃ᛶࡀᣦࡉࢀ࡚࠸ࡿ 11) 12)ࠋࡑࡢࡓࡵࠊ⒴⣽⬊≉␗ⓗ Ⓨ⌧ࡍࡿmiRNA ࢆ⏝ࡋࡓデ᩿ࡸ⸆ࢱ࣮ࢤࢵࢺࡋ࡚ miRNA ࡢ◊✲ࡶ㐍ࡵࡽࢀ ࡚࠸ࡿࠋ
5
11-1-2. ࣥࢳࢭࣥࢫἲ
1998 ᖺࠊୡ⏺Ⓨࡢ᰾㓟་⸆ရࡋ࡚ HIV ᝈ⪅ࡢࢧࢺ࣓࢞ࣟ࢘ࣝࢫࢆឤᰁࡼࡿ ⥙⭷⅖ࢆ⒪ࡍࡿࣥࢳࢭࣥࢫ་⸆ Vitraven ࡀ FDA ࡼࡾᢎㄆࡉࢀࡓࠋࡑࡋ࡚ࠊ2012 ᖺࡣᐙ᪘ᛶ㧗ࢥࣞࢫࢸ࣮ࣟࣝ⾑ࢆ⒪ࡍࡿ㌟ᢞྍ⬟࡞ࣥࢳࢭࣥࢫ་⸆ Kynamro ࡀᢎㄆࡉࢀࠊ᰾㓟་⸆㛤Ⓨࡢ୰࡛ࡶࣥࢳࢭࣥࢫ་⸆ࡣ⮫ᗋ◊✲ࡀ᭱ࡶ㐍ࢇ ࡛࠸ࡿᡭἲ࡛࠶ࡿࠋࣥࢳࢭࣥࢫἲࡣࠊDNAЍRNAЍࢱࣥࣃࢡ㉁ࡢࢭࣥࢺࣛࣝࢻࢢ ࣐ ࡢ ὶ ࢀ ࢆ ࣥ ࢳ ࢭ ࣥ ࢫ ᰾ 㓟 (ASO) ࡼࡾ㐽᩿ࡍࡿ㑇ఏᏊⓎ⌧ไᚚἲ࡛࠶ࡿ (Figure 22)ࠋࡇࡢࣥࢳࢭࣥࢫ᰾㓟ࡣ୍㔜㙐ࡢ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡛࠶ࡾࠊᶆⓗ mRNA 㓄ิ౫Ꮡⓗ㔜㙐ࢆᙧᡂࡋࠊᶆⓗRNA ࡢᶵ⬟ࢆไᚚࡍࡿ13) 14)ࠋࡇࢀࡣస⏝ᶵᗎ ࡼࡾࠊgapmer ᆺࢫࣉࣛࢩࣥࢢไᚚᆺูࡉࢀࡿ15)ࠋGapmer ᆺࣥࢳࢭࣥࢫ᰾㓟ࡣࠊ୧ᮎ➃ࡢ wing region RNA ࡢ⤖ྜຊࡀ㧗࠸ Ꮫಟ㣭యࢆᑟධࡋࠊ୰ኸࡢgap region DNA 㙐ࢆ⏝࠸ࡓ᰾㓟࡛࠶ࡿࠋࡑࡋ࡚ࠊ ᰾ෆᏑᅾࡍࡿDNA/RNA 㔜㙐ࢆㄆ㆑ࡋ࡚ RNA 㙐ࡢࡳࢆษ᩿ࡍࡿࢾࢡ࣮ࣞࢮ࡛࠶ ࡿRNase H ࡀ ASO/RNA 㔜㙐ࢆㄆ㆑ࡋࠊRNA 㙐ࡢࡳࢆษ᩿ࡍࡿࡇ࡛ࡑࡢ㑇ఏᏊ Ⓨ⌧ࢆᢚไࡍࡿࠋୖ㏙ࡋࡓKynamro ࡀ gapmer ᆺࣥࢳࢭࣥࢫ᰾㓟ࡢ௦⾲࡛࠶ࡾࠊ RNA ࡢ⤖ྜຊࢆ㧗ࡵࡿࡓࡵ wing region 2′-MOE RNA ࢆᑟධࡋࠊgap region DNA 㙐ࢆ⏝࠸ࡿࡇ࡛ຠ⋡ⓗ㑇ఏᏊⓎ⌧ࢆᢚไࡋ࡚࠸ࡿ16)ࠋ
6 ḟࠊࢫࣉࣛࢩࣥࢢไᚚᆺࡣࢫࣉࣛࢩࣥࢢࢆኚࡉࡏࠊmRNA ࡢ㓄ิࢆಟṇࡍࡿࡇ ࡛᭷ຠᛶࢆⓎࡍࡿࠋࡇࡢᡭἲࡣࠊRNA 㙐ࢆศゎࡍࡿᚲせࡀ࡞ࡃࠊᙉᅛᶆⓗ RNA ⤖ྜࡍࡿࡇ࡛స⏝ࡍࡿࡓࡵࠊRNA ࡢ⤖ྜぶᛶࡀᴟࡵ࡚㧗ࡃࠊRNase H ࡢᇶ㉁࡞ࡽ ࡞࠸Ꮫಟ㣭యࡀ⏝ࡉࢀࡿࠋ⌧ᅾࠊࢫࣉࣛࢩࣥࢢไᚚᆺࣥࢳࢭࣥࢫ᰾㓟࡛ࡣࢹࣗࢩ ࢙ࣥࢾᆺ➽ࢪࢫࢺࣟࣇ࣮ᑐࡍࡿ⒪⸆ࡢ㛤Ⓨẁ㝵ࡀ㐍ࢇ࡛࠸ࡿࠋࡇࢀࡣ࢚࢟ࢯࣥࢫ࢟ ࢵࣆࣥࢢࡢᶵᗎࡼࡗ࡚స⏝ࡋ࡚࠾ࡾࠊᏛಟ㣭యࡣRNase H ࡢᇶ㉁࡞ࡽ࡞࠸ࣔࣝࣇ࢛ ࣜࣀ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ (PMO) ࡸ 2′-MOE RNA
(Figure 33) ࡀࢃࢀ࡚࠸ࡿ17) 18)ࠋࡑࡋ࡚ࠊ2016 ᖺ 9 ᭶ࠊPMO ࢆᑟධࡋࡓࢫࣉࣛࢩࣥࢢไᚚᆺࣥࢳ ࢭࣥࢫ᰾㓟࡛࠶ࡿeteplirsen ࡀ FDA ᢎㄆࡉࢀࡓ ࡢࢆࡣࡌࡵࠊ12 ᭶ࡣ SMA ⒪⸆ࡢ SPINRAZA (ࢾࢩࢿࣝࢭࣥ) ࡀᢎㄆࡉࢀࡓࠋ Figure 33. Ꮫಟ㣭య
7
➨
➨
2 ⠇ ᰾㓟ࡢᏛಟ㣭
Ꮫಟ㣭యࡢ㛤Ⓨ
᰾㓟་⸆ࡢၥ㢟Ⅼࢆඞ᭹ࡍࡿࡓࡵࠊᵝࠎ࡞Ꮫಟ㣭యࡀ⪃ࡉࢀ࡚࠸ࡿࠋ࠼ࡤࠊ siRNA ࡸࣥࢳࢭࣥࢫ᰾㓟Ꮫಟ㣭యࢆᑟධࡍࡿࡇ࡛ࠊࢾࢡ࣮ࣞࢮ⪏ᛶࡀᖜ ྥୖࡋࠊయෆ࡛ࡢᏳᐃᛶࡣࡁࡃᨵၿࡋࡓࠋࡲࡓࠊᶆⓗRNA ࡢぶᛶࡸ⣽⬊ෆ ࡢྲྀࡾ㎸ࡳຠ⋡ࡶኳ↛ᆺẚ㍑ࡋ࡚ᨵ ၿࡋࡓࠋࡇࢀࡽࡢຠᯝࡼࡗ࡚ࠊsiRNA ࡸࣥࢳࢭࣥࢫ᰾㓟ࡣࡼࡾప⃰ᗘ࡛᭷ ຠᛶࢆᚓࡿࡇࡀྍ⬟࡞ࡾࠊ⣽⬊ẘ ᛶࡢၥ㢟ࡶపῶࡋࡘࡘ࠶ࡿࠋࡑࡢࡓࡵࠊ ᰾㓟་⸆ရࡗ࡚Ꮫಟ㣭యࡢᑟධ ࡣᴟࡵ࡚᭷⏝࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋ᰾ 㓟ࡢᏛಟ㣭ࡣࠊࣜࣥ㓟㒊ࠊ⢾㒊ࠊሷ ᇶ㒊ศ㢮࡛ࡁࡿ (Figure 44) 19) 20)ࠋ 1-2-1. ࣜࣥ㓟㒊ࡢಟ㣭 ࣜࣥ㓟ࢪ࢚ࢫࢸࣝ⤖ྜ㒊ࡢ௦⾲ⓗ࡞ಟ㣭ἲ (Figure 5) ࡣࠊ࣍ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ ྜࡢ㠀⤖ྜᛶ㓟⣲ཎᏊࢆ◲㯤ཎᏊ⨨ࡁ࠼ࡓ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺ (PS) ಟ㣭࡛࠶ࡿࠋ ᰾㓟᰾㓟ࢆࡘ࡞ࡄࣜࣥ㓟ࢪ࢚ࢫࢸࣝ⤖ྜ㒊ࡢಟ㣭࡛࠶ࡿࡇࡽࠊࢾࢡ࣮ࣞࢮ⪏ ᛶࡢ⋓ᚓࡁࡃᐤࡋࠊỈᛶࡀቑࡍࡇࡽ⣽⬊ෆࡢྲྀࡾ㎸ࡳࡶᨵၿࡋࡓࠋࡋ ࡋࠊኳ↛ᆺẚ㍑ࡋ࡚㠀≉␗ⓗ࡞ ࢱࣥࣃࢡ⤖ྜࡀቑຍࡋࠊ⣽⬊ẘᛶ ࡀㄏᑟࡉࢀࡿ࠸࠺ࡇࡀሗ࿌ࡉ ࢀ࡚࠾ࡾࠊ᰾㓟་⸆ࡢᐇ⏝ከ ᩘࡢPS ಟ㣭ࡢᑟධࡣ㞴ࡋ࠸21) 22)ࠋ ࡑࡢࡢಟ㣭ἲࠊ㠀⤖ྜᛶ㓟⣲ ࢆ➼㟁Ꮚࡢ࣎ࣛࣥ (-BH3) ⨨ࡁ ࠼ࡓ࣎ࣛࣀࣜࣥ㓟ಟ㣭ࡀ࠶ࡿࠋ siRNA BH3ಟ㣭ࢆᑟධࡍࡿࠊ 㓄ิࡼࡗ࡚ࡣ PS ಟ㣭ࡼࡾࡶ㧗 ࠸㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡍࡇࡶ Figure 55. ࣜࣥ㓟㒊ಟ㣭య PO 㦵᱁ PS 㦵᱁ BH3㦵᱁ Figure 44. ᰾㓟ࡢᏛಟ㣭8
࠶ࡾࠊࡲࡓࠊࣃࢵࢭࣥࢪ࣮ࣕ㙐ࡼࡾࡶ࢞ࢻ㙐ࡢᑟධࡢ᪉ࡀ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢྥୖ ࡘ࡞ࡀࡿ࠸࠺ࠊࡢᏛಟ㣭ࡣᑐ↷ⓗ࡞≉ᚩࢆ᭷ࡋ࡚࠸ࡿ23)ࠋ
11-2-2. ⢾㒊ࡢಟ㣭
siRNA ࡀ RNAi ㄏᑟάᛶࢆ᭷ࡍࡿࡓࡵࡸࠊࣥࢳࢭࣥࢫ᰾㓟ࡀ RNase H ษ᩿άᛶࢆ ច㉳ࡍࡿࡓࡵࡣࠊ㔜㙐ࡀA ᆺࡽࡏࢇᵓ㐀ࢆಖᣢࡋ࡚࠸ࡿࡇࡀ㔜せ࡛࠶ࡿ24) 25)ࠋ ࡋࡋࠊ2′Ỉ㓟ᇶࡣࡇࢀࡽάᛶᚲ㡲࡛ࡣ࡞࠸ࡇࡽࠊ2′ࡢᏛಟ㣭ࡢᑟධࡀከ ࡃሗ࿌ࡉࢀ࡚࠸ࡿ (Figure 66)ࠋࡑࡢ୰࡛ࡶ 2′-O-methyl (2′-OMe) RNA ࡣࠊ┦⿵㙐ࡢ ⤖ྜぶຊ࠾ࡼࡧࢾࢡ࣮ࣞࢮ⪏ᛶࢆྥୖࡉࡏࡿࡓࡵ 26)ࠊ᭷ຠ࡞᰾㓟ಟ㣭ἲࡋ࡚ỗ ⏝ࡉࢀ࡚࠸ࡿࠋࡋࡋࠊ㧗࠸㑇ఏᏊᢚไ⬟ࢆ⥔ᣢࡍࡿࡓࡵࡣࠊ2′-OMe RNA ࡢᑟධᩘ ࡸᑟධ㒊ไ㝈ࡀ࠶ࡿሗ࿌ࡉࢀ࡚࠸ࡿ 27)ࠋࡲࡓࠊ࣓ࢺ࢟ࢩ࢚ࢳࣝ (2′-O-MOE) ᇶ ࡸ࣮ࣜࣝ (2′-O-allyl) ᇶࡢᑟධࡶࢾࢡ࣮ࣞࢮ⪏ᛶࡢྥୖࡸ㧗࠸㑇ఏᏊⓎ⌧ᢚไ⬟ ࢆࡍࡿ28)ࠋࡇࡢ2′-O-MOE ಟ㣭ࡣ 2′-O-Me ಟ㣭ࡼࡾࡶ㧗࠸ࢾࢡ࣮ࣞࢮ⪏ᛶࢆᣢ ࡘ29)ࠋ ࡶࠊ2′ࡢࣇࢵ⣲ཎᏊࡢᑟධࡶ᭷ຠ࡞ಟ㣭ἲ࡛࠶ࡿࠋ2′㟁Ꮚ྾ᘬᛶࡢ⨨ ᇶࢆᑟධࡍࡿࢾࢡࣞ࢜ࢳࢻࡢ⢾㒊❧య㓄ᗙࡀ C2′-endo ᆺ (N ᆺ) ⛣⾜ࡍࡿࡇ ࡽࠊࡇࡢಟ㣭ࢆᑟධࡋࡓRNA ࡣ㔜㙐ᙧᡂ᭷࡛࠶ࡿࠋࡲࡓࠊ2′-F RNA ࡢ siRNA ࡢᑟධࡣࢾࢡ࣮ࣞࢮ⪏ᛶࡢྥୖຍ࠼࡚ࠊ㧗࠸㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࡓࠋࡉࡽࠊ ࡇࡢಟ㣭ἲࡣ࢞ࢻ㙐ࠊࣃࢵࢭࣥࢪ࣮ࣕ㙐ඹ࡚ಟ㣭ࡋ࡚ࡶࡑࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡀ ಖᣢࡉࢀࡓሗ࿌ࡉࢀ࡚࠸ࡿ 30) 31)ࠋḟࠊ࣮ࣜ࣎ࢫ⎔ࡢ㓟⣲ཎᏊࢆ◲㯤ཎᏊ⨨ࡋ ࡓ4′S-RNA ࡣࠊ┦⿵ RNA Ᏻᐃ࡞ A ᆺࡽࡏࢇᵓ㐀ࡢ㔜㙐ࢆᙧᡂࡋࠊ㧗࠸ࢾࢡࣞ ࣮ࢮ⪏ᛶࢆ᭷ࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࣃࢵࢭࣥࢪ࣮ࣕ㙐ࡢ4′S-RNA ࡢᑟධ ࡣ㑇ఏᏊᢚไάᛶᙳ㡪ࢆ࠼࡞࠸ࡀࠊ࢞ࢻ㙐ᑟධࡍࡿࠊ4′S-RNA ࡢᑟධ㒊 ࡸᩘࡼࡗ࡚㑇ఏᏊᢚไάᛶࡢపୗࡀᘬࡁ㉳ࡇࡉࢀࡿ32) 33) 34)ࠋ
୍᪉࡛ࠊ⢾㒊ࡢ❧య㓄ᗙࢆᅛᐃࡋࡓᯫᶫᆺ᰾㓟ࠊLNA (locked nucleic acid) 35) (Figure 77) ࡣ❧య㓄ᗙࡢᅛᐃࡼࡾࠊ㔜㙐ࡢ⇕ⓗᏳᐃᛶࢆᖜྥୖࡉࡏࡿࡇࡀ ሗ࿌ࡉࢀ࡚࠸ࡿࠋLNA ࡣ㧗࠸㔜㙐Ᏻᐃᛶࢾࢡ࣮ࣞࢮ⪏ᛶࢆ᭷ࡍࡿࡇࡽࠊᵝࠎ
2′-O-Me RNA 2′-O-MOE RNA 2′-O-allyl RNA 2′-F RNA 4′-S RNA
9
࡞siRNA ࡸࣥࢳࢭࣥࢫ᰾㓟ࡢᑟධࡀ⾜ࢃࢀ࡚࠸ࡿࠋࡑࢇ࡞ᯫᶫᆺᑐࡋ࡚ࠊPNA (polyamide nucleic acid) 36) ࡸ GNA (glycerol nucleic acid) 37) ࡢࡼ࠺࡞㛤⎔ᆺ᰾㓟 (Figure 77) ࡀ㛤ⓎࡉࢀࡓࠋPNA ࡣࣜࣥ㓟ࢪ࢚ࢫࢸࣝ⤖ྜࢆᣢࡓࡎࠊ㟁Ⲵࡀ࡞࠸ᵓ㐀࡛ ࠶ࡿࡓࡵ୰ᛶ࡛㔜㙐ᙧᡂ㟁ᏊⓎࡀ࡞࠸ࡇࡽࠊDNA 㧗࠸⇕ⓗᏳᐃᛶࢆ♧ࡍࠋ ࡑࡢ㠃ࠊ㟁Ⲵࡀ࡞࠸ࡓࡵỈ⁐ᛶࡀపୗࡋࠊ㛗㙐࡞ࡿỈ⁐ࡅ࡞࠸ḞⅬࡀ࠶ࡿࠋ ࡲࡓࠊGNA ࡣኳ↛ᆺ௨ୖࡢ⇕ⓗᏳᐃᛶࢆᣢࡘ GNA 㔜㙐ࢆᙧᡂࡍࡿࡇࡀ࡛ࡁࠊ RNA ࡣ㔜㙐ࢆᙧᡂࡍࡿࡀ DNA ࡣ㔜㙐ࢆᙧᡂࡋ࡞࠸38)ࠋ୍⯡ⓗࠊ㛤⎔ᆺ᰾ 㓟ࡣᵓ㐀ࡢ⮬⏤ᗘࡀ㧗࠸ࡓࡵࠊ㔜㙐ᙧᡂࡀ࢚ࣥࢺࣟࣆ࣮ⓗ࡞ࡾࡸࡍ࠸ഴྥࡀ ࠶ࡿ39)ࠋࡋࡋࠊGNA ࡣ୍㔜㙐࡛ࡶ㧗ḟᵓ㐀ࢆࡗ࡚࠸ࡿࡓࡵࠊ㔜㙐ᙧᡂࡢ࢚ࣥ ࢺࣟࣆ࣮ᦆኻࡀᑠࡉࡃࠊGNA 㔜㙐ᙧᡂ ࡣ࢚ࣥࢺࣟࣆ࣮ⓗ᭷࡛࠶ࡿࠋࡑࢀຍ ࠼࡚ࠊDNA ሷᇶ㛫ࡢ㊥㞳 GNA ሷᇶ㛫ࡢ ㊥㞳ࡣࡰྠࡌ࡛࠶ࡿࡓࡵࠊGNA ࡣ DNA ࡰྠ➼ࡢỈ⣲⤖ྜ⬟ࢆ᭷ࡋ࡚࠸ࡿ⪃ ࠼ࡽࢀࡿⅬࡀ GNA 㔜㙐ࡢ⇕ⓗᏳᐃᛶ ᐤࡋ࡚࠸ࡿሗ࿌ࡉࢀ࡚࠸ࡿ40) 41)ࠋ
1-2-3. ሷᇶ㒊ࡢಟ㣭
ሷᇶ㒊ࡣ┦⿵㙐ࡢ⤖ྜ≉㔜せ࡛࠶ࡿࡇࡽࠊ┦⿵㙐ᙧᡂࡀ๓ᥦ࡛࠶ࡿ RNA ᶆⓗࡢ᰾㓟་⸆ရ࠾࠸࡚ࢃࢀࡿࡣᑡ࡞࠸ࠋᡂຌࡋ࡚᭷ྡ࡞ಟ㣭ἲ (Figure 88) ࡣࠊ2-ࢳ࢜࢘ࣜࢪࣥࡸࢩࢳࢪࣥሷᇶᑐࢆᙧᡂࡍࡿࢩ࣮ࣗࢻ࢘ࣜࢪ࡛ࣥ࠶ࡿ42)ࠋ2-ࢳ࢜ ࢘ࣜࢪࣥཬࡧࢩ࣮ࣗࢻ࢘ࣜࢪࣥࡣ࣮ࣜ࣎ࢫ⎔ࡢ❧య㓄⨨ࢆ 3΄-endo ᆺᅛᐃࡉࡏࡿ ࡓࡵ㔜㙐ࡢ⇕ຊᏛⓗᏳᐃᛶࢆྥୖࡉࡏࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊࡇࡢ≉ᚩࢆ⏝ࡋࠊ siRNA ᑟධࡍࡿࡇ࡛㑇ఏᏊᢚไάᛶࡸࢱ࣮ࢤࢵࢺ≉␗ᛶࢆྥୖࡉࡏࡿࡇᡂຌ ࡋࡓࠋ ࡲࡓࠊ㏆ᖺࠊC5 ࣉࣟࣆࢽࣝཬࡧ࣓ࢳࣝᇶࢆࡶࡘ࢘ࣛࢩࣝࢆ siRNA ᑟධࡍࡿಟ 㣭ἲࡀሗ࿌ࡉࢀࡓ43) 44)ࠋ5-࣓ࢳࣝ࢘ࣛࢩࣝࡀᑟධࡉࢀࡓ siRNA ࡣኳ↛ᆺྠ➼ࡢ㑇ఏ ᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࡓࡀࠊ5-ࣉࣟࣆࢽࣝ࢘ࣛࢩࣝࡀᑟධࡉࢀࡓ siRNA ࡢ㑇ఏᏊᢚไ⬟ Figure 77. ⢾㒊ᨵኚᆺಟయLNA PNA GNA
Figure 88. ሷᇶ㒊ಟ㣭య
10 ࡣኳ↛ᆺࡼࡾࡶపୗࡋࡓࠋࡇࡢཎᅉࡋ࡚ࠊAgo ࢱࣥࣃࢡ㉁ࣉࣟࣆࢽࣝᇶࡢ㛫࡛ࡢ❧ యⓎࡀ⪃࠼ࡽࢀࡿࠋࡋࡋࠊࣉࣟࣆࢽࣝᇶࡀᑟධࡉࢀࡓsiRNA ࡣỈᛶ┦స⏝ ࡼࡿ࢚ࣥࢺࣟࣆ࣮ⓗ᭷ຍ࠼࡚ࠊȧȧ⤖ྜࡀሷᇶ㛫ࡢࢫࢱࢵ࢟ࣥࢢࢆቑᙉࡍࡿࡇ ࡼࡿ࢚ࣥࢱࣝࣆ࣮ⓗ᭷ࡀࡁࡃാࡁ㔜㙐ᙧᡂࢆ᭷ࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ ࡚࠸ࡿࠋ
11-2-4. ◊✲┠ⓗ
ᅇࠊRNA 㑅ᢥⓗ㔜㙐ࢆᙧᡂࡍࡿ GNA (1-2-2. ཧ↷) ࠊሷᇶ㛫ࡢࢫࢱࢵ࢟ ࣥࢢ┦స⏝ࡢቑᙉࢆ≺ࡗ࡚࢚ࢳࢽࣝᇶࢆࡋ࡚ሷᇶࢆ⤖ྜࡉࡏࡓ⢾㒊㛤⎔ᆺேᕤ᰾ 㓟ࢆタィࡋࡓࠋ⮬⏤ᗘࡀ㧗ࡃ㔜㙐ᙧᡂࡀ࢚ࣥࢺࣟࣆ࣮ⓗ࡞⢾㒊㛤⎔ᆺࢼࣟࢢ ࡢ࡞࡛ࡶࠊ࢚ࣥࢺࣟࣆ࣮ⓗ᭷㔜㙐ࢆᙧᡂࡍࡿGNA ๛┤࡞ᛶ㉁ࢆᣢࡘ࢚ࢳ ࢽࣝᇶࢆᑟධࡍࡿࡇ࡛ࠊGNA ௨ୖ RNA ⇕ⓗᏳᐃ࡞㔜㙐ࢆᙧᡂࡍࡿ⢾㒊㛤 ⎔ᆺேᕤ᰾㓟ࡢ〇ࢆᮇᚅࡋࡓ (Figure 99)ࠋ ᮏ◊✲࡛ࡣࠊタィࡋࡓ⢾㒊㛤⎔ᆺேᕤ᰾㓟ࡢྜᡂࡑࢀࡽࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ ࢳࢻࡢ≀⌮Ꮫⓗᛶ㉁ࡢ᳨ドࠊsiRNA ࡸࣥࢳࢭࣥࢫ᰾㓟࠸ࡗࡓ㑇ఏᏊⓎ⌧ᢚไࢶ ࣮ࣝࡢᛂ⏝ࡘ࠸᳨࡚ウࡋࡓࠋ (SS) --GNA㻌 Figure 99. ᅇタィࡋࡓ⢾㒊㛤⎔ᆺேᕤ᰾㓟 ⢾㒊㛤⎔ᆺ ேᕤ᰾㓟 DNA㻌 artificial DNA㻌11
➨
➨
2 ❶ ⢾㒊㛤⎔ᆺேᕤ᰾㓟ࡢྜᡂ
➨
1 ⠇ ⢾㒊㛤⎔ᆺ᰾㓟 (ࢳ࣑ࢪࣥࢼࣟࢢ) ࡢྜᡂ
2-1-1. ⢾㒊ྜᡂ
Meggers ࡽࡼࡿ GNA ࡢྜᡂ37) ྠᵝ (R)-(-)-glycidol ࢆฟⓎཎᩱࡋࠊịᾎୗࠊ Ỉ㓟ᇶࢆTBDPSCl ᛂࡉࡏࡿࡇࡼࡾࠊ➨୍⣭Ỉ㓟ᇶࢆ TBDPS ᇶ࡛ಖㆤࡋࡓ ྜ≀1 ࢆ⋡ 69%࡛ᚓࡓࠋ⥆࠸࡚ࠊࣝࢫ㓟Ꮡᅾୗࠊࢭࢳࣜࢻ࢜ࣥࡢồ᰾ຍ ࡼࡿ࢚࣏࢟ࢩࡢ㛤⎔ᛂࡼࡗ࡚ྜ≀2 ࢆ⋡ 93%࡛ᚓࡓࠋྜ≀ 2 ࡢ➨⣭Ỉ㓟 ᇶࢆTBDMS ᇶࡼࡾಖㆤࡋࡓྜ≀ 3 ࢆ⋡ 85%࡛ᚓࡓᚋࠊTMS ᇶࢆ NaOCH3࡛ ฎ⌮ࡍࡿࡇ࡛ࠊሷᇶ㒊ࡢ࢝ࢵࣉࣜࣥࢢ⏝࠸ࡿࣝ࢟ࣥㄏᑟయ࡛࠶ࡿྜ≀4 ࢆ ⋡99%࡛ྜᡂࡋࡓࠋ
2-1-2. ሷᇶ㒊ྜᡂ
࢘ࣛࢩࣝࢆฟⓎཎᩱࡋ࡚ࠊ࣊࢟ࢧ࣓ࢳࣝࢪࢩࣟ࢟ࢧࣥ (HMDS) ୰ࠊࢡࣟࣟࢺ࣓ࣜ ࢳࣝࢩࣛࣥ (TMSCl) ࡼࡾࢩࣜࣝࡋࡓᚋࠊCH3I ᛂࡉࡏࡿࡇ࡛ 1 ࡢ N ࢆ࣓ ࢳࣝࡋࡓྜ≀5 ࢆ⋡ 68%࡛ྜᡂࡋࡓ45)ࠋ⥆࠸࡚ࠊྜ≀5 ࢆ㓑㓟୰ࠊNIS ࡼ ࡾ࣮ࣚࢻࡋࡓྜ≀6 ࢆ⋡ 67%࡛ᚓࡓࠋScheme 1. Reagents and conditions: (a) TBDPSCl, imidazole, DMF, 12 h, 0 °C, 69%; (b) trimethylsilylacetylene, n-butyllithium, BF3㺃O(C2H5)2, THF, 12 h, -78 °C, 93%; (c) TBDMSCl, imidazole, DMF, 12 h, r.t., 85%; (d) 1 M NaOCH3 in MeOH, MeOH,
12
2-1-3. ࢳ࣑ࢪࣥࢼࣟࢢࡢྜᡂ
ࣝ࢟ࣥㄏᑟయ (ྜ≀ 4) ሷᇶ㒊࣮ࣚࢻయ (ྜ≀ 6) ࢆࣃࣛࢪ࣒࢘ゐ፹Ꮡᅾୗࠊ ᅬ㢌࢝ࢵࣉࣜࣥࢢᛂࡼࡾ㐃⤖ࡉࡏࡿࡇ࡛ྜ≀7 ࢆ⋡ 58%࡛ྜᡂࡋࡓ46)ࠋ⥆ ࠸࡚ࠊTBAF ࡼࡗ࡚ྜ≀ 7 ࡢྛࢩࣜࣝᇶࢆ㝖ཤࡍࡿࡇ࡛ࠊྜ≀ 8 ࢆ⋡ 86% ࡛ᚓࡓࠋࡑࡢᚋࠊྜ≀8 ࡢ➨୍⣭Ỉ㓟ᇶࢆ DMTr ᇶ࡛ಖㆤࡋࡓྜ≀ 9 ࢆ⋡ 92% ࡛ྜᡂࡋࡓࠋ DNA/RNA ⮬ື᰾㓟ྜᡂᶵࢆ⏝࠸࡚ࠊᅛ┦࣍ࢫ࣑࣍ࣟࢲࢺἲࡼࡾࢼࣟࢢࢆྵ ࡴ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆྜᡂࡍࡿࡓࡵࠊ࣑ࢲࢺࣘࢽࢵࢺࡢྜᡂࢆ⾜ࡗࡓࠋྜ≀9 ࢆTHF ୰ࠊ࣑ࢲࢺヨ⸆ሷᇶࢆຍ࠼ளࣜࣥ㓟ࡍࡿࡇ࡛ࠊ࣍ࢫ࣑࣍ࣟࢲࢺ య (ྜ≀ 10) ࢆ⋡ 88%࡛ྜᡂࡋࡓࠋ୍᪉ࠊྜ≀ 9 ࢆ THF ୰ࠊDMAP ࢆゐ፹ ࡋ࡚↓Ỉࢥࣁࢡ㓟ᛂࡉࡏࡿࡇࡼࡾࢫࢡࢩࢽࣝయࡋࡓᚋࠊCPG ᶞ⬡ࢆຍ࠼ ࡚⦰ྜࡉࡏࡿࡇ࡛ࢼࣟࢢࡢᅛ┦ᢸయ࡛࠶ࡿྜ≀11 ࢆᚓࡓ (ᶞ⬡άᛶ; 39 Pmol/g)ࠋScheme 22. Reagents and conditions: (a) 1. TMSCl, HMDS, 130 °C, 2 h, 2. CH3I, 60 °C, 12 h, 68%; (b) NIS, CH3COOH, 100 °C, 5 h, 67%.
13 S
Scheme 33. Reagents and conditions: (a) CuI, Pd(PPh3)4, Et3N, DMF, 40 °C, 12 h, 58%; (b) TBAF, THF, r.t., 24 h, 86%; (c) DMTrCl, pyridine, r.t., 4 h, 92%;
(d) chloro(2-cyanoethoxy)(N, N-diisopropylamino)phosphine, i -Pr2NEt, THF, r.t., 1 h, 88%; (e) 1. succinic anhydride, DMAP, pyridine, r.t., 72 h, 2. CPG resin, EDCl㺃HCl, DMF, r.t., 72 h, 39 μmol/g loading amount.
14
➨
➨
2 ⠇ ⢾㒊㛤⎔ᆺ᰾㓟 (ࢩࢳࢪࣥࢼࣟࢢ) ࡢྜᡂ
2-2-1. ሷᇶ㒊ྜᡂ
ࢯࢩࢺࢩࣥࢆฟⓎཎᩱࡋ࡚ࠊ1 M NaOH ୰ࠊࣚ࢘⣲ᛂࡉࡏࡿࡇࡼࡾ࣮ࣚ ࢻయ࡛࠶ࡿྜ≀12 ࢆ⋡ 96%࡛ྜᡂࡋࡓ47)ࠋ⥆࠸࡚ࠊྜ≀12 ࢆN,N-dimethylformamide dimethyl acetal (DMF-DMA) ࡛ฎ⌮ࡍࡿࡇ࡛ࠊ࣑ࣀᇶ ࢆࢪ࣓ࢳ࣑ࣝࣀ࣓ࢳࣞࣥᇶ࡛ಖㆤࡋࡓྜ≀13 ࢆ⋡ 66%࡛ྜᡂࡋࡓࠋ
2-2-2. ࢩࢳࢪࣥࢼࣟࢢࡢྜᡂ
ࣝ࢟ࣥㄏᑟయ (ྜ≀ 4) ሷᇶ㒊࣮ࣚࢻయ (ྜ≀ 13) ࢆࣃࣛࢪ࣒࢘ゐ፹Ꮡᅾୗࠊ ᅬ㢌࢝ࢵࣉࣜࣥࢢᛂࡼࡾ㐃⤖ࡉࡏࡿࡇ࡛ྜ≀14 ࢆ⋡ 67%࡛ྜᡂࡋࡓ46)ࠋ⥆ ࠸࡚ࠊTBAF ࡼࡗ࡚ྜ≀ 14 ࡢྛࢩࣜࣝᇶࢆ㝖ཤࡍࡿࡇ࡛ྜ≀ 15 ࢆ⋡ 45% ࡛ᚓࡓࠋࡑࡢᚋࠊྜ≀15 ࡢ➨୍⣭Ỉ㓟ᇶࢆ DMTr ᇶ࡛ಖㆤࡋࡓྜ≀ 16 ࢆ⋡ 69% ࡛ྜᡂࡋࡓࠋ DNA/RNA ⮬ື᰾㓟ྜᡂᶵࢆ⏝࠸࡚ࠊᅛ┦࣍ࢫ࣑࣍ࣟࢲࢺἲࡼࡾࢼࣟࢢࢆྵ ࡴ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆྜᡂࡍࡿࡓࡵ࣑ࢲࢺࣘࢽࢵࢺࡢྜᡂࢆ⾜ࡗࡓࠋྜ≀16 ࢆTHF ୰ࠊ࣑ࢲࢺヨ⸆ሷᇶࢆຍ࠼࡚ளࣜࣥ㓟ࡍࡿࡇ࡛ࠊ࣍ࢫ࣑࣍ࣟࢲ ࢺయ (ྜ≀ 17) ࢆ⋡ 89%࡛ྜᡂࡋࡓࠋScheme 4. Reagents and conditions: (a) I2, 1 M NaOH, 100 °C, 2 h, 96%; (b) DMF-DMA, DMF, 40 °C, 5 h, 66%.
15 S
Scheme 55.Reagents and conditions: (a) CuI, Pd(PPh3)4, Et3N, DMF, 40 °C, 12 h, 67%; (b) TBAF, acetic acid, THF, r.t., 24 h, 45%; (c) DMTrCl, pyridine, r.t., 4 h, 69%; (d) chloro(2-cyanoethoxy)(N, N-diisopropylamino)phosphine, i -Pr2NEt, THF,
16
➨
➨
3 ❶ RNA ᖸ΅ἲࡢᛂ⏝
➨
1 ⠇ siRNA/miRNA ࡢタィ
3-1-1. ࢜ࣇࢱ࣮ࢤࢵࢺຠᯝࡢᅇ㑊
siRNA ࡢ᰾㓟་⸆ရྥࡅ࡚ゎỴࡍࡁㄢ㢟ࡢࡦࡘࠊ࢜ࣇࢱ࣮ࢤࢵࢺ (off-target) ຠᯝࡢᅇ㑊ࡀ࠶ࡿࠋ࢜ࣇࢱ࣮ࢤࢵࢺຠᯝࡣࠊ⣽⬊ෆᑟධࡋࡓ siRNA ࡀ ᶆⓗmRNA ௨እࡢ mRNA ࡢ㑇ఏᏊⓎ⌧ࢆᢚไࡍࡿ⌧㇟ࡢࡇ࡛ࠊ⏕యෆ࡛㉳ࡁࡓሙ ྜࠊ㔜⠜࡞స⏝ࡘ࡞ࡀࡿ࠾ࡑࢀࡀ࠶ࡿࠋࡇࡢ࢜ࣇࢱ࣮ࢤࢵࢺຠᯝࡣࠊᶆⓗ mRNA ┦⿵ⓗ࡞siRNA ࡢ 19 ሷᇶࡢ࠺ࡕ 11 ሷᇶࡀྠࡌ࡛࠶ࢀࡤࠊࡑࡢ㑇ఏᏊⓎ⌧ᙳ㡪ࢆ ཬࡰࡋ࠺ࡿࡓࡵࠊࡢ㑇ఏᏊ┦ྠᛶࢆ♧ࡉ࡞࠸㡿ᇦࢆᶆⓗࡋ࡚㑅ࡪࡇࡀ㔜せ࡛࠶ ࡿ 48)ࠋ⌧ᅾࠊࣄࢺࢤࣀ࣒ࡢゎㄞࡀ⤊ࡋ࡚࠾ࡾࠊࢥࣥࣆ࣮ࣗࢱゎᯒࡼࡿ࢜ࣇࢱ࣮ ࢤࢵࢺຠᯝࢆᢚไࡍࡿ㓄ิࡢண ࡀྍ⬟࡞ࡗ࡚࠸ࡿࠋ ୍᪉ࠊsiRNA 㔜㙐ࡢ࠺ࡕᮏ᮶ࠊRISC ྲྀࡾ㎸ࡲࢀࡿࡁ࢞ࢻ㙐࡛ࡣ࡞ࡃ㏫㙐ࡢ ࣃࢵࢭࣥࢪ࣮ࣕ㙐ࡀ RISC ྲྀࡾ㎸ࡲࢀࡿࡇࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ࢜ࣇࢱ࣮ࢤࢵ ࢺຠᯝࡀ࠶ࡿࠋࡇࢀࢆᅇ㑊ࡍࡿࡓࡵࡣࠊRISC ࡢ࢞ࢻ㙐㑅ᢥᛶࡢྥୖࢆ┠ᣦࡋࡓ siRNA ࡢタィࡀᚲせ࡞ࡿࠋRISC ࡼࡿ㙐ࡢ㑅ᢥᛶࡣࠊ▷㙐㔜㙐 RNA ࡢ⇕ຊᏛⓗ 㠀ᑐ⛠ᛶᕥྑࡉࢀࡿࡇ ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (Figure 10) 49) 50)ࠋࢩࣙ࢘ࢪࣙ࢘ࣂ࢚ ࠾ࡅࡿRNA ᖸ΅ᶵᵓࡣࠊ 㔜㙐RNA ࡢ⇕ຊᏛⓗ㠀ᑐ ⛠ᛶࢆឤ▱ࡍࡿᶵ⬟ࢆᣢࡘ Dicer-2/R2D2 ࣊ࢸࣟࢲ࣐ ࣮ࡀᏑᅾࡋࠊࡼࡾ⇕ຊᏛⓗ Ᏻᐃ࡞㙐ࡢ 5′ᮎ➃㏆ Dicer-2 ࡀ⤖ྜࡍࡿࠋࡑࡋ࡚ࠊ ࡢ ᅉ Ꮚ ࡀ ຍ ࢃ ࡾ RLC (RISC loading complex) ࢆ ᙧ ᡂ ࡋ ࠊDicer-2 ࡀḟ➨ Ago2 ⨨ࡉࢀࠊsiRNA 㔜㙐ࡢ୍㔜㙐ࡢᕳࡁᡠࡋ ࡀ㉳ࡇࡿࠋࡑࡋ࡚ࠊRISC ࢆ17 ࡓ㙐 (࢞ࢻ㙐) ┦⿵ⓗ࡞㓄ิࢆྵࡴᶆⓗ mRNA ࡢⓎ⌧ࢆᢚไࡍࡿࡉࢀ࡚࠸ࡿ51)ࠋ ࡑࡢࡓࡵࠊsiRNA 㔜㙐ࡢ⇕ຊᏛⓗᏳᐃᛶࡣࠊRISC ࡢ࢞ࢻ㙐㑅ᢥᛶࡗ࡚㔜せ࡞ せ⣲࡞ࡿࠋ
3-1-2. ᅇ᳨ウࡍࡿ siRNA/miRNA
ᅇࠊࣃࢵࢭࣥࢪ࣮ࣕ㙐ࡼࡿ࢜ࣇࢱ࣮ࢤࢵࢺຠᯝࢆᅇ㑊ࡋࠊኳ↛ᆺ௨ୖࡢ㑇ఏᏊⓎ ⌧ᢚไ⬟ࢆ♧ࡍsiRNA ཬࡧ miRNA ࡢ〇ࢆ┠ⓗࡋࡓࠋྜᡂࡋࡓࢼࣟࢢTaࡣRNA 㔜㙐୰ᑟධࡍࡿࡇ࡛ࠊRNA 㔜㙐ࡢ⇕ⓗᏳᐃᛶࡀኚࡋ RISC ࡢ࢞ࢻ㙐㑅ᢥ ᛶᙳ㡪ࢆ࠼ࡿࡇࡀணࡉࢀࡓࠋࡑࡢࡓࡵࠊᮏ◊✲࡛ࡣRNA 㔜㙐୰ࡢ U ࡸ C ࢆTa⨨ࡋࡓsiRNA ཬࡧ miRNA (Figure 111) ࢆྜᡂࡋࠊࡑࡢ㔜㙐 RNA ࡢ⇕ⓗ Ᏻᐃᛶࡸ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ᳨ドࡋࡓࠋTaࡢRNA 㔜㙐ࡢᑟධࡀ RISC ࡢ࢞ࢻ㙐 㑅ᢥᛶࢆྥୖࡉࡏࠊ㧗࠸㑇ఏᏊⓎ⌧ᢚไ⬟ࡘ࡞ࡀࡿࡇࢆᮇᚅࡋࡓࠋFigure 111. ᅇ᳨ウࡍࡿ siRNA/miRNA ࡢࢹࢨࣥ
18
➨
➨
2 ⠇ ࢼࣟࢢ
T
aࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢྜᡂ≀⌮Ꮫⓗᛶ㉁
3-2-1. ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
RNA 㔜㙐ࡢ⇕ⓗᏳᐃᛶࡢ᳨ド
ࢼࣟࢢTa (Figure 112) ࢆ siRNA ࡢಟ㣭యࡋ࡚⏝ࡍࡿሙྜࠊTaࢆᑟධࡋࡓ RNA 㔜㙐ࡀᏳᐃ࡞㔜㙐ࢆᙧᡂࡍࡿᚲせࡀ࠶ࡿࠋࡲࡓࠊsiRNA 㔜㙐ࡢ⇕ⓗࠊ⇕ຊ ᏛⓗᏳᐃᛶࡀRISC ࡢ㙐ࡢ㑅ᢥᛶᙳ㡪ࢆ࠼ࡿࡇࡶሗ࿌ࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊ㙐ࡢ ୰ኸTaࢆᑟධࡋࡓRNA (ON 33) ┦⿵㙐 RNA (ON 22) ࡢ㔜㙐୪ࡧ࣑ࢫ࣐ࢵࢳሷᇶ (rU, rC, rG) ࢆᑟධࡋࡓ RNA (ON 44-66) ࡢ㔜㙐ࡘ࠸࡚ࠊ50%⼥ゎ ᗘ (Tm ್) ࢆ ᐃࡋࡓࠋࡑࡢ⤖ᯝࡼࡾࠊᅇྜᡂࡋࡓTaࢆᑟධࡋࡓRNA 㔜㙐ࡢ⇕ⓗᏳᐃᛶ
Taࡢሷᇶ≉␗ᛶࡢ᭷↓ࡘ࠸᳨࡚ウࡋࡓ (Table 22, Figure 112)ࠋ
Sample Abbreviation
of ON Sequencea
Duplex 11 ON 11 5'-r (AGA GAU ACA UUG ACC UUC) -3' ON 22 3'-r (UCU CUA UGU AAC UGG AAG) -5' Duplex 22 ON 33 5'-r (AGA GAU ACA TTaUG ACC UUC) -3'
ON 22 3'-r (UCU CUA UGU AAC UGG AAG) -5' Duplex 33 ON 33
ON 44
5'-r (AGA GAU ACA TTaUG ACC UUC) -3'
3'-r (UCU CUA UGU UAC UGG AAG) -5' Duplex 44 ON 33 5'-r (AGA GAU ACA TTaUG ACC UUC) -3'
ON 55 3'-r (UCU CUA UGU CAC UGG AAG) -5' Duplex 55 ON 33 5'-r (AGA GAU ACA TTaUG ACC UUC) -3'
ON 66 3'-r (UCU CUA UGU GAC UGG AAG) -5'
Table22. 㙐୰ኸTaaࢆᑟධࡋࡓRNA 㔜㙐ࡢ㓄ิ
aUnderlined letters indicate mismatched bases.
19
ᐃ᮲௳
ᐃ ᗘ⠊ᅖ; 20-100 °C ᐃἼ㛗; 260 nm
ࢧࣥࣉࣝ⃰ᗘ; 3.0 μM
buffer; 10 mM Na Phosphate and 100 mM NaCl (pH 7.0)
Figure 113 ࡼࡾࠊDuplex 11-55 ࡢྛ㔜㙐ࡣࢩࢢࣔࢻ᭤⥺ࢆᥥࡃ⇕ⓗᏳᐃ࡞㔜㙐 ࢆᙧᡂࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊDuplex 22-55 ࡢྛ㔜㙐ࡢ 50%⼥ゎ ᗘࡣኳ ↛ᆺ࡛࠶ࡿDuplex 11 (Tm = 67.9 °C ) ẚ㍑ࡋ࡚ࠊ3.6~9.3 °C పୗࡍࡿࡇࡀ♧ࡉࢀࡓࠋ ࡇࡢ⤖ᯝࡽࠊᅇྜᡂࡋࡓTaࢆ㙐ࡢ୰ኸᑟධࡋࡓRNA 㔜㙐ࡣኳ↛ᆺຎࡿࡶ ࡢࡢ⇕ⓗᏳᐃ࡞㔜㙐ࢆᙧᡂࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋ ࡋࡋࠊTaᑐ࡞ࡿሷᇶࡀࢹࢽࣥ (Duplex 22, Tm = 64.3 °C, ΔTm = -3.6 °C) ࡢሙ ྜࠊࢢࢽࣥ (Duplex 55, Tm = 63.7 °C, ΔTm = -4.2 °C) ࡸ࢘ࣛࢩࣝ (Duplex 33, Tm = 60.8 °C, ΔTm = -7.1 °C)ࠊࢩࢺࢩࣥ (Duplex 44, Tm = 58.6 °C, ΔTm = -9.3 °C) ࡢሙྜ ẚ㍑ࡋ࡚50%⼥ゎ ᗘࡢῶᑡᖜࡀᑠࡉ࠸ࠋࡇࡢࡇࡽࠊኳ↛ᆺ࡛࠶ࡿ U:rA ሷᇶᑐ ຎࡿࡶࡢࡢࠊTaࡶࢹࢽࣥሷᇶᑐࢆᙧᡂࡋ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ Sample Tm (°C) ΔTm (°C)a Duplex 11 67.9 - Duplex 22 64.3 -3.6 Duplex 33 60.8 -7.1 Duplex 44 58.6 -9.3 Duplex 55 63.7 -4.2 Figure113. 㙐୰ኸTaaࢆᑟධࡋࡓRNA 㔜㙐ࡢ 50%⼥ゎ ᗘ ᐃ⤖ᯝ
20
33-2-2. ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
RNA ࡢ࢚࢟ࢯࢾࢡ࣮ࣞࢮ⪏ᛶࡢ᳨ド
RNA ࡣࣄࢺ⾑Ύ୰࠾࠸࡚ࠊ 3′ᮎ➃ࡽຍỈศゎࡍࡿ㓝⣲࡛࠶ࡿ 3′-࢚࢟ࢯࢾࢡ ࣮ࣞࢮࡼࡗ࡚ศゎࡉࢀࡿࠋࡑࡇ࡛ࠊ3′-࢚࢟ࢯࢾࢡ࣮ࣞࢮ࡛࠶ࡿ⺬ẘ࣍ࢫ࣍ࢪ࢚ࢫ ࢸ࣮ࣛࢮ (Snake venom phosphodiesterase, SVPD) ࢆ⏝࠸࡚ࠊTaࢆᑟධࡋࡓ୍㔜㙐 RNA ࡢ࢚࢟ࢯࢾࢡ࣮ࣞࢮᑐࡍࡿᢠᛶࢆ᳨ドࡋࡓࠋSVPD ࡣ 3′-OH ᮎ➃ࡽ 3′ Ѝ5′ࡢ᪉ྥẁ㝵ⓗ࣍ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ྜࢆษ᩿ࡋࠊ5′-ࣔࣀࢾࢡࣞ࢜ࢳࢻࢆ⏘ฟࡍࡿ 㓝⣲࡛࠶ࡿࠋ 5′ᮎ➃ࢆ⺯ගᶆ㆑ࡋࡓ 21 ሷᇶࡽ࡞ࡿ ON 77 ࡑࢀ 3′ᮎ➃ࡢ࣮࢜ࣂ࣮ࣁࣥࢢ㒊 TaࢆᑟධࡋࡓON 88 ࢆྜᡂࡋ (Table 33)ࠊ୍㔜㙐≧ែ࠾࠸࡚ SVPD ࡛ฎ⌮ࡋࠊ ྛ㛫࡛ᛂࢆṆᚋࠊ㟁ẼὋືࡼࡗ࡚ࡑࡢRNA ศゎ≀ࢆゎᯒࡋࡓ (Figure 114)ࠋ ᛂ᮲௳ ᛂ ᗘ; 37 °C ࢧࣥࣉࣝ⃰ᗘ; 3.0 μM
buffer; 250 mM Tris-HCl and 50 mM MgCl2 (pH 8.0) SVPD; 0.5 unit
Sample Sequencea
ON 77 F-5'-r (CUU CUU CGU CGA GAC CAU G) dTdT-3' ON 88 F-5'-r (CUU CUU CGU CGA GAC CAU G) TTaTTa-3'
Table33. ࢚࢟ࢯࢾࢡ࣮ࣞࢮ⪏ᛶ᳨ド⏝࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ㓄ิ aF; Fluorescein. ON 77 0 1 5 10 20 30 45 1 3 [min] [h] 0 1 5 10 20 30 45 1 3 [min] [h] ON 88
21 Figure 114 ࡽࠊኳ↛ᆺ࡛࠶ࡿ ON 77 ࡀ 1 ศᚋࡣ 80%ࡀศゎࡉࢀ࡚࠸ࡓࡢᑐࡋࠊ 3′ᮎ➃ࡢ࣮࢜ࣂ࣮ࣁࣥࢢ㒊TaࢆᑟධࡋࡓON 88 ࡣ 1 ศᚋ࡛ࡶ 20%๓ᚋࡋศゎࡉ ࢀ࡚࠸࡞࠸ࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡲࡓࠊON 77 ࡢ༙ῶᮇࡀ 38 ⛊࡛࠶ࡿࡢᑐࡋࠊON 8 ࡣ 845 ⛊࡛࠶ࡾࠊࡇࡢࡇࡽ࢚࢟ࢯࢾࢡ࣮ࣞࢮᢠᛶࡣ 22.4 ಸቑᙉࡋࡓ⪃࠼ ࡽࢀࡿࠋᚑࡗ࡚ࠊ3′ᮎ➃ࡢ࣮࢜ࣂ࣮ࣁࣥࢢ㒊ࡢTaࡢᑟධࡣ3′-࢚࢟ࢯࢾࢡ࣮ࣞࢮ ⪏ᛶࡢྥୖ᭷⏝࡛࠶ࡿࡇࡀ♧ࡉࢀࡓࠋ
3-2-3. ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
RNA ࡢ࢚ࣥࢻࢾࢡ࣮ࣞࢮ⪏ᛶࡢ᳨ド
RNA 㔜㙐≧ែ࡛ࡢ࢚ࣥࢻࢾࢡ࣮ࣞࢮ⪏ᛶࢆࠊ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド࡛⏝࠸ࡿ HeLa ⣽⬊ᇵ㣴᮲௳ୗ᳨࡛ウࡋࡓࠋᮏ◊✲⏝ࡍࡿ HeLa ⣽⬊ࡣࠊD-MEMࠊ10% BS (࢘ࢩ⾑ΎࠊBovine serum) ࡛ᇵ㣴ࡋ࡚࠸ࡿࡓࡵࠊࡲࡎࠊࡇࡢ᮲௳ୗ࡛㔜㙐ࢆฎ⌮ࡋ ࡓࡀ㔜㙐ࡣࢇศゎࡉࢀ࡞ࡗࡓࠋࡑࡇ࡛ࠊBS ⃰ᗘࢆ 30%ୖࡆ࡚ࠊ5′ᮎ➃ࢆ ⺯ගᶆ㆑ࡋࡓኳ↛ᆺ࡛࠶ࡿDuplex 6 3′ᮎ➃ࡢ࣮࢜ࣂ࣮ࣁࣥࢢ㒊Taࢆᑟධࡋࡓ Duplex 7 (Table 44) ࢆ D-MEMࠊ30% BS ࡛ฎ⌮ࡋࠊྛ㛫࡛ᛂṆᚋࠊ㟁ẼὋື ࡼࡗ࡚RNA ศゎ⏘≀ࢆゎᯒࡋࡓ (Figure 115)ࠋSample Abbreviation
of ON Sequence Duplex 6 ON 77
ON 99
F-5'-r (CUU CUU CGU CGA GAC CAU G) dTdT-3' 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' Duplex 7 ON 88 F-5'-r (CUU CUU CGU CGA GAC CAU G) TTaTTa-3'
ON 99 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5'
Figure114.SVPD ࡼࡿ RNA ศゎ⬟ࡢ᳨ド
22 ᛂ᮲௳ ᛂ ᗘ; 37 °C ࢧࣥࣉࣝ⃰ᗘ; 4.0 μM buffer; ⣽⬊ᇵ㣴ᾮ (D-MEM 㧗ࢢࣝࢥ࣮ࢫ) ࢘ࢩ⾑Ύ (Bovine serum, BS) ⤊⃰ᗘ 30% Figure 115 ࡢ⤖ᯝࡼࡾࠊኳ↛ᆺ࡛࠶ࡿ Duplex 66 ࡀ 6 㛫ᚋࡰ㔜㙐ࡀศ ゎࡉࢀ࡚࠸ࡿࡢᑐࡋࠊ3′ᮎ➃ࡢ࣮࢜ࣂ࣮ࣁࣥࢢ㒊TaࢆᑟධࡋࡓDuplex 77 ࡛ࡣ 24 㛫ᚋ࡛ࡶ10%ࡀ㛗࡛㔜㙐ࡀṧᏑࡋ࡚࠸ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡇࡢࡇ ࡽࠊ⣽⬊ᇵ㣴᮲௳ୗ࡛TaࢆᑟධࡋࡓRNA 㔜㙐ࡣࢾࢡ࣮ࣞࢮᑐࡋ࡚ᏳᐃᏑᅾ ࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓࠋ Figure115.BS30%ࡼࡿ RNA ศゎ⬟ࡢ᳨ド 0 5 15 30 1 3 6 12 24 [min] [h] 0 5 15 30 1 30 6 12 24 [min] [h] Duplex 66 Duplex 77
23
➨
➨
3 ⠇ ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
siRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
3-3-1. ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
siRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
ᅇྜᡂࡋࡓࢼࣟࢢTaࢆᵝࠎ࡞㒊ᑟධࡋࡓRNA ࢆࠊᅛ┦࣍ࢫ࣑࣍ࣟࢲ ࢺἲᚑࡗ࡚DNA/RNA ⮬ື᰾㓟ྜᡂᶵࢆ⏝࠸࡚ྜᡂࡋࡓࠋࡑࡋ࡚ࠊ┦⿵ⓗ࡞㓄ิࢆ ᣢࡘRNA 㔜㙐ࢆᙧᡂࡉࡏࠊᵝࠎ࡞㒊TaࢆᑟධࡋࡓsiRNA 22-77 ࢆㄪ〇ࡋࡓ (Table 5)ࠋ Sample Abbreviation of ON SequencesiRNA 11 ON 110 5'-r (CUU CUU CGU CGA GAC CAU G) dTdT-3' ON 99 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' siRNA 22 ON 111 5'-r (CUU CUU CGU CGA GAC CAU G) TTaTTa-3'
ON 112 3'-TTaTTa(GAA GAA GCA GCU CUG GUA C) -5'
siRNA 33 ON 113 5'-r (TTaUU CUU CGU CGA GAC CAU G) dTdT-3' ON 99 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' siRNA 44 ON 114 5'-r (CTTaU CUU CGU CGA GAC CAU G) dTdT-3'
ON 99 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' siRNA 55 ON 115
ON 99
5'-r (CUU CUU CGU CGA GAC CATTa G) dTdT-3'
3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' siRNA 66 ON 110
ON 116
5'-r (CUU CUU CGU CGA GAC CAU G) dTdT-3' 3'-dTdT (GAA GAA GCA GCU CUG GTTaA C) -5'
siRNA 77 ON 110 5'-r (CUU CUU CGU CGA GAC CAU G) dTdT-3' ON 117 3'-dTdT (GAA GAA GCA GCU CUG GUA TTa) -5'
siRNA (Table 55) ࢆࣄࢺᏊᐑ⒴⏤᮶⣽⬊࡛࠶ࡿ HeLa ⣽⬊ࢆ⏝࠸࡚ࠊDual-luciferase reporter assay ࡼࡗ࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ᳨ドࡋࡓ (Figure 116)ࠋヲ⣽࡞ᐇ㦂᪉ἲࡣ ᐇ㦂㡯グ㍕ࡋࡓࠋ
24 Figure 116 ࡢ⤖ᯝࡼࡾࠊ3′ᮎ➃࣮࢜ࣂ࣮ࣁࣥࢢ㒊TaࢆᑟධࡋࡓsiRNA 22 ࡣኳ↛ ᆺ࡛࠶ࡿsiRNA 11 ẚ㍑ࡋ࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࡀྥୖࡋࡓࠋࡇࡢཎᅉࡋ࡚ࠊ3′ᮎ➃࢜ ࣮ࣂ࣮ࣁࣥࢢ㒊Taࢆᑟධࡋࡓࡇ࡛siRNA 22 ࡢࢾࢡ࣮ࣞࢮ⪏ᛶࡀྥୖࡋࡓࡓࡵ ⪃࠼ࡽࢀࡿ (3-2-3ࠊ3-2-4 ཧ↷)ࠋ ࡲࡓࠊࢭࣥࢫ㙐 5′ᮎ➃㏆ Taࢆᑟධࡋࡓ siRNA 33 siRNA 44 ࡣኳ↛ᆺ࡛࠶ࡿ siRNA 11 ẚ㍑ࡋ࡚ࠊ≉ప⃰ᗘ࠾࠸࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࡀపୗࡋࡓࠋࡇࢀࡣࠊTa ࢆᑟධࡋࡓࡇ࡛ࢭࣥࢫ㙐5′ᮎ➃㏆ࡀ⇕ⓗᏳᐃࡋࠊࢭࣥࢫ㙐ࡶ࢞ࢻ㙐ࡋ࡚ ྲྀࡾ㎸ࡲࢀࡸࡍࡃ࡞ࡿ࡞ࠊRISC ࡢ㙐ࡢ㑅ᢥᛶࡀపୗࡋࡓࡓࡵ⪃࠼ࡽࢀࡿࠋ≉ siRNA 33 ࡣ 5′ᮎ➃Taࢆᑟධࡋࡓࡇ࡛ࠊࢭࣥࢫ㙐5′ᮎ➃ࡢࣜࣥ㓟ࡀ㜼ᐖࡉࢀࠊࡇ ࢀࡼࡾ5′ᮎ➃ࡀࣜࣥ㓟ࡉࢀࡓࣥࢳࢭࣥࢫ㙐ࡢ RISC ࡢ㙐ࡢྲྀࡾ㎸ࡳࡀಁ㐍ࡉࢀ ࡿࡇࡀᮇᚅࡉࢀࡓࠋࡋࡋࠊsiRNA 33 ࡣኳ↛ᆺࢆୗᅇࡿ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࡓࡇ ࡽࠊTaࡢᑟධࡼࡿ㔜㙐ࡢ⇕ⓗᏳᐃࡣ㙐ࡢ 5′ᮎ➃ࡢࣜࣥ㓟ࡼࡾࡶ㙐ࡢ㑅 ᢥᛶࡁ࡞ᙳ㡪ࢆ࠼ࡿࡇࡀ♧၀ࡉࢀࡓࠋ ḟࠊࢭࣥࢫ㙐3′ᮎ➃㏆TaࢆᑟධࡋࡓsiRNA 55 ࡣ siRNA 11 ẚ㍑ࡋ࡚ࠊ㑇ఏᏊ Ⓨ⌧ᢚไ⬟ࡀⴭࡋࡃྥୖࡋࡓࠋsiRNA 55 ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢྥୖࡣࢭࣥࢫ㙐 3′ᮎ➃ ㏆Taࢆᑟධࡋࡓࡇ࡛ࠊࣥࢳࢭࣥࢫ㙐ࡢ 5′ᮎ➃㏆ࢆ⇕ⓗᏳᐃࡋࠊࣥࢳ ࢭࣥࢫ㙐ࡢRISC ࡢྲྀࡾ㎸ࡳࡀಁ㐍ࡉࢀࡓࡓࡵ⪃࠼ࡽࢀࡿࠋ
᭱ᚋࠊࣥࢳࢭࣥࢫ㙐5′ᮎ➃㏆TaࢆᑟධࡋࡓsiRNA 66 siRNA 77 ࡣ siRNA 11 ẚ㍑ࡋ࡚ࠊࡑࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡀపୗࡋࡓࠋࡇࡢ⤖ᯝࡽࠊࣥࢳࢭࣥࢫ㙐ࡢ 5′
Figure16. siRNA (Table 5) ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
control siRNA 1 siRNA 2 siRNA 3 siRNA 4 siRNA 5 siRNA 6 siRNA 7
Figure 16 siRNA (Table 5) ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
25 ᮎ➃㏆TTaࢆᑟධࡋ࡚㔜㙐ࢆ⇕ⓗᏳᐃࡍࡿࡼࡾࡶࠊsiRNA 55 ࡢࡼ࠺ࢭࣥ ࢫ㙐3′ᮎ➃㏆Taࢆᑟධࡋ࡚㛫᥋ⓗࣥࢳࢭࣥࢫ㙐ࡢ 5′ᮎ➃ࢆ⇕ⓗᏳᐃࡋ ࡚ࠊRISC ࡢ㙐ࡢྲྀࡾ㎸ࡳࢆಁ㐍ࡋࡓ᪉ࡀ᭷⏝࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡇࡢ⌮⏤ ࡋ࡚ࠊRISC ྲྀࡾ㎸ࡲࢀࡿࣥࢳࢭࣥࢫ㙐ࡢ seed 㡿ᇦࡢᏛಟ㣭యࡢᑟධࢆᅇ㑊 ࡋࡘࡘࠊࣥࢳࢭࣥࢫ㙐5′ᮎ➃ࡢ⇕ⓗᏳᐃࢆᘬࡁ㉳ࡇࡏࡿⅬࡀ⪃࠼ࡽࢀࡿࠋ
3-3-2. ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
siRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ドϩ
ࢼࣟࢢTaࢆᑟධࡋࡓsiRNA ࡼࡿ㑇ఏᏊⓎ⌧ᢚไ⬟ᑐࡍࡿຠᯝࡢ୍⯡ᛶࢆ᳨ウ ࡍࡿࡓࡵࠊ3-3-1.࡛⏝࠸ࡓ㓄ิࡣ␗࡞ࡿ㓄ิࡢ siRNA ࢆྜᡂࡋࡓ (Table 66)ࠋࡇࡢ siRNA ࡢ㓄ิࡣࠊࣥࢳࢭࣥࢫ㙐 5′ᮎ➃ഃ GC ሷᇶᑐࢆከࡃྵࡳࠊࢭࣥࢫ㙐 5′ᮎ➃ ഃAU ሷᇶᑐࢆከࡃྵࡴࡓࡵࠊࢭࣥࢫ㙐ࡀ RISC ྲྀࡾ㎸ࡲࢀࡸࡍ࠸ᛶ㉁ࢆᣢࡘࠋࡑ ࡢࡓࡵࠊࡇࡢsiRNA ࡣ࠶ࡲࡾ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡉ࡞࠸࠸࠺≉ᚩࢆ♧ࡍࠋ Sample Abbreviation of ON SequencesiRNA 88 ON 118 5'-r (UAA GAU GUU CAU CGA GUC C) dTdT-3' ON 119 3'-dTdT (AUU CUA CAA GUA GCU CAG G) -5' siRNA 99 ON 220 5'-r (TTaAA GAU GUU CAU CGA GUC C) dTdT-3'
ON 119 3'-dTdT (AUU CUA CAA GUA GCU CAG G) -5' siRNA 110 ON 221 5'-r (UAA GAU GUU CAU CGA GTTaC C) dTdT-3'
ON 119 3'-dTdT (AUU CUA CAA GUA GCU CAG G) -5' siRNA 111 ON 222 5'-r (TTaAA GAU GUU CAU CGA GTTaC C) dTdT-3'
ON 119 3'-dTdT (AUU CUA CAA GUA GCU CAG G) -5'
siRNA (Table 66) ࢆࣄࢺᏊᐑ⒴⏤᮶⣽⬊࡛࠶ࡿ HeLa ⣽⬊ࢆ⏝࠸࡚ࠊDual-Luciferase reporter assay ࡼࡗ࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ᳨ドࡋࡓ (Figure 117)ࠋ
26 Figure 117 ࡢ⤖ᯝࡼࡾࠊኳ↛ᆺ࡛࠶ࡿ siRNA 88 ࡛ࡶ 40%๓ᚋࡋ㑇ఏᏊⓎ⌧ࢆᢚไ ࡏࡎࠊࡲࡓ⃰ᗘ౫Ꮡⓗ࡞㑇ఏᏊⓎ⌧ᢚไ⬟ࡀほᐹࡉࢀ࡞ࡗࡓࠋࡇࡢ⤖ᯝࡽࠊᮏ㓄ิ ࡢsiRNA ࡣᙜึࡢண㏻ࡾ㙐ࡢ㑅ᢥᛶࡀపࡃࠊ㑇ఏᏊⓎ⌧ᢚไࡀప࠸ࡇࡀ♧၀ࡉࢀ ࡓࠋ ୍᪉࡛ࠊTaࢆᑟධࡋࡓsiRNA 99-111 ࡣ⃰ᗘ౫Ꮡⓗ࡞㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࡓࠋsiRNA 9 ࡣࢭࣥࢫ㙐 5′ᮎ➃ࡢTaࡢᑟධࡼࡾࠊࢭࣥࢫ㙐5′ᮎ➃ࡢࣜࣥ㓟ࢆ㜼ᐖࡉࢀࠊ ࣥࢳࢭࣥࢫ㙐5′ᮎ➃ࡀඃඛⓗࣜࣥ㓟ࡉࢀࡿࡇ࡛ࠊࣥࢳࢭࣥࢫ㙐ࡢ RISC ࡢྲྀ ࡾ㎸ࡳࡀಁ㐍ࡉࢀࡓࡓࡵࠊ㑇ఏᏊⓎ⌧ᢚไ⬟ࡀྥୖࡋࡓ⪃࠼ࡽࢀࡿࠋࡲࡓࠊsiRNA 110 ࡣࢭࣥࢫ㙐3′ᮎ➃㏆Taࢆᑟධࡋࡓࡇ࡛ࠊࣥࢳࢭࣥࢫ㙐 5′ᮎ➃㏆ࢆ⇕ⓗ Ᏻᐃࡋࠊࣥࢳࢭࣥࢫ㙐ࡢRISC ࡢྲྀࡾ㎸ࡳࡀಁ㐍ࡉࢀࡓࡓࡵ㑇ఏᏊⓎ⌧ᢚไ⬟ࡀ ྥୖࡋࡓ⪃࠼ࡽࢀࡿࠋࡋࡋࠊࡇࢀࡽࡢsiRNA ࢆ⤌ࡳྜࢃࡏࡓ siRNA 111 ࡣ siRNA 99ࠊ siRNA 10 ẚ㍑ࡋ࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࡀపୗࡋࡓࠋࡇࢀࡣࠊsiRNA Taࢆ2 ಶᑟධ ࡋࡓࡇࡼࡾࠊRNA 㔜㙐ࡢ⇕ⓗ࡞Ᏻᐃࡀ㐍⾜ࡋࡍࡂࡓࡓࡵ࡛ࡣ࡞࠸⪃࠼ ࡽࢀࡿࠋ ௨ୖࡢ⤖ᯝࡽࠊࢭࣥࢫ㙐3′ᮎ➃㏆ࡸࢭࣥࢫ㙐 5′ᮎ➃ࡢTaࡢᑟධࡣࠊ㑇ఏᏊⓎ ⌧ᢚไ⬟ࢆྥୖࡉࡏࡿࡓࡵ᭷⏝࡞ಟ㣭᪉ἲ࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊTaࡢᑟ ධࡣRNA 㔜㙐ࢆ⇕ⓗᏳᐃࡉࡏࡿࡓࡵࠊᩘከࡃᑟධࡍࡿࡇࡣྥࡁ࡛࠶ࡾࠊ 1 ಶ⛬ᗘࡢᑟධࡀ᭱㐺⪃࠼ࡽࢀࡿࠋ
control siRNA 8 siRNA 9 siRNA 10 siRNA 11
Figure117.siRNA (Table 66) ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
27
33-3-3.ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
siRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ドϪ
3-3-1.࡛㏙ࡓ࠾ࡾࠊsiRNA 55 ࡣ㧗࠸㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࡓࠋࡇࡢࢭࣥࢫ㙐 3′ ᮎ➃ࡽ4 ␒┠ࡢ⨨ࡢࢼࣟࢢTaࡢᑟධࡢ᭷⏝ᛶࢆࠊྛ࣑ࢫ࣐ࢵࢳሷᇶࢆᑟධࡋ ࡓsiRNA 112-114 ࢆ⏝࠸࡚㔜㙐ࡢ⇕ⓗᏳᐃᛶ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆẚ㍑ࡍࡿࡇ᳨࡛ ドࡋࡓࠋ ࡲࡎࠊsiRNA 5 ࡢTaᑟධ㒊ྠࡌ㒊ࠊTaࡢࡼ࠺㙐ࡢ⇕ⓗᏳᐃࢆᘬࡁ㉳ ࡇࡍ⪃࠼ࠊྛ࣑ࢫ࣐ࢵࢳሷᇶࢆᑟධࡋࡓsiRNA ࢆྜᡂࡋࡓ (Table 77)ࠋ Sample Abbreviation of ON SequenceasiRNA 11 ON 110 5'-r (CUU CUU CGU CGA GAC CAU G) dTdT-3' ON 99 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' siRNA 55 ON 115
ON 99
5'-r (CUU CUU CGU CGA GAC CATTa G) dTdT-3'
3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' siRNA 112 ON 223
ON 99
5'-r (CUU CUU CGU CGA GAC CAA G) dTdT-3' 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' siRNA 113 ON 224
ON 99
5'-r (CUU CUU CGU CGA GAC CAGG) dTdT-3' 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5' siRNA 114 ON 225 5'-r (CUU CUU CGU CGA GAC CAC G) dTdT-3'
ON 99 3'-dTdT (GAA GAA GCA GCU CUG GUA C) -5'
ࢭࣥࢫ㙐3′ᮎ➃ࡽ 4 ␒┠ࡢ⨨Taࡸྛ࣑ࢫ࣐ࢵࢳሷᇶࢆᑟධࡋࡓsiRNA 㔜㙐 ࡢ⇕ⓗᏳᐃᛶࢆẚ㍑ࡍࡿࡓࡵࠊ50%⼥ゎ ᗘ (Tm್) ࢆ ᐃࡋࡓ (Figure 118)ࠋ ᐃ᮲௳ ᐃ ᗘ⠊ᅖ; 20-100 °C ᐃἼ㛗; 260 nm ࢧࣥࣉࣝ⃰ᗘ; 3.0 μM
buffer; 10 mM Na Phosphate and 100 mM NaCl (pH 7.0)
aUnderlined letters indicate mismatched bases.
28
Figure 118 ࡼࡾࠊsiRNA 55ࠊ12-114 ࡣኳ↛ᆺ࡛࠶ࡿ siRNA 11 ẚ㍑ࡋ࡚ࠊࡰྠ⛬ᗘTm ್ࡀపୗࡍࡿࡇࡀ♧ࡉࢀࡓࠋࡇࡢ⤖ᯝࡽࠊࢭࣥࢫ㙐 3′ᮎ➃ࡽ 4 ␒┠ࡢ⨨ࡢ Taࡢᑟධࡣ࣑ࢫ࣐ࢵࢳሷᇶྠ⛬ᗘࡢ⇕ⓗᏳᐃࢆᘬࡁ㉳ࡇࡍࡇࡀ♧၀ࡉࢀࡓࠋ
ḟࠊsiRNA (Table 77) ࢆࣄࢺᏊᐑ⒴⏤᮶⣽⬊࡛࠶ࡿ HeLa ⣽⬊ࢆ⏝࠸࡚ࠊ
Dual-Luciferase reporter assay ࡼࡗ࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ᳨ドࡋࡓ (Figure 119)ࠋ
Sample Tm (°C) ΔTm (°C)a siRNA 11 73.2 - siRNA 55 72.3 -0.9 siRNA 112 72.6 -0.6 siRNA 113 72.2 -1.0 siRNA 114 72.1 -1.1 control
aΔTm = Tm (each duplex) – Tm (siRNA 11).
Figure118.siRNA 11, 55, 112-114 ࡢ 50%⼥ゎ ᗘ ᐃ⤖ᯝ
29
Figure 119 ࡼࡾࠊsiRNA 55ࠊ12-114 ࡣ࠸ࡎࢀࡶኳ↛ᆺ࡛࠶ࡿ siRNA 11 ௨ୖࡢ㑇ఏᏊⓎ ⌧ᢚไ⬟ࢆ♧ࡍࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡇࡢ⤖ᯝࡽࠊࢭࣥࢫ㙐3′ᮎ➃ࡽ 4 ␒┠ࡢ ⨨ࡢ Taࡸ࣑ࢫ࣐ࢵࢳሷᇶࡢࡼ࠺࡞ RNA 㔜㙐ࢆ⇕ⓗᏳᐃࡉࡏࡿࢼࣟࢢࡢ ᑟධࡣࠊ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢྥୖ᭷⏝࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋ ࡲࡓࠊ࣑ࢫ࣐ࢵࢳሷᇶࢆᑟධࡋࡓsiRNA 112-114 ᑐࡋ࡚TaࢆᑟධࡋࡓsiRNA 55 ࡣഹ ࡛ࡣ࠶ࡿࡀࠊ㧗࠸㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࡓࠋࡇࢀࡣࠊTaࡢᑟධࡼࡾࢾࢡ࣮ࣞ ࢮ⪏ᛶࡀྥୖࡋࡓࡓࡵ࡛࠶ࡿ⪃࠼ࡽࢀࡿࡓࡵࠊ5′ᮎ➃ࢆ⺯ගᶆ㆑ࡋࢭࣥࢫ㙐 3′ᮎ➃ ࡽ4 ␒┠ࡢ⨨ࡢTaࢆᑟධࡋࡓRNA ࢆྜᡂࡋࠊ୍㔜㙐≧ែ࠾࠸࡚ SVPD ᑐ ࡍࡿᢠᛶࢆ᳨ドࡋࡓࠋSVPD ࡛ฎ⌮ࡋࡓ RNA ࢆྛ㛫࡛ᛂṆᚋࠊRNA ศゎ⏘ ≀ࢆ㟁ẼὋືࡼࡗ࡚ゎᯒࡋࡓ (Table 88, Figure 220)ࠋ ᛂ᮲௳ ᛂ ᗘ; 37 °C ࢧࣥࣉࣝ⃰ᗘ; 3.0 μM
buffer; 250 mM Tris-HCl and 50 mM MgCl2 (pH 8.0) SVPD; 0.5 unit
Sample Sequencea
ON 77 F-5'-r (CUU CUU CGU CGA GAC CAUU G) dTdT-3' ON 226 F-5'-r (CUU CUU CGU CGA GAC CATTaU G) dTdT-3'
Figure 220. SVPD ࡼࡿ RNA ศゎ⬟ࡢ᳨ド 0㻌 1 5㻌 10㻌 20㻌 30㻌 45㻌 1 3㻌 [min]㻌 [h]㻌 ON 226 0 1 5 10 20 30 45 1 3 [min] [h] ON 77 Table88. ࢚࢟ࢯࢾࢡ࣮ࣞࢮ⪏ᛶ᳨ド⏝࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ㓄ิ aF; Fluorescein.
30 Figure 220 ࡼࡾࠊ3′ᮎ➃ࡽ 4 ␒┠ࡢ⨨TaࢆᑟධࡋࡓON 226 ࡣࠊኳ↛ᆺ࡛࠶ࡿ ON 77 ẚ㍑ࡋ࡚ࠊSVPD ᑐࡍࡿᢠᛶࡀᖜྥୖࡋࡓࠋON 226 ࡣᛂ㛤ጞ 1 㛫 ᚋࡶࡈࡃഹ࡛ࡣ࠶ࡿࡀ㙐㛗ࡢ RNA ࡀṧᏑࡋ࡚࠾ࡾࠊ3′ᮎ➃ Taࢆᑟධࡋࡓ ON 88 (3-2-3.ཧ↷) ࡰྠ⛬ᗘࡢࢾࢡ࣮ࣞࢮᢠᛶࢆᣢࡘࡇࡀ♧၀ࡉࢀࠊ3′ᮎ➃ ࡽ4 ␒┠ࡢ⨨TaࡢᑟධࡣSVPD ᑐࡍࡿᢠᛶࡢྥୖ᭷⏝࡛࠶ࡿࡇࡀ᫂ࡽ ࡞ࡗࡓࠋ ௨ୖࡢ⤖ᯝࡼࡾࠊࢭࣥࢫ㙐3′ᮎ➃ࡽ 4 ␒┠ࡢ⨨ࡢTaࡢᑟධࡣࠊ࣑ࢫ࣐ࢵࢳሷ ᇶᑟධྠ⛬ᗘࡢ㔜㙐ࡢ⇕ⓗᏳᐃࢆᘬࡁ㉳ࡇࡋࠊ࠸ࡎࢀࡶኳ↛ᆺ௨ୖࡢ㑇ఏᏊⓎ ⌧ᢚไ⬟ࢆ♧ࡍࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡲࡓࠊTaࢆᑟධࡋࡓsiRNA ࡣࢾࢡ࣮ࣞࢮ⪏ ᛶࡀྥୖࡍࡿࡓࡵࠊ࣑ࢫ࣐ࢵࢳሷᇶࢆᑟධࡋࡓsiRNA ࡼࡾࡶഹ࡛ࡣ࠶ࡿࡀ㧗࠸㑇ఏ ᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࡓࠋ
31
➨
➨
4 ⠇ ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
miRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
3-4-1. ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
miR-199a-3p ࡢྜᡂ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
ࢼࣟࢢ Taᑟධࡼࡿ㔜㙐ࡢ⇕ⓗᏳᐃࡼࡿ㙐ࡢ㑅ᢥᛶࡢྥୖࡀࠊ㑇ఏᏊⓎ ⌧ᢚไ⬟ࡢྥୖᙳ㡪ࢆ࠼ࡓ࠸࠺Ⅼࢆ᳨ドࡍࡿࡓࡵࠊ୧㙐ᶆⓗ㓄ิࢆᣢࡘ miRNA ࢆ⏝࠸᳨࡚ドࢆ⾜ࡗࡓࠋ ୧㙐ᶆⓗmRNA ࢆᣢࡘ miRNA ࡋ࡚ࠊC ᆺ⫢⅖࢘ࣝࢫ (HCV) ࡸ B ᆺ⫢⅖࢘ ࣝࢫ (HBV) ࡀឤᰁࡋࡓ⒴⣽⬊࠾࠸࡚Ⓨ⌧␗ᖖࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ miR-199a-5p/3p (Figure 221) ࢆ⏝࠸ࡓ52)ࠋ ࡲࡎࠊRISC ࡢ㙐ࡢ㑅ᢥᛶࡢྥୖ㉳ᅉ ࡍࡿ miR-199a-5p 㙐ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ ࡢྥୖࢆ┠ⓗࡋ࡚ࠊmiR-199a-3p 㙐ࡢ ᵝ ࠎ ࡞ 㒊 ࢼ ࣟ ࢢ Ta ࢆ ᑟ ධ ࡋ ࡓ miRNA 22-44 ࢆྜᡂࡋࡓࠋ㓄ิࡣ Table 99 ♧ࡋࡓࠋ Sample Abbreviation of ON SequencemiRNA 11 ON 227 5'-r (CCC AGU GUU CAG ACU ACC UGU UC) -3' ON 228 3'-r (AUU GGU UAC ACG UCU GAU GAC A) -5' miRNA 22 ON 227 5'-r (CCC AGU GUU CAG ACU ACC UGU UC) -3'
ON 229 3'-r (AUU GGTTa UAC ACG UCU GAU GAC A) -5' miRNA 33 ON 227 5'-r (CCC AGU GUU CAG ACU ACC UGU UC) -3'
ON 330 3'-r (AUU GGU UAC ACG TTaCU GAU GAC A) -5' miRNA 44 ON 227 5'-r (CCC AGU GUU CAG ACU ACC UGU UC) -3'
ON 331 3'-r (AUU GGU UAC ACG UCU GATTa GAC A) -5'
miRNA (Table 99) ࢆࣄࢺᏊᐑ⒴⏤᮶⣽⬊࡛࠶ࡿ HeLa ⣽⬊ࢆ⏝࠸࡚ࠊ
Dual-Luciferase reporter assay ࡼࡗ࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ᳨ドࡋࡓ (Figure 223)ࠋ siRNA ྠᵝホ౯ࡍࡿࡓࡵࠊpsiCHECKTM-2 vector ෆࡢRenila luciferase 㓄ิࡢୗ ὶ㒊ࡑࢀࡒࢀࡢᶆⓗ㓄ิࢆᤄධࡋࡓpsi199a-5p/3p vector ࢆ⏝ࡋࡓ (Figure 222) 53)ࠋヲ⣽࡞ᐇ㦂᪉ἲࡣᐇ㦂㡯グ㍕ࡋࡓࠋ
Figure 221. miR-199a-5p/3p
32
Figure 223 ࡢ⤖ᯝࡼࡾࠊኳ↛ᆺ࡛࠶ࡿ miRNA 11 ࡣ miR-199a-5p 㙐ẚ㍑ࡋ࡚ miR-199a-3p 㙐ࡀࢃࡎ㧗࠸㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࠊRISC ྲྀࡾ㎸ࡲࢀࡸࡍ࠸ࡇ ࡀ♧၀ࡉࢀࡿ⤖ᯝࢆᚓࡓࠋࡲࡓࠊmiRNA 22-44 ࡣ miR-199a-3p 㙐Taࢆᑟධࡋࡓࡓ ࡵmiR-199a-5p 㙐ࡀࡼࡾ RISC ྲྀࡾ㎸ࡲࢀࠊ㧗࠸㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡍࡇࢆᮇ ᚅࡋࡓࠋࡋࡋࠊmiR-199a-3p/5p ࡢ୧㙐ඹኳ↛ᆺྠ⛬ᗘࡢ㑇ఏᏊⓎ⌧ᢚไࡋ♧ ࡉ࡞ࡗࡓࡇࡽࠊTaࡢᑟධࡼࡿ㙐ࡢ㑅ᢥᛶࡢᙳ㡪ࢆほᐹࡍࡿࡇࡀ࡛ࡁ࡞ ࡗࡓࠋࡇࡢཎᅉࡋ࡚ࠊmiR-199a-5p/3p ࡀࣂࣝࢪ࢘ࢺ㒊ࡸ࣑ࢫ࣐ࢵࢳሷᇶᑐ࡞ 㔜㙐ࢆ⇕ⓗᏳᐃࡍࡿᵓ㐀ࢆᣢࡕྜࢃࡏ࡚࠸ࡿⅬࡀᣲࡆࡽࢀࡿࠋ
Figure23.miRNA (Table 9) ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド ( ) 㑇ఏ ᢚไ⬟ ᳨
psi199a-5p vector transfected in HeLa cells. psi199a-3p vector transfected in HeLa cells.
psi-5p reporter: 5′-GAA CAG GTA GTC TGA ACA CTG GG -3′
33
33-4-2. ࢼࣟࢢ
T
aࢆᑟධࡋࡓ
miR-199a-5p ࡢྜᡂ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
ḟࠊRISC ࡢ㙐ࡢ㑅ᢥᛶࡢྥୖ㉳ᅉࡍࡿ miR-199a-3p 㙐ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ ྥୖࢆ┠ⓗࡋ࡚ࠊmiR-199a-5p 㙐ࡢᵝࠎ࡞㒊ࢼࣟࢢTaࢆᑟධࡋࡓmiRNA 5-88 ࢆྜᡂࡋࡓࠋ㓄ิࡣTable 110 ♧ࡋࡓࠋ Sample Abbreviation of ON SequencemiRNA 1 ON 227 5'-r (CCC AGU GUU CAG ACU ACC UGU UC) -3' ON 228 3'-r (AUU GGU UAC ACG UCU GAU GAC A) -5' miRNA 55 ON 332 5'-r (CCC AGTTa GUU CAG ACU ACC UGU UC) -3'
ON 228 3'-r (AUU GGU UAC ACG UCU GAU GAC A) -5' miRNA 66 ON 333 5'-r (CCC AGU GUTTa CAG ACU ACC UGU UC) -3'
ON 228 3'-r (AUU GGU UAC ACG UCU GAU GAC A) -5' miRNA 77 ON 334 5'-r (CCC AGU GUU CAG ACTTa ACC UGU UC) -3'
ON 228 3'-r (AUU GGU UAC ACG UCU GAU GAC A) -5' miRNA 88 ON 335 5'-r (CCC AGU GUU CAG ACU ACC UGTTa UC) -3'
ON 228 3'-r (AUU GGU UAC ACG UCU GAU GAC A) -5'
miRNA (Table 110) ࢆࣄࢺᏊᐑ⒴⏤᮶⣽⬊࡛࠶ࡿ HeLa ⣽⬊ࢆ⏝࠸࡚ࠊ
Dual-Luciferase reporter assay ࡼࡗ࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ᳨ドࡋࡓ (Figure 224)ࠋ ヲ⣽࡞ᐇ㦂᪉ἲࡣᐇ㦂㡯グ㍕ࡋࡓࠋ
34
miRNA 55-88 ࡣ miR-199a-5p 㙐TaࢆᑟධࡋࡓࡓࡵࠊmiR-199a-3p 㙐ࡀࡼࡾ RISC ྲྀࡾ㎸ࡲࢀ㧗࠸㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡍࡇࢆᮇᚅࡋࡓࠋFigure 224 ࡼࡾࠊmiRNA 5-88 ࡣ miRNA 22-44 ࡢ⤖ᯝ (Figure 223) ␗࡞ࡾࠊ㙐ࡢ㑅ᢥᛶࡢྥୖࢆ♧၀ࡍࡿ⤖ᯝࢆ ᚓࡓࠋ
ࡲࡎࠊmiR-199a-5p 㙐ࡢ 5'ᮎ➃ഃTaࢆᑟධࡋࡓmiRNA 55 ࡣࠊmiR-199a-5p 㙐ࡢ 5'ᮎ➃ࢆ⇕ⓗᏳᐃࡍࡿࡇ࡛ RISC ྲྀࡾ㎸ࡲࢀࡸࡍ࠸ࡼ࠺タィࡋࡓࠋࡑࡢ≺࠸ ㏻ࡾࠊmiR-199a-3p 㙐ࡣࢇ RISC ྲྀࡾ㎸ࡲࢀࡎࠊ㑇ఏᏊⓎ⌧ᢚไࢆ♧ࡉ࡞ ࡗࡓࠋࡋࡋࠊ࢞ࢻ㙐࡞ࡿ miR-199a-5p 㙐ࡢ seed 㡿ᇦ Taࢆᑟධࡋࡓࡓࡵࠊ miR-199a-5p 㙐ࡶ࠶ࡲࡾ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡉ࡞ࡗࡓࠋ
ࡲࡓࠊmiR-199a-5p 㙐ࡢ୰ኸ㒊TaࢆᑟධࡋࡓmiRNA 66 ࡣኳ↛ᆺ࡛࠶ࡿ miRNA 1 ẚ㍑ࡋ࡚ࠊ㙐ࡢ㑅ᢥᛶࡢྥୖࡼࡿ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢྥୖࢆഹ♧ࡋࡓࠋ miRNA ࡢ୰ኸ㒊ࡢ࣑ࢫ࣐ࢵࢳࡣ RISC ࡢ㙐ࡢྲྀࡾ㎸ࡳࢆಁ㐍ࡉࡏࡿ࠸࠺▱ぢ54) ࡀ࠶ࡿࠋࡑࡢࡓࡵࠊmiR-199a-5p 㙐ࡢ୰ኸ㒊ࡢTaࡢᑟධࡣTaࡀሷᇶ≉␗ᛶࢆᣢࡘ ࡇࡶ࠶ࡾࠊ࣑ࢫ࣐ࢵࢳሷᇶㄆ㆑ࡉࢀࠊഹ࡛ࡣ࠶ࡿࡀmiRNA 1 ࢆୖᅇࡿ㑇ఏᏊⓎ ⌧ᢚไ⬟ࢆ♧ࡋࡓ⪃࠼ࡽࢀࡿࠋ
୍᪉࡛ࠊmiR-199a-5p 㙐ࡢ 3'ᮎ➃ഃTaࢆᑟධࡋࡓmiRNA 77-88 ࡣ miR-199a-3p 㙐 ࡛ኳ↛ᆺ௨ୖࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆ♧ࡋࠊmiR-199a-5p 㙐ࡣࢇ㑇ఏᏊⓎ⌧ᢚไ ⬟ࢆ♧ࡉ࡞ࡗࡓࠋ≉ࠊmiRNA 7 ࡛୧㙐ࡼࡿ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢᕪࡀࡁ࠸ࡇ
Figure224. TaaࢆᑟධࡋࡓmiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢ᳨ド
psi199a-5p vector transfected in HeLa cells. psi199a-3p vector transfected in HeLa cells.
35 ࡽࠊࣃࢵࢭࣥࢪ࣮ࣕ㙐࡞ࡿ㙐ࡢ3′ᮎ➃ࡽ 9 ሷᇶ┠ࡀ᭱ࡶ㙐ࡢ㑅ᢥᛶࡢྥୖຠᯝ ࡀ࠶ࡿTTaᑟධ㒊࡛࠶ࡿ♧၀ࡉࢀࡓࠋ௨ୖࡢ⤖ᯝࡼࡾࠊmiRNA ࠾࠸࡚ࡶ๓㡯࡛㏙ ࡓsiRNA ྠࡌࡃࣃࢵࢭࣥࢪ࣮ࣕ㙐ࡢ 3'ᮎ➃ഃTaࢆᑟධࡍࡿࡇ࡛㏫㙐࡛࠶ࡿ࢞ ࢻ㙐ࡢ5'ᮎ➃ࢆ⇕ⓗᏳᐃࡋࠊRISC ࡢ㙐ࡢྲྀࡾ㎸ࡳࢆಁ㐍ࡍࡿࡇࡀ᫂ࡽ ࡞ࡗࡓࠋ
36
➨
➨
4 ❶ ࣥࢳࢭࣥࢫἲࡢᛂ⏝᳨ウ
➨
1 ⠇ ࣥࢳࢭࣥࢫ᰾㓟
4-1-1. ࣥࢳࢭࣥࢫ᰾㓟ࡢᏛಟ㣭
ࣥࢳࢭࣥࢫ᰾㓟ࡣ⣽⬊ෆ࡛ᶆⓗmRNA ⤖ྜࡋࠊRNase H ࡼࡿ mRNA ࡢศゎ ࢆᘬࡁ㉳ࡇࡍࡇ࡛㑇ఏᏊⓎ⌧ࢆᢚไࡍࡿࠋࡋࡋࠊDNA ࢆᇶᮏ㦵᱁ࡍࡿࣥࢳࢭ ࣥࢫ᰾㓟ࡣ⣽⬊ෆࡢDNase ࡼࡗ࡚▷㛫࡛ศゎࡉࢀࡿࡓࡵࠊ᰾㓟་⸆ᛂ⏝ࡍࡿ㝿 ࡣࠊᏛಟ㣭యࡢᑟධࡼࡿࢾࢡ࣮ࣞࢮ⪏ᛶࡢቑᙉࡀᚲせ࡞ࡿࠋࡑࡇ࡛ࠊ᭱ึ ሗ࿌ࡉࢀࡓᏛಟ㣭ࡀ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺ (PS) 㦵᱁ࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡛ ࠶ࡿ (Figure 225) 18) 19)ࠋPS 㦵᱁ࢆᑟධࡋࡓࣥࢳࢭࣥࢫ᰾㓟ࡣࢾࢡ࣮ࣞࢮᑐࡋ࡚ ⪏ᛶࡀ࠶ࡾࠊࡲࡓࠊmRNA 㔜㙐ࢆᙧᡂࡋࠊRNase H άᛶࢆច㉳ࡋ࡚ᶆⓗ mRNA ࢆศゎࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋࡋࡋࠊࣥࢳࢭࣥࢫ᰾ 㓟୰ᑟධࡍࡿPS ⤖ྜࡢᩘࢆቑࡸࡍ⛬ mRNA ࡢぶ ᛶࡀపୗࡍࡿࠊ༶ࡕ㔜㙐ࡢ⇕ⓗᏳᐃᛶࡀపୗࡍࡿ࠸࠺ ၥ㢟Ⅼࡀ࠶ࡿࠋࡲࡓࠊPS 㦵᱁ࡢᑟධࡣ Toll-like receptor ┦స⏝ࡍࡿࡇ࡛චᛂ⟅ࢆᘬࡁ㉳ࡇࡍࠊࡶࡋࡃࡣ ࢱࣥࣃࢡ┤᥋⤖ྜࡍࡿࡓࡵ㔜⠜࡞స⏝ࢆᘬࡁ㉳ࡇࡍ ྍ⬟ᛶࡀᣦࡉࢀ࡚࠸ࡿࠋ ࡇࢀࡽࡢၥ㢟ⅬࢆゎỴࡍࡿࡓࡵࠊ㛤ⓎࡉࢀࡓᏛಟ㣭యࡀᶆⓗmRNA ࡢぶᛶྥ ୖࢆᣦྥࡋࡓ⢾㒊2′ࡢಟ㣭యࠊ2′-O-methyl (2′-OMe)
RNA ࡸ 2′-O-methoxyethyl (2′-MOE) RNA (Figure 226) 22) 23) ࡛࠶ࡿࠋࡇࢀࡽࢆᑟධࡋࡓࣥࢳࢭࣥࢫ᰾㓟ࡣRNA ⇕ⓗᏳᐃ࡞㔜㙐ࢆᙧᡂࡋࠊ㧗࠸ࢾࢡ࣮ࣞࢮ⪏ᛶࢆ♧ ࡍ୍᪉࡛ࠊRNA ࡢ㔜㙐ࡀ RNase H ࡢᇶ㉁࡞ࡽ࡞࠸ ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡑࡢࡓࡵࠊࡇࢀࡽᏛಟ㣭యࢆ ⏝ࡋࡓ gapmer ᆺࣥࢳࢭࣥࢫ᰾㓟 (Figure 227) ࡀ タィࡉࢀࡓࠋ Figure 255. ࣜࣥ㓟㒊ಟ㣭య PS 㦵᱁ PPO 㦵᱁ Figure 226. ⢾㒊2′ಟ㣭య
2′′-OOMe RRNA 2′′-MMOE RRNA
5′㻌 3′㻌
wing region 3-5 mer 22′-OMe/2′-MOE wing region
3-5 mer 2′′-OOMe/2′--MOE 7-11 mer DNA gap region
Figure 227. gapmer ᆺࣥࢳࢭࣥࢫ᰾㓟ࡢࢹࢨࣥ
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ࡇࡢgapmer ᆺࣥࢳࢭࣥࢫ᰾㓟ࡣࠊᏛಟ㣭యࢆᑟධࡋࡓ୧ᮎ➃ࡢ wing region DNA ࡽᡂࡿ୰ኸ㒊ࡢ gap region ࡛ᵓᡂࡉࢀ࡚࠸ࡿࠋGapmer ᆺࣥࢳࢭࣥࢫ᰾㓟ࡣࠊ ࡇࡢᏛಟ㣭యࢆᑟධࡋࡓwing region ࡼࡿ㧗࠸ࢾࢡ࣮ࣞࢮ⪏ᛶ㧗࠸ RNA ࡢ ぶᛶຍ࠼ࠊDNA ࡽᡂࡿ gap region ࡀᏑᅾࡍࡿࡇ࡛ࠊRNase H ࡼࡾ DNA/RNA 㔜㙐ࡀㄆ㆑ࡉࢀ RNA 㙐ࡢศゎࡉࢀࡿࡇ࡛ࠊ㑇ఏᏊⓎ⌧ࢆᢚไࡍࡿࠋ 2013 ᖺ Isis ♫ࡼࡾࠊ2′-MOE RNA ࢆ wing region ᑟධࡋࡓ gapmer ᆺࣥࢳࢭࣥ ࢫ᰾㓟ࡀ Kynamro ࠸࠺㌟ᢞྍ⬟࡞᰾㓟་⸆ရࡋ࡚ୖᕷࡉࢀ࡚࠸ࡿⅬࡽࡶࠊ ࡇࡢᵓ㐀ࡣ᭷⏝࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋ
ࡑࡋ࡚ࠊ᭦࡞ࡿRNA ࡢぶᛶࡢྥୖࢆᮇᚅࡋ࡚㛤ⓎࡉࢀࡓᏛಟ㣭యࡀ peptide nucleic acid (PNA) 36)ࠊlocked nucleic acid (LNA) 35) ࡸphosphoroamidate morpholino oligomer (PMO) 55) ࡞ࡢ⢾㒊ಟ㣭య࡛࠶ࡿ (Figure 228)ࠋࡇࢀࡽࡢಟ㣭యࢆᑟධࡋࡓ ࣥࢳࢭࣥࢫ᰾㓟┦⿵RNA ࡢ㔜㙐ࡣ RNase H ࡢᇶ㉁ࡣ࡞ࡽ࡞ࡗࡓࡀࠊRNA ࡢぶᛶࠊ⇕ⓗᏳᐃᛶࢆྥୖࡉࡏࡓࠋ
4-1-2. ᅇ᳨ウࡍࡿࣥࢳࢭࣥࢫ᰾㓟
4-1-1.ࡼࡾࠊRNA ࡢぶᛶࡢྥୖࡸࢾࢡ ࣮ࣞࢮ⪏ᛶࡢቑᙉࢆ≺ࡗࡓ gapmer ᆺࣥ ࢳࢭࣥࢫ᰾㓟ࡢ wing region ⢾㒊㛤⎔ᆺࢾ ࢡࣞ࢜ࢩࢻࢼࣟࢢ (Figure 229) ࢆᑟධࡋࡓ (Figure 330)ࠋ Figure 228. ⢾㒊ಟ㣭యPNA LNA PMO
Figure 229. ⢾㒊㛤⎔ᆺࢾࢡࣞ࢜ࢩࢻࢼࣟࢢ Ca Ta 5′㻌 3′㻌 wing region 3-5 mer TTa/CCa wing region 3-5 mer Ta/Ca gap region 7-11 mer DNA Figure 330. ᅇ᳨ウࡍࡿࣥࢳࢭࣥࢫ᰾㓟ࡢࢹࢨࣥ 17 mer ࣥࢳࢭࣥࢫ᰾㓟
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➨
➨
2 ⠇ ࢼࣟࢢࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢྜᡂ≀⌮Ꮫⓗᛶ㉁
4-2-1. ࢼࣟࢢࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢྜᡂ
୧ᮎ➃ࢼࣟࢢ (Figure 331) ࢆᑟධࡋ ࡓ17 ሷᇶࡢ ON ┦⿵㙐 RNA ࢆᅛ┦࣍ࢫ ࣑࣍ࣟࢲࢺἲᚑࡗ࡚ࠊDNA/RNA ⮬ື ᰾㓟ྜᡂᶵࡼࡗ࡚ྜᡂࡋࡓࠋ௨ୗྜᡂࡋ ࡓ ࢜ ࣜ ࢦ ࢾ ࢡ ࣞ ࢜ ࢳ ࢻ ࡢ 㓄 ิ ࢆ ♧ ࡋ ࡓ (Table 111)ࠋSample Abbreviation of ON Sequence
Duplex 8 ON 336 ON 337 3′-r (AAA GUGAUGAGGAU AAA) -5′ 5′-d (TTT CACTACTCCTA TTT) -3′ Duplex 99 ON 338 ON 337 5′-d (3′-r (AAA GUGAUGAGGAU AAA) -5′ TTaTTaTTa CACTACTCCTA TTaTTaTTa) -3′ Duplex 110 ON 339 ON 440 3′-r (AAAAA GAUGAGG AAAAA) -5′ 5′-d (TTTTT CTACTCC TTTTT) -3′ Duplex 111 ON 441 ON 440 5′-d (3′-r (AAAAA GAUGAGG AAAAA) -5′ TTaTTaTTaTTaTTa CTACTCC TTaTTaTTaTTaTTa) -3′ Duplex 112 ON 42 ON 443 3′-r (AGA GUGAUGAGGAU AGA) -5′ 5′-d (TCT CACTACTCCTA TCT) -3′ Duplex 13 ON 444 ON 443 5′-d (3′-r (AGA GUGAUGAGGAU AGA) -5′ TTaCCaTTa CACTACTCCTA TTaCCaTTa) -3′ Duplex 114 ON 45 ON 446 3′-r (AAA GUGGCUGCCGC AAA) -5′ 5′-d (TTT CACCGACGGCG TTT) -3′ Duplex 115 ON 447 ON 446 5′-d (3′-r (AAA GUGGCUGCCGC AAA) -5′ TTaTTaTTa CACCGACGGCG TTaTTaTTa) -3′ Duplex 16 ON 448 ON 449 3′-r (AAAAA GGCUGCC AAAAA) -5′ 5′-d (TTTTT CCGACGG TTTTT) -3′ Duplex 117 ON 550 ON 449 5′-d (3′-r (AAAAA GGCUGCC AAAAA) -5′ TTaTTaTTaTTaTTa CCGACGG TTaTTaTTaTTaTTa) -3′ Duplex 118 ON 551 ON 552 3′-r (AGA GUGGCUGCCGC AGA) -5′ 5′-d (TCT CACCGACGGCG TCT) -3′ Duplex 119 ON 553 ON 552 5′-d (3′-r (AGA GUGGCUGCCGC AGA) -5′ TTaCCaTTa CACCGACGGCG TTaCCaTTa) -3′
Figure 331. ⢾㒊㛤⎔ᆺࢾࢡࣞ࢜ࢩࢻࢼࣟࢢ
Ca Ta
39
44-2-2. ࢼࣟࢢࢆᑟධࡋࡓ ON/RNA 㔜㙐ࡢ⇕ⓗᏳᐃᛶࡢ᳨ド
ྜᡂࡋࡓON ┦⿵㙐 RNA ࡢ㔜㙐ࡢ⇕ⓗᏳᐃᛶࢆࠊ50%⼥ゎ ᗘࠊTm್ࢆ ᐃ ࡍࡿࡇ࡛ẚ㍑᳨ウࡋࡓ (Table 112ࠊ13)ࠋ ᐃ᮲௳ ᐃ ᗘ⠊ᅖ; 20-100 °C ᐃἼ㛗; 260 nm ࢧࣥࣉࣝ⃰ᗘ; 3.0 μMbuffer; 10 mM Na Phosphate and 100 mM NaCl (pH 7.0)
Table 112ࠊ13 ࡼࡾࠊኳ↛ᆺ࡛࠶ࡿ Duplex 88 (Tm = 56.5 °C) ࡸ Duplex 114 (Tm = 66.1 °C) ẚ㍑ࡋ࡚ࠊTaࢆᑟධࡋࡓON/RNA 㔜㙐࡛࠶ࡿ Duplex 99 (Tm = 52.0 °C, ΔTm = -4.5 °C) ࡸ Duplex 115 (Tm = 63.8 °C, ΔTm = -2.3 °C) ࡣ⇕ⓗᏳᐃᛶࡀపୗࡋࡓࠋ ຍ࠼࡚ࠊCaࢆᑟධࡋࡓON/RNA 㔜㙐࡛࠶ࡿ Duplex 113 (Tm = 53.1 °C, ΔTm = -7.2 °C) ࡸDuplex 119 (Tm = 61.9 °C, ΔTm = -7.4 °C) ࡢ⇕ⓗᏳᐃᛶࡶࡲࡓᑐᛂࡍࡿኳ↛ᆺẚ㍑ Sample Tm (°C) ΔTm (°C) a Duplex 88 56.5 - Duplex 99 52.0 -4.5 Duplex 110 56.3 - Duplex 111 47.1 -9.2 Duplex 112 60.4 - Duplex 113 53.1 -7.2 Sample Tm (°C) ΔTm (°C) a Duplex 114 66.1 - Duplex 115 63.8 -2.3 Duplex 116 57.6 - Duplex 117 44.2 -13.4 Duplex 118 69.3 - Duplex 119 61.9 -7.4 Table 112. 50%⼥ゎ ᗘ (Duplex 88-113) Table 113. 50%⼥ゎ ᗘ (Duplex 114-119)
aΔTm = Tm (DNA/RNA duplex) – Tm (corresponding ON/RNA duplex).
40 ࡋ࡚పୗࡋࡓࠋCCaࡣࢩࢳࢪࣥࢆᶍࡋ࡚࠾ࡾࠊࢳ࣑ࢪࣥࢆᶍࡋ࡚࠸ࡿTaࡼࡾࡶ㔜㙐ࡢ ⇕ⓗᏳᐃᛶࡢྥୖᐤࡍࡿ᥎ ࡋ࡚࠸ࡓࠋࡋࡋࠊCa ࢆᑟධࡋࡓ Duplex 13 (ΔTm = -7.2 °C ) ࡸ Duplex 19 (ΔTm = -7.4 °C ) ࡢᑐᛂࡍࡿኳ↛ᆺẚ㍑ࡋࡓ㝿ࡢ ΔTm ࡣࠊ Ta ࢆᑟධࡋࡓ Duplex 99 (ΔTm = -4.5 °C) ࡸ Duplex 115 (ΔTm = -2.3 °C) ẚ㍑ࡋ࡚ࡁ ࡗࡓࠋࡇࡢ⤖ᯝࡽࠊCaࡣ ON/RNA 㔜㙐ෆ࡛┦⿵ሷᇶ 3 ᮏࡢỈ⣲⤖ྜࡼࡿ Watson-Crick ᆺࡢሷᇶᑐࢆᙧᡂࡋ࡚࠸࡞࠸ࡇࡀ⪃࠼ࡽࢀࡿࠋࡇࢀ㛵ࡋ࡚ࠊSchlegel ࡽࡶGNA/RNA 㔜㙐 (A:T 10 mer) ୰ G:C ሷᇶᑐࢆቑࡸࡋ࡚࠸ࡃ㔜㙐ࡢ⇕ⓗ ᏳᐃᛶࡀᏳᐃࡍࡿࡇࢆሗ࿌ࡋ࡚࠸ࡿ 26)ࠋࡑࡢࡓࡵࠊᅇྜᡂࡋࡓ Ta Caࡢሷ ᇶ≉␗ᛶࡘ࠸᳨࡚ドࡍࡿࡇࡋࡓࠋ
![Table 3 3. ࢚࢟ࢯࢾࢡ࣮ࣞࢮ⪏ᛶ᳨ド⏝࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ㓄ิ aF; Fluorescein. ON 77 0 1 5 10 20 30 45 1 3 [min] [h] 0 1 5 10 20 30 45 1 3 [min] [h] ON 88](https://thumb-ap.123doks.com/thumbv2/123deta/10093422.1494007/26.892.463.720.795.1000/table-ࢯ-ࢡ-ࢮ-ᛶ-ド-ࢳࢻࡢ㓄-fluorescein.webp)
