RNA創薬を目指した塩基部欠損ヌクレオシドを有する核酸オリゴマーの合成

Title

RNA創薬を目指した塩基部欠損ヌクレオシドを有する核酸

_{オリゴマーの合成( 本文(Fulltext) )}

Author(s)

長屋, 優貴

Report No.(Doctoral

Degree)

博士(薬科学) 連創博甲第40号

Issue Date

2018-03-25

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/75253

※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。

RNA ๰

๰⸆ࢆ┠ᣦࡋࡓ

ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࢆ᭷ࡍࡿ

᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ

ᖹᡂ

_{29 ᖺᗘ}

┠

┠ḟ

⌮ㄽࡢ㒊

➨

1 ❶ ᗎㄽ

➨

2 ❶ ◊✲ࡢ⫼ᬒ

➨

1 ⠇ ᰾㓟་⸆

➨

2 ⠇ పศᏊᖸ΅ RNA (siRNA)

➨

3 ⠇ Argonaute ࢱࣥࣃࢡ㉁

➨

4 ⠇ siRNA ࡢ⮫ᗋᛂ⏝

➨

5 ⠇ ໬Ꮫಟ㣭᰾㓟࡜᰾㓟་⸆

➨

6 ⠇ ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡢ໬Ꮫಟ㣭

➨

7 ⠇ ሷᇶ㒊Ḟᦆࢹ࢜࢟ࢩࣜ࣎ࢾࢡࣞ࢜ࢩࢻ (dR

H

_{) ࡢᚑ᮶ྜᡂἲ}

➨

3 ❶ dR

H

_{ࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ࡜ᛶ㉁ホ౯}

➨

1 ⠇ ሷᇶ㒊⬺㞳཯ᛂࢆ฼⏝ࡋࡓ dR

H

_ࡢྜᡂ

➨

2 ⠇ dR

H

_{ྵ᭷᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ}

➨

3 ⠇ ᰾㓟ศゎ㓝⣲⪏ᛶホ౯

➨

4 ⠇ ⇕ⓗᏳᐃᛶホ౯

➨

5 ⠇ 㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯

➨

6 ⠇ ࢘ࢩ⾑Ύ୰ศゎ㓝⣲⪏ᛶホ౯

➨

4 ❶ 2 ఩ಟ㣭ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮

ࡢྜᡂ࡜ᛶ㉁ホ౯

➨

1 ⠇ 2 ఩࣋ࣥࢪࣝಟ㣭ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻ (R

HOBn

) ࢆ᭷ࡍࡿ RNA

➨

1 㡯 R

HOBn

ࡢྜᡂ

➨

2 㡯 R

HOBn

ྵ᭷

RNA ࡢྜᡂ

➨

3 㡯 ᰾㓟ศゎ㓝⣲⪏ᛶホ౯

➨

4 㡯 ⇕ⓗᏳᐃᛶホ౯

➨

5 㡯 㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯

➨

6 㡯 ࢘ࢩ⾑Ύ୰ศゎ㓝⣲⪏ᛶホ౯

➨

7 㡯 ࣄࢺ Argonaute2-PAZ ࢻ࣓࢖ࣥࢱࣥࣃࢡ㉁࡜ࡢぶ࿴ᛶホ౯

➨

2 ⠇ 2 ఩ࣉࣟࣃࣝࢠࣝಟ㣭ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻ (R

HOPg

) ࢆ᭷ࡍࡿ RNA

➨

1 㡯 R

HOPg

ࡢྜᡂ

➨

2 㡯 R

HOPg

ྵ᭷

RNA ࡢྜᡂ

➨

3 㡯 R

HOPg

ྵ᭷

RNA ࡜࢔ࢪࢻ໬ྜ≀࡜ࡢ CuAAC ཯ᛂ

➨

5 ❶ ⤖ㄽ

ㅰ㎡

6 7 2 9 21 26 30 24 22 1 35 15 19 11 31 12 41 43 36 8 25 32 29 28 33 37

ᐇ㦂ࡢ㒊

ᘬ⏝ᩥ⊩

62

1

➨

➨

1 ❶ ᗎㄽ

ᆅ⌫ୖ࡟Ꮡᅾࡍࡿ࡯ࡰ඲࡚ࡢ⏕≀ࡣࠊ㑇ఏ᝟ሗࢆDNA ࠿ࡽ RNAࠊࡑࡋ࡚ࢱࣥࣃࢡ㉁ࡢ㡰 ࡟ఏ㐩ࡍࡿࢭࣥࢺࣛࣝࢻࢢ࣐࡟ᚑ࠸ࠊ⏕࿨άືࢆ⥔ᣢࡋ࡚࠸ࡿࠋ᰾㓟࢜ࣜࢦ࣐࣮ࢆᇶᮏᵓ㐀 ࡟᭷ࡍࡿ᰾㓟་⸆ࡣࠊࢭࣥࢺࣛࣝࢻࢢ࣐࡟స⏝ࡋ࡚⸆⌮ຠᯝࢆ♧ࡍࡇ࡜࠿ࡽࠊḟୡ௦་⸆ရ ࡜ࡋ࡚ὀ┠ࡉࢀ࡚࠾ࡾࠊୡ⏺୰࡛㛤Ⓨࡀ㐍ࡵࡽࢀ࡚࠸ࡿࠋపศᏊᖸ΅RNA (small interfering RNA, siRNA) ࢆࡣࡌࡵ࡜ࡍࡿ▷㙐஧ᮏ㙐 RNA ࡣࠊRNA ᖸ΅ (RNA interference, RNAi) ࡟ ࡼࡗ࡚㓄ิ≉␗ⓗ࡟ᶆⓗࡢmRNA ࡟స⏝ࡋࠊ㑇ఏᏊࡢⓎ⌧ࢆᢚไࡍࡿࡇ࡜࠿ࡽࠊࡀࢇ࡞࡝ ࡢ㞴἞ᛶ⑌ᝈࡢ἞⒪࡬ࡢᛂ⏝ࡀᮇᚅࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋࠊsiRNA ࡟ࡣࠊi) ⏕యෆ᰾㓟ศゎ㓝 ⣲࡟ࡼࡾศゎࢆཷࡅࡿࠊii) ᶆⓗ⣽⬊࡬ࡢ㍺㏦ࢩࢫࢸ࣒ࡀ☜❧ࡉࢀ࡚࠸࡞࠸ࠊiii) ᶆⓗ࡜␗࡞ ࡿ㑇ఏᏊࡢⓎ⌧ࢆᢚไࡍࡿࠊ࡞࡝ࡢၥ㢟Ⅼࡀ࠶ࡾࠊsiRNA ࡢ⮫ᗋᛂ⏝࡟ࡣࡇࢀࡽࡢㄢ㢟ࢆ ඞ᭹ࡍࡿᚲせࡀ࠶ࡿࠋ ⴭ⪅ࡀᡤᒓࡍࡿ◊✲ᐊ࡛ࡣࠊsiRNA ࡢᵓ㐀ⓗ࡞≉ᚩ࡛࠶ࡿ 3′ᮎ➃ 2 ሷᇶ✺ฟ㒊఩࡟ⰾ㤶 ᪘໬ྜ≀࡞࡝᰾㓟ሷᇶ㒊ࢆᣢࡓ࡞࠸ศᏊࢆᑟධࡍࡿࡇ࡜࡛ࠊᮏ᮶ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆῶ ᙅࡍࡿࡇ࡜࡞ࡃࠊ᰾㓟ศゎ㓝⣲࡟ᑐࡍࡿ⪏ᛶࡀྥୖࡍࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࠋࡇࢀࡽࡢሗ࿌ ࠿ࡽⴭ⪅ࡣࠊsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ࡟࠾࠸࡚ 3′ᮎ➃࡟᰾㓟ሷᇶࡀᚲ㡲࡛࡞࠸࡜ண᝿ࡋࡓࠋ ࡑࡇ࡛ࠊᙜヱ㒊఩ࡢ᰾㓟ሷᇶࡢᙺ๭ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ᣦࡋࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻ ࢆ᭷ࡍࡿsiRNA ࡢྜᡂࢆ┠ᣦࡋࡓࠋሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࡢ໬Ꮫྜᡂἲࡣࡇࢀࡲ࡛࡟ᩘ ౛ሗ࿌ࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋࠊᚑ᮶ἲࡣ㐣㓞࡞཯ᛂ᮲௳ࢆᚲせ࡜ࡍࡿࠊ෌⌧ᛶࡀ࡞࠸ࠊ࡜࠸ࡗ ࡓၥ㢟ࡀ࠶ࡿࡇ࡜࠿ࡽࠊᮏ◊✲࡛ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࡢ⡆౽࠿ࡘᐇ⏝ⓗ࡞ྜᡂἲࢆ☜ ❧ࡋࡓࠋ㛤Ⓨᡭἲࢆ⏝࠸࡚ྜᡂࡋࡓᑐᛂࡍࡿሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࡣᑐᛂࡍࡿ᰾㓟⮬ື ྜᡂ⏝ㄏᑟయ࡟ኚ᥮ᚋࠊᅛ┦࣍ࢫ࣍ࣟ࢔࣑ࢲ࢖ࢺἲ࡛ 3′ᮎ➃࡟ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࢆ ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆྜᡂࡋࠊ┦⿵㙐࡜ࡢ࢔ࢽ࣮ࣜࣥࢢ࡟ࡼࡾsiRNA ࢆᵓ⠏ࡋࠊࡑ ࡢᛶ㉁ࢆホ౯ࡋࡓࠋ ᰾㓟་⸆ࡢ㛤Ⓨ࡟࠶ࡓࡾࠊ⏕యෆ᰾㓟ศゎ㓝⣲⪏ᛶࡢྥୖࢆ┠ⓗ࡜ࡋ࡚ siRNA ࡬ࡢಟ㣭 ᰾㓟ࡢᑟධࡀᗈࡃ᳨ウࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࡢ 2 ఩Ỉ㓟ᇶ࡬ࡢ⨨ ᥮ᇶᑟධ࡟ࡼࡿ᭦࡞ࡿศゎ㓝⣲⪏ᛶྥୖࢆᮇᚅࡋࠊ2 ఩Ỉ㓟ᇶࢆಟ㣭ࡋࡓሷᇶ㒊Ḟᦆࢾࢡࣞ ࢜ࢩࢻࢆࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟᭷ࡍࡿsiRNA ࡢྜᡂࠊᛶ㉁ホ౯ࢆ⾜ࡗࡓࠋ

2

➨

➨

2 ❶ ◊✲ࡢ⫼ᬒ

➨

1 ⠇ ᰾㓟་⸆

ࣄࢺࢤࣀ࣒ィ⏬ (Human Genome Project) ࢆ௦⾲࡜ࡍࡿࣄࢺࢤࣀ࣒ࢆᑐ㇟࡜ࡋࡓ◊✲࡟ ࡼࡾࠊࣄࢺࢤࣀ࣒ࡢ࠺ࡕࠊ㑇ఏᏊࡢᩘࡣ඲యࡢ⣙2%࡟࠶ࡓࡿ⣙ 2 ୓ 2 ༓࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿ ࡟࡞ࡗࡓ1)_{ࠋࢩࣟ࢖ࢾࢼࢬࢼࡢ㑇ఏᏊᩘࡣ⣙2 ୓ 6 ༓ࠊ࢖ࢿࡣ⣙ 3 ୓ 2 ༓࠶ࡾࠊࣄࢺࡼࡾ}

ࡶ㑇ఏᏊᩘࡀከࡃࠊ⏕≀ࡢ」㞧ࡉࡀ㑇ఏᏊᩘ࡟౫Ꮡࡋ࡚࠸࡞࠸ࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡾࠊ㑇ఏᏊ ᩘ௨እࡢせᅉࡀ㧗ᗘ࡞⏕࿨άືࢆ⥔ᣢࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡓࠋ

⏕≀ࡢ㧗➼ࡉ࡟㛵୚ࡍࡿࡶࡢ࡜ࡋ࡚ᶵ⬟ᛶRNA ࡸ non-coding RNA (ncRNA) ࡀぢฟࡉࢀ ࡓ (Figure 2-1)ࠋᙜึࡣࢱࣥࣃࢡ㉁ࡢ᝟ሗࢆᣢࡓ࡞࠸୙せ࡞ RNA ศᏊ࡜⪃࠼ࡽࢀ࡚࠸ࡓࡀࠊ ㌿෗ᚋࠊRNA ศᏊࡀᵝࠎ࡞ᶵ⬟ࢆ᭷ࡋ࡚࠾ࡾࠊncRNA ࡢാࡁ࡟ࡼࡗ࡚⏕࿨άືࡀ⥔ᣢࡉࢀ ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ௦⾲౛࡜ࡋ࡚ࠊࢱࣥࣃࢡ㉁ࡢ⩻ヂ᫬࡟ᶵ⬟ࢆ♧ࡍ㌿⛣ RNA (tRNA) ࡸࣜ࣎ࢯ࣮࣒ RNA (rRNA) ࡣᗈࡃ▱ࡽࢀ࡚࠸ࡿࠋ㌿෗ࡉࢀࡓᚋࠊ┦⿵ⓗ࡞ mRNA ࡜ ⤖ྜࡍࡿࡇ࡜࡛㑇ఏᏊࡢⓎ⌧ࢆᢚไࡍࡿmicro RNA (miRNA) ࡣ ncRNA ࡢ 1 ࡘ࡛࠶ࡾࠊ་ ⸆ရ࡬ࡢᛂ⏝ࡀᮇᚅࡉࢀࠊ⌧ᅾࠊୡ⏺୰࡛◊✲ࡀ⾜ࢃࢀ࡚࠸ࡿࠋࡲࡓࠊ⏕Ṫ⣽⬊ࡢࡳ࡛ࡳࡽ ࢀࡿ PIWI-interacting RNA (piRNA) ࡣࠊࢺࣛࣥࢫ࣏ࢰࣥࡢാࡁࢆᢚไࡍࡿࡇ࡜࡛ḟୡ௦࡬ ࡢ㑇ఏᏊࡢṇ☜࡞ఏ㐩࡟ᐤ୚ࡋ࡚࠸ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ

3 ᰾㓟་⸆ࡣncRNA ࡢࡼ࠺࡞᰾㓟ศᏊ࡛ᵓᡂࡉࢀࠊࢭࣥࢺࣛࣝࢻࢢ࣐࡟స⏝ࡋ⸆ຠࢆ♧ ࡍ་⸆ရࡢࡇ࡜ࢆᣦࡍࠋ᰾㓟་⸆ရࡣᚑ᮶ࡢపศᏊ་⸆ရ࡜␗࡞ࡿస⏝ᶵᗎ࡛⸆ຠࢆ♧ࡍ ࡓࡵࠊࡀࢇ࡞࡝ࡢ㞴἞ᛶ⑌ᝈ἞⒪⸆࡜࡞ࡾᚓࡿࠊࡲࡓࠊ᪤▱་⸆ရ࡜ే⏝ࡍࡿࡇ࡜࡛ࡼࡾ 㧗࠸ຠᯝࡀᮇᚅ࡛ࡁࡿࡇ࡜࠿ࡽࠊ⌧ᅾୡ⏺୰࡛⢭ຊⓗ࡟◊✲ࡀ⾜ࢃࢀ࡚࠸ࡿࠋ᰾㓟་⸆ࡣ ኱ࡁࡃ5 ✀࡟ศࡅࡽࢀࡿ (Table 2-1)ࠋDNA ࡢ㌿෗ᅉᏊ࡟స⏝ࡍࡿࡇ࡜࡛㌿෗ࢆ㜼ᐖࡍࡿ ࢹࢥ࢖᰾㓟ࠊmRNA ࡟స⏝ࡋ࡚⩻ヂࢆᢚไࡍࡿ miRNA ࡸ siRNAࠊ࢔ࣥࢳࢭࣥࢫ᰾㓟ࠊࢱ ࣥࣃࢡ㉁࡜┦஫స⏝ࡋຠᯝࢆ♧ࡍ࢔ࣉࢱ࣐࣮ࡢ5 ✀࡛࠶ࡿࠋ

Table 2-1. ᰾㓟་⸆ရࡢศ㢮࠾ࡼࡧస⏝ᶵᗎ

ࡋ࠿ࡋࠊୖᕷ࡟⮳ࡗ࡚࠸ࡿ᰾㓟་⸆ࡣࢃࡎ࠿ 6 ရ┠ࠊ✀㢮࡜ࡋ࡚ࡣ࢔ࣥࢳࢭࣥࢫ᰾㓟࡜ ࢔ࣉࢱ࣐࣮ࡢࡳ࡛࠶ࡿ (Table 2-2) 2)_ࠋ

4

ୡ⏺࡛ึࡵ࡚ୖᕷࡉࢀࡓ᰾㓟་⸆ရ࡛࠶ࡿ Vitravene (୍⯡ྡ Fomivirsen) ࡣࠊ࢔ࣥࢳࢭ ࣥࢫ᰾㓟࡛࠶ࡿࠋVitravene ࡣ࣍ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ྜࢆ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺ⤖ྜ࡟ಟ㣭ࡋࡓ ඲㛗21 ሷᇶ࡛ᵓᡂࡉࢀࠊImmediate early antigen 2 (IE2) mRNA ࢆᶆⓗ࡜ࡍࡿࢧ࢖ࢺ࣓࢞ ࣟ࢘࢖ࣝࢫ⥙⭷⅖἞⒪⸆࡛࠶ࡿࠋࢧ࢖ࢺ࣓࢞ࣟ࢘࢖ࣝࢫࡣ㏻ᖖࠊಖ⳦ࡋ࡚࠸࡚ࡶ⑕≧ࡣ࡞ࡃࠊ HIV ឤᰁ࡞࡝ච␿୙඲ࡢሙྜ࡟Ⓨ⑕ࡋࠊ⥙⭷⅖ࢆ⏕ࡌࡿࠋࢧ࢖ࢺ࣓࢞ࣟ࢘࢖ࣝࢫࡀቑṪࡍࡿ 㝿࡟IE2 ࡢⓎ⌧ࡀ㔜せ࡞ࡓࡵࠊVitravene ࡀ IE2 mRNA ࡟㑅ᢥⓗ࡟⤖ྜࠊࣜ࣎ࢯ࣮࣒ࡢ mRNA ࡢㄞࡳྲྀࡾࢆ㜼ᐖࡍࡿࡇ࡜࡛ IE2 ࡢⓎ⌧ࡀᢚไࡉࢀࠊࢧ࢖ࢺ࣓࢞ࣟ࢘࢖ࣝࢫࡢቑṪࢆ㜼ᐖ ࡍࡿࠋ1998 ᖺ࡟⡿ᅜ࡛ᢎㄆᚋࠊḢᕞ࡛ࡶᢎㄆࡉࢀࡓࡀࠊࢽ࣮ࢬࡀ࡞ࡃ࡞ࡾࠊ2011 ᖺ 6 ᭶࡟ ㈍኎ࡀ୰Ṇࡉࢀࡓࠋ 2004 ᖺ࡟ㄆྍࢆཷࡅ࡚ୖᕷࡉࢀࡓ Macugen (୍⯡ྡ Pegaptanib) ࡣࠊ඲㛗 27 ሷᇶࡢ࢔ ࣉࢱ࣐࣮࡛࠶ࡾࠊຍ㱋㯤ᩬ⑕἞⒪⸆࡛࠶ࡿࠋࣉࣜࣥࢾࢡࣞ࢜ࢩࢻ (adenosine, guanosine) ࡢ 2′఩Ỉ㓟ᇶࡀ࣓ࢳࣝ໬ࠊࣆ࣑ࣜࢪࣥሷᇶ (cytidine, uridine) ࡢ 2′఩Ỉ㓟ᇶࡀࣇࢵ⣲࡟⨨᥮ࡉ ࢀࠊ5′ᮎ➃࡟ 40 kDa ࡢ polyethylene glycol ࢆ௜ຍࡋ࡚࠸ࡿࠋ㧗㱋⪅ࡢኻ᫂ࡸどຊపୗࡢ୺ ࡞ཎᅉ࡛࠶ࡿຍ㱋㯤ᩬ⑕࡟ࡣⁱฟᆺ࡜ጤ⦰ᆺࡢ 2 ✀㢮ࡀ࠶ࡾࠊ≉࡟ⁱฟᆺࡣ῝้࡛ࠊ⥙⭷ ࡢୗࡢ⬦⤡⭷࡟࡛ࡁࡓࡶࢁ࠸⾑⟶࠿ࡽฟ⾑ࡍࡿࡇ࡜࠿ࡽ㉳ࡁࡿࠋMacugen ࡣ⾑⟶᪂⏕࡟㛵 ୚ࡍࡿ⾑⟶ෆ⓶⣽⬊ቑṪᅉᏊ165 (VEGF165) ࡟⤖ྜࡋࠊࡑࡢᶵ⬟ࢆ㜼ᐖࡍࡿࠋࡇࢀ࡟ࡼࡾ ⾑⟶᪂⏕ࡀ㜼ᐖࡉࢀࡿࡓࡵࠊどຊపୗࢆ⦆ࡸ࠿࡟ࡍࡿຠᯝࡀ࠶ࡿࠋ

2013 ᖺ࡟࢔࣓ࣜ࢝ࡢ FDA ࡣࠊKynamro (୍⯡ྡ Mipomersen) ࡟ᑐࡋ࡚㝈ᐃⓗ࡞ㄆྍࢆ ⾜ࡗࡓࠋKynamro ࡣ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺ⤖ྜ࡛඲࡚ࡢࢾࢡࣞ࢜ࢩࢻࡀ㐃⤖ࡋ࡚࠾ࡾࠊ඲㛗 20 ሷᇶࡢ࠺ࡕ 5′ᮎ➃࡜ 3′ᮎ➃ࡢ 5 ሷᇶศࡀ 2′఩ methoxyethoxy (MOE) ಟ㣭ࡉࢀࡓ࣮ࣜ࣎ ࢫࠊ୰ኸ㒊ࡀࢹ࢜࢟ࢩ࣮ࣜ࣎ࢫ࡛ᵓᡂࡉࢀ࡚࠸ࡿࢠࣕࣉ࣐࣮ᵓ㐀ࢆ࡜ࡗ࡚࠸ࡿࠋKynamro ࡣ lipoproteinn ࡢ 1 ࡘ࡛࠶ࡿ apolipoprotein B (ApoB) mRNA ࢆᶆⓗ࡜ࡋ࡚࠾ࡾࠊApoB mRNA ࡜஧ᮏ㙐ࢆᙧᡂࡋࡓᚋࠊRNA-DNA ࣁ࢖ࣈࣜࢵࢻ஧ᮏ㙐ࢆㄆ㆑ࡋ࡚ RNA 㙐ࢆษ᩿ࡍࡿ RNase H ࡢാࡁ࡟ࡼࡾ ApoB mRNA ࡀษ᩿ࡉࢀࡿࡇ࡜࡛ ApoB ࡢⓎ⌧ࢆᢚไࡍࡿࠋᐙ᪘ᛶ 㧗ࢥࣞࢫࢸ࣮ࣟࣝ⾑⑕ࡣlow density lipoprotein (LDL) ཷᐜయ㛵㐃㑇ఏᏊࡢኚ␗ࡀ⑓ᅉ࡛࠶ ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࠋࡇࢀࡲ࡛పศᏊ⸆࡛࠶ࡿࢫࢱࢳࣥ⣔་⸆ရࢆᑐ⑕⸆࡜ࡋ࡚ ⏝࠸࡚࠸ࡓࡀࠊLDL ཷᐜయ࡟ኚ␗ࢆ࡛࣍ࣔ᭷ࡋ࡚࠸ࡿ㔜⑕ᝈ⪅࡟ᑐࡋ࡚ࡣ༑ศ࡞ຠᯝࡀぢ ࡽࢀ࡞࠸ࡇ࡜ࡀከࡃࠊKynamro ࡜ࢫࢱࢳࣥ⣔་⸆ရࡢే⏝ࡍࡿࡇ࡜࡛ຠᯝⓗ࡞἞⒪ࡀྍ⬟ ࡟࡞ࡿࡇ࡜࠿ࡽୖᕷ࡟⮳ࡗࡓࠋ

Exondys 51 (୍⯡ྡ Eteplirsen) ࡣࠊ඲㛗 30 mer ࡢࣔࣝ࣍ࣜࣀ᰾㓟࡛ᵓᡂࡉࢀࡓ࢜ࣜࢦ ࢾࢡࣞ࢜ࢳࢻ࡛ࠊ࢔ࣥࢳࢭࣥࢫ᰾㓟࡜ࡋ࡚ᶵ⬟ࡍࡿࠋࢹࣗࢩ࢙ࣥࢾᆺ➽ࢪࢫࢺࣟࣇ࢕࣮ࡣࢪ ࢫࢺࣟࣇ࢕ࣥ㑇ఏᏊ࡟Ḟᦆ࡞࡝ࡀ⏕ࡌࡿࡇ࡜࡛ࢪࢫࢺࣟࣇ࢕ࣥࡀṇᖖ࡟ࡘࡃࡽࢀ࡞࠸ࡇ࡜ ࡀ⑓ᅉ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࠋExondys 51 ࡢᶆⓗࡣࢪࢫࢺࣟࣇ࢕ࣥ㑇ఏᏊ pre-mRNA ࡢ 51 ␒┠ࡢ࢚࢟ࢯ࡛ࣥ࠶ࡾࠊExondys 51 ࡀࢪࢫࢺࣟࣇ࢕ࣥ㑇ఏᏊ pre-pre-mRNA ࡟㑅 ᢥⓗ࡟స⏝ࡍࡿࡇ࡜࡛ࢫࣉࣛ࢖ࢩࣥࢢ᫬࡟࢚࢟ࢯࣥࡀㄞࡳ㣕ࡤࡉࢀࡿ (࢚࢟ࢯࣥࢫ࢟ࢵࣆ ࣥࢢ) ࡓࡵࠊ᏶඲㛗࡛ࡣ࡞࠸ࡶࡢࡢᶵ⬟ࢆ᭷ࡍࡿࢪࢫࢺࣟࣇ࢕ࣥࡀࡘࡃࡽࢀࡿࡇ࡜࠿ࡽࠊ᭷

5 ຠ࡞἞⒪⸆࡟࡞ࡾ࠺ࡿ࡜ᮇᚅࡉࢀ࡚࠸ࡿࠋ

Defitelio (୍⯡ྡ Defibrotide) ࡣࠊ2013 ᖺ࡟Ḣᕞ EMA ࠿ࡽࠊ2016 ᖺ 4 ᭶࡟࢔࣓ࣜ࢝ FDA ࠿ࡽㄆྍࢆཷࡅࡓ⫢୰ᚰ㟼⬦㛢ሰ⑕἞⒪⸆࡛࠶ࡿࠋDefitelio ࡣࣈࢱ⭠⤌⧊࠿ࡽᢳฟࡉࢀࡓ DNA ࠿ࡽㄪ〇ࡉࢀࡿ࣏ࣜࢾࢡࣞ࢜ࢳࢻ࡛࠶ࡾࠊ୍ᮏ㙐࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡀ 90%ࠊ2 ᮏ㙐 ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡀ 10%࡛ᵓᡂࡉࢀ࡚࠸ࡿࠋస⏝ᶵᗎ࡜ࡋ࡚ࠊ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ࣍ ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ྜ㒊఩ࡀ᭷ࡍࡿ㝜࢖࢜ࣥࡢࢱࣥࣃࢡ㉁࡜ࡢ㠀≉␗ⓗ࡞┦஫స⏝࡟ࡼࡿ࡜ ண᝿ࡉࢀ࡚࠸ࡿࠋ Spinraza (୍⯡ྡ Nusinersen) ࡣ඲㛗 18 ሷᇶࡢ࢔ࣥࢳࢭࣥࢫ᰾㓟࡟ศ㢮ࡉࢀࠊ࣍ࢫ࣍ࣟ ࢳ࢚࣮࢜ࢺ⤖ྜ࡛඲࡚ࡢࢾࢡࣞ࢜ࢩࢻࢆ㐃⤖ࡋࠊࡉࡽ࡟ࢾࢡࣞ࢜ࢩࢻࡢ2′఩Ỉ㓟ᇶࢆ MOE ಟ㣭ࠊcytidine ࡢ 5 ఩ࢆ࣓ࢳࣝ໬ࡋࡓಟ㣭᰾㓟ࢆ⏝࠸࡚࠸ࡿࠋஙඣᆺ⬨㧊ᛶ➽ⴎ⦰⑕ (SMA) ࡣSMN2 ࢱࣥࣃࢡ㉁ࡀṇᖖ࡟సࡽࢀ࡞࠸ࡇ࡜ࡀ⑓ᅉ࡛࠶ࡾࠊSpinraza ࡣ SMN2 mRNA ๓ 㥑యࡢ࢖ࣥࢺࣟࣥ 7 ࡟࠶ࡿ ISS-N1 ࢆㄆ㆑ࡋ࡚≉␗ⓗ࡟⤖ྜࡍࡿࡇ࡜࡛࢚࢟ࢯࣥࢫ࢟ࢵࣆ ࣥࢢࡀ㉳ࡇࡾࠊ᏶඲㛗࡛ࡣ࡞࠸ࡶࡢࡢᶵ⬟ࢆ᭷ࡍࡿࢱࣥࣃࢡ㉁ࡀ⏘⏕ࡉࢀࡿࡇ࡜࡛⑕≧ࡀ ᨵၿࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ2016 ᖺ 10 ᭶࡟࢔࣓ࣜ࢝ FDA ࡼࡾㄆྍࡉࢀࡓ᭱ࡶ᪂ࡋ࠸᰾㓟 ་⸆࡛࠶ࡿࠋ

6

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2 ⠇ పศᏊᖸ΅ RNA (siRNA)

RNA ᖸ΅ (RNA interference, RNAi) ࡣࠊ1998 ᖺ࡟ Fire ࡜ Mello ࡽ࡟ࡼࡗ࡚ሗ࿌ࡉࢀࡓ ⏕య཯ᛂ࡛࠶ࡿ3)_{ࠋRNAi ࡟ࡘ࠸࡚ࡣࠊࡇࢀࡲ࡛࡟௨ୗࡢࡼ࠺࡞ᶵᵓࢆ⤒ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟}

ࡉࢀ࡚࠸ࡿ (Figure 2-2) 4)_ࠋ

Figure 2-2. RNAi ᶵᵓ

 ⣽⬊ෆ࡟ᑟධࡉࢀࡓ㛗㙐஧ᮏ㙐RNA (double-stranded RNA, dsRNA) ࡣࠊRNase III ࣇ࢓ ࣑࣮ࣜࢱࣥࣃࢡ㉁࡛࠶ࡿ Dicer ࡟ࡼࡗ࡚ษ᩿ࡉࢀࠊࡑࢀࡒࢀࡢ㙐ࡢ 3′ᮎ➃࡟ 2 ሷᇶ✺ฟ㒊 (ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࠶ࡿ࠸ࡣ࣮࢜ࣂ࣮ࣁࣥࢢ) ࢆᣢࡘ 19-23 ሷᇶ⛬ᗘࡢపศᏊᖸ΅ RNA (small interfering RNA, siRNA) ࡜࡞ࡿࠋࡑࡢᚋࠊsiRNA ࡣ RNA ㄏᑟᆺࢧ࢖ࣞࣥࢩࣥࢢ」ྜ య (RNA induced silencing complex, RISC) ࡢ୺せࢱࣥࣃࢡ㉁࡛࠶ࡿ Argonaute 2 (Ago2) ࢱࣥࣃࢡ㉁࡬࡜㍺㏦ࡉࢀࡿࠋAgo2 ࢱࣥࣃࢡ㉁ࡣ㏻ᖖࠊ㛢ࡌࡓᵓ㐀ࢆ࡜ࡗ࡚࠸ࡿࡀࠊATP ౫ Ꮡⓗ࡞ศᏊࢩ࡛ࣕ࣌ࣟࣥ࠶ࡿHsp70/Hsc90 ࡢാࡁ࡟ࡼࡗ࡚ᵓ㐀ࡀኚ໬ࡋࠊsiRNA ࢆྲྀࡾ㎸ ࡴࠋ୍᪉ࡢ㙐 (ࢭࣥࢫ㙐) ࡣ Ago2 ࡢᣢࡘࢫࣛ࢖ࢧ࣮άᛶ࡟ࡼࡾษ᩿ࡉࢀࡓᚋ࡟ Ago2 ࠿ࡽ ᤼ฟࠊศゎࡉࢀࡿࠋAgo2 ෆ࡟ṧࡗࡓ㙐 (࢔ࣥࢳࢭࣥࢫ㙐) ࡣࠊAgo2 ࡜ඹ࡟ᡂ⇍ᆺ RISC ࢆ ᙧᡂࡍࡿࠋࢭࣥࢫ㙐࡜࢔ࣥࢳࢭࣥࢫ㙐ࢆ༊ูࡍࡿᶵᵓࡣࣄࢺ࡛ࡣ࠸ࡲࡔゎ᫂ࡉࢀ࡚࠸࡞࠸ࠋ ࡋ࠿ࡋࠊࢩࣙ࢘ࢪࣙ࢘ࣂ࢚࡟ࡘ࠸࡚ࡣヲ⣽࡟ሗ࿌ࡉࢀ࡚࠸ࡿࠋࢩࣙ࢘ࢪࣙ࢘ࣂ࢚࡛Ⓨ⌧ࡋ࡚ ࠸ࡿAgo2 ࡟ siRNA ࡀྲྀࡾ㎸ࡲࢀࡿ㝿ࠊsiRNA ࡣ Dicer-2 ཬࡧ஧ᮏ㙐 RNA ⤖ྜࢱࣥࣃࢡ࡛ ࠶ࡿR2D2 ࡜ඹ࡟ RISC–loading complex (RLC) ࢆᙧᡂࡋࠊ࡝ࡕࡽࡢ㙐ࡢ 5′ᮎ➃ࡀࡼࡾ୙ Ᏻᐃ࠿༊ูࡍࡿࠋࡑࡢ㝿ࠊ5′ᮎ➃ࡀࡼࡾ୙Ᏻᐃ࡞㙐ࢆ࢔ࣥࢳࢭࣥࢫ㙐ࠊࡼࡾᏳᐃ࡞㙐ࢆࢭࣥ ࢫ㙐࡜ࡋ࡚ㄆ㆑ࡍࡿࠋࣄࢺࡢሙྜࡶྠᵝ࡟ 5′ᮎ➃ࡢ୙Ᏻᐃᛶ࡟ࡼࡗ࡚㙐ࡀ༊ูࡉࢀࡿ࡜⪃ ࠼ࡽࢀ࡚࠸ࡿࠋᡂ⇍໬RISC ෆ࡟ṧࡗࡓ࢔ࣥࢳࢭࣥࢫ㙐ࡣ┦⿵ⓗ࡞㓄ิࢆᣢࡘᶆⓗ mRNA ࡜᏶඲┦⿵ⓗ࡟Ỉ⣲⤖ྜࡋࠊࢫࣛ࢖ࢧ࣮άᛶ࡟ࡼࡾ࢔ࣥࢳࢭࣥࢫ㙐ࡢ5′ᮎ➃࠿ࡽ 10 ሷᇶ┠ ࡜11 ሷᇶ┠࡟┦⿵ⓗ࡞ᶆⓗ mRNA 㙐ࡀษ᩿ࡉࢀࡿࡇ࡜࡛㑇ఏᏊⓎ⌧ࡀᢚไࡉࢀࡿࠋ

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3 ⠇ Argonaute ࢱࣥࣃࢡ㉁

RNAi ᶵᵓ࡟࠾࠸࡚㔜せ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠸ࡿ Ago2 ࡣࠊPIWI ࢻ࣓࢖ࣥࠊMID ࢻ࣓࢖ࣥࠊ PAZ ࢻ࣓࢖ࣥࠊN ᮎ➃ࢻ࣓࢖ࣥࡢ 4 ࡘࡢࢻ࣓࢖ࣥ࡜ L1ࠊL2 ࣮ࣜࣥ࢝ࡢ 2 ࡘࡢ࣮ࣜࣥ࢝࠿ࡽ ᵓᡂࡉࢀ࡚࠸ࡿ (Figure 2-3) 5)_ࠋ

Figure 2-3. ᡂ⇍ᆺ RISC ࡟࠾ࡅࡿ Ago2 ࢱࣥࣃࢡ㉁

PIWI ࢻ࣓࢖ࣥࡣ RNase H ࡟㢮ఝࡋࡓᵓ㐀ࢆࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊࢫࣛ࢖ࢧ࣮άᛶࢆ᭷ࡋ ࡚࠸ࡿ5)_{ࠋsiRNA ࡀ Ago2 ࡟ྲྀࡾ㎸ࡲࢀ࡚ᡂ⇍ᆺ RISC ࡀᙧᡂࡉࢀࡓᚋࠊᡂ⇍ᆺ RISC ෆࡢ}

࢔ࣥࢳࢭࣥࢫ㙐ࡀ┦⿵ⓗ࡞㓄ิࢆᣢࡘᶆⓗmRNA ࡜᏶඲┦⿵ⓗ࡟Ỉ⣲⤖ྜࡋࡓ㝿ࠊPIWI ࢻ ࣓࢖ࣥࡀ᭷ࡍࡿࢫࣛ࢖ࢧ࣮άᛶ࡟ࡼࡗ࡚ᶆⓗmRNA 㙐ࡀษ᩿ࡉࢀࡿࠋࡲࡓࠊAgo2 ࡢ siRNA ྲྀࡾ㎸ࡳ᫬ࡢ࢔ࣥࢳࢭࣥࢫ㙐5′ᮎ➃ࡢㄆ㆑࠾ࡼࡧᅛᐃ࡟ PIWI ࢻ࣓࢖ࣥ㏆ഐࡢࣜࢪࣥṧᇶࡀ 㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࠋ MID ࢻ࣓࢖ࣥࡣ PIWI ࢻ࣓࢖ࣥ࡜ࡢቃ⏺㡿ᇦ࡛࢔ࣥࢳࢭࣥࢫ㙐ࡢ 5′ᮎ➃ࣜࣥ㓟ᇶࢆㄆ㆑ࠊ ┦஫స⏝ࡍࡿࡇ࡜࡟ࡼࡾAgo2 ࡟࢔ࣥࢳࢭࣥࢫ㙐ࢆᅛᐃࡍࡿ6)_{ࠋཎ᰾⏕≀࡜┿᰾⏕≀࡛ࡣ࢔} ࣥࢳࢭࣥࢫ㙐ࡢᅛᐃᵝᘧࡀ␗࡞ࡾࠊཎ᰾⏕≀࡛ࡣ࣐ࢢࢿࢩ࣒࢘࢖࢜ࣥࢆ௓ࡋ࡚ࣜࣥ㓟ᇶ࡜ ┦஫స⏝ࡋ࡚ᅛᐃ໬ࡉࢀ࡚࠸ࡿࡢ࡟ᑐࡋࠊ┿᰾⏕≀ࡢሙྜࡣ┿᰾⏕≀࡟ࡢࡳಖᏑࡉࢀ࡚࠸ ࡿࣜࢪࣥṧᇶࡢ࢔࣑ࣀᇶࡀࣜࣥ㓟ᇶ࡜┦஫స⏝ࡍࡿࡇ࡜࡛ᅛᐃ໬ࡀ⾜ࢃࢀ࡚࠸ࡿࠋ PAZ ࢻ࣓࢖ࣥࡣࠊⰾ㤶᪘࢔࣑ࣀ㓟ࡀᐦ㞟ࡋࡓ␯Ỉᛶ࣏ࢣࢵࢺࢆ᭷ࡋ࡚࠾ࡾࠊsiRNA ࡢᵓ 㐀ⓗ࡞≉ᚩ࡛࠶ࡿ 3′ᮎ➃ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡜┦஫స⏝ࡍࡿ 7)_{ࠋᡂ⇍໬ RISC ࡢ≧ែ࡛ࡣ} PAZ ࢻ࣓࢖ࣥࡀࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ㒊ࢆྲྀࡾ㎸ࢇ࡛࠸ࡿࡀࠊᶆⓗ mRNA ㄆ㆑᫬ࡣᅛᐃࡋ࡚ ࠸ࡓࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ㒊ࢆᨺฟࡍࡿࡓࡵࠊ࢔ࣥࢳࢭࣥࢫ㙐ࡣᶆⓗmRNA ࡜᏶඲┦⿵ⓗ࡟ Ỉ⣲⤖ྜࡍࡿࠋPAZ ࢻ࣓࢖ࣥࡣࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ㒊ࡢᅛᐃ࡜ᨺฟࡢ 2 ┦≧ែࢆ⧞ࡾ㏉ࡍ ࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠾ࡾࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡜PAZ ࢻ࣓࢖ࣥࡢ┦஫స⏝ࡢᙉࡉࡀ㑇ఏ ᏊⓎ⌧ᢚไ⬟࡟ᙳ㡪ࢆ୚࠼ࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿ (Figure 2-4)ࠋ Figure 2-4. PAZ ࢻ࣓࢖ࣥ 2 ┦≧ែ

8 N ᮎ➃ࢻ࣓࢖ࣥࡣ Ago2 ࡢ୰ᚰ㒊࡟ࡏࡾฟࡍᵓ㐀ࢆ࡜ࡗ࡚࠸ࡿ8)_{ࠋAgo2 ࡀ Hsp70/Hsc90} ࡢാࡁ࡟ࡼࡗ࡚ᵓ㐀ኚ໬ࡋࠊsiRNA ࢆྲྀࡾ㎸ࢇࡔ㝿ࠊsiRNA ࡢ஧ᮏ㙐㛫࡟ࡡࡌ㎸ࡴࡼ࠺࡟ N ᮎ➃ࢻ࣓࢖ࣥࡀ఩⨨ࡋ❧య㞀ᐖࢆ୚࠼ࡿࡇ࡜࠿ࡽࠊ஧ᮏ㙐 siRNA ࡢゎ㞳ࠊ୍ᮏ㙐໬ࢆಁ 㐍ࡍࡿാࡁࡀ࠶ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ

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4 ⠇ siRNA ࡢ⮫ᗋᛂ⏝

1998 ᖺ࡟ Fire ࡜ Mello ࡽ࡟ࡼࡗ࡚ RNAi ࡀⓎ⾲ࡉࢀ࡚࠿ࡽ⌧ᅾ࡟⮳ࡿࡲ࡛ࠊsiRNA ࡣ་ ⸆ရ࡜ࡋ࡚ᐇ⏝໬ࡉࢀ࡚࠸࡞࠸ࠋsiRNA ࡀ࠸ࡲࡔ་⸆ရ໬࡟⮳ࡗ࡚࠸࡞࠸ཎᅉ࡜ࡋ࡚ࠊi) ⏕ యෆ᰾㓟ศゎ㓝⣲࡟ࡼࡾศゎࢆཷࡅࡿࠊii) ᶆⓗ⣽⬊࡬ࡢ㍺㏦ࢩࢫࢸ࣒ࡀ☜❧ࡉࢀ࡚࠸࡞࠸ࠊ iii) ᶆⓗ࡜␗࡞ࡿ㑇ఏᏊࡢⓎ⌧ࢆᢚไࡍࡿࠊ࡞࡝ࡢၥ㢟Ⅼࡀᣲࡆࡽࢀࡿࠋࡋ࠿ࡋࠊ㏆ᖺ࡟࡞ ࡾ୺࡟siRNA ࡢ㍺㏦ἲࡢ㛤Ⓨ࡟ࡼࡾࠊ⮫ᗋẁ㝵࡟⮳ࡗ࡚࠸ࡿ siRNA ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ 㑇ఏᛶATTR ࢔࣑ࣟࢻ࣮ࢩࢫࡣࠊࢺࣛࣥࢫࢧ࢖ࣞࢳࣥ (TTR) 㑇ఏᏊࡢኚ␗ࡀཎᅉ࡛࠶ࡿ ᖖᰁⰍయඃᛶࡢ㑇ఏᏊ⑌ᝈࡢࡇ࡜࡛ࠊࡇࢀࡲ࡛⫢⛣᳜ࡀ᭷ຠ࡞἞⒪ἲ࡜ࡋ࡚⾜ࢃࢀ࡚ࡁࡓࠋ ࡋ࠿ࡋ㏆ᖺࠊ㑇ఏᛶ⑌ᝈ࡟㛵ࡍࡿ◊✲ࡀ㐍ࡳࠊ㑇ఏᛶATTR ࢔࣑ࣟࢻ࣮ࢩࢫࡢཎᅉ㑇ఏᏊ࡛ ࠶ࡿTTR 㑇ఏᏊ mRNA ࡢⓎ⌧ࢆᢚไࡍࡿࡇ࡜࡛ࠊ᫂ࡽ࠿࡞ࣇ࢙ࣀࢱ࢖ࣉࡀぢࡽࢀ࡞࠿ࡗ ࡓࡇ࡜࠿ࡽࠊTTR 㑇ఏᏊ mRNA ࢆᶆⓗ࡜ࡋࡓ siRNA ࢆ⏝࠸ࡓ㑇ఏᏊ἞⒪ࡢ⮫ᗋࡀ⾜ࢃࢀ ࡚࠸ࡿ9)_{ࠋ⮫ᗋヨ㦂ࡣAlnylam Pharmaceuticals ♫ࢆ୰ᚰ࡟⾜ࢃࢀ࡚࠾ࡾࠊ⬡㉁ࢼࣀ⢏Ꮚࢆ} ⏝࠸࡚siRNA ࢆຠ⋡ⓗ࡟㍺㏦ࡍࡿࡇ࡜࡛ࠊsiRNA ࢆ⏕యෆ᰾㓟ศゎ㓝⣲࡟ࡼࡿศゎࢆᅇ㑊 ࡋ࡞ࡀࡽᶆⓗ࡜࡞ࡿ⫢⮚࡬㍺㏦ࡍࡿࡇ࡜࡟ᡂຌࡋ࡚࠸ࡿࠋ⌧ᅾࠊPhase 3 ࡢẁ㝵࡟࠶ࡿࠋ ࡲࡓࠊAlnylam ♫ࡣ N-࢔ࢭࢳࣝ࢞ࣛࢡࢺࢧ࣑ࣥ࡜⫢⣽⬊⾲㠃࡟Ⓨ⌧ࡋ࡚࠸ࡿ࢔ࢩ࢔ࣟ⢾ ࢱࣥࣃࢡ㉁ཷᐜయ࡜ࡢ㧗࠸ぶ࿴ᛶࢆ฼⏝ࡋࠊsiRNA ࡢᮎ➃㒊࡟ N-࢔ࢭࢳࣝ࢞ࣛࢡࢺࢧ࣑ࣥ ࢆ௜ຍࡍࡿࡇ࡜࡛⬡㉁ࢼࣀ⢏Ꮚࡢᵝ࡞㍺㏦ศᏊࢆ⏝࠸ࡿࡇ࡜࡞ࡃࠊsiRNA ࡢ⫢⣽⬊࡬ࡢ㍺ ㏦ࢆྍ⬟࡟ࡋࡓ10)_{ࠋN-࢔ࢭࢳࣝ࢞ࣛࢡࢺࢧ࣑ࣥಟ㣭ࡣ siRNA ࡢ඲㌟ᢞ୚ࢆྍ⬟࡟ࡋࠊ⌧ᅾࠊ} 㧗ࢥࣞࢫࢸ࣮ࣟࣝ⾑⑕἞⒪⸆࡜ࡋ࡚Phase 2 ࡢẁ㝵࡟࠶ࡿࠋ

9

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5 ⠇ ໬Ꮫಟ㣭᰾㓟࡜᰾㓟་⸆

ࡇࢀࡲ࡛࡟ୖᕷࡉࢀࡓ᰾㓟་⸆ࡣࠊ࠸ࡎࢀࡶ໬Ꮫಟ㣭ࡀ᪋ࡉࢀ࡚࠸ࡿࠋ᰾㓟ࡢᵓᡂ༢఩࡛ ࠶ࡿࢾࢡࣞ࢜ࢳࢻࡣሷᇶ㒊ࠊ⢾㒊ࠊ࣍ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ྜ㒊ࡢ኱ࡁࡃ 3 ࡘ࡟ศ㢮࡛ࡁࡿ (Figure 2-5)ࠋࡋ࠿ࡋࠊ᰾㓟་⸆࡬ࡢ฼⏝ࢆ┠ⓗ࡜ࡋࡓಟ㣭ἲࡣ⏬୍ⓗ࡞ࡶࡢ࡛ࡣ࡞ࡃࠊ᭱ 㐺࡞ಟ㣭ἲࢆศᏊࡈ࡜࡟⏝࠸ࡿᚲせࡀ࠶ࡿ11)_ࠋ Figure 2-5. ࢾࢡࣞ࢜ࢳࢻࡢಟ㣭㒊఩ Watson-Crick ሷᇶᑐ࡟ࡼࡿỈ⣲⤖ྜ࡟ࡼࡗ࡚᰾㓟࢜ࣜࢦ࣐࣮ࡣ஧ᮏ㙐ࢆᙧᡂࡍࡿࡇ࡜࠿ ࡽࠊỈ⣲⤖ྜ࡟ᙳ㡪ࢆ୚࠼ࡿሷᇶ㒊ࡢ໬Ꮫಟ㣭ࡣ࠶ࡲࡾ⾜ࢃࢀ࡚࠸࡞࠸ࠋࡋ࠿ࡋࠊSpinraza ࡢ5′ᮎ➃࡟ࡣࠊ5-methylcytidine ࡀᑟධࡉࢀ࡚࠸ࡿ (Figure 2-6)ࠋ⣽⬊ࡀ࢜ࣜࢦࢾࢡࣞ࢜ࢳ ࢻࢆྲྀࡾ㎸ࡴ㝿ࠊToll ᵝཷᐜయ 9 (TLR9) ࡀ 5′ᮎ➃࡟ CpG (cytidine-࣍ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ྜ-guanosine) ࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆㄆ㆑ࡋࠊච␿ᛂ⟅ࢆ♧ࡍࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋ Spinraza ࡣ 5′ᮎ➃࡟ CpG ࢆ᭷ࡍࡿ㓄ิ࡛࠶ࡿࡇ࡜࠿ࡽࠊ5′ᮎ➃ cytidine ࡢ 5 ఩ࢆ࣓ࢳࣝ໬ ࡍࡿࡇ࡜࡛ࠊTLR9 ࡢάᛶ໬ࢆᅇ㑊ࡋ࡚࠸ࡿࠋ Figure 2-6. cytidine ࡢ 5 ఩࣓ࢳࣝ໬ ⢾㒊ಟ㣭࡜ࡋ࡚2′఩Ỉ㓟ᇶࡢಟ㣭ࡀᗈࡃ⾜ࢃࢀ࡚࠸ࡿࠋMacugen ࡛ࡣ 2′఩࣓ࢺ࢟ࢩ໬ࡋ ࡓࣉࣜࣥࢾࢡࣞ࢜ࢩࢻ (adenosine, guanosine) ࡜ 2′఩ࣇࢵ⣲໬ࡋࡓࣆ࣑ࣜࢪ࣮ࣥࣜ࣎ࢫ (adenosine, uridine) ࢆ⏝࠸࡚࠸ࡿ (Figure 2-7)ࠋࡲࡓࠊKynamro ࡜ Spinraza ࡣ 2′఩ࢆ MOE ಟ㣭ࡋࡓಟ㣭᰾㓟ࢆᑟධࡋ࡚࠸ࡿࠋ⢾㒊2′఩Ỉ㓟ᇶࢆಟ㣭ࡍࡿࡇ࡜࡟ࡼࡗ࡚ࠊ⏕యෆ࡟Ꮡᅾ ࡍࡿ᰾㓟ศゎ㓝⣲࡟ᑐࡍࡿ⪏ᛶࡀྥୖࡍࡿࠋຍ࠼࡚ࠊExondys 51 ࡟࠾࠸࡚ࡣ⢾㒊ࢆ࣮ࣜ࣎ ࢫ⢾࠶ࡿ࠸ࡣࢹ࢜࢟ࢩ࣮ࣜ࣎ࢫ⢾࡛ࡣ࡞ࡃࠊࣔࣝ࣍ࣜࣀ᰾㓟ࢆ⏝࠸࡚࠸ࡿࠋࣔࣝ࣍ࣜࣀ᰾㓟 ࡶ⢾㒊2 ఩Ỉ㓟ᇶಟ㣭࡜ྠᵝ࡟ࠊ᰾㓟ศゎ㓝⣲⪏ᛶࡢྥୖࢆ┠ⓗ࡜ࡋ࡚౑⏝ࡉࢀ࡚࠸ࡿࠋ

10 Figure 2-7. 2′఩Ỉ㓟ᇶಟ㣭ἲ࠾ࡼࡧࣔࣝ࣍ࣜࣀ᰾㓟ࡢศᏊᵓ㐀 ࣍ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ྜ㒊ࡢಟ㣭ἲ࡜ࡋ࡚ࠊ㓟⣲ཎᏊ (O) ࢆ◲㯤ཎᏊ (S) ࡟⨨᥮ࡋࡓ࣍ࢫ ࣍ࣟࢳ࢚࣮࢜ࢺ⤖ྜࡀᗈࡃ⏝࠸ࡽࢀ࡚࠸ࡿ (Figure 2-8)ࠋVitravene ࡣࡍ࡭࡚ࡢ࣍ࢫ࣍ࢪ࢚ ࢫࢸࣝ⤖ྜࢆ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺ⤖ྜ࡟⨨᥮ࡋ࡚࠸ࡿࠋࡲࡓࠊKynamro ࡜ Spinraza ࡟࠾࠸ ࡚ࡶ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺಟ㣭ࡀ⾜ࢃࢀ࡚࠸ࡿࠋ᰾㓟ศゎ㓝⣲ࡣࣜࣥ㓟⤖ྜࢆㄆ㆑ࡋࠊຍỈศ ゎࡍࡿࡇ࡜࡛᰾㓟ศᏊࢆศゎࡍࡿࠋ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺ⤖ྜ࡬ࡢ⨨᥮࡟ࡼࡗ࡚⤖ྜ㒊ࡀ᰾ 㓟ศゎ㓝⣲࡟ㄆ㆑ࡉࢀ࡟ࡃ࠸ࠊ࠿ࡘࠊศゎࡉࢀ࡟ࡃࡃ࡞ࡿࡓࡵᗈࡃ⏝࠸ࡽࢀࡿಟ㣭ἲ࡜࡞ࡗ ࡓࠋ Figure 2-8. ࣍ࢫ࣍ࣟࢳ࢚࣮࢜ࢺಟ㣭

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6 ⠇ ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡢ໬Ꮫಟ㣭

ⴭ⪅ࡢᡤᒓࡍࡿ◊✲ᐊ࡛ࡣࠊsiRNA ࡢᵓ㐀ⓗ≉ᚩ࡛࠶ࡿࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟╔┠ࡋࠊ ✀ࠎࡢࢲࣥࢢࣜࣥࢢ࢚ࣥࢻಟ㣭siRNA ࢆྜᡂࡋࠊᙜヱ㒊఩ࡢ໬Ꮫಟ㣭ࡀ RNA ᖸ΅࡟ཬࡰ ࡍᙳ㡪࡟ࡘ࠸࡚ㄪᰝࢆ㐍ࡵ࡚ࡁࡓࠋࡇࢀࡲ࡛࡟ࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ㒊఩࡟ሷᇶ㒊Ḟᦆࢾࢡ ࣞ࢜ࢩࢻࡸⰾ㤶᪘໬ྜ≀ࢆᑟධࡍࡿࡇ࡜࡟ࡼࡾࠊ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆపୗࡉࡏࡿࡇ࡜࡞ࡃ ᰾㓟ศゎ㓝⣲⪏ᛶࢆ኱ᖜ࡟ᨵၿ࡛ࡁࡿࡇ࡜ࢆぢฟࡋ࡚࠸ࡿ (Figure 2-9) 12) – 16)_{ࠋ௚࡟ࡶࢲࣥ} ࢢࣜࣥࢢ࢚ࣥࢻ࡜PAZ ࢻ࣓࢖ࣥ࡜ࡢ┦஫స⏝࡟㛵ࡍࡿヲ⣽࡞◊✲ࡀ⾜ࢃࢀ࡚࠾ࡾࠊࢲࣥࢢ ࣜࣥࢢ࢚ࣥࢻ㒊఩ࡢ໬Ꮫಟ㣭࡟ࡼࡗ࡚᰾㓟ศゎ㓝⣲⪏ᛶࡢᨵၿࡔࡅ࡛࡞ࡃࠊ㑇ఏᏊⓎ⌧ᢚ ไάᛶࡶྥୖࡍࡿ࡜࠸࠺ሗ࿌ࡀ࡞ࡉࢀ࡚࠸ࡿ17) – 20)_ࠋ Figure 2-9. ᡤᒓ◊✲ᐊࡀሗ࿌ࡋࡓ໬Ꮫಟ㣭ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ

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7 ⠇ ሷᇶ㒊Ḟᦆࢹ࢜࢟ࢩࣜ࣎ࢾࢡࣞ࢜ࢩࢻ (dR

H

_{) ࡢᚑ᮶ྜᡂἲ}

ሷᇶ㒊Ḟᦆࢹ࢜࢟ࢩࣜ࣎ࢾࢡࣞ࢜ࢩࢻ (dRH_{) ࡢྜᡂἲ࡜ࡋ࡚ࡣࡇࢀࡲ࡛࡟௨ୗ࡟♧ࡍ} [1]~[4] ࡢᡭἲࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ [1] 2-Deoxy-D-ribose ࢆฟⓎཎᩱ࡜ࡋ࡚ࠊ࢔ࣝࢹࣄࢻᇶࢆ㑏ඖᚋࠊ㓟ᛶ᮲௳໬ຍ⇕ࡍࡿࡇ࡜࡛ 2 ᕤ⛬࡛ dRH_{ࢆྜᡂࡋ࡚࠸ࡿ (Scheme 2-1)} 21), 22)_{ࠋ⤒㊰A ࡣࠊ࢔ࣝࢹࣄࢻᇶࡢ㑏ඖ࡟Ỉ⣲} ໬࣍࢘⣲ࢼࢺ࣒ࣜ࢘ࠊ㓟ᛶ᮲௳࡟2M ሷ㓟ࢆ⏝࠸࡚࠸ࡿ (඲཰⋡ 54%)ࠋ⤒㊰ B ࡣࢽࢵࢣࣝ ゐ፹ୗࠊ㧗 㧗ᅽ᮲௳࡛᥋ゐỈ⣲໬ࢆ⾜࠺ࡇ࡜࡛࢔ࣝࢹࣄࢻᇶࢆ㑏ඖࡋࠊ㓟ᛶ᮲௳࡜ࡋ࡚ p-ࢺ࢚ࣝࣥࢫࣝ࣍ࣥ㓟ࢆ⏝࠸ࡿࡇ࡜࡛⤒㊰A ࡼࡾ㧗཰⋡࡛ dRH_{ࢆᚓ࡚࠸ࡿ (඲཰⋡ 68%)ࠋ⤒} ㊰AࠊB ࡜ࡶ࡟ᑡ࡞࠸ᕤ⛬ᩘ࡛ dRH_{ࡀྜᡂྍ⬟࡛࠶ࡿࡀࠊ཯ᛂ᮲௳ࡀ㐣㓞࡛࠶ࡿࡇ࡜ࡀၥ} 㢟Ⅼ࡛࠶ࡿࠋ ⤒㊰ A ⤒㊰ B Scheme 2-1. 㛢⎔཯ᛂ࡟ࡼࡿ dRH_ྜᡂἲ [2] ࡲࡓࠊ2-Deoxy-D-ribose ࡢ 1 ఩ࡢỈ㓟ᇶࢆ⬺㞳ᇶ࡟ኚ᥮ࡋࡓᚋ࡟ࠊỈ⣲࡜⨨᥮ࡍࡿᡭἲࡀ ሗ࿌ࡉࢀ࡚࠸ࡿ (Scheme 2-2, 2-3) 23)_ࠋ

2-Deoxy-D-ribose ࡢ 1 ఩Ỉ㓟ᇶࢆ࣓ࢳࣝ໬ࡋࠊ3 ఩ཬࡧ 5 ఩Ỉ㓟ᇶࢆ Tol ᇶ࡛ಖㆤࡋࡓᚋ

࡟ࢺ࢚ࣜࢳࣝࢩࣛࣥࢆస⏝ࡉࡏࡿࡇ࡜࡛ 1 ఩ࢆ⬺㓟⣲໬ࡋࠊTol ᇶࢆ㝖ཤࡍࡿࡇ࡜࡟ࡼࡾ dRH_{ࢆᚓ࡚࠸ࡿ (඲ 4 ᕤ⛬ࠊ඲཰⋡ 61%)ࠋࡲࡓࠊ1 ఩࣓ࢺ࢟ࢩయࢆሷ⣲࡟⨨᥮ࡋࡓ 1 ఩ࢡ}

ࣟࣟయࢆ⤒⏤ࡋࡓᡭἲࡶሗ࿌ࡉࢀ࡚࠸ࡿ (඲ 5 ᕤ⛬ࠊ඲཰⋡ 41%)ࠋScheme 2-2ࠊ2-3 ࡟♧ ࡋࡓྜᡂἲࡣ཯ᛂ᮲௳ࡀ✜ࡸ࠿࡛࠶ࡿࡀࠊ཰⋡ࡀప࠸ࡇ࡜ࡀㄢ㢟࡛࠶ࡿࠋ

13 Scheme 2-2. 1 ఩Ỉ⣲ᑟධ࡟ࡼࡿ dRH_ྜᡂἲ Scheme 2-3. 1 ఩ࢡࣟࣟయࢆ⤒⏤ࡋࡓ 1 ఩Ỉ⣲ᑟධ࡟ࡼࡿ dRH_ྜᡂἲ [3] ࡲࡓࠊࢺ࣓ࣜࢳࣝࢩࣜࣝ໬๣ࢆ⏝࠸࡚ 1 ఩࣓ࢺ࢟ࢩᇶࡢ⬺㞳⬟ࢆྥୖࡉࡏࡓᡭἲࡀሗ࿌ ࡉࢀ࡚࠸ࡿ (Scheme 2-4) 24)_{ࠋ3 ఩ཬࡧ 5 ఩Ỉ㓟ᇶࡢಖㆤࢆ⾜ࢃࡎ 2 ᕤ⛬࡛ dR}H_{ࡢྜᡂࡀ࡛} ࡁࡿࠋࡲࡓࠊ඲཰⋡ 86%࡜཰⋡ࡶ㧗࠸ࡀࠊ⢭〇ࡢ㝿࡟㐣๫㔞ࡢ㝧࢖࢜ࣥ஺᥮ᶞ⬡ࢆ⏝࠸ࡿ ᚲせࡀ࠶ࡿࠋ Scheme 2-4. ࢺ࣓ࣜࢳࣝࢩࣜࣝ໬๣ࢆ⏝࠸ࡓ dRH_ྜᡂἲ  [4] ኳ↛᰾㓟ࡢ୍ࡘ࡛࠶ࡿ࢔ࢹࣀࢩࣥࢆฟⓎཎᩱ࡜ࡋࡓ dRH _{ྜᡂἲࡶሗ࿌ࡉࢀ࡚࠸ࡿ} (Scheme 2-5) 25)_{ࠋ⢾㒊Ỉ㓟ᇶࢆ࣋ࣥࢰ࢖ࣝᇶ࡛ಖㆤࡋࡓᚋࠊ㓑㓟୰↓Ỉ㓑㓟ࢆస⏝ࡉࡏࠊ} 100 ºC ࡟ຍ⇕ࡍࡿࡇ࡜࡛ሷᇶ㒊ࢆ㝖ཤ࡜࡜ࡶ࡟ 1 ఩࡟࢔ࢭࢺ࢟ࢩᇶࢆᑟධࡋࡓࠋ⥆࠸࡚ࠊ ࢔ࢭࢺ࢟ࢩᇶࢆࢳ࢚࢜ࢳࣝᇶ࡟⨨᥮ࡋࡓᚋࠊࣛࢿ࣮ࢽࢵࢣࣝࢆ⏝࠸ࡓ㑏ඖⓗ⬺◲཯ᛂ࡟ࡼ ࡗ࡚1 ఩ࢆỈ⣲໬ࡋ࡚࠸ࡿࠋᕤ⛬ᩘࡀ 5 ᕤ⛬࡜ከࡃࠊ඲཰⋡ 41%࡜୰⛬ᗘ࡛࠶ࡿࠋ

14

15

➨

➨

3 ❶ dR

H

_{ࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ࡜ᛶ㉁ホ౯}

➨

1 ⠇ ሷᇶ㒊⬺㞳཯ᛂࢆ฼⏝ࡋࡓ dR

H

_ࡢྜᡂ

ሷᇶ㒊Ḟᦆࢹ࢜࢟ࢩࣜ࣎ࢾࢡࣞ࢜ࢩࢻ (dRH_{; 1) ࡢ⡆౽ྜᡂἲࢆ㛤Ⓨࡍࡿ࡟࠶ࡓࡾࠊᮏ◊} ✲࡛ࡣCameron ࡽࡀ㛤Ⓨࡋࡓሷᇶ㒊⬺㞳཯ᛂ࡟ࡼࡗ࡚ᚓࡽࢀࡿࣇࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝࢆ⤒ ⏤ࡋࡓᡭἲࢆཧ⪃࡟ࡍࡿࡇ࡜࡟ࡋࡓ 26)_{ࠋࢳ࣑ࢪࣥ (2) ࡢ 3'఩࠾ࡼࡧ 5'఩Ỉ㓟ᇶࢆ TBDPS} ᇶ࡛ಖㆤࡋࠊ3',5'-di-O-TBDPS-ࢳ࣑ࢪࣥ (3) ࢆᚓࡓᚋࠊ◲㓟࢔ࣥࣔࢽ࣒࢘ᏑᅾୗࠊHMDS ୰ ຍ⇕㑏ὶࡋࡓࠋࡑࡢ⤖ᯝࠊ┠ⓗࡢࣇࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝ 4 ࡣ඲ࡃᚓࡽࢀࡎࠊ4 ࡢ 4 ఩Ỉ⣲ ࡜3 ఩ࢩࣜࣝ࢜࢟ࢩᇶࡀ⬺㞳ࡋࡓࣇࣛࣥㄏᑟయ 5 ࡀ 38%࡛ᚓࡽࢀࡓ (Scheme 3-1)ࠋ Scheme 3-1. 3 ࡢྜᡂ࡜ሷᇶ㒊⬺㞳཯ᛂ࡟ࡼࡿࣇࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝ 4 ࡬ࡢኚ᥮᳨ウ ࡇࡢ⌮⏤࡜ࡋ࡚ࠊ3',5'-di-O-TBDPS-ࢳ࣑ࢪࣥ (3) ࡢሷᇶ㒊⬺㞳཯ᛂ࡟ࡼࡗ࡚⏕ᡂࡋࡓࣇ ࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝ4 ࡀ HMDS ࡜཯ᛂࡋࠊ3 ఩⨨᥮ᇶୖ㓟⣲ࡢ TMS ໬ࡀ㐍⾜ࡍࡿࡇ࡜࡛ࠊ Ᏻᐃ࡞ࣇࣛࣥ࡬ࡢⰾ㤶⎔໬ࡀ㥑ືຊ࡜࡞ࡾࠊ4 ఩Ỉ⣲ࡢࣉࣟࢺࣥ࡜ࡋ࡚ࡢ⬺㞳ࢆకࡗ࡚ 3 ఩ ࢩࣜࣝ࢜࢟ࢩᇶࡀࢪࢩ࢚࣮ࣜࣝࢸࣝ࡜ࡋ࡚እࢀࡿࡇ࡜࡟ࡼࡾࣇࣛࣥㄏᑟయ 5 ࡀ⏕ᡂࡋࡓ࡜ ⪃࠼ࡽࢀࡓ (Scheme 3-2)ࠋ Scheme 3-2. ᝿ᐃࡉࢀࡿࣇࣛࣥㄏᑟయ 5 ࡢ⏕ᡂᶵᵓ

16  ࡑࡇ࡛ࠊ3 ఩ࡢ⬺ࢩࣜࣝ࢜࢟ࢩ໬ࢆᢚไࡍࡿࡓࡵ࡟ 5'఩Ỉ㓟ᇶࡢࡳࢆ TBDPS ᇶ࡛ಖㆤࡋ ࡓ 5'-O-TBDPS-ࢳ࣑ࢪࣥ (6) ࢆྜᡂࡋࠊୖグ࡜ྠ᮲௳ୗ࡛ሷᇶ㒊⬺㞳཯ᛂࢆ᳨ウࡋࡓ (Scheme 3-3)ࠋࡑࡢ⤖ᯝࠊTLC ศᯒ࡟࡚ᑐᛂࡍࡿࣇࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝ 7 ࡜ᛮࢃࢀࡿ໬ྜ ≀ࡢ⏕ᡂࡀ☜ㄆ࡛ࡁࡓࠋࡋ࠿ࡋࠊᢳฟᚋࡢࢩࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡿ⢭〇 ᫬࡟ࣇࣛࣥㄏᑟయ 5 ࡬ࡢኚ᥮ࡀ㐍⾜ࡋࠊ┠ⓗࡢ 7 ࡢ༢㞳ࠊ⢭〇ࡣᅔ㞴࡛࠶ࡗࡓࠋ Scheme 3-3. 6 ࡢྜᡂ࡜ሷᇶ㒊⬺㞳཯ᛂ࡟ࡼࡿࣇࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝ 7 ࡬ࡢኚ᥮཯ᛂ ࡑࡇ࡛ࠊ5'-O-TBDPS-ࢳ࣑ࢪࣥ (6) ࠿ࡽࡢሷᇶ㒊⬺㞳཯ᛂᚋ࡟㐃⥆ࡋ࡚ 3 ఩⨨᥮ᇶࡢ⬺ TMS ໬ࢆ⾜ࡗࡓᚋ࡟⢭〇ࡍࡿࡇ࡜࡟ࡋࡓࠋ ࡲࡓࠊࡇࢀࡲ࡛ሷᇶ㒊⬺㞳཯ᛂࡢ཯ᛂ᫬㛫ࡣCameron ࡽࡢሗ࿌ࢆཧ⪃࡟ࡋ࡚࠸ࡓࡀࠊ⏕ ᡂࡋࡓࣇࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝ7 ࡀ཯ᛂ⣔ෆ࡛᫬㛫⤒㐣࡜࡜ࡶ࡟ᑐᛂࡍࡿࣇࣛࣥㄏᑟయ 5 ࡟ ኚ᥮ࡉࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊHMDS ࡢ㔞ࡀᑡ࡞ࡃࠊ཯ᛂ⁐ᾮࡢ⃰ᗘࡀ㧗࠸ሙ ྜ࡟ࡶࣇࣛࣥㄏᑟయ࡬ࡢኚ᥮ࡀ㐍⾜ࡋࡸࡍ࠸ࡇ࡜ࡀุ᫂ࡋࡓࠋࡑࡇ࡛ࠊ཯ᛂ⁐ᾮࡢ⃰ᗘ࡞ࡽ ࡧ࡟཯ᛂ᫬㛫ࡢ᭱㐺໬ࢆ᳨ウࡋࠊࡑࡢ⤖ᯝ࠿ࡽ125 μMࠊ1.5 ᫬㛫ࡢ⤌ࡳྜࢃࡏࢆ᭱㐺᮲௳ ࡜ࡋࡓࠋ᭱㐺᮲௳ୗࠊࢳ࣑ࢪࣥㄏᑟయ 6 ࠿ࡽࢢ࣮ࣜ࢝ࣝ 7 ࡟ኚ᥮ࡋࡓᚋࠊⅣ㓟࣒࢝ࣜ࢘ࡢ ࣓ࢱࣀ࣮ࣝᠱ⃮ᾮࢆస⏝ࡉࡏ࡚3 ఩Ỉ㓟ᇶయ 8 ࡟ㄏᑟࡋࡓࠋࡋ࠿ࡋࠊ 8 ࡶ 7 ࡜ྠᵝ࡟ࢩ ࣜ࢝ࢤ࣒ࣝ࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ࡢ⢭〇᫬࡟ࣇࣛࣥㄏᑟయ 5 ࡟ኚ᥮ࡉࢀ࡚ࡋ࡚ࡋࡲ ࠸ࠊ༢㞳ࠊ⢭〇ࡣᅔ㞴࡛࠶ࡗࡓ (Scheme 3-4)ࠋ Scheme 3-4. 6 ࡢሷᇶ㒊⬺㞳཯ᛂ࡜㐃⥆ⓗ⬺ TMS ໬࡟ࡼࡿࣇࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝ 8 ࡢྜᡂ

17 ࡑࡇ࡛ࠊ3 ఩⨨᥮ᇶࡢ⬺ TMS ໬࡟ࣇࣛࣀ࢖ࢻࢢ࣮ࣜ࢝ࣝ 8 ࡢ༢㞳ࡣ⾜ࢃࡎࠊ㐃⥆ⓗ࡟᥋ ゐỈ⣲໬ࢆ⾜࠸ࠊdRH_{ㄏᑟయ9 ࡟ㄏᑟࡍࡿࡇ࡜࡟ࡋࡓࠋ5'-O-TBDPS-ࢳ࣑ࢪࣥ (6) ࠿ࡽ 8 ࡟} ኚ᥮ࡋࡓᚋࠊPd/C ࢆ⏝࠸ࡓ࣓ࢱࣀ࣮ࣝ୰࡛ࡢ୍⯡ⓗ࡞᥋ゐỈ⣲໬᮲௳࡟࡚஧㔜⤖ྜࡢ㑏ඖ ࢆ⾜ࡗࡓ࡜ࡇࢁࠊᡤᮃ9 ࡀ 36%ࡢ཰⋡࡛ᚓࡽࢀࡿ࡜࡜ࡶ࡟ࠊ⁐፹ࡢ࣓ࢱࣀ࣮ࣝ࡟ࡼࡿ Ferrier ᆺ཯ᛂࡀేⓎࡋࠊ1 ఩࡟࣓ࢺ࢟ࢩᇶࡀ⨨᥮ࡋࡓࢸࢺࣛࣄࢻࣟࣇࣛࣥㄏᑟయ 10 ࡀ཰⋡ 31%࡛ ᚓࡽࢀࡓ (Scheme 3-5)ࠋ Scheme 3-5. Pd/C ࢆ⏝࠸ࡓ࣓ࢱࣀ࣮ࣝ୰࡛ࡢ᥋ゐỈ⣲໬࡟ࡼࡿ஧㔜⤖ྜࡢ㑏ඖ཯ᛂ  ཯ᛂ⁐፹ࢆ࣓ࢱࣀ࣮ࣝ࠿ࡽᔞ㧗ࡃồ᰾ᛶࡀప࠸ࣉࣟࢺࣥᛶ⁐፹࡛࠶ࡿ࢖ࢯࣉࣟࣃࣀ࣮ࣝ ࡟ኚ᭦ࡋࡓ࡜ࡇࢁࠊ๪཯ᛂࡣ඲ࡃ㐍⾜ࡏࡎࠊ஧㔜⤖ྜࡢ㑏ඖ཯ᛂࡢࡳ㐍⾜ࡋࡓ (Scheme 3-6)ࠋ Scheme 3-6. Pd/C ࢆ⏝࠸ࡓ࢖ࢯࣉࣟࣃࣀ࣮ࣝ୰࡛ࡢ᥋ゐỈ⣲໬࡟ࡼࡿ஧㔜⤖ྜࡢ㑏ඖ཯ᛂ  ࡉࡽ࡟ࠊⅣ㓟࣒࢝ࣜ࢘ࡢ࣓ࢱࣀ࣮ࣝᠱ⃮ᾮࢆ⏝࠸ࡓ3 ఩ TMS ᇶࡢ㝖ཤ࠾ࡼࡧ஧㔜⤖ྜࡢ 㑏ඖ཯ᛂࡢ཯ᛂ᫬㛫ࡢ᭱㐺໬ࢆ⾜࠸ࠊ5'-O-TBDPS-ࢳ࣑ࢪࣥ (6) ࠿ࡽሷᇶ㒊⬺㞳཯ᛂࠊ3 ఩ TMS ᇶࡢ㝖ཤࠊPd/C-࢖ࢯࣉࣟࣃࣀ࣮ࣝࢆ⏝࠸ࡓ᥋ゐỈ⣲໬ࢆ㐃⥆ⓗ࡟⾜࠺ࡇ࡜࡛ࠊ5'-O-TBDPS-dRH _{(9) ࢆ཰⋡ 94%࡛ᚓࡿࡇ࡜ࡀ࡛ࡁࡓ (Scheme 3-7)ࠋ}

18 Scheme 3-7. 6 ࠿ࡽࡢ 5-O-TBDPS-dRH _{(9) ࡢྜᡂ} ᭱ᚋ࡟ࠊTBAF ࢆ⏝࠸࡚ TBDPS ᇶࡢ㝖ཤࢆ⾜࠸ࠊdRH _{(1) ࢆ཰⋡ 97%࡛ᚓࡓ (Scheme} 3-8)ࠋ Scheme 3-8. TBDPS ᇶࡢ㝖ཤ ᮏ◊✲࡛㛤ⓎࡋࡓdRH_{ࡢྜᡂ⤒㊰ࢆ௨ୗ࡟♧ࡍ (Scheme 3-9)ࠋ} ᕤ⛬ᩘ㸸5 ඲཰⋡㸸83% Scheme 3-9. ᮏ◊✲࡛㛤Ⓨࡋࡓ dRH_{ࡢྜᡂ⤒㊰}

19

➨

➨

2 ⠇ dR

H

_{ྵ᭷᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ}

 ๓⠇࡛dRH_{ࡢ⡆౽ྜᡂἲ࡟ࡘ࠸࡚㏙࡭ࡓࠋᮏᡭἲࢆ⏝࠸࡚ྜᡂࡋࡓdR}H _{(1)ࢆ⏝࠸ࠊᐃἲ} ࡟ᚑࡗ࡚᰾㓟⮬ືྜᡂ⏝ㄏᑟయ࡟ኚ᥮ࡋࡓ (Scheme 3-10)ࠋ1 ࡢ 5 ఩Ỉ㓟ᇶࢆ DMTr ᇶ࡛ 㑅ᢥⓗ࡟ಖㆤࡋࠊ5-O-DMTr-dRH _{(11) ࢆᐃ㔞ⓗ࡟ᚓࡓᚋࠊTHF ⁐፹୰ DIPEA ࢆሷᇶ࡜ࡋ࡚} ⏝࠸࡚ 3 ఩Ỉ㓟ᇶࡢளࣜࣥ㓟໬ࢆ⾜࠺ࡇ࡜࡟ࡼࡾࠊ᰾㓟⮬ືྜᡂ⏝࣍ࢫ࣍ࣟ࢔࣑ࢲ࢖ࢺయ 12 ࢆᚓࡓࠋࡲࡓࠊࢺࣜࢳࣝయ 11 ࡟ࣆࣜࢪࣥ⁐፹࡛↓Ỉࢥࣁࢡ㓟ࢆస⏝ࡉࡏࡿࡇ࡜࡛ 3 ఩ Ỉ㓟ᇶࢆࢫࢡࢩࢽࣝ໬ࡋࠊ⥆࠸࡚ࠊDMF ⁐፹୰ࠊEDC·HCl ࢆ⏝࠸࡚⬺Ỉ⦰ྜ࡟ࡼࡾ CPG ᶞ⬡ࢆ㐃⤖ࡋࡓࠋ᭱ᚋ࡟ࣆࣜࢪࣥ⁐፹୰DMAP ඹᏑୗ࡛↓Ỉ㓑㓟ࢆస⏝ࡉࡏࠊCPG ᶞ⬡ୖ ࡢᮍ཯ᛂࡢ࢔࣑ࣀᇶࢆ࢔ࢭࢳࣝᇶ࡛࢟ࣕࢵࣉࡍࡿࡇ࡜࡟ࡼࡾᡤᮃࡢdRH_{㐃⤖ᅛ┦ᢸయ14 ࢆ} ᚓࡓࠋ Scheme 3-10. ᰾㓟⮬ືྜᡂ⏝ dRH_{ㄏᑟయࡢྜᡂ} ᚓࡽࢀࡓ᰾㓟⮬ືྜᡂ⏝ㄏᑟయ (11ࠊ13) ࢆ⏝࠸ࠊ᰾㓟⮬ືྜᡂᶵ࡟࡚ 3′ᮎ➃࡟ dRH_ࢆ ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆྜᡂࡋࡓࠋsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢホ౯ࢆ Dual-luciferase reporter assay system ࢆ⏝࠸࡚⾜࠺ࡓࡵࠊRenilla luciferase mRNA ࢆᶆⓗ࡜ࡋࡓ siRNA ࡢ ࢔ࣥࢳࢭࣥࢫ㙐ཬࡧࢭࣥࢫ㙐㸦࢔ࣥࢳࢭࣥࢫ㙐࡟┦⿵ⓗ࡞㓄ิࢆ᭷ࡍࡿ㙐㸧ࢆㄪ〇ࡋࡓࠋࡲ ࡓࠊ᰾㓟ศゎ㓝⣲࡟ᑐࡍࡿ⪏ᛶホ౯⏝࡟࢔ࣥࢳࢭࣥࢫ㙐ࡢ 5′ᮎ➃࡟⺯ගศᏊ࡛࠶ࡿࣇࣝ࢜ ࣞࢭ࢖ࣥࢆᑟධࡋࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡶྜᡂࡋࡓࠋᚓࡽࢀࡓᅛ┦ୖࡢ࢜ࣜࢦࢾࢡࣞ࢜ࢳ ࢻࡣࠊ࢔ࣥࣔࢽ࢔Ỉ࡛ᅛ┦ᢸయ࠿ࡽࡢษࡾฟࡋ࡜ሷᇶ㒊ಖㆤᇶࡢ㝖ཤࢆ⾜ࡗࡓᚋࠊTBAF ࡟ ࡼࡾ⢾㒊ࡢಖㆤᇶࢆ㝖ཤࡋࡓࠋࡑࡢᚋࠊኚᛶ࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻ㟁ẼὋື࠾ࡼࡧ HPLC ࡟ ࡼࡿ⢭〇ࢆ㡰ḟ⾜ࡗࡓࠋMALDI-TOF/MS ࢆ⏝࠸ࡓ㉁㔞ศᯒ࡟ࡼࡗ࡚ࠊᵓ㐀ࢆ☜ㄆࡋࠊࡑࡢ ᚋࡢホ౯࡟⏝࠸ࡓ (Figure 3-1)ࠋ

20

21

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3 ⠇ ᰾㓟ศゎ㓝⣲⪏ᛶホ౯

 5′ᮎ➃࡟ࣇࣝ࢜ࣞࢭ࢖ࣥࠊ3′ᮎ➃࡟ dRH_dRH_{ࢆ᭷ࡍࡿ࢔ࣥࢳࢭࣥࢫ㙐࡟ᑐࡋࠊ3′→5′࢚࢟ࢯ} ࢾࢡࣞ࢔࣮ࢮࡢ୍ࡘ࡛࠶ࡿ࣊ࣅẘ࣍ࢫ࣍ࢪ࢚ࢫࢸ࣮ࣛࢮ (SVPD) ࢆస⏝ࡉࡏ࡚ࠊ୍ᮏ㙐᰾ 㓟ࡢ᰾㓟ศゎ㓝⣲⪏ᛶࢆホ౯ࡋࡓ (Figure 3-2)ࠋẚ㍑ᑐ↷࡜ࡋ࡚ࠊ3′ᮎ➃࡟ࢳ࣑ࢪࣥ (TT) ཬ ࡧሷᇶ㒊Ḟᦆࣜ࣎ࢾࢡࣞ࢜ࢩࢻ (RH_RH_{) ࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࢆ⏝࠸ࡓࠋ} Conditions Buffer: PBS (−) Concentration: 3 μM

Nuclease: 1 unit/mL snake venom phosphodiesterase Incubation temperature: 37 ºC Figure 3-2. ᰾㓟ศゎ㓝⣲⪏ᛶホ౯ࡢ⤖ᯝ  ホ౯ࡢ⤖ᯝࠊdRH_dRH_{ࢆ3′ᮎ➃࡟᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ᰾㓟ศゎ㓝⣲⪏ᛶࡣࠊTT ࠾} ࡼࡧRH_RH_{ࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡜ẚ㍑ࡋ࡚ඃࢀ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡇࡢ} ࡇ࡜࠿ࡽࠊ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡢ 3′ᮎ➃࡟ࢹ࢜࢟ࢩ࣮ࣜ࣎ࢫ⢾ࢆ㦵᱁࡜ࡍࡿಟ㣭᰾㓟ࢆᑟ ධࡍࡿࡇ࡜࡛࢚࢟ࢯࢾࢡࣞ࢔࣮ࢮ࡟ᑐࡍࡿ⪏ᛶࡀࡼࡾྥୖࡍࡿ࡜⪃࠼ࡽࢀࡓࠋ

22

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4 ⠇ ⇕ⓗᏳᐃᛶホ౯

᰾㓟ሷᇶࡣ260 nm ࡢගࢆ྾཰ࡍࡿᛶ㉁ࡀ࠶ࡿࠋࡋ࠿ࡋࠊᑐᛂࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ྠኈࡀ Ỉ⣲⤖ྜࢆᙧᡂࡋࠊ஧ᮏ㙐໬ࡋࡓሙྜࠊ᰾㓟ሷᇶ㛫ࡢ π-π ࢫࢱࢵ࢟ࣥࢢ┦஫స⏝࡟ࡼࡗ࡚ ஧ᮏ㙐᰾㓟඲యࡢ྾ගᗘࡢ⥲࿴ࡣࠊ᰾㓟ሷᇶࡑࢀࡒࢀࡢ྾ගᗘࡢ⥲࿴ࡼࡾపࡃ࡞ࡿࠋࡇࡢ᫬ࠊ ஧ᮏ㙐᰾㓟ࢆຍ⇕ࡍࡿ࡜ࠊỈ⣲⤖ྜࡀษ᩿ࡉࢀ୍࡚ᮏ㙐໬ࡍࡿࡓࡵࠊ᰾㓟ሷᇶࡀࡑࢀࡒࢀග ࢆ྾཰ࡋࠊ྾ගᗘࡀୖ᪼ࡍࡿࠋࡇࡢຍ⇕࡟ࡼࡿ஧ᮏ㙐᰾㓟ࡢ୍ᮏ㙐໬ࡢࡇ࡜ࢆ⼥ゎ࡜࿧ࡪࠋ ࡲࡓࠊ྾ගᗘࡢኚ໬ࢆ ᗘኚ໬࡜࠶ࢃࡏ࡚ࣉࣟࢵࢺࡍࡿࡇ࡜࡛⼥ゎ᭤⥺ࡀᚓࡽࢀࠊࢩࢢࣔ࢖ ࢻ᭤⥺ࢆ♧ࡍࠋ஧ᮏ㙐᰾㓟඲యࡢ50㸣ࡀ୍ᮏ㙐໬ࡍࡿ᫬ࡢ ᗘࢆ 50%⼥ゎ ᗘ (Tm) ࡜࿧ ࡧࠊ஧ᮏ㙐᰾㓟ࡢ⇕࡟ᑐࡍࡿᏳᐃᛶࡢᣦᶆ࡜࡞ࡿࠋ ሷᇶ㒊Ḟᦆࢹ࢜࢟ࢩࢾࢡࣞ࢜ࢩࢻ (dRH_{) ࢆ᭷ࡍࡿ siRNA ࡢ⇕ⓗᏳᐃᛶࢆホ౯ࡍࡿࡓࡵࠊ} siRNA ࡢ᪼ ࡟࡜ࡶ࡞࠺྾ගᗘࡢኚ໬ࢆ ᐃࡋࡓࠋẚ㍑ᑐ↷࡜ࡋ࡚ࠊ3′ᮎ➃࡟ࢳ࣑ࢪࣥ (TT) ཬࡧሷᇶ㒊Ḟᦆࣜ࣎ࢾࢡࣞ࢜ࢩࢻ (RH_RH_{) ࢆ᭷ࡍࡿ siRNA ࢆ⏝࠸ࡓࠋ} Conditions

Buffer: 10 mM NaH2PO4∙Na2HPO4 (pH 7.0)ࠊ100 mM NaCl

Concentration: 3 μM Figure 3-3. ⇕ⓗᏳᐃᛶホ౯ࡢ⤖ᯝ Table 3-1. ⟬ฟࡋࡓ Tm್ XX Tm (ºC) ΔTm (ºC) TT 82.7 㸫 RH_RH _{81.8 -0.9} dRH_dRH _{81.2 -1.5}

23 ࢩࢢࣔ࢖ࢻ᭤⥺ࡀᚓࡽࢀࡓࡇ࡜࠿ࡽࠊ3′ᮎ➃࡟ dRH_{ࢆ᭷ࡍࡿsiRNA ࡣ஧ᮏ㙐ࢆᙧᡂࡋ࡚} ࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊ୰⥺ἲ࡟࡚Tm್ࢆ⟬ฟࡋࡓ⤖ᯝࠊTm್ࡣࡑࢀࡒࢀ82.7 ºC (TT)ࠊ81.8 ºC (RH_RH_{)ࠊ81.2 ºC (dR}H_dRH_{) ࡛࠶ࡾࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࡢᑟධ࡟ࡼ} ࡗ࡚⇕ⓗᏳᐃᛶࡣࢃࡎ࠿࡟୙Ᏻᐃ໬ࡋࡓࠋࡇࢀࡣሷᇶ㒊ࡢḞᦆ࡟ࡼࡾ᰾㓟ሷᇶ㛫ࡢ π-π ࢫ ࢱࢵ࢟ࣥࢢ┦஫స⏝ࡀᙧᡂࡉࢀ࡞࠸ࡇ࡜࡟㉳ᅉࡍࡿ࡜⪃࠼ࡽࢀࡓࠋࡋ࠿ࡋࠊ3′ᮎ➃࡟ dRH_ࢆ ᭷ࡍࡿsiRNA ࡣẚ㍑ᑐ↷࡜ࡋ࡚⏝࠸ࡓ siRNA ࡜ྠᵝ࡟ᐊ ࡛஧ᮏ㙐ᵓ㐀ࢆ⥔ᣢࡋ࡚࠸ࡿࡇ ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ ௨ୖࡢ⤖ᯝ࠿ࡽࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࡢsiRNA3′ᮎ➃࡬ࡢᑟධࡣࠊsiRNA ࡢ஧ᮏ㙐ᵓ 㐀࡟኱ࡁ࡞ᙳ㡪ࢆཬࡰࡉ࡞࠸ࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ

24

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5 ⠇ 㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯

 ⇕ⓗᏳᐃᛶホ౯ࡢ⤖ᯝࡼࡾࠊሷᇶ㒊Ḟᦆಟ㣭యࢆᑟධࡋࡓsiRNA ࡣᐊ ࡛஧ᮏ㙐ᵓ㐀ࢆ ⥔ᣢࡋ࡚࠸ࡿࡇ࡜ࡀุ᫂ࡋࡓࡓࡵࠊ⥆࠸࡚ HeLa ⣽⬊ࢆ⏝࠸ࡓ Dual-Luciferase reporter assay system ࡟ࡼࡾࠊdRH_{ಟ㣭siRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࢆホ౯ࡋࡓࠋẚ㍑ᑐ↷࡜ࡋ࡚ࠊ}

3′ᮎ➃࡟ TTࠊRH_RH_{ࢆ᭷ࡍࡿsiRNA ࢆ⏝࠸ࡓࠋ}

Assay kit

Dual-Glo Luciferase Assay System (Promega) Vector: psiCHECK-2

Transfection reagent: Transfast Buffer: Opti-MEM Conditions Transfection time: 1 h Incubation time: 24 h Figure 3-4. 㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯ࡢ⤖ᯝ  㑇ఏᏊⓎ⌧ᢚไ⬟ࢆホ౯ࡋࡓ⤖ᯝࠊ3′ᮎ➃࡟ dRH_dRH_{ࢆ᭷ࡍࡿ siRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ} ⬟ࡣTT ࠾ࡼࡧ RH_RH_{ࢆ᭷ࡍࡿsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟࡜ྠ⛬ᗘࡔࡗࡓࠋ}

௨ୖࡢࡇ࡜࠿ࡽࠊsiRNA ࡢ 3′ᮎ➃࡬ࡢ dRH_{ࡢᑟධࡣࠊsiRNA ࡢ RNAi άᛶ࡟࡯࡜ࢇ࡝ᙳ}

25

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6 ⠇ ࢘ࢩ⾑Ύ୰ศゎ㓝⣲⪏ᛶホ౯

 ⾑ᾮ୰࡟ࡣᵝࠎ࡞ࢱࣥࣃࢡ㉁ࡀྵࡲࢀ࡚࠾ࡾࠊ᰾㓟ศᏊࢆศゎࡍࡿ᰾㓟ศゎ㓝⣲ࡶྵࡲ ࢀ࡚࠸ࡿࠋ᰾㓟ศゎ㓝⣲࡜ࡋ࡚࢚ࣥࢻࢾࢡࣞ࢔࣮ࢮ࠾ࡼࡧ࢚࢟ࢯࢾࢡࣞ࢔࣮ࢮࡀྵࡲࢀ࡚ ࠸ࡿࡓࡵࠊ᰾㓟་⸆ရࡢᐇ⏝໬࡟࠾࠸࡚ࠊ⾑ΎෆᏳᐃᛶࡢྥୖࡣᚲ㡲࡜ࡉࢀ࡚࠸ࡿࠋ ࡑࡇ࡛ࠊ50%࢘ࢩ⾑Ύࢆ⏝࠸࡚ྜᡂࡋࡓ siRNA ࡢ⏕యෆᏳᐃᛶࢆホ౯ࡋࡓࠋ㑇ఏᏊⓎ⌧ ᢚไ⬟ホ౯࡜ྠᵝ࡟dRH_dRH_{ಟ㣭siRNA ࢆ 50%࢘ࢩ⾑Ύ୰࡟ 37 ºC ࡛࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࠊ}

ྛ᫬㛫࡟࠾ࡅࡿṧᏑsiRNA 㔞ࢆ㠀ኚᛶ PAGE ࠾ࡼࡧ GelRed ᰁⰍࢆ⾜࠺ࡇ࡜࡟ࡼࡗ࡚ホ౯ ࡋࡓࠋ

Conditions

Buffer: D-MEM (Phenol Red free) Concentration: 3 μM Incubation temperature: 37 ºC Figure 3-5. 50%࢘ࢩ⾑Ύ୰࡟࠾ࡅࡿᏳᐃᛶホ౯ࡢ⤖ᯝ  3′ᮎ➃࡟ TTࠊRH_RH_{ࢆ᭷ࡍࡿsiRNA ࢆẚ㍑ᑐ↷࡜ࡋ࡚ホ౯ࡋࡓ⤖ᯝࠊdR}H_dRH_{ಟ㣭ᆺsiRNA} ࡢ࢘ࢩ⾑Ύ୰ᏳᐃᛶࡣࠊTT ಟ㣭ᆺ࡜ྠ⛬ᗘ࡛࠶ࡗࡓࠋ୍᪉࡛ࠊRH_RH_{ಟ㣭ᆺsiRNA ࡣ 60 ศ} ࡛ࡶ⣙40%ṧᏑࡋ࡚࠾ࡾࠊ᭱ࡶᏳᐃ࡛࠶ࡗࡓࠋ  ௨ୖࡢ⤖ᯝࡼࡾࠊ࢘ࢩ⾑Ύ୰ࡢᏳᐃᛶࡣࢹ࢜࢟ࢩ࣮ࣜ࣎ࢫ㦵᱁ࡼࡾࡶ࣮ࣜ࣎ࢫ㦵᱁ࡢ᪉ ࡀඃࢀ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

26

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4 ❶ 2 ఩ಟ㣭ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡢྜᡂ࡜

ᛶ㉁ホ౯

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1 ⠇ 2 ఩࣋ࣥࢪࣝಟ㣭ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻ (R

HOBn

)

ࢆ᭷ࡍࡿ

RNA

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1 㡯 R

HOBn

ࡢྜᡂ

 ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࡢ2 ఩Ỉ㓟ᇶࢆಟ㣭ࡋࡓ໬ྜ≀࡜ࡋ࡚ࠊ2 ఩࣋ࣥࢪࣝಟ㣭ሷᇶ㒊 Ḟᦆࢾࢡࣞ࢜ࢩࢻ (RH_{OBn) ࡀሗ࿌ࡉࢀ࡚࠸ࡿ} 27)_ࠋRH_{OBnࡣ࣮ࣜ࣎ࢫࢆฟⓎཎᩱ࡜ࡋ࡚ሷᇶ} 㒊Ḟᦆࣜ࣎ࢾࢡࣞ࢜ࢩࢻ (RH_{) ࢆᚓࡓᚋࠊ3 ఩࠾ࡼࡧ 5 ఩Ỉ㓟ᇶࢆ㑅ᢥⓗ࡟ಖㆤࡋࡓᚋࠊሷ} ᇶ࡛࠶ࡿBEMP Ꮡᅾୗ࣋ࣥࢪࣝࣈ࣐ࣟ࢖ࢻࢆస⏝ࡉࡏࡿࡇ࡜࡛ 2 ఩Ỉ㓟ᇶࢆ࣋ࣥࢪࣝ໬ࡋࠊ ᭱ᚋ࡟ࢩࣜࣝಖㆤᇶࢆ㝖ཤࡍࡿࡇ࡜࡛ᚓࡓ (Scheme 4-1)ࠋ඲ 9 ᕤ⛬࡛ᚓࡽࢀࡓ RH_OBnࡣ siRNA ࡢෆ㒊࡟ᑟධࡉࢀࠊ᰾㓟ศゎ㓝⣲⪏ᛶࡀྥୖࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋࠊ RH_{OBnࢆsiRNA ࡢࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟ᑟධࡋࡓሗ࿌ࡣ࡞࠸ࠋ} Scheme 4-1. ࣮ࣜ࣎ࢫࢆฟⓎཎᩱ࡜ࡍࡿ RH_OBnྜᡂἲ

27 ᡤᒓ◊✲ᐊ࡛ࡣࡇࢀࡲ࡛࡟ࠊsiRNA ࡢࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟ⰾ㤶᪘໬ྜ≀ࢆᑟධࡍࡿࡇ ࡜࡛ࠊsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟࡟ᙳ㡪ࢆཬࡰࡍࡇ࡜࡞ࡃ᰾㓟ศゎ㓝⣲⪏ᛶࡀྥୖࡍࡿࡇ ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࠋࡇࡢࡇ࡜࠿ࡽⴭ⪅ࡣࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻ࡟ⰾ㤶⎔ࢆ௜ຍࡍࡿࡇ࡜ ࡛ࡼࡾ᰾㓟ศゎ㓝⣲⪏ᛶࡀྥୖࡍࡿ࡜ண᝿ࡋࡓࠋࡑࡇ࡛ࠊsiRNA ࡢᙜヱ㒊఩࡟ RH_OBnࢆᑟ ධࡍࡿࡇ࡜࡛ࠊ᭦࡞ࡿ᰾㓟ศゎ㓝⣲⪏ᛶࡢྥୖࢆ┠ᣦࡋࡓࠋ RH_{OBn (15) ࢆྜᡂࡍࡿ࡟࠶ࡓࡾࠊཎᩱ࡜ࡋ࡚ࠊᡤᒓ◊✲ᐊ࡛ࡇࢀࡲ࡛࡟⡆౽ྜᡂἲࢆ㛤Ⓨ} ࡋ࡚࠸ࡿሷᇶ㒊Ḟᦆࣜ࣎ࢾࢡࣞ࢜ࢩࢻ (RH_{, 16) ࢆ⏝࠸ࡓࠋࡲࡎ R}H_{ࡢ3 ఩࠾ࡼࡧ 5 ఩Ỉ㓟} ᇶࢆ 1,1,3,3-ࢸࢺࣛ࢖ࢯࣉࣟࣆࣝࢪࢩࣟ࢟ࢧࢽࣜࢹࣥᇶ (TIPDS ᇶ) ࡟࡚ಖㆤࡋࠊ3,5-O-TIPDS-RH _{(17) ࢆᚓࡓ (Scheme 4-2)ࠋ} Scheme 4-2. RH_{ࡢ3 ఩࠾ࡼࡧ 5 ఩Ỉ㓟ᇶࡢ TIPDS ಖㆤ} ḟ࡟ࠊ⎔≧ࢩࣜࣝಖㆤయ17 ࡟Ỉ⣲໬ࢼࢺ࣒ࣜ࢘Ꮡᅾୗࠊ࣋ࣥࢪࣝࣈ࣐ࣟ࢖ࢻࢆస⏝ࡉࡏ ࡚ࠊ3,5-O-TIPDS-RH_{OBn (18) ࢆᚓࡓᚋࠊ⥆ࡅ࡚ TBAF ࢆ⏝࠸࡚ TIPDS ᇶࡢ㝖ཤࢆ⾜ࡗࡓ࡜}

ࡇࢁࠊ27%࡜ప཰⋡࡞ࡀࡽ RH_{OBn (15) ࡀᚓࡽࢀࡓ (Scheme 4-3)ࠋࡇࡢ᫬ࠊ2 ఩Ỉ㓟ᇶࡢ࣋}

ࣥࢪࣝ໬ࡢ㝿࡟TIPDS ᇶࡢ⬺㞳ࡀేⓎࡋࡓࠋࡑࡇ࡛ࠊ㓟ᛶ᮲௳ୗ࡛ࡢࢺ࢚ࣜࢳࣝࢩࣛࣥ࠾ ࡼࡧ࣋ࣥࢬ࢔ࣝࢹࣄࢻࢆ⏝࠸ࡓ࣋ࣥࢪࣝ໬ࢆ᳨ウࡋࡓࠋ✀ࠎ᳨ウࡋࡓ⤖ᯝࠊゐ፹㔞ࡢሷ໬㕲 (III)Ꮡᅾୗ࡛࣋ࣥࢪࣝ໬ࢆ⾜࠸ࠊ⥆ࡅ࡚ࢺ࢚ࣜࢳࣝ࢔࣑࣭ࣥ୕ࣇࢵ໬Ỉ⣲ሷࢆ⏝࠸࡚ࢩࣜࣝ ᇶࢆ㝖ཤࡍࡿࡇ࡜࡟ࡼࡾࠊ2 ᕤ⛬࡛཰⋡ 47%࡜཰⋡ࢆᨵၿࡍࡿࡇ࡜ࡀ࡛ࡁࡓ (Scheme 4-4)ࠋ

Scheme 4-3. BnBr/NaH ࢆ⏝࠸ࡓ 2 ఩Ỉ㓟ᇶࡢ࣋ࣥࢪࣝ໬ࢆ⤒ࡓ RH_OBnࡢྜᡂ

28

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2 㡯 R

HOBn

ྵ᭷

RNA ࡢྜᡂ

 RH_{OBn (15) ࢆᐃἲ࡟ᚑࡗ࡚᰾㓟⮬ືྜᡂ⏝ㄏᑟయ࡟ኚ᥮ࡋࡓࠋ5 ఩Ỉ㓟ᇶࢆ DMTr ᇶ࡛㑅}

ᢥⓗ࡟ಖㆤࡋࠊ5-O-DMTr-RH_{OBn (19) ࢆᚓࡓᚋࠊTHF ⁐፹୰ DIPEA ࢆሷᇶ࡜ࡋ࡚⏝࠸࡚ 2}

఩Ỉ㓟ᇶࢆளࣜࣥ㓟໬ࡋࠊ᰾㓟⮬ືྜᡂ⏝࣍ࢫ࣍ࣟ࢔࣑ࢲ࢖ࢺయ 20 ࢆ཰⋡ 97%࡛ᚓࡓ (Scheme 4-5)ࠋ

Scheme 4-5. ᰾㓟⮬ືྜᡂ⏝ RH_{OBnㄏᑟయࡢྜᡂ}

➨ 3 ❶࡛㏙࡭ࡓ dRH_{ࡢሙྜ࡜ྠᵝ࡟20 ࢆ⏝࠸࡚᰾㓟⮬ືྜᡂᶵ࡟࡚ Renilla luciferase}

mRNA ࢆᶆⓗ࡜ࡋࡓ siRNA ࡢ࢔ࣥࢳࢭࣥࢫ㙐ཬࡧࢭࣥࢫ㙐ࡢࡑࢀࡒࢀࡢ 3′ᮎ➃࡟ RH_OBnRH_{OBnࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆྜᡂࡋࡓ (Figure 4-1)ࠋࡲࡓࠊ᰾㓟ศゎ㓝⣲࡟ᑐ}

ࡍࡿ⪏ᛶホ౯⏝࡟࢔ࣥࢳࢭࣥࢫ㙐ࡢ 5′ᮎ➃࡟⺯ගศᏊ࡛࠶ࡿࣇࣝ࢜ࣞࢭ࢖ࣥࢆᑟධࡋࡓ࢜ ࣜࢦࢾࢡࣞ࢜ࢳࢻࡶྜᡂࡋࡓࠋᅛ┦ᢸయ࠿ࡽษࡾฟࡋ࡜ྛ✀ಖㆤᇶࢆ㝖ཤࡋࠊ⢭〇ᚋ࡟ MALDI-TOF/MS ࢆ⏝࠸ࡓ㉁㔞ศᯒ࡟ࡼࡗ࡚ࠊᵓ㐀ࢆ☜ㄆࡋࠊࡑࡢᚋࡢホ౯࡟⏝࠸ࡓࠋ

29

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3 㡯 ᰾㓟ศゎ㓝⣲⪏ᛶホ౯

5′ᮎ➃࡟ࣇࣝ࢜ࣞࢭ࢖ࣥࠊ3′ᮎ➃࡟ RH_OBnRH_{OBnࢆ᭷ࡍࡿ࢔ࣥࢳࢭࣥࢫ㙐࡟ᑐࡋࠊ࣊ࣅẘ࣍} ࢫ࣍ࢪ࢚ࢫࢸ࣮ࣛࢮ (SVPD) ࢆస⏝ࡉࡏࠊ᰾㓟ศゎ㓝⣲⪏ᛶࢆホ౯ࡋࡓ (Figure 4-2)ࠋẚ㍑ ᑐ↷࡜ࡋ࡚ࠊ3′ᮎ➃࡟ TT ࠾ࡼࡧ RH_RH_{ࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࢆ⏝࠸ࡓࠋ} Conditions Buffer: PBS (−) Concentration: 3 μM

Nuclease: 1 unit/mL snake venom phosphodiesterase Incubation temperature: 37 ºC Figure 4-2. ᰾㓟ศゎ㓝⣲⪏ᛶホ౯  ホ౯ࡢ⤖ᯝࠊRH_OBnRH_{OBnࢆ3′ᮎ➃࡟᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮ࡣࠊTT ࠾ࡼࡧ R}H_RH_ࢆ᭷ࡍࡿ ᰾㓟࢜ࣜࢦ࣐࣮࡜ẚ㍑ࡋ࡚㧗࠸ศゎ㓝⣲⪏ᛶࢆ♧ࡋࠊRH_OBnRH_{OBnᆺࡢ༙ῶᮇࡣTT ᆺࡢ 5 ಸ} ௨ୖ࡛࠶ࡗࡓࠋ௨ୖࡢࡇ࡜࠿ࡽࠊሷᇶ㒊Ḟᦆࣜ࣎ࢾࢡࣞ࢜ࢩࢻࡢ 2 ఩Ỉ㓟ᇶ࡬ࡢⰾ㤶⎔ࡢ ௜ຍ࡟ࡼࡾࠊ࢚࢟ࢯࢾࢡࣞ࢔࣮ࢮ⪏ᛶࡀ኱ᖜ࡟ྥୖࡍࡿࡇ࡜ࡀᙉࡃ♧၀ࡉࢀࡓࠋ

30

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4 㡯 ⇕ⓗᏳᐃᛶホ౯

ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡢࢾࢡࣞ࢜ࢩࢻࢆRH_{OBn࡟⨨᥮ࡋࡓsiRNA ࡢ⇕ⓗᏳᐃᛶࢆホ౯ࡍࡿ} ࡓࡵࠊsiRNA ࡢ᪼ ࡟࡜ࡶ࡞࠺྾ගᗘࡢኚ໬ࢆ ᐃࡋࡓࠋẚ㍑ᑐ↷࡜ࡋ࡚ࠊ3′ᮎ➃࡟ࢳ࣑ࢪ ࣥ (TT) ࢆ᭷ࡍࡿ siRNA ࢆ⏝࠸ࡓࠋ Conditions

Buffer: 10 mM NaH2PO4∙Na2HPO4 (pH 7.0)ࠊ100 mM NaCl

Concentration: 3 μM Figure 4-3. ⇕ⓗᏳᐃᛶホ౯ Table 4-1. ⟬ฟࡋࡓ Tm್ XX Tm (ºC) ΔTm (ºC) TT 77.0 㸫 RH_OBnRH_{OBn 76.4 -0.6} ࢩࢢࣔ࢖ࢻ᭤⥺ࡀᚓࡽࢀࡓࡇ࡜࠿ࡽࠊ3′ᮎ➃࡟ RH

OBnRHOBnࢆ᭷ࡍࡿsiRNA ࡀ஧ᮏ㙐ࢆᙧ

ᡂࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊ୰⥺ἲ࡟࡚Tm್ࢆ⟬ฟࡋࡓ⤖ᯝࠊTm್ࡣࡑࢀࡒ ࢀ77.0 ºC (TT)ࠊ76.4 ºC (RH

OBnRHOBn) ࡛࠶ࡾࠊRHOBnࡢᑟධ࡟ࡼࡾࢃࡎ࠿࡟୙Ᏻᐃ໬ࡋࡓࠋ

ࡇࢀࡣ➨4 ❶࡛㏙࡭ࡓ dRH_{ࡢሙྜ࡜ྠᵝࡢ⤖ᯝ࡛࠶ࡾࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ㒊఩ࡢሷᇶᑐ}

㛫ࡢπ-π ࢫࢱࢵ࢟ࣥࢢ┦஫స⏝ࡢḞዴ࡟ࡼࡾ⇕ⓗᏳᐃᛶࡀⱝᖸపୗࡍࡿࡶࡢࡢࠊࢲࣥࢢࣜ ࣥࢢ࢚ࣥࢻࡢࢾࢡࣞ࢜ࢩࢻࢆRH

OBn࡟⨨᥮ࡋࡓsiRNA ࡶᐊ ࡛஧ᮏ㙐ᵓ㐀ࢆ⥔ᣢࡋ࡚࠸ࡿ

31

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5 㡯 㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯

HeLa ⣽⬊ࢆ⏝࠸ࡓ Dual-Luciferase reporter assay system ࡟ࡼࡾࠊRH_{OBnಟ㣭siRNA ࡢ}

㑇ఏᏊⓎ⌧ᢚไ⬟ࢆホ౯ࡋࡓࠋẚ㍑ᑐ↷࡜ࡋ࡚ࠊ3′ᮎ➃࡟ TT ࢆ᭷ࡍࡿ siRNA ࢆ⏝࠸ࡓࠋ Assay kit

Dual-Glo Luciferase Assay System (Promega) Vector: psiCHECK-2

Transfection reagent: ScreenFect A Plus

Buffer: ScreenFect A Plus diluted transfection reagent Conditions

Transfection time: 1 h Incubation time: 24 h

Figure 4-4. 㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯

 㑇ఏᏊⓎ⌧ᢚไ⬟ࢆホ౯ࡋࡓ⤖ᯝࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟ TT ࢆ᭷ࡍࡿ siRNA ࡜ RH_OBnRH_{OBnࢆ᭷ࡍࡿsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ࡣྠ⛬ᗘࡔࡗࡓࠋ}

௨ୖࡢࡇ࡜࠿ࡽࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡢࢾࢡࣞ࢜ࢩࢻࡢRH_{OBn࡬ࡢ⨨᥮ࡣࠊ㑇ఏᏊⓎ⌧ᢚ}

32

➨

➨

6 㡯 ࢘ࢩ⾑Ύ୰ศゎ㓝⣲⪏ᛶホ౯

ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟RH_OBnRH_{OBnࢆ᭷ࡍࡿsiRNA ࡢ⏕యෆᏳᐃᛶࢆホ౯ࡋࡓࠋR}H_OBnಟ

㣭siRNA ࢆ 50%࢘ࢩ⾑Ύ୰࡟ 37 ºC ࡛࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࠊྛ᫬㛫࡟࠾ࡅࡿṧᏑ siRNA 㔞 ࢆ㠀ኚᛶPAGE ࠾ࡼࡧ GelRed ᰁⰍࢆ⾜࠺ࡇ࡜࡟ࡼࡗ࡚ホ౯ࡋࡓࠋ

Conditions

Buffer: DMEM (Phenol Red free) Concentration: 3 μM

Incubation temperature: 37 ºC

Figure 4-5. 50%࢘ࢩ⾑Ύ୰ࡢᏳᐃᛶホ౯

 ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟RH_OBnRH_{OBnࢆ᭷ࡍࡿsiRNA ࡣ 12 ᫬㛫ᚋ࡛ࡶ 20%௨ୖṧᏑࡋ࡚࠾}

33

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7 㡯 ࣄࢺ Argonaute2-PAZ ࢻ࣓࢖ࣥࢱࣥࣃࢡ㉁࡜ࡢぶ࿴ᛶホ౯

RNAi ᶵᵓࡢ୰࡛ࠊsiRNA ࡢࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡀ Ago2 ࢱࣥࣃࢡ㉁ࡢ PAZ ࢻ࣓࢖ࣥ (Ago2-PAZ) ࡜┦஫స⏝ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊsiRNA ࡢࢲࣥࢢࣜࣥ ࢢ࢚ࣥࢻࡢࢾࢡࣞ࢜ࢩࢻࡢ RH_{OBn࡬ࡢ⨨᥮ࡀ Ago2-PAZ ࡜ࡢぶ࿴ᛶ࡟ᙳ㡪ࡍࡿ࠿ホ౯ࡍࡿ}

ࡇ࡜࡟ࡋࡓࠋ

ࣄࢺAgo2-PAZ ࢱࣥࣃࢡ㉁ࡢ㑇ఏ᝟ሗࢆ᭷ࡍࡿ pET15b ࣋ࢡࢱ࣮ࢆタィࡋ (Figure 5-6)ࠊ ேᕤ㑇ఏᏊྜᡂ࡟࡚┠ⓗ࡜࡞ࡿ࣋ࢡࢱ࣮ࢆᚓࡓࡢࡕࠊ኱⭠⳦BL21 (DE3) ᰴࢆ⏝࠸࡚Ⓨ⌧ࠊ ⢭〇ࡍࡿࡇ࡜࡛His-tag ⼥ྜ Ago2-PAZ ࢱࣥࣃࢡ㉁ࢆᚓࡓ20)_ࠋ

Figure 4-6. タィࡋࡓ Ago2-PAZ ࢻ࣓࢖ࣥࢱࣥࣃࢡ㉁ࡢ᝟ሗࢆ᭷ࡍࡿ࣋ࢡࢱ࣮࣐ࢵࣉ  ࡣࡌࡵ࡟ࠊBL21 (DE3) ࡢࢥࣥࣆࢸࣥࢺࢭࣝࢆస〇ࡋࠊAgo2-PAZ ࢱࣥࣃࢡ㉁ࡢ㑇ఏ᝟ሗ ࢆ᭷ࡍࡿpET15b ࣋ࢡࢱ࣮ࢆᑟධࠊⓎ⌧ࡋࡓࠋⓎ⌧ᚋࠊᚓࡽࢀࡓΰྜ≀ࢆ Talon Resin ࢝ࣛ ࣒࠾ࡼࡧ㝜࢖࢜ࣥ஺᥮ᶞ⬡ࢆ⏝࠸࡚⢭〇ࡋࠊ┠ⓗ࡛࠶ࡿAgo2-PAZ ࢱࣥࣃࢡ㉁ࢆᚓࡓࠋᚓࡽ ࢀࡓࢱࣥࣃࢡ㉁ࡢ⣧ᗘࢆSDS-PAGE ࡟ࡼࡗ࡚☜ㄆࡋࠊศᏊ㔞ࢆ MALDI-TOF/MS ࡛ ᐃࡋࠊ ྠᐃࡋࡓࠋAgo2-PAZ ࢱࣥࣃࢡ㉁ࡣᇵ㣴ᾮ 1 L ࠶ࡓࡾ 0.2 mg ࡢ཰㔞࡛ᚓࡓࠋ

 siRNA ࡣࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡀ୧ᮎ➃࡟ᏑᅾࡋࠊsiRNA1 ศᏊ࡟ᑐࡋ࡚ࢱࣥࣃࢡ㉁ࡀ 2 ศᏊ⤖ྜࡍࡿࡇ࡜ࡀண᝿࡛ࡁࡓࡓࡵࠊAgo2-PAZ ࢱࣥࣃࢡ㉁࡜ RH_OBnRH_{OBnಟ㣭siRNA ࡢぶ}

࿴ᛶࢆ⾜࠺࡟࠶ࡓࡾࠊࢭࣥࢫ㙐19 ሷᇶ࡜࢔ࣥࢳࢭࣥࢫ㙐 21 ሷᇶࢆ஧ᮏ㙐໬ࡋࠊ୍᪉ࡢᮎ ➃ࢆᖹ⁥࡟ࡋࡓ஧ᮏ㙐 RNA (dsRNA) ࢆᵓ⠏ࡋࡓ (Figure 5-7)ࠋస〇ࡋࡓ஧ᮏ㙐࡜ Ago2-PAZ ࢱࣥࣃࢡ㉁ࢆ 37 ºC ࡛ 10 ศ㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࠊྛ᫬㛫࡟࠾ࡅࡿ dsRNA 㔞ࢆ㠀ኚᛶ PAGE ࠾ࡼࡧ GelRed ᰁⰍࢆ⾜࠸ࠊホ౯ࡋࡓࠋẚ㍑ᑐ↷࡜ࡋ࡚ࠊ3′ᮎ➃࡟ࢳ࣑ࢪࣥ (TT) ࢆ ᭷ࡍࡿ஧ᮏ㙐RNA ࢆ⏝࠸ࡓࠋ Figure 4-7. ホ౯࡟⏝࠸ࡓ dsRNA ࡢ㓄ิ ColE1 origin T7 LacO 6xHis tag2Thrombin

hAgo2-PAZ.ape 6146 bp

hAgo2/PAZ

34

Figure 4-8. Ago2-PAZ ࢱࣥࣃࢡ㉁࡜ dsRNA ࡢぶ࿴ᛶホ౯

 ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟TT ࠾ࡼࡧ RH_OBnRH_{OBnࢆ᭷ࡍࡿdsRNA ࡜ Ago2-PAZ ࢱࣥࣃࢡ㉁}

࡜ࡢぶ࿴ᛶࡣྠ⛬ᗘ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋࡇࡢࡇ࡜࠿ࡽࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡢࢾ ࢡࣞ࢜ࢩࢻࡢRH_{OBn࡬ࡢ⨨᥮ࡣࠊAgo2-PAZ ࢻ࣓࢖ࣥ࡜ࡢぶ࿴ᛶ࡟኱ࡁ࡞ᙳ㡪ࢆཬࡰࡉ࡞࠸}

ࡇ࡜ࡀ♧၀ࡉࢀࠊRNAi ᶵᵓࡢ㐍⾜࡬ࡢᙳ㡪ࡀᑠࡉ࠸ࡓࡵ㑇ఏᏊⓎ⌧ᢚไ⬟ࡢῶᙅࡀぢࡽࢀ ࡞࠸࡜⪃࠼ࡽࢀࡓࠋ

35

➨

➨

2 ⠇ 2 ఩ࣉࣟࣃࣝࢠࣝಟ㣭ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻ (R

HOPg

)

ࢆ᭷ࡍࡿ

RNA

➨

1 㡯 R

HOPg

ࡢྜᡂ

ᡤᒓ◊✲ᐊ࡛ࡣࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࢆ໬Ꮫಟ㣭ࡋࡓᵝࠎ࡞ siRNA ࡢྜᡂ࠾ࡼࡧᛶ㉁ホ౯ ࢆ⾜ࡗ࡚ࡁࡓࠋࡑࡢ୰࡛ࠊ࣍ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ྜࢆ࢘ࣞࢱࣥ⤖ྜࡸ࢘ࣞ࢔⤖ྜ࡟⨨᥮ࠊ࠶ࡿ ࠸ࡣࢾࢡࣞ࢜ࢩࢻࢆⰾ㤶᪘໬ྜ≀࡟⨨᥮ࡋࡓsiRNA ࡀᮍಟ㣭 siRNA ࡜ྠ⛬ᗘࡢ㑇ఏᏊⓎ⌧ ᢚไ⬟ࢆ♧ࡍࡇ࡜ࢆぢฟࡋ࡚࠸ࡿࠋຍ࠼࡚ࠊ๓⠇ࡲ࡛ࡢ⤖ᯝ࠿ࡽࠊsiRNA ࡢࢲࣥࢢࣜࣥࢢ࢚ ࣥࢻ㒊఩ࢾࢡࣞ࢜ࢩࢻࡢሷᇶ㒊Ḟᦆࡣ㑇ఏᏊⓎ⌧ᢚไ⬟࡟ᙳ㡪ࢆཬࡰࡉ࡞࠸ࡇ࡜ࡀᙉࡃ♧ ၀ࡉࢀࡓࠋ௨ୖࡢ▱ぢ࠿ࡽࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡢࢾࢡࣞ࢜ࢩࢻ㛫ࡢ࣍ࢫ࣍ࢪ࢚ࢫࢸࣝ⤖ྜ ࡞ࡽࡧ࡟᰾㓟ሷᇶࡣsiRNA ࡢ㑇ఏᏊⓎ⌧ࡢᢚไ࡟ᚲ㡲࡛ࡣ࡞࠸ࡇ࡜ࡀண᝿ࡉࢀࡓࠋ ࡑࡇ࡛ࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࡢ 2 ఩࡟ࣉࣟࣃࣝࢠࣝᇶࢆᑟධࡋࡓሷᇶ㒊Ḟᦆࢾࢡࣞ ࢜ࢩࢻ (RH_{OPg) ࡢྜᡂࢆ┠ᣦࡋࡓࠋ3′ᮎ➃࡟ R}H_{OPgࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡣࠊCu(I)}

ゐ፹Ꮡᅾୗᵝࠎ࡞࢔ࢪࢻ໬ྜ≀ࢆ⏝࠸ࡿࡇ࡜࡛ࠊCu-Catalyzed Azide Alkyne Cycloaddition (CuAAC) ཯ᛂ࡟ࡼࡗ࡚ࢺࣜ࢔ࢰ࣮ࣝ⎔ࢆ௓ࡋ࡚✀ࠎࡢ⨨᥮ᇶࢆᑟධྍ⬟࡛࠶ࡿࡇ࡜࠿ࡽ (Figure 4-9)ࠊsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯ࡸ Ago2-PAZ ࡜ࡢぶ࿴ᛶホ౯࡟ࡼࡿ໬Ꮫಟ㣭 ࢲࣥࢢࣜࣥࢢ࢚ࣥࢻࡢ⥙⨶ⓗゎᯒ࡟฼⏝ྍ⬟࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋࡲࡓࠊ๓⠇࡛㏙࡭ࡓࡼ࠺ ࡟ࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻRH_{ࡢ2 ఩Ỉ㓟ᇶࢆ࣋ࣥࢪࣝ໬ࡋࡓ R}H_{OBnࢆ3′ᮎ➃࡟᭷ࡍࡿ࢜} ࣜࢦࢾࢡࣞ࢜ࢳࢻࡣ᰾㓟ศゎ㓝⣲࡟ᑐࡋ࡚ඃࢀࡓ⪏ᛶࢆ♧ࡋࡓࠋࡇࡢࡇ࡜࠿ࡽࠊ࢔ࢪࢻ໬ྜ ≀࡜ࡢCuAAC ཯ᛂ࡛ࡢ࣏ࢫࢺಟ㣭࡟ࡼࡾᚓࡽࢀࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡣࠊRH_{OBnಟ㣭࢜ࣜ} ࢦࢾࢡࣞ࢜ࢳࢻ࡜㢮ఝࡋࡓᵓ㐀ࢆࡶࡘࡇ࡜࠿ࡽࠊ㧗࠸᰾㓟ศゎ㓝⣲⪏ᛶࢆ᭷ࡍࡿ࡜⪃࠼ࡽ ࢀࡿࠋ

Figure 4-9. 3′ᮎ➃࡟ RH_{OPgࢆ᭷ࡍࡿ᰾㓟࢜ࣜࢦ࣐࣮࡜࢔ࢪࢻ໬ྜ≀ࢆ⏝࠸ࡓCuAAC ཯ᛂ}

 ๓⠇࡛ࠊ2 ఩Ỉ㓟ᇶࢆ࣋ࣥࢪࣝ໬ࡍࡿ㝿࡟ࣝ࢖ࢫ㓟ࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡾ཰⋡ࢆྥୖ࡛ࡁࡓ ࡀࠊᑐᛂࡍࡿ࢔ࣝࢹࣄࢻ໬ྜ≀ࡢධᡭࡸྜᡂࡀᅔ㞴࡛࠶ࡿࡇ࡜࠿ࡽࠊࡇࡇ࡛ࡣỈ⣲໬ࢼࢺࣜ ࣒࢘ࢆ⏝࠸࡚ࣉࣟࣃࣝࢠࣝᇶࢆᑟධࡋࡓࠋඛ࡟ྜᡂࡋࡓ 3,5-O-TIPDS-RH _{(17) ࡟Ỉ⣲໬ࢼ}

ࢺ࣒ࣜ࢘Ꮡᅾୗࠊࣉࣟࣃࣝࢠࣝࣈ࣐ࣟ࢖ࢻࢆస⏝ࡉࡏ࡚ࠊ3,5-O-TIPDS-RH_{OPg (20) ࡟ㄏᑟ}

ࡋࡓᚋࠊ⥆ࡅ࡚ TBAF ࢆ⏝࠸࡚ TIPDS ᇶࡢ㝖ཤࢆ⾜࠸ࠊRH_{OBn (21) ࢆ཰⋡ 38%࡛ᚓࡓ}

36

Scheme 4-6. RH_OPgࡢྜᡂ

➨

➨

2 㡯 R

HOPg

ྵ᭷

RNA ࡢྜᡂ

RH_{OPg (21) ࢆᐃἲ࡟ᚑࡗ࡚᰾㓟⮬ືྜᡂ⏝ㄏᑟయ࡟ኚ᥮ࡋࡓࠋ5 ఩Ỉ㓟ᇶࢆ DMTr ᇶ࡛㑅}

ᢥⓗ࡟ಖㆤࡋࠊ5-O-DMTr-RH_{OPg (22) ࢆᚓࡓᚋࠊTHF ⁐፹୰ DIPEA ࢆሷᇶ࡜ࡋ࡚⏝࠸࡚ 2}

఩Ỉ㓟ᇶࢆளࣜࣥ㓟໬ࡋࠊ᰾㓟⮬ືྜᡂ⏝࣍ࢫ࣍ࣟ࢔࣑ࢲ࢖ࢺయ 23 ࢆᚓࡓ (Scheme 4-7)ࠋ

Scheme 4-7. ᰾㓟⮬ືྜᡂ⏝ RH_{OBnㄏᑟయࡢྜᡂ}

➨ 3 ❶࡛㏙࡭ࡓ dRH_{ࡢሙྜ࡜ྠᵝ࡟23 ࢆ⏝࠸࡚᰾㓟⮬ືྜᡂᶵ࡟࡚ Renilla luciferase}

mRNA ࢆᶆⓗ࡜ࡋࡓ siRNA ࡢ࢔ࣥࢳࢭࣥࢫ㙐ࡢ 3′ᮎ➃࡟ RH_{OPgࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳ}

ࢻࢆྜᡂࡋࡓ (Figure 4-10)ࠋᅛ┦ᢸయ࠿ࡽࡢษࡾฟࡋ࡜ྛ✀ಖㆤᇶࢆ㝖ཤࡋࠊ⢭〇ᚋ࡟ MALDI-TOF/MS ࢆ⏝࠸ࡓ㉁㔞ศᯒ࡟ࡼࡗ࡚ࠊᵓ㐀ࢆ☜ㄆࡋࠊࡑࡢᚋࡢホ౯࡟⏝࠸ࡓࠋ

37

➨

➨

3 㡯 R

HOPg

ྵ᭷

RNA ࡜࢔ࢪࢻ໬ྜ≀࡜ࡢ CuAAC ཯ᛂ

࢔ࢪࢻ໬ྜ≀࡜ࡋ࡚ࠊ࣋ࣥࢪࣝ࢔ࢪࢻ (24) ࡜ 4-ࣇࣝ࢜ࣟ࣋ࣥࢪࣝ࢔ࢪࢻ (25)ࠊ1-࢔ࢪࢻ -1-ࢹ࢜࢟ࢩ-β-ࢸࢺࣛ࢔ࢭࢳࣝࢢࣝࢥ࣮ࢫ (26)ࠊ1-࢔ࢪࢻ-1-ࢹ࢜࢟ࢩ-β-ࢢࣝࢥ࣮ࢫ (27)ࠊ 1-࣓ࢳࣝ࢔ࢪࢻࢼࣇࢱࣞࣥ (28) ࡢ 5 ࡘࡢ໬ྜ≀ࢆ⏝࠸࡚ࠊ3′ᮎ➃࡟ RH_{OPgࢆ᭷ࡍࡿ࢜ࣜࢦ} ࢾࢡࣞ࢜ࢳࢻ࡜ࡢ CuAAC ཯ᛂࡢ᳨ウࢆ⾜ࡗࡓ (Figure 4-11)ࠋᐜ᫆࡟ධᡭྍ⬟࡞࣋ࣥࢪࣝ ࢔ࢪࢻ (24) ௨እࡣ௨ୗࡢ⤒㊰࡛ࡑࢀࡒࢀྜᡂࡋࡓ (Scheme 4-8)ࠋ Figure 4-11. ᮏ◊✲࡛౑⏝ࡋࡓ࢔ࢪࢻ໬ྜ≀ Scheme 4-8. ࢔ࢪࢻ໬ྜ≀ࡢྜᡂ⤒㊰  4-ࣇࣝ࢜ࣟ࣋ࣥࢪࣝ࢔ࢪࢻ (25) ࡣࠊ4-ࣇࣝ࢜ࣟ࣋ࣥࢪࣝࢡࣟࣛ࢖ࢻ࡟ࣚ࢘໬ࢼࢺ࣒ࣜ࢘ Ꮡᅾୗ࢔ࢪ໬ࢼࢺ࣒ࣜ࢘ࢆస⏝ࡉࡏ࡚཰⋡79%࡛ᚓࡓ28)_{ࠋ1-࢔ࢪࢻ-1-ࢹ࢜࢟ࢩ-β-ࢸࢺࣛ࢔} ࢭࢳࣝࢢࣝࢥ࣮ࢫ (26)࡜ 1-࢔ࢪࢻ-1-ࢹ࢜࢟ࢩ-β-ࢢࣝࢥ࣮ࢫ (27)ࡣ࣌ࣥࢱ࢔ࢭࢳࣝࢢࣝࢥ ࣮ࢫ (30) ࠿ࡽྜᡂࡋࡓࠋ30 ࢆゐ፹㔞ࡢሷ໬㕲(III)࡜ࢺ࣓ࣜࢳࣝࢩࣜࣝ࢔ࢪࢻ࡜ᐊ ࡛᧠ᢾ ࡍࡿࡇ࡜࡛β 㑅ᢥⓗ࡟࢔ࢪࢻࢆᑟධࡋࠊ26 ࢆᚓࡓ29)_{ࠋࡲࡓࠊ26 ࡟࢔ࣥࣔࢽ࢔ᛶ࣓ࢱࣀ࣮ࣝ} ࢆస⏝ࡉࡏࡿࡇ࡜࡛࢔ࢭࢳࣝᇶࡢ㝖ཤࢆ⾜࠸ࠊ27 ࡟ኚ᥮ࡋࡓࠋ1-࣓ࢳࣝ࢔ࢪࢻࢼࣇࢱࣞࣥ (28) ࡣࠊ1-ࢼࣇࢱࣝ࢔ࣝࢹࣄࢻ࡟Ỉ⣲໬࣍࢘⣲ࢼࢺ࣒ࣜ࢘ࢆస⏝ࡉࡏࡓᚋࠊ㐃⥆ⓗ࡟Ỉ㓟 ᇶࡢ⮯⣲໬࡜࢔ࢪࢻ໬ࢆྠ᫬࡟⾜࠸ࠊ2 ᕤ⛬཰⋡ 78%࡛ྜᡂࡋࡓ30)_ࠋ

38  ᡤᒓ◊✲ᐊ࡛㛤Ⓨࡋࡓᡭἲࢆ⏝࠸࡚ࠊ࢔ࢪࢻ໬ྜ≀࡜๓㡯࡛ྜᡂࡋࡓ 3′ᮎ➃࡟ RH_OPgࢆ ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡜ࡢCuAAC ཯ᛂࡢ᳨ウࢆ⾜ࡗࡓ 31)_{ࠋ3′ᮎ➃ R}H_{OPgྵ᭷࢜ࣜࢦࢾ} ࢡࣞ࢜ࢳࢻ࠾ࡼࡧ࢔ࢪࢻ໬ྜ≀ࠊ◲㓟㖡ࠊ࢔ࢫࢥࣝࣅࣥ㓟ࢼࢺ࣒ࣜ࢘ࢆຍ࠼ࠊ⃭ࡋࡃΰ࿴ࡋ ࡓᚋࠊᐊ ࡛15 ศ㟼⨨ࡋࡓࠋ㉸⣧Ỉࢆຍ࠼࡚཯ᛂ⁐ᾮࢆᕼ㔘ᚋࠊHPLC ࡛཯ᛂࢆゎᯒࡋࡓࠋ MALDI-TOF/MS ࢆ⏝࠸࡚㉁㔞ศᯒࢆ⾜ࡗࡓࠋ ࣋ ࣋ࣥࢪࣝ࢔ࢪࢻ (24)

 ཯ᛂᚋࡢࣆ࣮ࢡ (Retention time: 10 min, 12 min, 13 min) ࢆศྲྀࡋࡓ (Figure 5-12)ࠋ㉁ 㔞ศᯒࡢ⤖ᯝࠊಖᣢ᫬㛫13 ศࡢࣆ࣮ࢡࡀ┠ⓗࡢಟ㣭࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡛࠶ࡗࡓࠋಖᣢ᫬ 㛫10 ศ࠾ࡼࡧ 12 ศࡢࣆ࣮ࢡࡣศྲྀࡋࡓࡶࡢࡢࠊ㔞ࡀᑡ࡞ࡃࠊ㉁㔞ศᯒࡀᅔ㞴࡛࠶ࡾࠊྠ ᐃ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋ

Figure 4-12. RH_{OPgྵ᭷࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡜24 ࡜ࡢ CuAAC ཯ᛂ}

4-ࣇࣝ࢜ࣟ࣋ࣥࢪࣝ࢔ࢪࢻ (25)

 ཯ᛂᚋࡢࣆ࣮ࢡ (Retention time: 17 min, 25 min) ࢆศྲྀࡋࡓ (Figure 5-13)ࠋ㉁㔞ศᯒࡢ ⤖ᯝࠊಖᣢ᫬㛫25 ศࡢࣆ࣮ࢡࡀ┠ⓗࡢಟ㣭࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡛࠶ࡗࡓࠋಖᣢ᫬㛫 17 ศ ࡢࣆ࣮ࢡࡣศྲྀࡋࡓࡶࡢࡢࠊ㔞ࡀᑡ࡞ࡃࠊ㉁㔞ศᯒࡀᅔ㞴࡛࠶ࡾࠊྠᐃ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋ

39

1-࢔࢔ࢪࢻ-1-ࢹ࢜࢟ࢩ-β-ࢸࢺࣛ࢔ࢭࢳࣝࢢࣝࢥ࣮ࢫ (26)

཯ᛂᚋࡢࣆ࣮ࢡ (Retention time: 11 min, 13.5 min, 15 min) ࢆศྲྀࡋࡓ (Figure 5-14)ࠋ㉁ 㔞ศᯒࡢ⤖ᯝࠊಖᣢ᫬㛫15 ศࡢࣆ࣮ࢡࡀ┠ⓗࡢಟ㣭࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡛࠶ࡗࡓࠋಖᣢ᫬ 㛫11 ศ࠾ࡼࡧ 13.5 ศࡢࣆ࣮ࢡࡣศྲྀࡋࡓࡶࡢࡢࠊ㔞ࡀᑡ࡞ࡃࠊ㉁㔞ศᯒࡀᅔ㞴࡛࠶ࡾࠊྠ ᐃ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋ

Figure 4-14. RH_{OPgྵ᭷࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡜26 ࡜ࡢ CuAAC ཯ᛂ}

1-࢔ࢪࢻ-1-ࢹ࢜࢟ࢩ-β-ࢢࣝࢥ࣮ࢫ (27)

ཎᩱ࡛࠶ࡿ 3′ᮎ➃ RH_{OPgྵ᭷࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡜ྠ⛬ᗘࡢಖᣢ᫬㛫࡟ࣆ࣮ࢡࡀ☜ㄆ࡛}

ࡁࡓ (Figure 5-15)ࠋศྲྀࡋࡓᚋ࡟㉁㔞ศᯒࢆ⾜࠸ࠊ┠ⓗࡢಟ㣭࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡛࠶ࡿ ࡇ࡜ࢆ☜ㄆࡋࡓࠋ

40 1-࣓࣓ࢳࣝ࢔ࢪࢻࢼࣇࢱࣞࣥ (28)

཯ᛂᚋࡢࣆ࣮ࢡ (Retention time: 11 min, 15 min, 17.5 min) ࢆศྲྀࡋࡓ (Figure 5-16)ࠋ㉁ 㔞ศᯒࡢ⤖ᯝࠊಖᣢ᫬㛫17.5 ศࡢࣆ࣮ࢡࡀ┠ⓗࡢಟ㣭࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡛࠶ࡗࡓࠋಖᣢ ᫬㛫11 ศ࠾ࡼࡧ 15 ศࡢࣆ࣮ࢡࡣศྲྀࡋࡓࡶࡢࡢࠊ㔞ࡀᑡ࡞ࡃࠊ㉁㔞ศᯒࡀᅔ㞴࡛࠶ࡾࠊ ྠᐃ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋ

Figure 4-16. RH_{OPgྵ᭷࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡜28 ࡜ࡢ CuAAC ཯ᛂ}

௨ୖࡢ⤖ᯝ࠿ࡽࠊ3′ᮎ➃࡟ RH_{OPgࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡣ◲㓟㖡࡜࢔ࢫࢥࣝࣅࣥ㓟}

ࢼࢺ࣒ࣜ࢘Ꮡᅾୗ࡛࠸ࡎࢀࡢ࢔ࢪࢻ໬ྜ≀࡜ࡶ㏿ࡸ࠿࡟཯ᛂࡋࠊᑐᛂࡍࡿࢺࣜ࢔ࢰ࣮ࣝ⎔ ࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࡀᚓࡽࢀࡓࠋ

41

➨

➨

5 ❶ ⤖ㄽ

siRNA ࢆ་⸆ရ࡜ࡋ࡚฼⏝ࡍࡿ࡟࠶ࡓࡾࠊi) siRNA ࡀ⏕యෆ᰾㓟ศゎ㓝⣲࡟ࡼࡗ࡚ศゎ ࡉࢀࡿࡇ࡜࡛ᮏ᮶ࡢάᛶࢆ♧ࡉ࡞࠸ࠊii) ᶆⓗ⣽⬊ࡲ࡛ࡢ siRNA ࡢ㍺㏦ࢩࢫࢸ࣒ࡀ☜❧ࡋ࡚ ࠸࡞࠸ࠊiii) ᶆⓗ࡜␗࡞ࡿ㑇ఏᏊࡢⓎ⌧ࢆᢚไࡍࡿࠊ࡞࡝ࡢၥ㢟ࡀᐇ⏝໬ࡢጉࡆ࡟࡞ࡗ࡚࠸ ࡿࠋᡤᒓ◊✲ᐊ࡛ࡣࠊsiRNA ࡢᵓ㐀ⓗ≉ᚩ࡛࠶ࡿࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟╔┠ࡋࠊᙜヱ㒊఩ࡢ ໬Ꮫಟ㣭ࡀRNAi ࡟ཬࡰࡍຠᯝ࡟ࡘ࠸᳨࡚ドࢆ⾜ࡗ࡚ࡁࡓࠋࡑࡢ୰࡛᰾㓟ሷᇶࢆࡶࡓ࡞࠸໬ ྜ≀ࢆᑟධࡋ࡚ࡶ RNAi άᛶࡀపୗࡋ࡞࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡑࡇ࡛ⴭ⪅ࡣࠊᙜヱ㒊 ఩ࢾࢡࣞ࢜ࢩࢻ୰ࡢ᰾㓟ሷᇶࡣᚲࡎࡋࡶᚲせ࡛ࡣ࡞࠸࡜ண᝿ࡋࠊdRH_{ࢆ᭷ࡍࡿsiRNA ࡢ} ྜᡂ࠾ࡼࡧᛶ㉁ホ౯ࢆ⾜ࡗࡓࠋࡲࡓࠊ᭦࡞ࡿᶵ⬟ᛶྥୖࢆᮇᚅࡋࠊRH _{ࡢ 2 ఩Ỉ㓟ᇶࢆ࣋ࣥ} ࢪࣝ໬ࡋࡓRH_{OBnࢆ᭷ࡍࡿsiRNA ࢆྜᡂࡋࠊᛶ㉁ࢆホ౯ࡋࡓࠋ௨ୗࠊᚓࡽࢀࡓ▱ぢࢆせ⣙} ࡍࡿࠋ ࡇࢀࡲ࡛࡟dRH_{ྜᡂἲࡀᩘ౛ሗ࿌ࡉࢀ࡚࠸ࡿࡶࡢࡢࠊ㐣㓞࡞཯ᛂ᮲௳ࡀᚲせࡔࡗࡓࡓࡵࠊ} ฟⓎཎᩱ࡟dT ㄏᑟయࢆ⏝࠸ࡓࢢ࣮ࣜ࢝ࣝᙧᡂ཯ᛂࢆ฼⏝ࡋࡓᐇ⏝ⓗྜᡂἲࢆ㛤Ⓨࡋࡓࠋᐃ ἲ࡟ᚑࡗ࡚᰾㓟⮬ືྜᡂᶵ⏝ㄏᑟయ࡟ኚ᥮ࡋࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟ dRH_{ࢆ᭷ࡍࡿ࢜ࣜࢦ} ࢾࢡࣞ࢜ࢳࢻࢆྜᡂࡋࡓࠋ3′→5′࢚࢟ࢯࢾࢡࣞ࢔࣮ࢮࡢ୍ࡘ࡛࠶ࡿ࣊ࣅẘ࣍ࢫ࣍ࢪ࢚ࢫࢸࣛ ࣮ࢮࢆ⏝࠸࡚᰾㓟ศゎ㓝⣲⪏ᛶホ౯ࢆ⾜ࡗࡓ⤖ᯝࠊỗ⏝ࡉࢀ࡚࠸ࡿࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟ dT ࢆ᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻ࡜ẚ㍑ࡋ࡚ඃࢀࡓ⪏ᛶࢆ♧ࡋࡓࠋᑐᛂࡍࡿ┦⿵㙐ࢆ⏝࠸࡚ siRNA ࢆᵓ⠏ᚋࠊHeLa ⣽⬊ࢆ⏝࠸࡚㑇ఏᏊⓎ⌧ᢚไ⬟ࢆホ౯ࡋࡓ࡜ࡇࢁࠊdT ಟ㣭య࡜࡯ ࡰྠ➼࡛࠶ࡾࠊሷᇶ㒊Ḟᦆ᰾㓟ㄏᑟయࡢᑟධࡣRNAi άᛶ࡟ᙳ㡪ࢆཬࡰࡉ࡞࠸ࡇ࡜ࡀุ᫂ࡋ ࡓࠋ RH_{ࢆฟⓎཎᩱ࡜ࡋ࡚R}H_{OBnࢆྜᡂࡋࡓࠋࡑࡢ㝿ࠊࣝ࢖ࢫ㓟Ꮡᅾୗ࡛࣋ࣥࢬ࢔ࣝࢹࣄࢻ࡜} ࢺ࢚ࣜࢳࣝࢩࣛࣥࢆ⏝࠸࡚ 2 ఩Ỉ㓟ᇶࡢ࣋ࣥࢪࣝ໬ࢆ⾜࠺ࡇ࡜࡟ࡼࡾࠊ๓㥑య࠿ࡽࡢኚ᥮ ຠ⋡ࢆᨵၿࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟ RH_{OBnࢆ᭷ࡍࡿಟ㣭࢜ࣜࢦࢾࢡࣞ࢜} ࢳࢻࢆྜᡂࡋࠊ᰾㓟ศゎ㓝⣲࡟ᑐࡍࡿ⪏ᛶホ౯ࢆ⾜ࡗࡓ࡜ࡇࢁࠊRH_{OBnࡢᑟධ࡟ࡼࡗ࡚ศゎ} 㓝⣲⪏ᛶࡀ኱ᖜ࡟ྥୖࡋࡓࠋ50%࢘ࢩ⾑Ύࢆ⏝࠸࡚ RH_{OBnಟ㣭siRNA ࡢ⾑Ύ୰Ᏻᐃᛶࢆホ} ౯ࡋࡓ⤖ᯝࠊඃࢀࡓᏳᐃᛶࢆ♧ࡋࡓࠋࡲࡓࠊRNAi άᛶࢆホ౯ࡋࡓ࡜ࡇࢁࠊdT ಟ㣭 siRNA ࡜࡯ࡰྠ➼ࡢάᛶࢆ♧ࡋࡓࡇ࡜࠿ࡽࠊRH_{OBnಟ㣭ࡀ siRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟࡟ᙳ㡪ࢆཬ} ࡰࡉ࡞࠸ࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ ࡉࡽ࡟ࠊࢲࣥࢢࣜࣥࢢ࢚ࣥࢻ࡟ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࢆᑟධࡋࡓ siRNA ࡢ Ago2 ࢱࣥ ࣃࢡ㉁࡜ࡢぶ࿴ᛶࢆホ౯ࡍࡿࡓࡵࠊࣄࢺAgo2 ࡢ PAZ ࢻ࣓࢖ࣥࢱࣥࣃࢡ㉁ (Ago2–PAZ) ࡢ 㑇ఏᏊࢆࢥ࣮ࢻࡍࡿ࣋ࢡࢱ࣮ࢆ኱⭠⳦࡟ᑟධࠊᙧ㉁㌿᥮ࡋࠊAgo2–PAZ ࢱࣥࣃࢡ㉁ࢆᚓࡓࠋ ಟ㣭siRNA ࡜ Ago2–PAZ ࢱࣥࣃࢡ㉁࡜ࡢぶ࿴ᛶホ౯ࢆ⾜ࡗࡓ⤖ᯝࠊdT ಟ㣭య࡜ RH_OBnಟ

㣭య㛫࡛ぶ࿴ᛶ࡟኱ࡁ࡞ᕪ␗ࡣࡳࡽࢀ࡞࠿ࡗࡓࠋ 

42 ຍ࠼࡚ࠊ㖡Ꮡᅾୗ࢔ࢪࢻ໬ྜ≀࡜ࡢCuAAC ཯ᛂ࡟ࡼࡿ࣏ࢫࢺಟ㣭ࡀྍ⬟࡞ 2 ఩ࣉࣟࣃࣝ ࢠࣝಟ㣭RH _(RH_{OPg) ࢆ 3′ᮎ➃࡟᭷ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆྜᡂࡋࠊྛ✀࢔ࢪࢻ໬ྜ≀࡜} ࡢ CuAAC ཯ᛂ࡟ࡼࡾᑐᛂࡍࡿᵝࠎ࡞ 3′ᮎ➃ಟ㣭࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆᚓࡿࡇ࡜࡟ᡂຌࡋ ࡓࠋ ௨ୖᮏ◊✲࡛ࡣࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻㄏᑟయࡢᐇ⏝ⓗྜᡂἲࢆ☜❧ࡋࡓࠋࡲࡓࠊ3′ᮎ ➃࡟ሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻࢆ᭷ࡍࡿsiRNA ࡢᛶ㉁ホ౯ࢆ⾜࠸ࠊሷᇶ㒊Ḟᦆࢾࢡࣞ࢜ࢩࢻ ࡢᑟධࡀ㑇ఏᏊⓎ⌧ᢚไ⬟࡟ᙳ㡪ࢆཬࡰࡍࡇ࡜࡞ࡃ᰾㓟ศゎ㓝⣲࡟ᑐࡍࡿ⪏ᛶࡢྥୖ࡟ࡘ ࡞ࡀࡿࡇ࡜ࠊ≉࡟RH_{OBnࢆ᭷ࡍࡿsiRNA ࡣ㢧ⴭ࡞⾑Ύ୰Ᏻᐃᛶࢆ♧ࡍࡇ࡜ࢆぢฟࡋࡓࠋࡉ} ࡽ࡟ࠊsiRNA ࡢ㑇ఏᏊⓎ⌧ᢚไ⬟ホ౯ࡸ Ago2-PAZ ࡜ࡢぶ࿴ᛶホ౯࡟ࡼࡿ໬Ꮫಟ㣭ࢲࣥࢢ ࣜࣥࢢ࢚ࣥࢻࡢ⥙⨶ⓗゎᯒ࡟฼⏝ྍ⬟࡞2 ఩ࣉࣟࣃࣝࢠࣝಟ㣭 RH _(RH_{OPg) ࢆ㛤Ⓨࡋࡓࠋࡋ} ࡓࡀࡗ࡚ࠊᮏᡭἲࡣ᰾㓟་⸆๰〇ࡢࡓࡵࡢ᭷⏝࡞ࢶ࣮ࣝ࡟࡞ࡿ࡜ᮇᚅࡉࢀࡿࠋ

43

ㅰ

ㅰ㎡

 ᮏ◊✲ࡢᐇ᪋࡟࠶ࡓࡾࠊ⤊ጞࠊ⇕ᚰ࡞ᚚᣦᑟ࡜ᚚ㠴᧡ࢆ㈷ࡾࡲࡋࡓᒱ㜧኱Ꮫྡ㄃ᩍᤵ (ᒱ 㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆◊✲⛉๰⸆⛉Ꮫᑓᨷࠊ⌧ࠊឡ▱ᕤᴗ኱ᏛᕤᏛ㒊ᩍᤵ)࣭໭ฟᖾኵᩍᤵࠊ ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉๰⸆⛉Ꮫᑓᨷ࣭㉥ᑿᖾ༤ᩍᤵ࡟῝⏒࡞ࡿឤㅰࡢព ࢆ⾲ࡋࡲࡍࠋ  ᮏ✏ࡢసᡂ࡟㝿ࡋࠊ᭷┈࡞ᚚຓゝ࡜ᚚ㧗㜀ࢆ㈷ࡾࡲࡋࡓᒱ㜧⸆⛉኱Ꮫ⸆Ꮫ㒊 (ᒱ㜧኱Ꮫ኱ Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉๰⸆⛉Ꮫᑓᨷ)࣭Ᏹ㔝ᩥ஧ᩍᤵࠊᒱ㜧኱Ꮫ་Ꮫ㒊 (ᒱ㜧኱Ꮫ኱ Ꮫ㝔㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉་⒪᝟ሗᏛᑓᨷ)࣭୹⩚㞞அᩍᤵ࡟῝ࡃᣏㅰ⮴ࡋࡲࡍࠋ ᮏ◊✲ࡢᐇ᪋࡞ࡽࡧ࡟᥎㐍࡟࠶ࡓࡾࠊ⇕ᚰ࡞ᚚᣦᑟ࡜ᚚウㄽࢆ㈷ࡾࡲࡋࡓᒱ㜧኱ᏛᕤᏛ 㒊 (ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ๰⸆◊✲⛉๰⸆⛉Ꮫᑓᨷ)࣭ụ⏣ᑗᩍᤵ࡟ㅽࢇ࡛ᚚ♩⏦ࡋୖࡆࡲࡍࠋ ᮏ◊✲ࡢᐇ᪋࡞ࡽࡧ࡟ᮏㄽᩥࡢᚚᣦᑟ࡜ᚚウㄽࢆ㈷ࡾࡲࡋࡓᒱ㜧኱ᏛᕤᏛ㒊࣭႐ከᮧᚨ ᫛ຓᩍ࡟῝ㅰ⮴ࡋࡲࡍࠋ ᮏ◊✲ࡢ㐙⾜࡟࠶ࡓࡾ᭷┈࡞ᚚຓゝ୪ࡧ࡟ᚚ⃭ບࢆ㡬ࡁࡲࡋࡓᒱ㜧኱ᏛᕤᏛ㒊࣭ᰘ⏣⥤ ຓᩍ࡟ឤㅰ⏦ࡋୖࡆࡲࡍࠋ ⣽⬊ᐇ㦂ࡢ㐙⾜࡟࠶ࡓࡾࠊ⇕ᚰ࡞ᚚᣦᑟ࡜᭷┈࡞ᚚຓゝࢆ㡬ࡁࡲࡋࡓᒱ㜧኱ᏛᕤᏛ㒊 (ᒱ 㜧኱Ꮫ㐃ྜ๰⸆་⒪᝟ሗ◊✲⛉๰⸆⛉Ꮫᑓᨷ)࣭᳃⏣ὒᏊᩍᤵࠊ኱ᶫ᠇ኴ㑻෸ᩍᤵ࡟῝ࡃᣏ ㅰ⮴ࡋࡲࡍࠋ ㉁㔞ศᯒᶵჾࡢ౑⏝࡟࠶ࡓࡾࠊᚚᣦᑟ࡜ᚚ༠ຊࢆ㡬ࡁࡲࡋࡓᒱ㜧኱Ꮫ⏕࿨⛉Ꮫ⥲ྜ◊✲ ᨭ᥼ࢭࣥࢱ࣮࣭ࢤࣀ࣒◊✲ศ㔝࠾ࡼࡧᶵჾศᯒศ㔝ࡢⓙᵝ࡟ឤㅰ⮴ࡋࡲࡍࠋ  ᐇ㦂࡟㝿ࡋ࡚ࠊከ኱࡞ࡿᚚ༠ຊ࡜ᚚຓゝࢆ㡬ࡁࡲࡋࡓ∦ᒸ㈆≉ู◊✲ဨ (ᒱ㜧⸆⛉኱Ꮫྡ ㄃ᩍᤵࠊඖᶓ὾⸆⛉኱Ꮫᩍᤵ)ࠊཎ᫂≉ู◊✲ဨ (ඖᒱ㜧⸆⛉኱Ꮫᩍᤵ)ࠊ᳃♩⨾≉ู◊✲ဨࠊ Ἑฟ⨾࿴◊✲ဨࠊᑠ⦩⥤༤ኈ࡟ឤㅰ⮴ࡋࡲࡍࠋ ࡲࡓࠊ᪥ኪ⇕ᚰ࡟ᐇ㦂ࢆ⾜࠸ࠊ⚾ࢆዧ㉳ࡉࡏࡓᒱ㜧኱ᏛᕤᏛ㒊⮬↛⛉Ꮫ◊✲⛉⏕࿨⛉Ꮫ࣭ ໬Ꮫᑓᨷụ⏣◊✲ᐊࡢⓙᵝ࡟ឤㅰࡍࡿ࡜࡜ࡶ࡟ࠊᑛᩗࡢពࢆ⾲ࡋࡲࡍࠋ  ᭱ᚋ࡟ࠊ⤊ጞࠊ◊✲⏕άࡢບࡳ࡜࡞ࡗࡓ୧ぶ࡟῝ࡃឤㅰࡋࡲࡍࠋ