DESIGN OF AN OPEN SYSTEM IN THE THERMODYNAMIC SENSE FOR THE
CHEMICAL EVOLUTION OF BIOPOLYMERS. 1. INFLUENCE OF THE STEPWISE
ADDITION OF ACTIVATED NUCLEOTIDE MONOMER TO THE TEMPLATE-DIRECTEDFORMATION OF OLIGOGUANYLATE
Kunio Kawamura* and Akira Nakamura
Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University,
Gakuen-cho 1-1, Sakai, Osaka 599-8531, Japan.
Fax: +81-72-254-9910; Email: kawamura@chem.osakafu-u.ac.jp(Received: November 17, 2006
Accepted: December 25, 2006)(Abstract)
To construct a flow reactor system, which enables to simulate the chemical evolution of biopolymers in anopen system in the thermodynamic sense, the influence of stepwise addition of the 2-methylimidazolide ofguanosine 5’-monophosphate (2MeImpG) to the template-directed formation of oligoguanylate (oligoG) on
polycytidylic acid (polyC) template (TD reaction) was investigated. Both the experiment and the computational
simulation of the addition of 2MeImpG showed that the higher oligoGs were accumulated by the addition of2MeImpG. In addition, the elongation of oligoG was slightly effective when the activated nucleotide monomer
was added within a shorter interval period, where the total amount of the addition of the activated monomer waskept as constant. This fact indicates that the continuous supplement of 2MeImpG would facilitate the formationof oligoG rather than the stepwise addition of 2MeImpG when the total amount of guanosine unit was increased.
The present study showed that the computer simulations concerning the TD reaction can be used to evaluate
experimental data of the TD reaction.
(Keywords)
open system, experimental design, nucleotide, biopolymer, thermodynamics, kinetics,⇍ ງᏕ Ⓩ ࡝ 㛜 ᨲ ⣌ࡡ ໩ Ꮥ 㐅 ໩ ᐁ㥺 ⣌ ࡡ モ ష ࡛ࡐ ࡡ よ ᯊ㸯㸝 㸦 㸞࢛ ࣛ ࢥ ࢡ ࢓ ࢼࣜ 㓗 ࡡ 㗢 ᆵᣞ ♟ ⏍ᠺ ཬᚺ ࢅ ⏕ ࠷ ࡾ ࣓ࢿ ࣏ ࣭ ࡡ ㏪ ḗ౩ ⤝ ࡡ ᙫ 㡢ᕖᮟ㑝⏠*㸡୯ᮟකኬ㜨ᗋ❟ኬᏕኬᏕ㝌ᕝᏕ◂✪⛁ᚺ⏕໩Ꮥฦ㔕
ࠚ599-8531 ኬ㜨ᗋሗᕰᏕᅧ⏣ 1-1
Fax: 072-254-9910; Email: kawamura@chem.osakafu-u.ac.jp
㸦㸣 ᗆㄵ⏍∸ࡢ࢙ࢾ࣭࡛ࣜ࢟∸㈻ࢅ⎌ሾ࡛ࡡ㛣࡚ஹᥦࡌࡾࡆ࡛࡞ࡻࡖ࡙⏍࿤Ὡິࢅ⥌ᣚࡊ࡙࠷ࡾ㸣ࡗࡱࡽ㸡⏍࿤⣌ࡢ⇍ງᏕⓏ࡝㛜ᨲ⣌࡚࠵ࡾ㸣ᚉࡖ࡙໩Ꮥ㐅໩ࡡ◂✪࡚ࡢ㸡⏍࿤࡞Ⓠᒈࡊࡒ໩Ꮥཬᚺ⣌ࡢ⇍ງᏕⓏ࡝㛜ᨲ⣌࡚࠵ࡾ࡛࠷࠹っⅤࢅᣚࡔ㎲ࡳࡆ࡛࠿ᚪさ࡚࠵ࡾ㸣ࡆࡡࡻ࠹࡝᮪௲ࢅណᅒࡊ࡙⾔ࡖࡒᐁ㥺Ⓩ࡝◂✪ࡢᑛ࡝࠷㸣ౚ࠻ࡣ㸡Miller ࡡᐁ㥺[1]ࡷ௑஬࣬ᮿ㔕ࡼࡡ῕ᾇᗇࡡ⇍Ềᄂฝཾࢅᶅࡊࡒᐁ㥺[2]ࡢ㸡ᨲ㞹࠵ࡾ࠷ࡢ⇍࢙ࢾ࣭ࣜ࢟ࢅአ࠾ࡼ౩⤝ࡌࡾ⣌࡚࠵ࡾ࡛ぜ࡝ࡌࡆ࡛࠿࡚ࡀࡾ㸣ࡊ࠾ࡊ㸡ࡆࡿࡼࡡౚࡡࡻ࠹࡞⇍ࡷᨲᑏ⥲ࢅአ㒂࠾ࡼຊ࠻ࡾࡆ࡛ࡢẒ㍉Ⓩ⠾༟࡞࡚ࡀࡾ࠿㸡∸㈻ࢅὮฝ
ථࡈࡎࡾ⣌࡚ࡊ࠾ࡵ⁈ᾦ⣌ࡡ໩Ꮥ㐅໩ࡡᐁ㥺ࡢᑛ࡝࠷㸣Ferris ࡼࡢ㖌∸り፳ࢅ⏕࠷࡙࣓ࢿ࣏࣭ࢅᏽ᭿Ⓩ࡞῟ຊࡊ࡙㛏㙈ࡡ࢛ࣛࢥࢽࢠ࢛ࣝࢲࢺࡷ࢛ࣛࢥ࣋ࣈࢲࢺࢅ⏍ᠺࡈࡎࡒ[3]㸣ࡱࡒ㸡RNA ࡡin vitro selection ࡢཋᩩࢅṹ㝭Ⓩ࡞౩⤝ࡊ୘さ∸ࢅᤴฝࡌࡾࡡ࡚㸡ୌ⛸ࡡ㛜ᨲ⣌࡛ぜࡾࡆ࡛࠿࡚ࡀࡾࡓࢀ࠹㸣ࡒࡓࡊ㸡⏍∸⏜ᮮࡡ㓕⣪ࡡຐࡄ࠿ᚪさ࡚࠵ࡽ㸡໩Ꮥ㐅໩࡞ࡻࡖ࡙⏍ᠺࡊᚋࡾ㟸⏍∸Ⓩ࡝⣪ᮞࢅ౐ࡖ࡙ᵋᠺࡊࡒ⣌ࡢࡱࡓぜ࠵ࡒࡼ࡝࠷㸣ࡊ࠾ࡊ㸡ౚ࠻ࡣ໩Ꮥᕝᴏࣈࣚࣤࢹ࡚⏕࠷ࡼࡿࡾᵬᆵ
Ὦ㏳ཬᚺჹࡷ᚜⏍∸ࡡ◂✪ࡡࡒࡴࡡ chemostat ࡚ࡢ㸡⣌࡫ࡡ∸㈻ࡡᑙථ࣬ᑙฝࢅ㏻⤾Ⓩ࡞᡽࠹ࡆ࡛
࠿࡚ࡀࡾ࠿[4]㸡໩Ꮥ㐅໩ࡡᐁ㥺࡚ࡢ࡮࡛ࢆ࡜⏕࠷ࡼࡿ࡙ࡆ࡝࠾ࡖࡒ㸣ᚉࡖ࡙ RNA ࡷࢰࣤࣂࢠ㈻Viva Origino 35 (2007) 45 - 51
© 2007 by SSOEL Japanࡡ໩Ꮥ㐅໩ࡡ◂✪࡚ࡵ㸡㛜ᨲ⣌࡛࠷࠹っⅤ࡞࡙ࡼࡊ࡙◂✪ࡌࡾࡆ࡛࠿ᚪさ࡚࠵ࡾ㸣ࡐࡡࡒࡴ࡞ࡢ㸡㐲ᙔ࡝ཬᚺ⣌ࢅ㐽ࡦཋᩩࢅ㏛ຊࡊ⏍ᠺ∸࠾ࡼ୘さ∸ࢅᤴฝ࡚ࡀࡾᐁ㥺⣌ࢅᵋ⠇ࡊ࡝ࡄࡿࡣ࡝ࡼ࡝࠷㸣ࡱࡒࡆࡡ⛸ࡡᐁ㥺⣌࡚ࡢཬᚺ⣌ࢅ㛜ᨲ⣌࡞ࡌࡾຝᯕࢅㄢ࡬ࡾ࡞㊂ࡾ༎ฦ࡝⢥ᗐ࠿ᚪさ࡚࠵ࡾ㸣ࡐࡆ࡚ᮇ◂✪࡚ࡢࡆࡡ⛸ࡡᐁ㥺⣌ࢅᵋ⠇ࡌࡾ◂✪ࡡ➠ୌṹ㝭࡛ࡊ࡙㸡Ὡᛮ໩ࢽࢠ࢛ࣝࢲࢺࢅ⏕࠷ࡾ࢛ࣛࢥࢽࢠ࢛ࣝࢲࢺࡡ㗢ᆵᣞ♟ཬᚺ[5]ࢅ౐ࡖ࡙㸡ᏽ᭿Ⓩ࡞ཋᩩ࣓ࢿ࣏࣭ࢅ౩⤝ࡌࡾᐁ㥺ࢅ⾔ࡖࡒ㸣ࡱࡒࡆࡡཬᚺ⣌ࡡ྘⤊㊪ࡡ㏷ᗐᏽᩐࢅỬᏽࡊ㸡ࡐࡿࡼࡡೋ࡞ᇱࡘ࠷࡙࣓ࢿ࣏࣭ࢅ౩⤝ࡌࡾຝᯕࢅࢨ࣐࣭ࣖࣝࢨࣘࣤࡊࡒ㸣ࡐࡡ⤎ᯕ㸡⣌හࡡࢽࢠ࢛ࣝࢲࢺࡡሲᇱ࠵ࡒࡽࡡ⥪⃨ᗐ࠿ୌᏽ࡞ಕࡒࡿࡾ⣌࡚ࡢ࣓ࢿ࣏࣭ࢅᏽ᭿Ⓩ࡞౩⤝ࡌࡾຝᯕࡢᑚࡈࡂ㸡ࡱࡒ⣌හࡡࢽࢠ࢛ࣝࢲࢺࡡሲᇱ࠵ࡒࡽࡡ⥪⃨ᗐ࠿ኬࡀࡂ࡝ࡾ⣌࡚ࡢ࣓ࢿ࣏࣭ࢅ῟ຊࡌࡾ᫤㛣㛣㝰ࢅᑚࡈࡂࡌࡾ࡛࢛ࣛࢥࢽࢠ࢛ࣝࢲࢺࡡ⏍ᠺ㔖࠿ᑛࡊቌຊࡊࡒ㸣ࡈࡼ࡞㸡ࡆࡿࡼࡡ⌟㇗ࢅゝ⟤ᶭࢨ࣐࣭ࣖࣝࢨࣘࣤࢅ⏕࠷࡙᳠ッࡊ㸡ࡆࡡཬᚺ⣌ࡢ㛜ᨲ⣌ࢅᵋ⠇ࡌࡾࡒࡴࡡ⣪ᮞ࡛ࡊ࡙༎ฦ࡝⢥ᗐ්࡛⌟ᛮࢅᣚࡖ࡙࠷ࡾࡆ࡛ࢅ᪺ࡼ࠾࡞ࡊࡒ㸣㸧㸣ᐁ㥺࠽ࡻࡦࢨࢪࢷ࣑ࡡスゝ㸧㸣㸦㸣モⷾ

- 45 -

Viva Origino 35 (2007) 45 - 51
ࢡ࢓ࢿࢨࣤ 5’-࣓ࢿࣛࣤ㓗 2-࣒ࢲࣜ࢕࣐ࢱࢯࣛ
ࢺ㸝2MeImpG㸞ࡢ᪜ሒࡡ᪁Ἢ[6]࡞ᚉࡖ࡙ྙᠺࡊࡒ 㸣 ࣛ ࣍ ࢽ ࢠ ࣝ ࢓ ࣭ ࢭ A 㸝 RNaseA 㸡 Bovine
Pancrease ㉫″ R-5500 Type VII-A㸞ࡢ SIGMA ♣⿿ࡡࡵࡡ㸡࠽ࡻࡦ࣎ࣛࢨࢲࢩࣜ㓗㸝polyC㸞ࡢ⏍໩Ꮥᕝᴏ♣⿿ࡡࡵࡡࢅ⏕࠷ࡒ㸣ࡐࡡ௙ࡡモⷾࡢධ࡙≁⣥ဗࢅ⏕࠷ࡒ㸣㸧㸣㸧㸣⏍ᠺ∸ࡡฦᯊ⏍ᠺ∸ࡢ㧏㏷ᾦమࢠ࣏ࣞࢹࢡࣚࣆ࢔࣭
㸝 HPLC 㸞 ࡞ 㝔 ࢕ ࢛ ࣤ ஹ ᥦ ࢜ ࣚ ࣑ 㸝 TOSOH 㸡DNA-NPR㸡4.6 mm12.5 mm㸞࠵ࡾ࠷ࡢ㏣┞ฦ㒼࣑࢜ࣚ㸝ODS-2㸡GL ࢦ࢕࢙ࣤࢪ㸡4.6 mm15 mm㸞
ࢅࡗࡄ࡙ฦᯊࡊࡒ[7]㸣࢕࢛ࣤஹᥦ HPLC ࡢ㸡⁈㞫ᾦ࡛ࡊ࡙ A: pH=11.0 Ề⁈ᾦ㸡B: 1.5 M NaCl㸡
pH=11.0 ࢅ⏕࠷㸡B ࡡ๪ྙ࠿ 20%㸝t = 0 min㸞࠾ࡼ 100%㸝t = 70 min㸞࡛࡝ࡾ├⥲ࢡࣚࢩ࢙ࣤࢹࢅ⏕࠷࡙ฦᯊࡊࡒ㸣Ὦ㏷ࡢ 0.75 mL/min㸡࣑࢜ࣚῺᗐ 35Υ㸡᳠ฝࡢ 253 nm ࡚⾔ࡖࡒ㸣࢛ࣛࢥࢡ࢓ࢼ
ࣜ㓗㸝oligoG㸞ࡡ⏍ᠺ㔖ࡢ࢕࢛ࣤஹᥦ HPLC ࡡ⤎ᯕ࠾ࡼゝ⟤ࡊࡒ㸣ࡒࡓࡊࡆࡡ᪁Ἢ࡚ࡢ 2MeImpG㸡
ࢡ࢓ࢿࢨࣤ 5’-࣓ࢿࣛࣤ㓗 (5’pG)㸡࠽ࡻࡦ polyC
ࡡฦよ⏍ᠺ∸ࡡࢨࢲࢩࣤ 3’-࣓ࢿࣛࣤ㓗㸝C3’p㸞ࢅฦ㞫࡚ࡀ࡝࠷㸣ࡐࡆ࡚㏣┞ฦ㒼 HPLC ࢅ⏕࠷࡙㸡⁈㞫ᾦ࡛ࡊ࡙ A:0.005 M NaH2PO4 㸡pH 3.5㸡B:
0.01 M NaH2PO4 㸡40 % CH3OH㸡pH 4.0 ࢅ⏕࠷㸡Bࡡ๪ྙ࠿ 0%㸝t = 0 min㸞࠾ࡼ 40%㸝t = 30 min㸞㸡
100%㸝t = 45 min㸞࡛࡝ࡾࡻ࠹࡞୕オࡡ⿞⨠ࡡࢨࢪࢷ࣑ࢤࣤࢹ࣭࣭ࣞࣚࡡ㸧ࡡ᭜⥲ࢡࣚࢩ࢙ࣤࢹࢅ⏕࠷࡙ࡆࡿࡼࡡᠺฦࢅฦᯊࡊࡒ㸣Ὦ㏷ࡢ 1.0mL/min㸡࣑࢜ࣚῺᗐ 35Υ㸡᳠ฝࡢ 253 nm ࡚⾔ࡖࡒ㸣㸧㸣㸨㸣㗢ᆵᣞ♟ཬᚺ࠵ࡼ࠾ࡋࡴ 2.59 mg ࡡ࣎ࣛࢨࢲࢩࣜ㓗㸝polyC㸞ࢅࣈࣚࢪࢲࢴࢠࣁ࢕࢓ࣜ࡞࡛ࡽ 0.25 mL ࡡⵠ⏻Ề࡞⁈࠾ࡊ㸡ࡈࡼ࡞ 2.26 mg ࡡ 2MeImpG ࢅຊ࠻࡙⁈よࡊࡒ㸣ࡆࡡ⁈ᾦ࡞ 2.0 M NaCl㸡0.4 M MgCl2㸡
0.2 M HEPES (pH = 8.0) ࢅྱ ࡳ ⁈ᾦ 㸝HEPESbuffer㸞ࢅ 0.25 mL ຊ࠻࡙ࡻࡂ᧘ᢶࡊ㸡ࡇࡂ᚜ᑚమ✒ࡡሲ㓗࠵ࡾ࠷ࡢỀ㓗໩ࢻࢹ࣑ࣛࢗ⁈ᾦࢅຊ
࠻࡙ pH ࢅ 8.0 ࡞ㄢ⟿ࡊࡒ㸣ࡆࡡ⁈ᾦ࡞ࡢ 0.01 M
ࡡ 2MeImpG ࡛ 0.015 M ࡡ polyC㸡1.0 M NaCl㸡0.2
M MgCl2㸡0.1 M HEPES㸝pH = 8.0㸞࠿ྱࡱࡿࡾ㸣ྜྷᵕ࡞ࡊ࡙ 2MeImpG ࠵ࡾ࠷ࡢ polyC ࡡ⃨ᗐ࠿␏࡝ࡾ⁈ᾦࢅㄢ⿿ࡊࡒ㸣ࡆࡿࡼࡡ⁈ᾦࢅ 25ࠤ50Υ
࡚ 3 ᪝㛣ཬᚺࡈࡎࡒ㸣モᩩࢅᏽ᭿Ⓩ࡞᤿ཱིࡊᾦమ
✽⣪࡚█᫤࡞෼⤎ࡊࡒ㸣ཬᚺᚃ࡞⁈ᾦ୯ࡡ polyC
ࢅฦよࡌࡾࡒࡴ࡞㸡モᩩ 0.03 mL ࡞ᑊࡊ࡙ 0.0112
mL ࡡ RNaseA ⁈ᾦ
㸝3600 unit㸞࠽ࡻࡦ 0.2 M EDTA⁈ᾦ 0.018 mL ࢅຊ࠻࡙㸡37Υ࡚ 18 h 㛣ฌ⌦ࡊࡒ㸣ࡆࡿࢅཬᚺ A ࡛ࡌࡾ㸣ୌ᪁㸡ࡆࡿ࡛ྜྷᵕࡡ᪁Ἢ࡚ㄢ⿿ࡊࡒモᩩ⁈ᾦࢅཬᚺࡈࡎ㸡ᡜᏽ᫤㛣ࡇ࡛࡞モᩩࢅ᤿ཱིࡊࡒᚃ࡞
ཋᩩ➴ࢅྱࡳ⁈ᾦࢅ῟ຊࡌࡾ௧ୖࡡࡻ࠹࡝ᐁ㥺ࢅ⾔ࡖࡒ㸣ཬᚺ B㸯モᩩࢅ 12 h ࡇ࡛࡞ 50 µL ᤿ཱིࡊ㸡
0.01 M ࡡ 2MeImpG ࠽ࡻࡦ 0.015 MpolyC ࢅྱࡳ⁈ᾦࢅ 50 µL ῟ຊࡊࡒ㸣ࡆࡆ࡚ࡢࢡ࢓ࢼࣤࡡ࣓ࢿ࣏࣭༟న࡚ࡡ⃨ᗐࡢୌᏽ࡚࠵ࡽ㸡ࡱࡒ௙ࡡᠺฦࡡ⃨ᗐࡵୌᏽ࡚࠵ࡾ㸣ཬᚺ C1㸯モᩩࢅ 24 h ࡇ࡛࡞ 100 µL ᤿ཱིࡊ㸡0.02 M ࡡ 2MeImpG ࠽ࡻࡦ 0.015 M
polyC ࢅྱࡳ⁈ᾦࢅ 100 µL ῟ຊࡊࡒ㸣ࡌ࡝ࢂࡔ 2MeImpG ⃨ᗐࡢቌຊࡊ㸡ࡐࡡ௙ࡡᠺฦࡡ⃨ᗐࡢୌᏽ࡚࠵ࡾ㸣ཬᚺ C2㸯モᩩࢅ 12 h ࡇ࡛࡞ 50 µL ᤿ཱིࡊ㸡
0.02 M ࡡ 2MeImpG ࠽ࡻࡦ 0.015 M
polyC ࢅྱࡳ⁈ᾦࢅ 50 µL ῟ຊࡊࡒ㸣ࡌ࡝ࢂࡔ 2MeImpG ⃨ᗐࡢቌຊࡊ㸡ࡐࡡ௙ࡡᠺฦࡡ⃨ᗐࡢୌᏽ࡚࠵ࡾ㸣㸧㸣㸩㸣ゝ⟤ᶭࢨ࣐࣭ࣖࣝࢨࣘࣤ
 ཬᚺ㏷ᗐᏽᩐࡡỬᏽ࠽ࡻࡦཬᚺᣪິࡡࢨ࣐ࣖ
࣭ࣝࢨࣘࣤ࡞ࡢ SIMFIT[8]ࢅ⏕࠷ࡒ㸣㸨㸣⤎ᯕ࠽ࡻࡦ⩻ᐳ㸨㸣㸦㸣㗢ᆵᣞ♟ཬᚺࢅ⏕࠷ࡾ࣓ࢿ࣏࣭ࢅ㏪ḗ౩⤝ࡌࡾຝᯕ㗢ᆵᣞ♟ཬᚺ㸝Scheme 1㸞࡚ཋᩩࢅ῟ຊࡌࡾຝᯕࢅㄢ࡬ࡾ᮪௲࡛ࡊ࡙㸡ཬᚺῺᗐ࡞ࡗ࠷࡙᳠ゞࡊࡒ㸣㗢ᆵᣞ♟ཬᚺࡢ 25Υ࡚ࡢ⣑ 3 ᪝㛣࡚ᩐ 10㸚㐅⾔ࡌࡾ࠿㸡ཬᚺ࠿ᩐ᫤㛣࡚⤂஡ࡌࡾ⛤ᗐࡡ㏷ࡈ
ࢅᣚࡗ᮪௲ࡡ᪁࠿㏛㊟ࡊࡷࡌ࠷㸣ࡐࡆ࡚ཬᚺ㏷ᗐࢅᑛࡊ㧏ࡴࡾࡒࡴ࡞ 40Υ࠽ࡻࡦ 50Υ࡚㗢ᆵᣞ♟ཬᚺࢅモࡲࡒ㸣ࡊ࠾ࡊཬᚺຝ⋙ࡢ㟸ᖏ࡞఩ࡂ࡝ࡖ
ࡒ㸣ࡆࡿࡢ᪜ሒࡡ 2MeImpG ࡡ㗢ᆵᣞ♟ཬᚺࡡῺᗐ౪Ꮛᛮࡡ◂✪࡛ୌ⮬ࡊ࡙࠷ࡾ㸣࡛ࡆࢀ࠿㸡Zn(II)り፳Ꮛᅹୖ࡚ࢡ࢓ࢿࢨࣤ 5’-࣓ࢿࣛࣤ㓗࢕࣐ࢱࢯࣛࢺ㸝ImpG㸞࠾ࡼ oligoG ࠿⏍ᠺࡌࡾ㗢ᆵᣞ♟ཬᚺࡢ 60Υ࡚ࡵ࠾࡝ࡽཬᚺຝ⋙ࡢ㧏ࡂ㸡ᮇ⤎ᯕ࡛␏࡝ࡖ࡙࠷ࡾ[7]㸣2MeImpG ࢅ࣓ࢿ࣏࣭࡛ࡊ࡙⏕࠷ࡾ࡛ࡀ࡞ཬᚺຝ⋙࠿఩ࡂ࡝ࡾཋᅄ࡛ࡊ࡙௧ୖࡡ⌦⏜࠿⩻࠻ࡼࡿࡾ㸣㗢ᆵᣞ♟ཬᚺ࡚ࡢ 2ࠤ3 ฦᏄࡡὩᛮ໩ࢽࢠ࢛ࣝࢲࢺ࣓ࢿ࣏࣭࠿㗢ᆵ polyC୕࡞ఌྙࡊ࡙㒼าࡊ㸝Scheme 1㸞㸡㗢ᆵ୕࡚ࡐࡿࡼ࠿⦨ྙࡊ࡙ࣛࣤ㓗ࢩ࢙ࢪࢷࣜ⤎ྙ࠿⏍ᠺࡌࡾ[9]㸣ࡆࡡఌྙࡢ࣓ࢿ࣏࣭㛣ࡡ␧Ềᛮ┞பష⏕㸡ȧ-ȧ┞பష⏕࠽ࡻࡦ㗢ᆵ࡛ࡡỀ⣪⤎ྙ࠾ࡼ࡝ࡾ࡛⩻࠻ࡼࡿࡾࡡ࡚㸡Ὼᗐ୕᪴࡞ఔࡖ࡙ࡆࡿࡼࡡ┞பష⏕࠿ᘽࡱࡾࡡ࡚㗢ᆵᣞ♟ཬᚺࡡຝ⋙࠿఩ୖࡌࡾ࡛஢᝷ࡈࡿࡾ㸣ୌ᪁㸡ImpG ⣌࡚ࡢ Zn(II)ࡡ
഼ࡀ࡞ࡻࡖ࡙ࡆࡿࡼ࣓ࢿ࣏࣭㛣࠽ࡻࡦఘ㛏ࡌࡾoligoG ࡛ࡡఌྙ࠿⿭ᙁࡈࡿ 2MeImpG ⣌ࡻࡽࡵ㧏Ὼୖ࡚ oligoG ࠿ຝ⋙ࡻࡂ⏍ᠺࡊࡒࡵࡡ࡛᥆ᏽࡈ
ࡿࡾ㸣
2MeImpG ⣌࡚ࡢ Zn(II)ࢅྱࡱ࡝࠷ࡡ࡚ࡆࡡ
ࡻ࠹࡝ຝᯕ࠿ᘽࡂ㸡Ὼᗐ୕᪴࡞ࡻࡖ࡙ཬᚺຝ⋙࠿ኬࡀࡂ΅ᑛࡊࡒࡵࡡ࡛⩻࠻ࡼࡿࡾ㸣ࡆࡿࡼࡡ⤎ᯕ㸡ᮇ◂✪࡚ࡢῺᗐࢅ 25Υ࡛ࡊࡒ㸣ࡱࡒ㸡㗢ᆵᣞ♟ཬᚺࡡຝ⋙ࡢ࣓ࢿ࣏࣭࡛㗢ᆵ polyC ⃨ᗐ࡞ࡵ౪Ꮛࡌࡾࡡ࡚ 2MeImpG ࢅ 0.005ࠤ0.015 M㸡polyC
ࢅ 0.01ࠤ0.02 M ࡡ⠂ᅑ࡚⏕࠷ࡾࡆ࡛࡛ࡊࡒ㸣Scheme 1- 46 -Viva Origino 35 (2007) 45 - 51

Fig. 1. Anion-exchange HPLC chromatograms for the template-directed reaction ofoligoG on a polyC template.
Initial reaction conditions: [2MeImpG] = 0.01 M, [polyC] = 0.015 M, 1.0 M [NaCl] =1.0 M, [MgCl2] = 0.2 M, [HEPES] = 0.1 M, pH = 8.0.
A: control; B: A 50 µL sample solution was withdrawn at every 12 h and then a 50 µL
solution containing 0.01 M 2MeImpG and 0.015 M polyC was added; C1: A 100 µL
sample solution was withdrawn at every 24 h and then a 100 µL sample containing 0.02
M 2MeImpG and 0.015 M polyC was added; C2: A 50 µL sample solution waswithdrawn at every 12 h and then a 50 µL sample containing 0.02 M 2MeImpG and0.015 M polyC was added.

Fig. 2a.

Fig. 2c.

Fig. 2b.

Fig. 2d.

Fig. 2. Reaction curves for the template-directed reactions of oligoG on a polyC template.
Reaction conditions are the same as shown in Fig. 1.
a: reaction A, b: reaction B, c: reaction C1, d: reaction C2; open circles: 2MeImpG, closedcircles: 5’pG.

ࡆࡿࡼࡡ᮪௲ࢅ⩻៎ࡊ࡙ᐁ㥺㡧࡞♟ࡊࡒ᮪௲ୖ࡚㗢ᆵᣞ♟ཬᚺࢅ⾔ࡖࡒ㸣࢕࢛ࣤஹᥦ HPLC ࡞ࡻࡾࢠ࣏ࣞࢹࢡ࣑ࣚࢅ Fig. 1a – 1d ࡞♟ࡌ㸣ࡆࡆ࡚㸡࣓ࢿ࣏࣭࠽ࡻࡦ polyC ⃨ᗐ᮪௲ࡢ᪜Ꮛࡡ◂✪࡚⏕࠷ࡼࡿ࡙ࡀࡒ௥⾪Ⓩ࡝᮪௲ࡻࡽࡵ఩࠷࠿㸡oligoG ࡢຝ⋙ࡻࡂ⏍ᠺࡊࡒ㸣ཬᚺ A㸝Fig. 1a㸞ࡢ㏳ᖏࡡ㗢ᆵᣞ♟ཬᚺ࡚㸡ཬᚺ B㸝Fig. 1b㸞࡚ࡢཬ
ᚺ⁈ᾦࡡ 10㸚㸝50 µL㸞ࢅ 12 h ࡇ࡛࡞᤿ཱིࡊ㸡ࡐࡡ࡛ࡀิ᭿⃨ᗐࡡ 2MeImpG㸝0.01 M㸞࠽ࡻࡦ
polyC㸝0.015 M㸞ࢅྱࡳ⁈ᾦࢅ 50 µL ࡍࡗ⿭ඖࡊࡒ㸣ᚉࡖ࡙ཬᚺ B ࡚ࡢ࣓ࢿ࣏࣭༟న࡚ࡡࢡ࢓ࢼ⃨ࣤᗐࡢୌᏽ࡚࠵ࡾ㸣ࡆࡡ㸧ࡗࡡ⣌࡚ࡢ oligoGࡡ⏍ᠺ㔖ࡢཬᚺ A ࡚ࢂࡍ࠾࡞ኬࡀ࠾ࡖࡒ㸣ୌ᪁㸡ཋᩩࢅ౩⤝ࡌࡾཬᚺ C1 ࠽ࡻࡦ C2 ࡡ୦᪁࡚⏍ᠺ∸ࡡ㔖ࡢቌຊࡊࡒ㸝Fig. 1c, 1d㸞
㸣C1 ࠽ࡻࡦ C2 ࡚ࡢ 24 h ࠵ࡒࡽ࡚አ㒂࠾ࡼ῟ຊࡌࡾ࣓ࢿ࣏࣭ࡡ⥪㔖ࢅྜྷࡋ࡛ࡊࡒ࠿㸡ཚᐠ࡞ࡢ 24 h ࡇ࡛࡚ࡢࢡ࢓ࢿࢨ࣓ࣤࢿ࣏࣭༟న࠵ࡒࡽࡡ㔖ࡢ C1 ࡡ᪁࠿0.23%ኬࡀ࠷㸣ࡒࡓࡊᮇᐁ㥺࡚ࡢࡆࡡᕣࡢ↋っ࡚ࡀࡾ㸣ࡈࡼ࡞レ ⣵࡞㆗ㄵࡌࡾ ࡒࡴ࡞ࡆࡿ ࡼࡡHPLC ࡞ࡻࡾฦᯊ࡞ᇱࡘ࠷࡙㸡྘ཬᚺ⤊᫤ን໩ࢅ㏛㊟ࡊࡒ㸣
2MeImpG ࡡ΅ᑛ࡛ 5’pG ࡡ⏍ᠺ㸝Fig. 2a– 2d㸞㸡࠽ࡻࡦ oligoG ࡡ⏍ᠺᣪິࢅぜࡾࡒࡴ࡞5mer㸡10mer ࠽ࡻࡦ 15mer ࡡ⤊᫤ን໩ࢅㄢ࡬ࡒ
㸝Fig. 3a – 3c㸞㸣ࡆࡿࡼࡡ⤎ᯕ࠾ࡼ㸡ཬᚺ A ࠽ࡻ
ࡦ B ࡞࠽࠷࡙ࡢ㙈㛏࠿ᑚࡈ࠷ oligoG ࡡ⏍ᠺ⋙ࡢཬᚺ B ࡚ཬᚺ A ࡻࡽࡵࡷࡷ఩ࡂ㸡㙈㛏࠿ኬࡀࡂ࡝ࡾ࡛ཬᚺ B ࡡ᪁࠿ oligoG ࡡ⏍ᠺ⋙ࡢࡷࡷ㧏࠷࡛࠷࠹ലྡྷ࠿ヾࡴࡼࡿࡒ㸣ཬᚺ B㸡C1㸡࠽ࡻࡦC2 ࡚ࡢ 2MeImpG ࡡ΅ᑛ㸝Fig. 2b – 2d㸞࡞࠽࠷࡙㸡- 47 -

Viva Origino 35 (2007) 45 - 51µL ࡞ᑊࡊ࡙㸡㸭㹝ࡇ࡛࡞モᩩࢅ 0.05 mL ᤿ཱིࡊ0.375 M ࡡ 2MeImpG ࡛ 0.02 M ࡡ polyC ࢅ 50 µLຊ࠻ࡾ㸝ཬᚺ D1㸞㸡ࡱࡒࡢ㸡 16㹝ࡇ࡛࡞モᩩࢅ
100 µL ᤿ཱིࡊ 0.375 M ࡡ 2MeImpG ࡛ 0.02 M ࡡpolyC ⁈ᾦࢅ 100 µL ຊ࠻㸝ཬᚺ D2㸞㸡ཬᚺᣪິࢅ㏛㊟ࡊࡒ㸣ࡆࡡሔྙࡡ 4mer ࠽ࡻࡦ 14mer ࡡ oligoGࡡ⏍ᠺ㔖ࡢཬᚺ D1 ࡚ D2 ࡻࡽࡵ㧏ࡂ㸡୕オࡡ⣌࡚ࡡ⤎ᯕ࡛ྜྷᵕ࡚࠵ࡖࡒ㸣௧୕ࡡࡻ࠹࡞㸡㗢ᆵᣞ♟ཬᚺ⣌࡚ࡢࢽࢠ࢛ࣝࢲࢺࡡሲᇱ࠵ࡒࡽࡡ⥪⃨ᗐ࠿ୌᏽࡡሔྙ࡞ࡢ࣓ࢿ࣏࣭ࢅ౩⤝ࡌࡾ࡛ oligoG ࡡ⏍ᠺ㔖࠿ࡷࡷ΅ᑛࡊࡒ㸣ୌ᪁ࢽࢠ࢛ࣝࢲࢺࡡሲᇱ࠵ࡒࡽࡡ⥪⃨ᗐ࠿ቌຊࡌࡾሔྙ࡞ࡢ࣓ࢿ࣏࣭ࢅᏽ᭿Ⓩ࡞㏛ຊࡊ࠾ࡗࡐࡡ᫤㛣㛣㝰ࢅᑚࡈࡂࡌࡾ࡛ oligoG ࡡ⏍ᠺ㔖ࡢቌຊࡊࡒ㸣ࡊ࠾ࡊࡆࡿࡼࡡຝᯕࡢ᚜ᑛ࡚࠵ࡽ㸡ࡱࡒᮇᐁ㥺⣌ࡢཬᚺ⏍ᠺ∸ࡡฦᯊ࡛よᯊ࠿々㞟࡝ࡡ࡚㸡ཬᚺ B ࠵ࡾ࠷ࡢཬᚺ C1 ࡷ C2 ࡚ᐁ㥺࡞ࡻࡖ࡙ヾࡴࡼࡿࡒ㐢࠷࠿ጂᙔ࡚࠵ࡾࡡ࠾࡜࠹࠾
࡞ࡗ࠷࡙ࡈࡼ࡞᳠ッࡌࡾࡆ࡛࠿ᚪさ࡚࠵ࡾ㸣ࡐࡆ࡚㸡ᮇᐁ㥺ࢅゝ⟤ᶭࢨ࣐࣭ࣖࣝࢨࣘࣤࡊ࡙ཬᚺᣪ
ິࢅẒ㍉ࡌࡾࡆ࡛࡞ࡊࡒ㸣

Fig. 3a.

Fig. 3b.

Fig. 3c.

Fig.
3.
Reactioncurvesforthetemplate-directed reactions of oligoG on a
polyC template.
Reaction conditions are the same as shown inFig. 1.
a: formation of 5mer, b: formation of 10mer,
c: formation of 15mer. Reactions: opencircles: A, closed circles: B, open squares:
C1, closed squares: C2 as shown in thecaption of Fig. 1.

モᩩࢅ᤿ཱིࡊ࡙᩺ࡒ࡞አ㒂࠾ࡼ 2MeImpG ࢅ῟ຊࡊࡒࡆ࡛࡞ࡻࡖ࡙ 2MeImpG ⃨ᗐ࠿㝭ṹ≟ࡡን໩࡛ࡊ࡙⌟ࡿ࡙࠷ ࡾ㸣ཬᚺ᫤㛣 ࠿▯࠷⠂ᅑ ࡚ࡢoligoG ࡡ⏍ᠺ㔖ࡢ㸡ཬᚺ C1 ࠽ࡻࡦ C2 ࡢཬᚺ A࠽ࡻࡦཬᚺ B ࡛Ẓ࡬࡙࠵ࡱࡽ㐢ࢂ࡝࠾ࡖࡒ࠿㸡ཬᚺ᫤㛣࠿ 40 h ⛤ᗐ࠾ࡼࡢཬᚺ C1 ࠽ࡻࡦ C2 ࡚ኬࡀࡂ࡝ࡖࡒ㸣ཬᚺ C1 ࠽ࡻࡦ C2 ࡡ⣌࡚ࡢ࣓ࢿ࣏࣭ࢅ㏛ຊࡌࡾࡡ࡚㸡ࢽࢠ࢛ࣝࢲࢺࡡ⥪⃨ᗐࡢቌຊࡌࡾࡡ࡚ oligoG ࡡ⏍ᠺ㔖࠿ኬࡀࡂ࡝ࡾࡆ࡛ࡢ⮤↓࡚࠵ࡾ㸣ୌ᪁㸡24 h ࡇ࡛࡞Ẓ࡬ࡾ࡛ཬᚺ C1 ࡛ C2 ࡚ࡢྜྷ㔖ࡡ 2MeImpG ࢅ㏛ຊࡊࡒࡆ࡛࡞࡝ࡾ࠿㸡⏍ᠺ㔖ࡢཬᚺ C2 ࡡ᪁࠿ኬࡀࡂ࡝ࡾലྡྷ࠿ヾࡴࡼࡿࡾ㸣ࡆࡿࡢ㸡࣓ࢿ࣏࣭ࢅ㏪ḗ῟ຊࡌࡾ᫤㛣㛣㝰ࢅን࠻ࡾ࡛⏍ᠺ㔖࠿ን໩ࡌࡾࡆ࡛࡚࠵ࡽ⮾࿝῕࠷㸣ࡆࡡ⌟㇗ࡢ㸡ཬᚺ C1 ࠽ࡻࡦ C2 ࡛ྜྷᵕ࡞ࡊ࡙␏࡝ࡾ⃨ᗐ࡛࿔᭿࡚モᩩࢅ᤿ཱིࡊ࣓ࢿ࣏࣭ࢅ㏛ຊࡊࡒሔ ྙ ࡞ ࡵ ヾ ࡴ ࡼ ࡿ ࡒ 㸣 ౚ ࠻ ࡣ 0.0125 M ࡡ2MeImpG ࡛ 0.02 M ࡡ polyC ࢅྱࡳ㸝௙ࡡሲ㢦➴ࡡ⃨ᗐࡢཬᚺ C1 ࠽ࡻࡦ C2 ࡛ྜྷࡋ㸞ཬᚺ⁈ᾦ 50㸨㸣㸧㸣 ゝ⟤ᶭࢨ࣐࣭ࣖࣝࢨ࡛ࣘࣤࡡẒ㍉୕オࡡཬᚺ A㸡B㸡C1㸡࠽ࡻࡦ C2 ࡞ࡗ࠷࡙௧
ୖࡡ࣓ࢸࣜ࡞ᚉࡖ࡙ oligoG ࡡ⏍ᠺ㔖ࢅゝ⟤ࡊࡒ㸣
2MeImpG
Ѝ 2MeIm 㸠 pG
2MeImpG
㸠2MeImpG Ѝ (pG)2(pG)2  
㸠2MeImpG Ѝ (pG)3 ࣬ ࣬ ࣬(pG)18 
㸠2MeImpG Ѝ (pG)19ࡐࡿࡑࡿࡡ㐛⛤ࡡ㏷ᗐᏽᩐ࡛ࡊ࡙ Kanavarioti ࡼࡡᩝ⊡ೋ[9]㸡khy = 6.4 x 10-3 h-1㸡k2 = 0.11 M-1 h-1㸡
k3 = 29.0 M-1 h-1㸡k4 = k5 = ͐ = k19 = 65 M-1 h-1 ࢅ᭩ิ࡞⏕࠷ࡒ㸣ࢨ࣐࣭ࣖࣝࢨࣘࣤࡢ࡝ࡾ࡬ࡂ㙈㛏ࡡኬࡀ࡝ࡵࡡࡱ࡚ࢅᏽ⩇ࡊࡒ᪁࠿ᐁ㥺࣓ࢸࣜ࡞㎾ࡂ࡝ࡾ࡛⩻࠻ࡼࡿࡾࡡ࡚[7]㸡ࡆࡆ࡚ࡢ 19mer ࡱ࡚ࢅᏽ⩇ࡊࡒ㸣ᐁ㥺࡛ࡐࡿ࡞ᑊᚺࡌࡾࢨ࣐࣭ࣖࣝ
ࢨࣘࣤࡡ⤎ᯕࢅẒ㍉ࡊࡒ࠿㸡Ὡᛮ໩ࢽࢠ࢛ࣝࢲࢺࡡ΅ᑛࡡലྡྷࡢࡻࡂୌ⮬ࡊࡒ࠿㸡࡮࠾ࡡᣪິࡢ࠵ࡱࡽୌ⮬ࡊ࡝࠾ࡖࡒ㸣ࡆࡿࡢ㸡㗢ᆵᣞ♟ཬᚺࡡ㏷ᗐᏽᩐࡢࢽࢠ࢛ࣝࢲࢺ࣓ࢿ࣏࣭⃨ᗐ㸡㗢ᆵ⃨ᗐࡷῺᗐ࡝࡜ࡡᐁ㥺᮪௲࡞ࡻࡖ࡙ን໩ࡌࡾࡒࡴ࡚࠵ࡾ࡛⩻࠻ࡼࡿࡾ㸣ࡐࡆ࡚㸡ཬᚺ A ࡡᐁ㥺⤎ᯕ࠾ࡼࡆࡿࡼࡡ㏷ᗐᏽᩐࢅᨭࡴ࡙ SIMFIT ࡚Ửᏽࡊ㸡ࡐࡡೋ࡞ᇱࡘ࠷࡙ࢨ࣐࣭ࣖࣝࢨࣘࣤࢅ⾔࠹ࡆ࡛࡞ࡊࡒ㸣
ᚋࡼࡿࡒ㏷ᗐᏽᩐࡢ㸡khy = (9.43 ± 0.01) x 10-3-1
h 㸡k2 = (3.38 ± 0.05) x 10-2 M-1 h-1㸡k3 = (5.72 ± 0.18)
M-1 h -1㸡k4 = k5 = ͐ = k19 = (19.6 ± 0.9) M-1 h-1 ࡚࠵ࡖࡒ㸣ᮇᐁ㥺࡚ᚋࡼࡿࡒ㏷ᗐᏽᩐ࡛ Kanavariotiࡼࡡ⤎ᯕࢅẒ㍉ࡌࡾ࡛㸡2MeImpG ࡡຊỀฦよ㏷ᗐ࡞ࡗ࠷࡙ࡢ୦⩽ࡢẒ㍉Ⓩ㎾࠷ೋ࡚࠵ࡖࡒ࠿㸡ᮇᐁ㥺࡚ᚋࡒ௙ࡡ㏷ᗐᏽᩐࡢ Kanavarioti ࡼࡡ⤎ᯕ࡛Ẓ࡬࡙ᑚࡈ࠷ലྡྷ࠿ヾࡴࡼࡿࡒ㸣ᮇ◂✪࡚ᚋࡒ㏷ᗐᏽᩐࢅ⏕࠷࡙ࢨ࣐࣭ࣖࣝࢨࣘࣤࡊࡒ⤎ᯕࢅ
Fig. 4a – 4d㸡࠽ࡻࡦ 5a – 5c ࡞♟ࡌ㸣2MeImpG ࡡ΅ᑛ࡞ࡗ࠷࡙ࡢᐁ㥺ೋ࡛ゝ⟤ೋ࡚࠾࡝ࡽⰃࡂୌ⮬ࡊࡒ㸣ࡱࡒ oligoG ࡡ⏍ᠺ࡞ࡗ࠷࡙ࡵ㸡5mer ࡚ࡢゝ⟤ೋࡡ᪁࠿ᑚࡈ࠷ലྡྷ࠿ぜࡼࡿࡾ࠿㸡௙࡚ࡢ⏍ᠺᣪິࡢ࠽࠽ࡳࡠⰃࡂୌ⮬ࡊࡒ㸣

- 48 -

Viva Origino 35 (2007) 45 - 51

Fig. 4a.

Fig. 5a.

Fig. 5b.

Fig. 4b.

Fig. 5c.

Fig. 4c.

Fig. 5. Simulated reaction curves of thetemplate-directed reactions corresponding
to Fig. 3.
a: formation of 5mer, b: formation of10mer, c: formation of 15mer. Solid lines
from top to bottom: a: C1, C2, A, B; b andc: C2, C1, A, B; dashed line in b: reaction
conditions are the same as C1 and C2, but
the interval period is 6 h.

Fig. 4d.

Fig. 4. Simulated reaction curves of thetemplate-directed reactions corresponding
to Fig. 2.
a: reaction A, b: reaction B, c: reaction
C1, d: reaction C2; top lines at time 0:2MeImpG, bottom lines at time 0: 5’pG.

࡛ࡆࢀ࡚㸡ゝ⟤ᶭࢨ࣐࣭ࣖࣝࢨ࡚ࣘࣤࡢ࠵ࡾ㙈㛏ࡱ࡚ࡡ oligoG ࡡ⏍ᠺࢅ࠵ࡼ࠾ࡋࡴᏽ⩇ࡊ࡝ࡄࡿࡣ࡝ࡼ࡝࠷࠿㸡ᐁ㝷ࡡᐁ㥺࡞࠽࠷࡙ oligoG ࡡ⏍ᠺࡡ㙈㛏ࡡ㝀⏲ࢅᏽ⩇ࡌࡾࡆ࡛ࡢ↋ណ࿝࡚࠵ࡾ㸣ᮇ࣓ࢸ࡚ࣜࡢ 4mer ௧୕ࡡ⏍ᠺ㏷ᗐᏽᩐࢅୌ
ᏽ࡛௫ᏽࡊࡒ࠿㸡᭩㛏㙈㛏㸝19mer㸞ࡡ oligoG ࡢఘ㛏ཬᚺ࠿ᏽ⩇ࡈࡿ࡝࠷ࡡ࡚⏍ᠺ㔖࠿ቌኬࡌࡾലྡྷ࠿࠵ࡾ㸣ࡌ࡝ࢂࡔ㸡4mer ௧୕ࡡ⏍ᠺ㏷ᗐࢅ
ୌᏽ࡛ࡊ࡙ࢨ࣐࣭ࣖࣝࢨࣘࣤࡌࡾሔྙ࡞ࡢ㙈㛏ࡡቌຊ࡛࡛ࡵ࡞ oligoG ࡡ㔖ࡢ΅ᑛࡌࡾ࠿㸡19merࡡ⏍ᠺ㔖ࡓࡄࡢ 18mer ࡻࡽࡵኬࡀࡂ࡝ࡾലྡྷ࠿࠵ࡾ㸣ࡊ࠾ࡊ㸡ᮇ⣌࡚ࡢ୕㏑ࡡࡻ࠹࡞㛏࠷㙈㛏ࡱ࡚ࢅ௫ᏽࡊ࡙ࢨ࣐࣭ࣖࣝࢨࣘࣤࡌࡾࡆ࡛࡚ࡐࡡ
ᙫ㡢ࢅᑚࡈࡂࡌࡾࡆ࡛࠿࡚ࡀࡒ㸣oligoG ࡡ⏍ᠺ㔖ࡢ㸡ཬᚺ C2㸳C1㸳A㸳B ࡡᗆา࡚΅ᑛࡊ࡙࠽ࡽࡆࡡലྡྷࡢᐁ㥺ೋ࡛ゝ⟤ೋ࡚ୌ⮬ࡊࡒ㸣≁࡞㸡A
࡛ B㸡࠵ࡾ࠷ࡢ C1 ࡛ C2 ࡡ㐢࠷ࡢᑚࡈ࠷࡞ࡵ㛭ࢂࡼࡍ㸡ゝ⟤࡚ࡐࡡലྡྷࢅ්⌟࡚ࡀࡒࡆ࡛ࡢᮇ◂✪ࡡᐁ㥺⤎ᯕ࠽ࡻࡦᐁ㥺࣓ࢸࣜࡡよᯊἪ࡛ࢨ࣐࣭ࣖࣝࢨࣘࣤ࠿ጂᙔ࡚࠵ࡾࡆ࡛ࢅ♟ࡊ࡙࠷ࡾ㸣ࡌ࡝ࢂࡔ oligoG ࡡ⏍ᠺ㔖ࡢ㸡モᩩࢅ᤿ཱིࡊ࡙ࡗࡁ㊂ࡌ᫤㛣㛣㝰ࢅ▯ࡂࡌࡾ࡛ 12 h ࡇ࡛࡞ࡗࡁ㊂ࡊ
ࡒ᪁࠿ 24 h ࡇ࡛࡞ࡗࡁ㊂ࡌࡻࡽࡵࢂࡍ࠾࡞ኬࡀ
ࡂ࡝ࡾࡆ࡛ࢅࢨ࣐࣭ࣖࣝࢨ࡚ࣘࣤ᳠ッ࡚ࡀࡒ㸣ྜྷ᫤࡞㸡ゝ⟤࡞ࡻࡖ࡙ࡵࡖ࡛ࢸࣛࢢ࣭ࢹ࡝஢ῼࢅࡌ
ࡾࡆ࡛ࡵྊ⬗࡚࠵ࡾࡆ࡛ࢅ♟ြࡊ࡙࠷ࡾ㸣ࡐࡆ࡚㸡
C1 ࠽ࡻࡦ C2 ࡛ྜྷࡋཬᚺ⣌࡚᤿ཱི࡛῟ຊࡡࢰ࢕࣐ࣤࢡࢅ 6 h ࡞ࡊࡒሔྙࡡࢨ࣐࣭ࣖࣝࢨࣘࣤࢅ⾔ࡖࡒ㸣ࡐࡡ⤎ᯕ㸡6 h ࡡሔྙ࡞ࡢ 12 h 㛣㝰࡚ཋᩩࢅ㏛ຊࡌࡾሔྙࡻࡽࡵࡈࡼ࡞ oligoG ࡡ⏍ᠺ㔖࠿ኬࡀ࠷ࡆ࡛࠿஢ῼࡈࡿࡒ㸝Fig. 5b Ⅴ⥲㸞㸣 ࡆࡡ஥ᐁࡢ㸡᫤㛣㛣㝰ࢅᑚࡈࡂࡌࡾ㸡ࡌ࡝ࢂࡔ㏻⤾Ⓩ࡞ཋᩩࢅ౩⤝ࡊᤴฝࡌࡾሔྙ࡞ oligoG ࡡ⏍ᠺ㔖ࡢࡈࡼ࡞ቌຊࡌࡾྊ⬗ᛮࢅ♟ြࡊ࡙࠷ࡾ㸣ୌ᪁㸡ཬᚺ B ࡚ࡢᐁ㥺࡚ࡢ oligoG ࡡ⏍ᠺ㔖ࡢཬᚺ A ࡞Ẓ࡬࡙ᑚࡈࡂ࡝ࡾࡆ࡛࠿ヾࡴࡼࡿࡒ࠿㸡ࡆࡿࡢࢽࢠ࢛ࣝࢲࢺࡡሲᇱ࠵ࡒࡽࡡ⥪⃨ᗐ࠿ୌᏽ࡞ಕࡒࡿ࡙࠷࡙ࡵ㸡࣓ࢿ࣏࣭ࢅ㏪ḗ౩⤝ࡊ࡙⏍ᠺ∸ࢅ㝎ཡࡌࡾ⣌࡚ࡢ oligoG ࡡ⏍ᠺຝ⋙࠿఩ࡂ࡝ࡾࡆ࡛ࢅ♟ြࡊ࡙࠷ࡾ㸣ࡐࡆ࡚㸡ᏽ᭿Ⓩ࡞࣓ࢿ
࣏࣭ࢅຊ࠻ࡾ࡛ࡀࡡ᫤㛣㛣㝰ࢅን࠻࡙ oligoG ࡡ⏍ᠺ㔖࠿࡜ࡡࡻ࠹࡞ን໩ࡌࡾ࠾ゝ⟤ᶭࢨ࣐ࣖࣝ
࣭ࢨ࡚ࣘࣤㄢ࡬ࡒ㸝Fig. 6a – 6c㸞㸣㏪ḗ࣓ࢿ࣏࣭ࢅ῟ຊࡌࡾ᫤㛣㛣㝰ࢅ㸡0 h㸡36 h㸡24 h㸡࠽ࡻࡦ
12 h ࡛ࡊ࡙ゝ⟤ࡊẒ㍉ࡊࡒ㸝Fig. 6a – 6c ࡡࡐࡿࡑ- 49 -

Viva Origino 35 (2007) 45 - 51ࡿ⣵࠷Ⅴ⥲㸡⣵࠷ᐁ⥲㸡ኯ࠷Ⅴ⥲㸡ኯ࠷ᐁ⥲㸞㸣ࡐࡡ⤎ᯕ㸡᫤㛣㛣㝰ࢅኬࡀࡂࡊࡒ᪁࠿ᡜᏽ᫤㛣ࡇ࡛ࡡ oligoG ࡡ⏍ᠺ㔖ࡢኬࡀࡂ࡝ࡾࡆ࡛࠿஢ῼࡈࡿࡒ㸣15mer ࡚ࡢ῟ຊࡌࡾ᫤㛣㛣㝰࡞ࡻࡾ㐢࠷ࡢ㟸ᖏ࡞ᑚࡈࡂ࡝ࡖࡒ㸣ࡆࡿࡼࡢཬᚺ C1 ࠽ࡻࡦ C2ࡡሔྙ࡛ࡢୌぜ␏࡝ࡾലྡྷ࡚࠵ࡾ㸣ࢽࢠ࢛ࣝࢲࢺࡡ⥪⃨ᗐ࠿ୌᏽ࡞ಕࡒࡿࡾሔྙ࡞ࡢ㸡࣓ࢿ࣏࣭ࢅ῟ຊࡌࡾࡆ࡛࡚ oligoG ࡡ⏍ᠺ㔖࠿ቌຊࡌࡾຝᯕࡻࡽࡵ㸡⏍ᠺࡊࡒ oligoG ࢅ⣌࠾ࡼཱིࡽ㝎ࡂຝᯕࡡ᪁࠿ኬࡀ࠷ࡆ࡛ࢅ♟ࡊ࡙࠷ࡾ㸣ᚉࡖ࡙㸡ཬᚺ B࡞࠽࠷࡙࣓ࢿ࣏࣭ࢅ῟ຊࡌࡾ᫤㛣㛣㝰ࢅ↋㝀ᑚ࡞ࡊࡒሔྙ㸡ࡌ࡝ࢂࡔ㏻⤾Ⓩ࡝ᏰධΊྙཬᚺᵬ࡞
࠽࠷࡙౩⤝ࡈࡿࡾ࣓ࢿ࣏࣭⁈ᾦ࠿ཬᚺ⁈ᾦ࡛ྜྷࡋ࡚㸡࠾ࡗ౩⤝࡛ᤴฝࡡὮ㏷࠿ྜྷࡋሔྙ࡞ࡢ㸡oligoG ࡡ⏍ᠺ㔖ࡢᑚࡈࡂ࡝ࡾࡆ࡛ࢅ♟ြࡊ࡙࠷ࡾ㸣
Fig. 6a.

㸨㸣㸨㸣ࡱ࡛ࡴ࡛௑ᚃࡡㄚ㢗௧୕ࡡᐁ㥺࡛ࢨ࣐࣭ࣖࣝࢨࣘࣤ࡞ࡻࡖ࡙ᮇ㗢ᆵᣞ♟ཬᚺࢅ⏕࠷࡙ཋᩩࢅࡗࡁ㊂ࡌཬᚺ࡞ࡗ࠷࡙௧ୖࡡࡆ࡛࠿ฦ࠾ࡖࡒ㸣ࡱࡒ㸡㛜ᨲ⣌࡚㗢ᆵᣞ♟ཬᚺࢅࡌࡾሔྙࡡၡ㢗Ⅴࡵ஢ῼࡈࡿࡒ㸣㸦㸞࣓ࢿ࣏࣭ࢅᏽ᭿Ⓩ࡞౩⤝ࡊ⏍ᠺ∸ࢅྱࡳ⁈ᾦࢅᤴฝࡌࡾ࡛ࡀ㸡ࢽࢠ࢛ࣝࢲࢺࡡሲᇱ࠵ࡒࡽࡡ⥪⃨ᗐ࠿ୌᏽ࡞ಕࡒࡿࡾ࡛ࡀ㸡oligoG ࡡ⏍ᠺ㔖ࡢᑚࡈࡂ࡝ࡾ㸣ࡱࡒ౩⤝ࡌࡾ᫤㛣㛣㝰ࢅᑚࡈࡂࡌ
ࡾ࡛ oligoG ࡡ⏍ᠺ㔖ࡢࡻࡽᑚࡈࡂ࡝ࡾ㸣㸧㸞ྜྷᵕ࡞ࡊ࡙㸡ཋᩩ࣓ࢿ࣏࣭ࢅᏽ᭿Ⓩ࡞౩⤝ࡊࢽࢠ࢛ࣝࢲࢺࡡሲᇱ࠵ࡒࡽࡡ⥪⃨ᗐ࠿ኬࡀࡂ࡝ࡾ࡛ࡀ࡞ࡢ㸡㙈㛏ࡡኬࡀ࡝ oligoG ࡡ⏍ᠺ㔖ࡢቌຊࡌࡾ㸣oligoG ࡡ⏍ᠺ㔖ࡡቌຊࡢࢽࢠ࢛ࣝࢲࢺࡡධమ㔖ࡡቌຊ࡞ఔ࠹ࡵࡡ࡚࠵ࡾ࠿㸡ࡐࡡሔྙ࡞ࡢモᩩࢅ᤿ཱིࡊ࣓ࢿ࣏࣭ࢅ㏛ຊࡌࡾ᫤㛣㛣㝰ࢅᑚࡈࡂࡌࡾ࡮࡜ oligoG ࡡ⏍ᠺ㔖ࡢኬࡀࡂ࡝ࡖࡒ㸣㸨㸞ゝ⟤ᶭࢨ࣐࣭ࣖࣝࢨ࡚ࣘࣤࡢ࢛ࣛࢥࢽࢠ࢛ࣝࢲࢺࡡ⏍ᠺࡢ࠵ࡾ㙈㛏ࡱ࡚ࡊ࠾ᏽ⩇࡚ࡀ࡝࠷࡛࠷࠹ၡ㢗࠿࠵ࡾ࠿㸡ࢨ࣐࣭ࣖࣝࢨ࡚ࣘࣤᏽ⩇ࡌࡾ㙈㛏ࢅኬࡀࡂࡌࡾࡆ࡛࡚࡮࡯よỬ࡚ࡀࡾ㸣㸩㸞ཬᚺ⣌࡞࣓ࢿ࣏࣭࡝࡜ࢅ㏪ḗ౩⤝ࡌࡾຝᯕࢅᐁ㥺࡞ࡻࡖ࡙ㆉื࡚ࡀࡾࡆ࡛࠿ฦ࠾ࡖࡒ㸣ࡊ࠾ࡊ㸡ᮇ◂✪࡚♟ࡊࡒཬᚺ A ࡛ཬᚺ B ࡡ㐢࠷ࡷ㸡ཬᚺ C1 ࡛ཬᚺ C2 ࡡ㐢࠷ࡢ᚜ᑚ࡚࠵ࡾ㸣ᚉࡖ࡙㸡ࣆ࣭ࣞ⣌࡝࡜ࡡࡻࡽ々㞟࡝⣌ࡡᣪິࢅㄢ࡬ࡾሔྙ࡞ࡢᐁ㥺࡛ࢨ࣐࣭ࣖࣝࢨࣘࣤࢅె⏕ࡊ࡙フ౮ࡌࡾࡆ࡛࠿᭻ࡱࡊ࠷ࡆ࡛ࢅ▩ࡖࡒ㸣

Fig. 6b.

ㅨ㎙
SIMFIT ࡢ von Kiedrowsiki ᩅ᤭࡞࠷ࡒࡓࡀࡱࡊࡒ㸣ࡆࡆ࡞ㅨណࢅ⾪ࡊࡱࡌ㸣

Fig. 6c.

Fig. 6. Simulated reaction curves of the thetemplate-directed reactions corresponding
to Fig. 2a and 2b and its variations.
a: disappearance of 2MeImpG andformation of pG, b: formation of 5mer, c:formation of 10mer.
Lines: thin dashed lines: no monomer wasadded; solid lines: a 150 µL sample solutionwas withdrawn at every 36 h and then a 150µL solution containing 0.01 M 2MeImpG
and 0.015 M polyC was added; bold dashedlines: a 100 µL sample solution waswithdrawn at every 24 h and then a 100 µLsolution containing 0.01 M 2MeImpG and0.015 M polyC was added; bold solid lines:
reaction B.

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