4章 困難な課題への挑戦 核酸結晶学
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1 I. a. DNA DNA RNA Watson & Crick 1953 b. DNA DNA DNA DNA RNA Cech et al RNA in vitro selection DNA Breaker & Joyce % 50% 30% International Human Genome Sequencing Consortium
2 DNA RNA RNA RNA rrna trna mrna snrna RNA rrna 50S V Garrett & Rodriguez-Fonseca 1996 c. - DNA G, C, A, T RNA G, C, A, U DNA C3'-endo C2'-endo A B C D E B Z RNA 2
3 C3'-endo A RNA DNA II. a. DNA RNA DNA12 d(cgcgaattcgcg) Wing et al B P DNA12 Chatake et al. 1999a, 3
4 1999b; Hossain et al. 2001a, 2001b; Tsunoda et al. 2001, 2002 A:T A:U G:C II HDV GAAA Ferré-D'Amaré et al Scott et al RNA Anderson et al I II Cate et al. 1996; Zhang & Doudna 2002 II-e U1A RNA RNA RNA U1A U1A-P RNA RNA U1A-RNA U1A-P 1 U1A-P U1A-RNA K d ~10-11 M; van Gelder et al. 1993) U1A-P MAD RNA RNA HDV Ferré-D'Amaré & Doudna 2000; Rupert & Ferré-D'Amaré
5 1 U1A-RNA HDV A T A U G C G:C A:T A:U G:C G G G 2 MFOLD Jacobson & Zuker 1993 Vienna RNA package Hofacker 2003 RNA secondary structure prediction Brodsky et al MFOLD Vienna RNA package RNA secondary structure prediction 5
6 2 MAD Å DNA/RNA Ennifar et al G:C A:T A:U G:C C 5 pk a 2 Sunami et al C:C + b. DNA RNA Caruthers et al DNA/RNA 6
7 3' 5' DNA RNA DNA 2 - t- TBDMS 2-3'- DNA TBDMS 2 - RNA - 3:1 TBDMS -n- II-c RNA RNA in vitro RNA Summers & Siegel 1970 T7 RNA in vitro in vitro 1mol 100mol RNA in vitro RNA DNA DNA (i) RNA (ii) RNA (iii) DNA T7 DNA 80 DNA DNA T7 RNA G Davanloo et al. 1984) 5 G RNA 50µl 1ml 7
8 DNA 1 RNA II-c DNA DNA PCR Saiki et al PCR DNA in vitro DNA Taq DNA PCR DNA DNA (i) 20 (ii) GC 45~60% (iii) 5' 3' T m (vi) 5' 3' (v) PCR DNA Taq 10-3 ~10-5 DNA Taq DNA Pfu DNA Vent DNA Taq DNA c. HPLC HPLC 8
9 HPLC 18 5' DMTr 5'- DMTr A 0.1M (ph 7.0) B HPLC DEAE 25mM (ph6.0) 25mM (ph6.0) 1M HPLC PAGE 7M 1kbp 1 TBE 89mM 89mM 2.5mM EDTA ph8.3 40cm 30cm 10 C BPB XC 260nm 9
10 UV shadowing 1 (% w/v) ( ) ( ) ~ ~ ~ ~ ~ ~ C 8 10
11 d. PAGE 0.05% 7M 0.1~1 pmol/0.5~1 µl MALDI Matrix Assisted Laser Desorption Ionization TOF-MS Time of Flight mass spectroscopy MALDI-TOF-MS MALDI TOF-MS 11
12 MALDI-TOF-MS Na Ragas et al RNA MALDI-TOF-MS PAGE 230nm 260nm 280nm 320nm A 260 /A 280 A 260 /A 280 A 260 /A 280 = A 260 /A 230 = A 260 /A nm A 260 /A nm 275nm e. II-a Fedor 2002 ph 100mM EDTA 7M 12
13 II-d 32 P 33 P 2 32 P Nowakowski et al. 1999, 2000 Mg 2+ 4 Kondo & Takénaka 2000 PAGE Mg 2+ PAGE PAGE TBE ph 1mM II-d EtBr 1µg/ml EtBr 260nm EtBr 590nm DNA EtBr 13
14 4 PAGE (a) Mg 2+ (b) 10 mm Mg 2+ (c) 10 mm Mg C mer 2 17mer mer RNA 5 12mer RNA f. DNA DNA RNA DNA RNA RNA DNA RNA DNA DNA RNA III. 14
15 ph DNA RNA - a ,4- MPD PEG trna (NH 4 ) 2 SO 4 Li 2 SO 4 pk a ph pk a ph5.0 C:C + C ph 4.7~6.8 Robinson et al. 1992, 1993; Inman, R.B RNA ph ph 5.0~7.4 ph 7.0~8.0 15
16 20~100mM ph 100~300mM Mikol et al Mg 2+ G Tsunoda et al Mg 2+ Co 2+ Mn 2+ Ennifar et al Sr 2+ Ba 2+ Ennifar et al Mg 2+ Pb 2+ Zn 2+ Cd 2+ Fedor 2002 K + G Pilch et al trna Young et al Ladner et al trna 16
17 Sauter et al Mg 2+ G Sunami et al. 2004; Kondo et al Å MAD Sunami et al Sunami et al T U Sunami et al RNA U1A-RNA U1A HDV Ferré-D'Amaré & Doudna 2000; Rupert & Ferré-D'Amaré 2001 III-d MAD CD 1 17
18 b. T m 4~37 C DNA I-c A B 4 C 100K RNA DNA 20~37 C RNA RNA X α- c. 18
19 B DNA Arai et al nm Gray et al mer (1) 5 neaa neac neag neat (2) A C G T U nea nec neg net neu (3) 2 ε ( DNA ) = 2 [ neaa ε ( daa) + neac ε( dac) + neag ε( dag) + neat ε ( dat) +...] [ nea ε ( da) + nec ε ( dc) + neg ε ( dg) + net ε ( dt) ] ( RNA ) = 2 [ neaa ε( raa) + neac ε( rac) + neag ε( rag) + neau ε( rau) +...] [ nea ε ( ra) + nec ε ( rc) + neg ε ( rg) + neu ε ( ru) ] ε (1) A C G T U nea nec neg net neu (2) 2 ε ( DNA) = nea ε ( da) + nec ε ( dc) + neg ε ( dg) + net ε (dt) ε ( RNA) = nea ε ( ra) + nec ε ( rc) + neg ε ( rg) + neu ε (ru) Berger et al = 24mM ( 20mM) / 19
20 DNA mM / 24 = 1mM 2 DNA RNA A AG CU TC C AT GA TG G AU GC TT T CA GG UA U CC GT UC AA CG GU UG AC CT TA UU CD 2 Berger Scott Nucleic acid mini screen TM Natrix Hampton research 0~200mM CD 1 20~1000mM CD 3 Kondo & Takénaka 2002 Crystal screen TM 20
21 ph Mg mM Na + Sr 2+ d E. coli DNA dt 4 DNA RNA K d 10-5 M RNA DNA 21
22 ph DNase I DNA Suk et al X 1.9Å Richmond & Davey 2003 trna Asp aspartyl-trna trna Asp aspartyl-trna 2:1 Lorber et al PEG MPD (NH 4 ) 2 SO 4 RNA- (NH 4 ) 2 SO 4 MgCl 2 CaCl 2 e. X DNA10 Arai et al
23 B DNA 5 Solubility curve II Kondo & Takénaka RNA 23
24 RNA MPD 0.5% (w/v) MPD 6 IV. 24
25 - - HPLC HPLC CL-2 CL-4 CL-3Cm HPLC 7 8 Takénaka et al (a) (b) (c) (b) 8 330~380nm Ultra Violet 400~440nm Blue Violet UV BV 9 25
26 (a) (b) (c) (d) 9 HIV-1 RNA (b) BV (d) UV 10 UV 460~480nm BV 480~500nm (a) UV (b) BV 11 DNA ( 300~800nm) 26
1 1 H Li Be Na M g B A l C S i N P O S F He N Cl A e K Ca S c T i V C Mn Fe Co Ni Cu Zn Ga Ge As Se B K Rb S Y Z Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb T e
No. 1 1 1 H Li Be Na M g B A l C S i N P O S F He N Cl A e K Ca S c T i V C Mn Fe Co Ni Cu Zn Ga Ge As Se B K Rb S Y Z Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb T e I X e Cs Ba F Ra Hf Ta W Re Os I Rf Db Sg Bh
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