1
Electronic Supplementary Material
2
Title
3
Assembly of zinc finger motif-fused enzymes on a dsDNA scaffold for catalyzing consecutive reactions
4
with a proximity effect
5
6
Authors
7
Hisakage Funabashi, Satoshi Yanagi, Shigeya Suzuki, Masayasu Mie, Eiry Kobatake
8
9
10
List of contents
Supplementary materials and methods
11
Plasmid construction for protein production
12
Protein expression and purification
13
DNA scaffold production
14
Measurement of bioluminescence
15
16
17
Supporting results and discussions
Evaluation of the biding specificity to the DNA scaffold
Reference
18
19
20
1
21
Supplementary materials and methods
22
All chemicals utilized in the experiments were of analytical grade, unless otherwise specified in the
23
manuscript. Synthesized oligonucleotides were obtained from Texas genomics and are listed in Table S1.
24
Restriction endonucleases and DNA-modifying enzymes for plasmid constructions were obtained from
25
TaKaRa Bio (Shiga, Japan). Polymerase chain reactions (PCR) were carried out with either KOD or KOD
26
–plus–DNA polymerase from Toyobo (Osaka, Japan).
27
28
Supplementary Table 1. The sequence of synthesized oligonucleotides used in the research
Sequence (5' to 3')
Name
O1 AAGGTACCTAGATCTGAACGCCCATATGCTTGCCCTGTCG
O2 CGTCTAGACGGATCCACCGCCGTCCTTCTGTCTTAAATGGATTTTGG
AAGGTACCTAGATCTGAGAAGCCCTATGCTTGTCCGGAATGTGGTAAGTCCTTCAGCCAGCGCGCAAACCTGC
O3
GCGCCCACCAGCGTACC
TAGTCAGGTGATCGCTGCGGCTAAAAGATTTGCCGCACTCTGGGCATTTATACGGTTTTTCACCCGTGTGGGT
O4
ACGCTGGTGGGCGCGCA
CCGCAGCGATCACCTGACTACCCATCAACGCACTCATACTGGCGAGAAGCCATACAAATGTCCAGAATGTGGC
O5
AAGTCTTTCAGTCGCAG
CGTCTAGACGGATCCACCGCCATCTTTTTCACCGGTGTGAGTACGTTGGTGGCGCACCAGCACATCGCTGCGA
O6
CTGAAAGACTTGCC
O7 AAGGTACCTGAATTCATGCCGAAGTACGTTTACGACTTCACCG
O8 CGTCTAGACGCTAGCACCGCCTCGGGTGTCGGACGCCGCGCACGTCAGCG
O9 AAGGTACCTAGATCTATGGAAGACGCCAAAAACATAAAG
O10 CCCAAGCTTACAATTTGGACTTTCCGCCCTTCTTGGCC
O11 GATCCGCGTGGGCGGCGTGGGCGTCGACTTAAGAATTCCATGGCTAGC
O12 GGCCGCTAGCCATGGAATTCTTAAGTCGACGCCCACGCCGCCCACGCG
O13 AATTGTGAGCGGATAACAA
O14 GGGATGTGCTGCAAGGCGA
O15 GATTTAGAGCTTGACGGGG
O16 GGCCGCATGCTGCAGGTACCTCGAGTTTCCACACTTTCCACACAGATCTGCT
O17 CTAGAGCAGATCTGTGTGGAAAGTGTGGAAACTCGAGGTACCTGCAGCATGC
O18 GGGAGAAAGGCGGACAGG
O19 GATCCGCGTGGGCGGCGTGGGCGGTGTGGAAAGTGTGGAAAAGATC
O20 GATCTTTTCCACACTTTCCACACCGCCCACGCCGCCCACGCGGATC
O21 GATCCGCGTGGGCGGCGTGGGCGTCGACTCGAGGTGTGGAAAGTGTGGAAAAGATC
O22 GATCTTTTCCACACTTTCCACACCTCGAGTCGACGCCCACGCCGCCCACGCGGATC
O23 GATCCGCGTGGGCGGCGTGGGCGTCGACTTAAGAATTCTCGAGGTGTGGAAAGTGTGGAAAAGATC
O24 GATCTTTTCCACACTTTCCACACCTCGAGAATTCTTAAGTCGACGCCCACGCCGCCCACGCGGATC
GATCCGCGTGGGCGGCGTGGGCGTCGACTTAAGAATTCCATGGGTACCTCGAGGTGTGGAAAGTGTGGAAAAG
O25
ATC
GATCTTTTCCACACTTTCCACACCTCGAGGTACCCATGGAATTCTTAAGTCGACGCCCACGCCGCCCACGCGG
O26
ATC
O27 GTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGA
O28 TCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATAC
29
30
31
32
2
33
Plasmid construction for protein production
34
Zif268
35
The Zif268 (Pavletich and Pabo 1991) gene was amplified by PCR using the cDNA library as a template,
36
made from neural and glial cells that were induced from Mouse Embryonal Carcinoma P19 cells by the
37
addition of retinoic acid. The mRNAs were first extracted from the cultured cells with TRIzol (Invitrogen)
38
and donated for reverse transcription to generate the cDNA libraries utilizing Super Script II Reverse
39
Transcriptase (Invitrogen). Each process was carried out following the manufacturers’ protocols. The
40
Zif268 gene was then cloned into the Kpn I and Xba I sites of pBluescript II SK (+) (Toyobo) after PCR
41
amplification with the cDNA library as a template, and O1 and O2 primers, generating pBS-zif268.
42
43
Tandem Zif268
44
The zif268 gene fragment was cut out from pBS-zif268 by treatment with Bgl II and Xba I digestions.
45
The purified zif268 fragment was then re-inserted into the BamH I and Xba I sites of pBS-zif268, producing
46
pBS-zif268-zif268. This plasmid encodes two Zif268 motifs connected by the GGGS amino acid sequence
47
as a linker.
48
49
PBSII
50
The PBSII (Blancafort et al. 2004; Ooi et al. 2006) gene was artificially generated using PCR with 4
51
synthesized oligos (O3 to O6) as a template. The amplified fragment was cloned into pBluescript II SK(+)
52
after Kpn I and Xba I digestions, generating pBS-PBSII.
53
54
55
3
56
Tandem PBSII
57
The PBSII gene was cut out from pBS-PBSII by treatment with Bgl II and Xba I restriction enzymes. The
58
PBSII fragment was then re-inserted into the BamH I and Xba I sites of pBS-PBSII, producing pBS-PBSII-
59
PBSII. This plasmid encodes two PBSII motifs connected by the GGGS amino acid sequence as a linker.
60
61
PPDK- Zif268-Zif268
62
First, the gene of PPDK (Eisaki et al. 1999) was amplified by PCR from pPPDK23 with O7 and O8, and
63
cloned into pBluescript II SK (+) after Kpn I and Xba I digestions, generating pBS-PPDK. The gene
64
fragment of PPDK was cut out by EcoR I digestion. The edge of the fragment was blunted and then treated
65
with the Not I restriction enzyme. The fragment was inserted between the Not I site and the blunted BamH
66
I site of pGEX-5X-1 (GE Healthcare), generating pGEX-PPDK. The gene fragment of zif268-zif268 was
67
cut out from pBS-zif268-zif268 by treatment with Bgl II, followed by the blunting reaction and Not I. This
68
fragment was inserted between the Not I and blunted Nhe I restriction sites, producing pGEX-PPDK-
69
zif268-zif268.
70
71
PBSII-PBSII-fLuc
72
First, the gene fragment of PBSII-PBSII was cut out from pBS-PBSII-PBSII by treatment with Bgl II,
73
followed by the blunting reaction and Not I. This fragment was inserted between the Not I and blunted
74
BamH I restriction sites of pGEX-5X-1, generating pGEX-PBSII-PBSII. Next, the gene of thermostabilized
75
fLuc was amplified by PCR from pMALU7 (Ebihara et al. 1999) with O9 and O10, and cloned into
76
pBluescript II SK (+) after Kpn I and Xba I digestions, generating pBS-fLuc. The gene fragment of fLuc
77
was cut out by Bgl II and Not I digestion, and was inserted between the BamH I and Not I sites of pGEX-
78
PBSII-PBSII, generating pGEX-PBSII-PBSII-fLuc.
79
80
Protein expression and purification
4
81
The E. coli strains BL21 and JM109 were transformed with pGEX-PBSII-PBSII-fLuc and pGEX-PPDK-
82
zif268-zif268. Both strains were cultured in 200 ml of fresh LB broth with the following supplements: 50
83
μg/ml ampicilin, 100 μg/ml ZnCl2 and an aliquot of Antifoam A (Sigma-Aldrich) at 30˚C until the OD at
84
660 nm was between 0.6-1.0. Then, the bacteria were further cultured for 3 h at 16˚C after the addition of
85
1 mM IPTG to induce protein expression. The cells were collected by centrifugation and stored at -80˚C
86
until being subjected to further purification procedures. The cellular pellets were dissolved in phosphate
87
buffered saline (PBS) for PBSII-PBSII-fLuc and PBS with 250 mM of ammonium sulfate (PBS-A) for
88
PPDK-Zif268-Zif268, and were lysed by ultrasonication at a power of 200 W for 240 s (INSONATOR
89
201M, KUBOTA, Japan). After centrifugation at 25,000 g for 15 min, the supernatants were subjected to
90
further protein purification steps with Glutathione Sepharose 4B (GE Healthcare), following the
91
manufacturers’ protocol. For all processes, PBS and PBS-A–based buffer systems were utilized for the
92
purification of PBSII-PBSII-fLuc and PPDK-Zif268-Zif268, respectively. Purified proteins were analyzed
93
by SDS-PAGE. As shown in Fig. S1, GST-PBSII-PBSII-fLuc (107.5 kDa) was collected as a relatively
94
pure sample, while GST-PPDK-Zif268-Zif268 (144.1 kDa) exhibited two major bands. Because GST-
95
PPDK-Zif268-Zif268 tended to express as an insoluble fraction, the by-product might be attributed to the
96
chaperonin proteins that bind to the protein to keep it in its soluble form. Also, PPDK has the Factor Xa
97
digestion site for the removal of the GST-tag. Therefore, we utilized these proteins for the following
98
experiments without further purification. The proteins were stored in fLuc/PPDK binding buffer (10 mM
99
Tris-HCl (pH 6.8), 50 mM NaCl, 15 mM MgCl2, 1 mM DTT, 0.1 mM ZnCl2, 0.05% Nonidet P-40, 5%
100
glycerol) at a concentration of 1 μM for GST-PBSII-PBSII-fLuc, and a total protein concentration of 300
101
μg/ml for GST-PPDK-Zif268-Zif268.
5
102
103
Supplementary Fig. 1. SDS PAGE analysis of the proteins. Lane 1; GST-PBSII-PBSII-fLuc (107.5 kDa).
104
Lane 2; GST-PPDK-Zif268-Zif268 (144.1 kDa).
105
106
DNA scaffold production
107
Zif268-Zif268 binding scaffold
108
Two synthesized oligos, O11 and O12, coding tandem Zif268 binding sites, were annealed and inserted
109
into the BamH I and Not I cloning sites of pBluescript II SK(+), generating pBS-Zif268-Zif268-BS. DNA
110
scaffolds of differing lengths, coding for tandem Zif268 binding sites, were synthesized from pBS-Zif268-
111
Zif268-BS by PCR with the primer combinations of O13 and O14, and O13 and O15, for the 324 bp and
112
577 bp scaffolds, respectively. These scaffolds were utilized for the following gel-shift assay.
113
114
PBSII-PBSII binding scaffold
115
Two synthesized oligos, O16 and O17, coding for tandem PBSII binding sites, were annealed and inserted
116
into the Xba I and Not I cloning sites of pBluescript II SK (+), generating pBS-PBSII-PBSII-BS. DNA
117
scaffolds of differing lengths, coding for tandem PBSII binding sites, were synthesized from pBS-PBSII-
118
PBSII-BS by PCR with the primer combinations of O13 and O14, and O13 and O15, for 359 bp and 594
119
bp scaffolds, respectively. These scaffolds were utilized for the following gel-shift assay.
6
120
121
122
Non-specific binding scaffold for gel-shift analysis
The non-specific binding DNA scaffold (1078 bp) used for the following gel-shift analysis was produced
by PCR with pBluescript II SK (+) as a template, and O15 and O18.
123
124
DNA scaffolds to control the distance between enzymes
125
The gap-distance-programmed DNA scaffolds were generated via the annealing of synthesized oligos.
126
Two oligos at a concentration of 100 M were heated to 95˚C for 5 min, and gradually cooled to room
127
temperature in TE buffer solution. O19 and O20, O21 and O22, O23 and O24, and O25 and O26 were used
128
for Zif268-Zif268-(0 bp)-PBSII-PBSII, Zif268-Zif268-(10 bp)-PBSII-PBSII, Zif268-Zif268-(20 bp)-
129
PBSII-PBSII, and Zif268-Zif268-(30 bp)-PBSII-PBSII scaffolds, respectively. The dsDNA scaffold (56
130
bp), annealed with O27 and O28, was used for the control experiment, as a non-specific binding experiment.
131
132
Measurement of bioluminescence
133
First, the zinc finger motif-fused proteins and DNA scaffolds were mixed at room temperature for 3 to 5
134
hours. One hundred and eighty microliters of the solution containing 0.1 μM of GST-PBSII-PBSII-fLuc,
135
30 μg/ml of GST- PPDK-Zif268-Zif268, 5 μM of the DNA scaffold, 5 μM of the non-specific binding DNA
136
scaffold, 330 μM of D-luciferin, 10 mM of phosphoenolpyruvate and 50 μM of inorganic diphosphate in
137
fLuc/PPDK binding buffer (10 mM Tris-HCl (pH 6.8), 50 mM NaCl, 15 mM MgCl2, 1 mM DTT, 0.1 mM
138
ZnCl2, 0.05% Nonidet P-40, 5% glycerol) was put into a black 96 well ELISA plate. Each concentration
139
indicates the final concentration after injection of 20 μl of energy solution, namely adenosine
140
monophosphate (AMP) or adenosine-5'-triphosphate (ATP). The bioluminescence was monitored by
141
LUMINOUS CT-9000D (Mitsubishi Kagaku Iatron Inc., Tokyo, Japan) after the injection of energy
142
solution and evaluated by integrating the light emission for 98 sec after the injection.
143
144
Supporting results and discussions
7
145
Evaluation of the biding specificity to the DNA scaffold
146
The binding specificity to the DNA scaffold was evaluated using a gel-shift assay with 1 pmole of DNA
147
scaffold (about 350 bp) coding for the appropriate binding site of the zinc finger motif, and DNA scaffold
148
coding for another zinc finger motif (about 600 bp), and the 1078 bp of a nonspecific DNA fragment.
149
Almost all of the DNA scaffolds were shifted when 4 pmole of the protein was added (Fig. S2a), and the
150
similar gel shift patterns attributed to the PBSII-PBSII DNA scaffold were observed even in the mixture of
151
the DNA fragments (Fig. S2b).
A
1
0 0.5
2
B
4
1
0 0.5
2
4
Nonspecific DNA
Zif268-Zif268 binding site
PBSII-PBSII binding site
PBSII-PBSII binding site
152
153
Supplementary Fig 2. Agarose gel electrophoresis analysis for the binding specificity of GST-PBSII-
154
PBSII-fLuc with the appropriate DNA scaffold only (a) and with other DNA scaffolds (b). The numbers
155
above mentioned are the molar ratio of the protein against the DNA scaffold.
156
157
For GST-PPDK-Zif268-Zif268, when 8 μl of protein solution was added, about the half of the DNA
158
scaffold coding Zif268-Zif268 binding site disappeared (Fig. S3a). The similar gel shift patterns of the
159
Zif268-Zif268 DNA scaffold were observed even in the mixture of the DNA fragments (Fig. S3b).
160
In each case, the shifted band patterns were not simple. One reason for this is considered as the
161
oligomerizations of proteins attributed to the GST tag. Although further study is definitely needed to
162
analyze the conjugated formation, at least these results confirmed the specific binding of each protein to
163
the appropriate DNA scaffold.
8
A
0
1
2
4
B
8
0
1
2
4
8
Nonspecific DNA
PBSII-PBSII binding site
Zif268-Zif268 binding site
Zif268-Zif268 binding site
164
165
Supplementary Fig 3. Agarose gel electrophoresis analysis for the binding specificity of GST-PPDK-
166
Zif268-Zif268 with the appropriate DNA scaffold only (a) and with other DNA scaffolds (b). The numbers
167
above mentioned are the volume of the protein solution.
168
169
Reference
170

171
172
discovery. Mol Pharmacol 66:1361-1371

173
174
Blancafort P, Segal DJ, Barbas CF 3rd (2004) Designing transcription factor architectures for drug
Ebihara T, Takayama H, Yanagida Y, Kobatake E, Aizawa M (2002) Thermostabilization of protein
A-luciferase fusion protein by single amino acid mutation. Biotechnol Lett 24:147–149

Eisaki N, Tatsumi H, Murakami S, Horiuchi T (1999) Pyruvate phosphate dikinase from a
175
thermophilic actinomyces Microbispora rosea subsp. aerata: purification, characterization and
176
molecular cloning of the gene. Biochim Biophys Acta 1431:363-373
177

178
179
180
Ooi AT, Stains CI, Ghosh I, Segal DJ (2006) Sequence-enabled reassembly of beta-lactamase (SEERLAC): a sensitive method for the detection of double-stranded DNA. Biochem 45:3620-3625

Pavletich NP, Pabo CO (1991) Zinc finger-DNA recognition: crystal structure of a Zif268-DNA
complex at 2.1 Å. Science 252:809-817
9