supplementary information - biorxiv · 1/20/2017 · 2 23 supplementary methods 24 cloning of the...
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Supplementary Information 1
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DNA-dependent RNA cleavage by the Natronobacterium gregoryi Argonaute 3
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Sunghyeok Ye1,2, Taegeun Bae1,2, Kyoungmi Kim1, Omer Habib1, Seung Hwan Lee1, Yoon 5
Young Kim3, Kang-In Lee3, Seokjoong Kim3, and Jin-Soo Kim1,2,4 6
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1 Center for Genome Engineering, Institute for Basic Science, Seoul 08826, South Korea 8
2 IBS School, University of Science and Technology, Daejeon 34113, South Korea 9
3 ToolGen, Inc., Seoul 08501, South Korea 10
4 Department of Chemistry, Seoul National University, Seoul 08826, South Korea 11
Correspondence should be addressed to J.-S.K. ([email protected]). 12
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Table of Contents 14
Supplementary Methods 15
Supplementary figure 1. NgAgo purification under denaturing condition. 16
Supplementary figure 2. NgAgo metal dependency. 17
Supplementary figure 3. NgAgo cleavage point. 18
Supplementary figure 4. Multiple turnover of NgAgo. 19
Supplementary figure 5. In vitro cleavage assay with NgAgo mutants. 20
Supplementary figure 6. Amino-acid sequence of the expressed version of NgAgo 21
Supplementary Table 1. Oligonucleotides used in this study 22
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Supplementary Methods 23
Cloning of the NgAgo gene 24
The E. coli codon-optimized NgAgo-coding sequence (Supplementary Fig.6) was synthesized 25
using oligonucleotides and cloned into pET-28a expression plasmid (Novagen). In this vector, 26
NgAgo is expressed under the control of the T7 promoter and is fused to a poly-histidine tag 27
and the human influenza hemagglutinin (HA) epitope at the N-terminus. QuikChange II Site-28
Directed Mutagenesis Kit (Agilent) was used to make NgAgo mutants. These plasmids will be 29
available from Addgene. 30
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Purification of the NgAgo protein 32
The recombinant NgAgo protein was expressed in E. coli strain BL21 (DE3) cultured in LB 33
medium at 18 °C overnight after induction with 0.5 mM IPTG. Cells were harvested and 34
resuspended in a buffer containing 20 mM Bis-Tris (pH 8.0), 20 mM NaCl, 2 mM MgCl2, 1 mM 35
PMSF, 1 mM DTT, 1 mg/ml lysozyme, 1% (vol/vol) Triton X-100, and 10% glycerol. Cells were 36
lysed via sonication and centrifuged for 30 min at 15000 x g. After centrifugation, the NgAgo 37
protein was purified from the soluble fraction and the pellet. The soluble lysate was treated with 38
0.04% polyethylenimine (Sigma) for 10 min at 4°C and centrifuged again for 30 min at 5000 x g. 39
The NgAgo protein in the supernatant was bound to Ni-NTA agarose resin (Qiagen), washed 40
with a buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM KCl, and 20 mM imidazole, and 41
eluted with a buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM KCl, and 250 mM imidazole. 42
The eluted protein was subjected to size exclusion chromatography using Superdex 200 column 43
(GE Life Sciences) in 20 mM HEPES (pH 7.5), 300 mM KCl, 1 mM DTT, and 5% (vol/vol) 44
glycerol. The purified NgAgo protein was dialyzed against 20 mM HEPES (pH 7.5), 300 mM 45
KCl, 1 mM DTT and 40% (vol/vol) glycerol. 46
The NgAgo protein was also purified from inclusion bodies. After removing the soluble fraction 47
in the cell lysate via centrifugation, the pellet containing the NgAgo protein was resuspended in 48
a buffer containing 100 mM NaH2PO4 (pH 8.0), 10 mM Tris-Cl, and 6 M guanidine-HCl. The 49
denatured NgAgo protein was bound in batch to Ni-NTA agarose (Qiagen) and washed with a 50
buffer containing 100 mM NaH2PO4 (pH 8.0), 10 mM Tris-Cl, 1 M NaCl, and 6 M guanidine-HCl. 51
The NgAgo protein still attached to the agarose resin was refolded in a buffer containing 100 52
mM NaH2PO4 (pH 8.0), 10 mM Tris-Cl, 2 M NaCl at 4°C for 20 min and eluted from the resin 53
with a buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM KCl, and 250 mM Imidazole. The 54
purified NgAgo protein was dialyzed against 20 mM HEPES (pH 7.5), 300 mM KCl, 1 mM DTT, 55
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and 40% glycerol and analyzed by SDS-PAGE. The refolded NgAgo protein was more efficient 56
than the protein purified from the soluble cell lysate, possibly because the refolded protein was 57
free of single stranded DNA co-purified from E. coli, and was used throughout this study. 58
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RNA substrates 60
The RNA substrate (Table S1) encoding exon 11 of the human DYRK1A gene was synthesized 61
by in vitro transcription using T7 RNA polymerase. The plasmid DNA encoding the RNA 62
substrate was mixed with T7 RNA polymerase in a reaction buffer containing 40 mM Tris-HCl 63
(pH 7.9), 6 mM MgCl2, 10 mM DTT, 10 mM NaCl, 2 mM spermidine, NTP, and RNase inhibitor. 64
The reaction mixture was incubated at 37 °C for 8 h. The RNA substrate was purified using PCR 65
purification kits (Macrogen) and quantified using NanoDrop (Thermo Fisher). Small RNAs of ≤ 66
30 nt in length were chemically synthesized at IDT. 67
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In vitro cleavage assay 69
RNA, single-stranded DNA, and double-stranded DNA substrates (0.1 μM each) were treated at 70
37 °C for 20 min in a reaction buffer containing 20 mM bis-tris (pH 7.5), 300 mM KCl, 10 μM 71
MnCl2, and 2 mM DTT with the NgAgo protein (0.5 μM), which had been pre-incubated with 72
guide ODN or RNA (5 μM) at 37°C for 30 min. The reaction was stopped by adding an equal 73
volume of formamide gel loading buffer supplemented with 0.4% SDS and 40 mM EDTA. 74
Cleavage products were subjected to electrophoresis using a urea-polyacrylamide (6% or 15%) 75
gel (Invitrogen) and stained by SYBR gold (Thermo Fisher). 76
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NgAgo cleavage point detection 78
RNA product which was digested by NgAgo (0.5uM) with gODN (5uM) for 1hr was isolated 79
using an easy-spin TM Total RNA extraction Kit (iNtRON, South Korea) according to the 80
manufacturer’s protocol. Purified RNA was treated with E.coli poly (A) polymerase (NEB) at 81
37’C for 30min. Poly (A) tailed RNA was then reverse transcribed with Oligo dT using 82
Superscript II (Enzynomics, South Korea). PCR was performed using Phusion High-Fidelity 83
DNA polymerase (Thermo Fisher) with following primers.: 5’-CCAACGAATAGCTCCT - 3’ 84
(forward) , 5’ - TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT – 3’ (reverse). PCR product was 85
cloned into TA vector using TOPcloner TM XL TA kit (Enzynomics, South Korea). Sanger 86
sequencing was performed by Macrogen company with following primers : 5’ – 87
CGGCTCGTATGTTGTGTGGA – 3’ (forward) , 5’ – TGGGTAACGCCAGGGTTTTC – 3’. 88
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Supplementary Figures 89
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Supplementary Figure 1. NgAgo purification under denaturing conditions. 91
(a) The NgAgo protein was purified from E. coli and analyzed via SDS-PAGE. (b) Purified wild-92
type and mutant NgAgo proteins.93
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Supplementary Figure 2. Cations required for NgAgo-mediated RNA cleavage. 94
(a) NgAgo failed to cleave RNA in the presence of EDTA. (b) The RNA substrate was treated 95
with NgAgo in a reaction buffer supplemented with various metal ions. Eight divalent cations 96
(Mn2+, Mg2+, Co2+, Zn2+, Ca2+, Cu2+, Ni2+, and Fe2+) and one trivalent cation (Fe3+) were tested.97
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b
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Supplementary Figure 3. NgAgo cleavage positions in an RNA substrate. 98
(a) Schematic representation of the experimental workflow. (b) Sanger sequencing was used to 99
determine cleavage positions. Red arrows indicate positions of cleaved phophodiester bonds. It 100
is ambiguous whether the RNA substrate was cut at G-A (solid line) or A-G (broken line) bond, 101
owing to poly(A) tailing. 102
b a
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Supplementary Figure 4. NgAgo is a multiple-turnover enzyme. 103
The RNA substrate (0.1 μM) was treated at 37 °C for 0.5 to 24 h with the NgAgo protein (0.03 104 μM) in the presence of a guide ODN (5 μM). 105
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Supplementary Figure 5. In vitro cleavage assay with NgAgo mutants. 106
A target RNA was incubated with wild-type and mutant NgAgo proteins and then subjected to 107
electrophoresis. Red arrows indicate cleaved RNA products. 108
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Supplementary Figure 6. Amino-acid sequence of the recombinant NgAgo protein with a 109 poly-histidine tag at the N terminus. 110
The amino-acid sequence of the recombinant NgAgo protein expressed in E. coli. using pET-111 28a plasmid is shown below. 112
MGHHHHHHSSHHHHHHVYPYDVPDYAELPGIRIPTVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPRMSLAFEQDNGERRYITLWKNTTPKDVFTYDYATGSTYIFTNIDYEVKDGYENLTATYQTTVENATAQEVGTTDEDETFAGGEPLDHHLDDALNETPDDAETESDSGHVMTSFASRDQLPEWTLHTYTLTATDGAKTDTEYARRTLAYTVRQELYTDHDAAPVATDGLMLLTPEPLGETPLDLDCGVRVEADETRTLDYTTAKDRLLARELVEEGLKRSLWDDYLVRGIDEVLSKEPVLTCDEFDLHERYDLSVEVGHSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTTYRPRRGHIVWGLRDECATDSLNTLGNQSVVAYHRNNQTPINTDLLDAIEAADRRVVETRRQGHGDDAVSFPQELLAVEPNTHQIKQFASDGFHQQARSKTRLSASRCSEKAQAFAERLDPVRLNGSTVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAHPDETFSKGIVNPPESFEVAVVLPEQQADTCKAQWDTMADLLNQAGAPPTRSETVQYDAFSSPESISLNVAGAIDPSEVDAAFVVLPPDQEGFADLASPTETYDELKKALANMGIYSQMAYFDRFRDAKIFYTRNVALGLLAAAGGVAFTTEHAMPGDADMFIGIDVSRSYPEDGASGQINIAATATAVYKDGTILGHSSTRPQLGEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGFMNEDLDPATEFLNEQGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNEPRATVATFGAPEYLATRDGGGLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHNSTARLPITTAYADQASTHATKGYLVQTGAFESNVGFLRDPYVSK
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Supplementary Tables 114
Supplementary Table 1. Oligonucleotides used in this study 115
a 116
Sequence (5’-3’)
RNA target
GCCTCTACCCAAGATTCTATGGAGGTTGGCCACAGTCACCACTCCATGACATCCCTGTCTTCCTCAACGACTTCTTCCTCGACATCTTCCTCCTCTACTGGTAACCAAGGCAATCAGGCCTACCAGAATCGCCCAGTGGCTGCTAATACCTTGGACTTTGGACAGAATGGAGCTATGGACGTTAATTTGACCGTCTACTCCAATCCCCGCCAAGAGACTGGCATAGCTGGACATCCAACATACCAATTTTCTGCTAATACAGGTCCTGCACATTACATGACTGAAGGACATCTGACAATGAGGCAAGGGGCTGATAGAGAAGAGTCCCCCATGACAGGAGTTTGTGTGCAACAGAGTCCTGTAGCTAGCTCGTGACTACATTGAAACTTGAGTTTGTTTCTTGTGTGTTTTTATAGAAGTGGTGTTTTTTTTC
CAAAAACAAAGTGCAAAGCTGAAAAAA
ssDNA target GACTTTGGACAGAATGGAGCTATGGACGTTAATTTGACCGTCTACTCCAATCCCCGCCAAG
AGACTGGCATAGCTGGACATCCAACATA
dsDNA target
CAGGCCAAGGGTGAAATTAATACGACTCACTATAGCCTCTACCCAAGATTCTATGGAGGTTGGCCACAGTCACCACTCCATGACATCCCTGTCTTCCTCAACGACTTCTTCCTCGACATCTTCCTCCTCTACTGGTAACCAAGGCAATCAGGCCTACCAGAATCGCCCAGTGGCTGCTAATACCTTGGACTTTGGACAGAATGGAGCTATGGACGTTAATTTGACCGTCTACTCCAATCCCCGCCAAGAGACTGGCATAGCTGGACATCCAACATACCAATTTTCTGCTAATACAGGTCCTGCACATTACATGACTGAAGGACATCTGACAATGAGGCAAGGGGCTGATAGAGAAGAGTCCCCCATGA
CAGGAGTTTGTGTGCAACAGAGTCCTGTAGCTAGCTCGTGACTACATTGAAACTTGAGTTTGTTTCTTGTGTGTTTTTATAGAAGTGGTGTTTTTTTTCCAAAAACAAAGTGCAAAGCTGAAAAA
A
117
b 118
Experiment Oligo name 5’ end
chemical residue
Sequence (5’-3’)
gODN sense,
antisense
5’-OH-gODN-sense OH TACCAGAATCGCCCAGTGGCTG
5’-P-gODN-sense P TACCAGAATCGCCCAGTGGCTG
5’-OH-gODN-antisense OH CAGCCACTGGGCGATTCTGGTA
5’-P-gODN-antisense P CAGCCACTGGGCGATTCTGGTA
Site specific cleavage
5’-OH-gODN-1 OH TTGCCTTGGTTACCAGTAGAGG
5’-OH-gODN-2 OH TATTAGCAGCCACTGGGCGATT
5’-OH-gODN-3 OH AGTCCAAGGTATTAGCAGCCAC
5’-OH-gODN-4 OH TAACGTCCATAGCTCCATTCTG
5’-OH-gODN-5 OH TTAACGTCCATAGCTCCATTCT
5’-OH-gODN-6 OH AATTAACGTCCATAGCTCCATT
5’-OH-gODN-7 OH TGGAGTAGACGGTCAAATTAAC
NgAgo guide preference
Study
5’-OH-gODN OH TGGAGTAGACGGTCAAATTAAC
5’-P-gODN P TGGAGTAGACGGTCAAATTAAC
5’-OH-gRNA OH UGGAGUAGACGGUCAAAUUAAC
5’-P-gRNA P UGGAGUAGACGGUCAAAUUAAC
gODN length test
5’-OH-gODN-Length 11 OH TGGAGTAGACG
5’-OH-gODN-Length 12 OH TGGAGTAGACGG
5’-OH-gODN-Length 13 OH TGGAGTAGACGGT
5’-OH-gODN-Length 14 OH TGGAGTAGACGGTC
5’-OH-gODN-Length 15 OH TGGAGTAGACGGTCA
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5’-OH-gODN-Length 16 OH TGGAGTAGACGGTCAA
5’-OH-gODN-Length 17 OH TGGAGTAGACGGTCAAA
5’-OH-gODN-Length 18 OH TGGAGTAGACGGTCAAAT
5’-OH-gODN-Length 19 OH TGGAGTAGACGGTCAAATT
5’-OH-gODN-Length 20 OH TGGAGTAGACGGTCAAATTA
5’-OH-gODN-Length 21 OH TGGAGTAGACGGTCAAATTAA
5’-OH-gODN-Length 22 OH TGGAGTAGACGGTCAAATTAAC
5’-OH-gODN-Length 23 OH TGGAGTAGACGGTCAAATTAACG
5’-OH-gODN-Length 24 OH TGGAGTAGACGGTCAAATTAACGT
5’-OH-gODN-Length 25 OH TGGAGTAGACGGTCAAATTAACGTC
5’-OH-gODN-Length 26 OH TGGAGTAGACGGTCAAATTAACGTCC
5’-OH-gODN-Length 27 OH TGGAGTAGACGGTCAAATTAACGTCCA
5’-OH-gODN-Length 28 OH TGGAGTAGACGGTCAAATTAACGTCCAT
5’-OH-gODN-Length 29 OH TGGAGTAGACGGTCAAATTAACGTCCATA
5’-OH-gODN-Length 30 OH TGGAGTAGACGGTCAAATTAACGTCCATAG
5’-OH-gODN-Length 40 OH TGGAGTAGACGGTCAAATTAACGTCCATAGCTCCATTCTG
5’-OH-gODN-Length 50 OH TGGAGTAGACGGTCAAATTAACGTCCATAGCTCCATTCTGTCCAAAGTCC
5’-OH-gODN-Length 60 OH TGGAGTAGACGGTCAAATTAACGTCCATAGCTCCATTCTGTCCAAAGTCCAAGGTATTAG
5’-OH-gODN-Length 70 OH TGGAGTAGACGGTCAAATTAACGTCCATAGCTCCATTCTGTCCAAAGTCCAAGGTATTAGCAGCCACTGG
NgAgo mismatch
tolerance test
1-nt mismatch
5’-OH-gODN OH TGGAGTAGACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-1 OH AGGAGTAGACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-2 OH TCGAGTAGACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-3 OH TGCAGTAGACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-4 OH TGGTGTAGACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-5 OH TGGACTAGACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-6 OH TGGAGAAGACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-7 OH TGGAGTTGACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-8 OH TGGAGTACACGGTCAAATTAAC
5’-OH-gODN-1nt Mis-9 OH TGGAGTAGTCGGTCAAATTAAC
5’-OH-gODN-1nt Mis-10 OH TGGAGTAGAGGGTCAAATTAAC
5’-OH-gODN-1nt Mis-11 OH TGGAGTAGACCGTCAAATTAAC
5’-OH-gODN-1nt Mis-12 OH TGGAGTAGACGCTCAAATTAAC
5’-OH-gODN-1nt Mis-13 OH TGGAGTAGACGGACAAATTAAC
5’-OH-gODN-1nt Mis-14 OH TGGAGTAGACGGTGAAATTAAC
5’-OH-gODN-1nt Mis-15 OH TGGAGTAGACGGTCTAATTAAC
5’-OH-gODN-1nt Mis-16 OH TGGAGTAGACGGTCATATTAAC
5’-OH-gODN-1nt Mis-17 OH TGGAGTAGACGGTCAATTTAAC
5’-OH-gODN-1nt Mis-18 OH TGGAGTAGACGGTCAAAATAAC
5’-OH-gODN-1nt Mis-19 OH TGGAGTAGACGGTCAAATAAAC
5’-OH-gODN-1nt Mis-20 OH TGGAGTAGACGGTCAAATTTAC
5’-OH-gODN-1nt Mis-21 OH TGGAGTAGACGGTCAAATTATC
5’-OH-gODN-1nt Mis-22 OH TGGAGTAGACGGTCAAATTAAG
NgAgo mismatch
tolerance test
2-nt mismatch
5’-OH-gODN OH TGGAGTAGACGGTCAAATTAAC
5’-OH-gODN-2nt Mis-1 OH ACGAGTAGACGGTCAAATTAAC
5’-OH-gODN-2nt Mis-2 OH TCCAGTAGACGGTCAAATTAAC
5’-OH-gODN-2nt Mis-3 OH TGCTGTAGACGGTCAAATTAAC
5’-OH-gODN-2nt Mis-4 OH TGGTCTAGACGGTCAAATTAAC
5’-OH-gODN-2nt Mis-5 OH TGGACAAGACGGTCAAATTAAC
5’-OH-gODN-2nt Mis-6 OH TGGAGATGACGGTCAAATTAAC
5’-OH-gODN-2nt Mis-7 OH TGGAGTTCACGGTCAAATTAAC
5’-OH-gODN-2nt Mis-8 OH TGGAGTACTCGGTCAAATTAAC
5’-OH-gODN-2nt Mis-9 OH TGGAGTAGTGGGTCAAATTAAC
5’-OH-gODN-2nt Mis-10 OH TGGAGTAGAGCGTCAAATTAAC
5’-OH-gODN-2nt Mis-11 OH TGGAGTAGACCCTCAAATTAAC
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5’-OH-gODN-2nt Mis-12 OH TGGAGTAGACGCACAAATTAAC
5’-OH-gODN-2nt Mis-13 OH TGGAGTAGACGGAGAAATTAAC
5’-OH-gODN-2nt Mis-14 OH TGGAGTAGACGGTGTAATTAAC
5’-OH-gODN-2nt Mis-15 OH TGGAGTAGACGGTCTTATTAAC
5’-OH-gODN-2nt Mis-16 OH TGGAGTAGACGGTCATTTTAAC
5’-OH-gODN-2nt Mis-17 OH TGGAGTAGACGGTCAATATAAC
5’-OH-gODN-2nt Mis-18 OH TGGAGTAGACGGTCAAAAAAAC
5’-OH-gODN-2nt Mis-19 OH TGGAGTAGACGGTCAAATATAC
5’-OH-gODN-2nt Mis-20 OH TGGAGTAGACGGTCAAATTTTC
5’-OH-gODN-2nt Mis-21 OH TGGAGTAGACGGTCAAATTATG
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