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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. (jskim01@snu.ac.kr). 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

b a

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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

a

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

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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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