Supporting Online Information

Supporting Online Information Isolation of Human Genomic DNA Sequences with Expanded Nucleobase Selectivity Preeti Rathi, Sara Maurer, Grzegorz Kubik and Daniel Summerer* Department of Chemistry and Chemical Biology Technical University of Dortmund CP-02-143 Otto-Hahn-Str. 4a 44227 Dortmund *Daniel.Summerer@tu-dortmund.de S1

Table of Contents List of Oligonucleotides... 3 Figure SI 1: Protein Sequences of Exemplary TALE proteins with N-terminal TRX and N- or C-terminal GFP domain... 7 Figure SI 2: SDS PAGE Analysis of Exemplary Ni-NTA-Purified TALE Protein Expression (TALE_BRCA1)... 8 Figure SI 3: Agarose gel analysis of human whole genome amplified DNA randomly sheared by sonication... 9 Figure SI 4: Off-Target sequence of TALE_MGMT in Hey2 locus... 10 Figure SI 5: Exemplary Sanger Sequencing traces of PCR products from bisulfite converted zebrafish DNA before and after enzymatic CpG methylation... 11 Figure SI 6: Sanger Sequence traces of PCR products from bisulfite converted human DNA before and after enzymatic CpG methylation... 12 Figure SI 7: Denaturing PAGE analyses of exemplary primer extension assays for analysis of 5mC dependent DNA binding of TALES... 13 Figure SI 8: Genomic Affinity Enrichment Assay with TALE_BRCA_1 using different types of beads... 14 Figure SI 9: Genomic Affinity Enrichment assay with TALE_CDKN2A carrying the His-tag at the N or C terminal of the protein... 15 Figure SI 10: Raw Data of Electromobility shift assay with data shown in Figure 2a 16 SI Figure 11: Representative raw data of selectivity profiles of designed TALE repeats shown in Figure 6b... 17 SI Figure 12 Principle of DNA Polymerase Accessibility Assay... 18 References... 19 S2

List of Oligonucleotides All Oligos were ordered from Sigma-Aldrich. 5-methyl-C nucleotides are marked as C. 5-methylhydroxy-C nucleotides are marked as X. 5-formyl-C nucleotides are marked as Y. 5-carboxyl-C nucleotides are marked as Z. Quantitative real time PCR BRCA1 Fw (oprr1060) GGG GAT TGG GAC CTC Rv (oprr1114) AAA GTG CCT GCC CTC TA CDKN2A Fw (oprr1064) TCG AAG CGC TAC CTG AT Rv (oprr1065) TAG AGG AGG TGC GGG MGMT Fw (oprr1457) GAA AAG GTA CGG GCC A Rv (oprr1459) GCC TGA CCC GGA TG BRCA1 Fw (oprr1570) CAA TCC AGA GCC CCG Rv (oprr1577) GGA CTC TAC TAC CTT TAC C ZAP-70 Fw (oprr1759) GCT CTG GGA GAC CTG Rv (oprr1764) CGG TTC TGG GTG AGG Zebrafish Fw (odas903) GGA ACT GAA GTG GGA GCG T Rv (odas904) GAT GAT ATC AAA GCA TAC ATC TCA Electromobility shift assay Hey2c_fw (ogrk702) ACA TTT AAA TCC AAC ATT TAA AAC GCT CCC ACT TCA GTT CCC CAC GGT CGG TAT GGT TTA CTG CTG CTC CCG CT Hey2c_rv (ogrk787) GCA GCG GGA GCA GCA GTA AA S3

DNA polymerase accessibility assay BRCA 1 Non-methylated oligo (oprr1077) AAA CTC AGG TAG AAT TCT TCC TCT TCC GTC TCT TTC CTT TTA CGT CAT CCG GGG GCA GA Double Methylated oligo (oprr1076) AAA CTC AGG TAG AAT TCT TCC TCT TCC GTC TCT TTC CTT TTA CGT CAT CCG GGG GCA GA Single methylation 3 (oprr1184) AAA CTC AGG TAG AAT TCT TCC TCT TCC GTC TCT TTC CTT TTA CGT CAT CCG GGG GCA GA Single methylation 5 (oprr1185) AAA CTC AGG TAG AAT TCT TCC TCT TCC GTC TCT TTC CTT TTA CGT CAT CCG GGG GCA GA Radiolabelled primer (oprr1078) CTG CCC CCG GAT GAC GTA AAA GGA AAG AGA CDKN2A Non-methylated oligo (oprr1080) ACC CGA CCC CGG GCC GCG GCC GTG GCC AGC CAG TCA GCC GAA GGC TCC ATG CTG CTC CC Methylated oligo (oprr1079) ACC CGA CCC CGG GCC GCG GCC GTG GCC AGC CAG TCA GCCGAA GGC TCC ATG CTG CTC CC Radiolabelled primer (oprr1082) GGG AGC AGC ATG GAG CCT TCG GCT GA MGMT Non-methylated oligo (oprr1084) CTT CGG CCG GTA CAA GCC GGG CGG CGC CTT CCC AGC TTC CGC CTG AGG CTC TGT GCC TT Methylated oligo (oprr1083) CTT CGG CCG GTA CAA GCC GGG CGG CGC CTT CCC AGC TTC CGC CTG AGG CTC TGT GCC TT Radiolabelled primer (oprr1085) AAG GCA CAG AGC CTC AGG CGG AAG CTG GGA Zebrafish Non-methylated oligo (oprr1826) TTCAGCCCCAGCGTTACAGCATCTTCAGTGGCTTCTTCCACCGTGAGCTCTTCCGTTTCCACATCCACCACAT CCCAAC Methylated oligo (oprr1827) TTC AGC CCC AGC GTT ACA GCA TCT TCA GTG GCT TCT TCC ACC GTG AGC TCT TCC GTT TCC ACA TCC ACC ACA TCC CAA C Radiolabelled primer (oprr1828) GTT GGG ATG TGG TGG ATG TGG AAA CGG ZAP-70 Non-methylated oligo (oprr1805) GAG GAT GAA CCC CCC CCA TCC ATG AGT GAG AAA CCC TGG CGG GGT GTG ACA TCC TCC CC Methylated oligo (oprr1835) GAG GAT GAA CCC CCC CCA TCC ATG AGT GAG AAA CCC TGG CGG GGT GTG ACA TCC TCC CC Radiolabelled primer (oprr1803) ATG TCA CAC CCC GCC AGG GTT TCT CA S4

Bisulphite PCR / Sequencing BRCA1 Fw1 (oprr1307) GAA ATT GGA GAT TTT TAT TAG GG Rv1 (oprr1308) TAT CTA AAA AAC CCC ACA ACC TAT C Fw2 (oprr1256) TGG GGG ATT GGG ATT TTT TTT Rv2 (oprr1257) TTA ACC ACC CAA TCT ACC CCC Zebrafish Fw1 (odas754) GTT TGA GGT TTT TTA AAG AGG T Rv1 (odas751) CCC AAA ATA TTA ACT TAA ATT TAA C Fw1 (odas756) TAG TAG TTG TTG TAG TTG TAG TTT AAA TTT Rv1 (odas757) CTA CTT TCC TTT ATA TAC AAA ACA TAA AAA Spike-in PCRs Zebrafish Spike-in1 (ogrk476) TGG ATT CCC ACT CTT CAG CCC CAG CGT TAC AGC ATC TTC AGT GGC TTC TTC CAC CGT GAG CTC TTC CGT TTC CAC ATC C Spike-in 2 (ogrk1056) TGG ATT CCC ACT CTT CAG CCC CAG CGT TAC AGC ATC TTC AGT GGC TTC TTC CAC CGT GAG CTC TTC CGT TTC CAC ATC C Spike-in 3 (ogrk465) TGG ATT CCC ACT CTT CAG CCC CAG CGT TAC AGC ATC TTC AGT GGC TTC TTC CAC CGT GAG CTC TTC CGT TTC CAC ATC C Spike-in 4 (ogrk1057) TGG ATT CCC ACT CTT CAG CCC CAG CGT TAC AGC ATC TTC AGT GGC TTC TTC CAC CGT GAG CTC TTC CGT TTC CAC ATC C Spike- in hmc (ogrk520) TGG ATT CCC ACT CTT CAG CCC CAG CGT TAC AGC ATC TTC AGT GGC TTC TTC CAC CGT GAG CTC TTC XGT TTC CAC ATC C Spike-in fc (ogrk1423) TGG ATT CCC ACT CTT CAG CCC CAG CGT TAC AGC ATC TTC AGT GGC TTC TTC CAC CGT GAG CTC TTC YGT TTC CAC ATC C Spike-in cac (ogrk1422) TGG ATT CCC ACT CTT CAG CCC CAG CGT TAC AGC ATC TTC AGT GGC TTC TTC CAC CGT GAG CTC TTC ZGT TTC CAC ATC C Common reverse primer: (ogrk1597) TTG TAT AAA TCG GAG AAG ATC CTG TCT CCC CCC AAA CAA ACA GTA G BRCA1 Spike-in Fw1 (ogrk1516) CTT CCT CTT CCG TCT CTT TCC TTT TAC GTC ATC CGG GGG CAG ACT Spike-in Fw2 (ogrk1518) CTT CCT CTT CCG TCT CTT TCC TTT TAC GTC ATC CGG GGG CAG ACT Common Spike-in Rv (ogrk1601) GTC AAA GAA TAC CCA TCT GTC AGC TTC GGA AAT CCA CTC TCC CAC BRCA1_a S5

Spike-in Fw1 (ogrk1599) AGT CTG CCC CCG GAT GAC GTA AAA GGA AAG AGA CGG AAG AGG AAG Spike-in Fw2 (ogrk1600) AGT CTG CCC CCG GAT GAC GTA AAA GGA AAG AGA CGG AAG AGG AAG Common Spike-in Rv (ogrk1529) GCACAGGTCTCCAATCTATCCACTGGATTTCCGTGAGAATTGTGCCCG TALE cloning Gibson psab339 Fw1 (oani1366) GGC ATG GAC GAG CTG TAC AAG ACA CTC GGT TAT TCG CAA CAG C Gibson psab339 Rv1 (oani1311) GAA CAG CTC CTC GCC CTT GCT CAT ATG TAT ATC TCC TTC TTA A Gibson primer extension Fw1 (odas1143) CTG GAA GTT CTG TTC CAG GGG ATC GAG GGA AGG CTC GAA AAT CTT TAT TTT CAG TCT CTC GAG ATG GAT CCC TCC Gibson primer extension Rv1 (odas1144 CTG TTG CGA ATA ACC GAG TGT GCG TAG ATC CAC CTG CGC GGC CGG CGA AGC GTC GGA GGG ATC CAT CTC GAG AGA CTG MBP-tag Fw1 (odas1139) TTT AAG AAG GAG ATA TAC ATA TGG ATT ACA AAG ATG ATG ATG AT MBP-tag Rv1 (odas1140) ATT TTC GAG CCT TCC CTC GAT CCC CTG GAA CAG AAC TTC CAG GFP-tag Fw1 (oani1310) TTA AGA AGG AGA TAT ACA TAT GAG CAA GGG CGA GGA GCT GTT C GFP-tag Rv1 (oani1365) GCT GTT GCG AAT AAC CGA GTG TCT TGT ACA GCT CGT CCA TGC C N-terminal 6HisInsert Fw (oprr1916) TTT TCA TAT GGG CCA TCA CCA TCA CCA TCA CAG CAA GGG CGA GGA GCT GTT CAC CG N-terminal 6HisInsert Rv (oprr1917) AAA AGC GGC CGC TCA TTC CAG CAG ATG GTC GTT GGA GAC GAC C S6

Figure SI 1: Protein Sequences of Exemplary TALE proteins with N-terminal TRX and N- or C-terminal GFP domain TRX_TALE_Hey2b MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLL LFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGERQHMDSPDLGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGH GFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKR GGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQR LLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLT PDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLL PVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPD QVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPV LCQDHGLTPDQVVAIASHDGGKQALESIVAQLSRPDPALAALTNDHLLEHHHHHH* GFP_TALE_Hey2b MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQ ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPI GDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHP AALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQV VAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQD HGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQR LLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGK QALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALESIVAQL SRPDPALAALTNDHLLEHHHHHH* GFP_TALE_CDKN2A_N-terminal 6His MGHHHHHHSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSR YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM ADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGM DELYKTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQ WSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQ RLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGL TPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNN GGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRL LPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTP DQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGG KQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLP VLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQ VVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQ ALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVL CQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVV AIASHDGGKQALESIVAQLSRPDPALAALTNDHLLE* GFP_TALE_CDKN2A_C-terminal 6His MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQ HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG IKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKTL GYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARAL EALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLC QDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALE TVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQD HGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETV QRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQR LLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLT PDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALESIVAQLSRPDPALAALTNDHLLEHHHHHH* S7

Figure SI 2: SDS PAGE Analysis of Exemplary Ni-NTA-Purified TALE Protein Expression (TALE_BRCA1) Lane 1: non-induced Lane 2: purified first elution of expression culture transformed with pet_gfp_tale_brca1 induced with 1mM IPTG Lane 3: purified second elution of expression culture transformed with pet_gfp_tale_brca1 Lane 4: protein ladder S8

Figure SI 3: Agarose gel analysis of human whole genome amplified DNA randomly sheared by sonication Lane 1: 2 log DNA ladder Lane 2: whole genome amplified human DNA (100ng) S9

Figure SI 4: Off-Target sequence of TALE_MGMT in Hey2 locus Sequence in the HEY2 locus with highest similarity to target sequence of TALE_MGMT (+/- 500 bp up- and downstream of the primer binding sites used in HEY2 qpcr) TALE_MGMT HEY2 -----------------------------TCCCAGCTTCCGCCTGAGGCTCTGTG----- TTCCTGCAGCAGAGCGGACTTCCCTCCTCAGAGAGCTCCTCCGGCAGGCTGTCTGAGGCT S10

Figure SI 5: Exemplary Sanger Sequencing traces of PCR products from bisulfite converted zebrafish DNA before and after enzymatic CpG methylation Upper Trace: original D. rerio DNA Lower Trace: Methylated D. rerio DNA Relevant CpG in the target sequence of TALE_Hey_2a marked blue Upper Trace: original D. rerio DNA Lower Trace: Methylated D. rerio DNA Relevant CpG in the target sequence of TALE_Hey_2b marked blue S11

Figure SI 6: Sanger Sequence traces of PCR products from bisulfite converted human DNA before and after enzymatic CpG methylation Upper Trace: original H. sapiens DNA Lower Trace: Methylated H. sapiens DNA Relevant CpG in the target sequence of TALE_BRCA1 marked blue S12

Figure SI 7: Denaturing PAGE analyses of exemplary primer extension assays for analysis of 5mC dependent DNA binding of TALES Reactions were conducted with 250 mu of Klenow Fragment (exo - ) S13

Figure SI 8: Genomic Affinity Enrichment Assay with TALE_BRCA_1 using different types of beads S14

Figure SI 9: Genomic Affinity Enrichment assay with TALE_CDKN2A carrying the Histag at the N or C terminal of the protein S15

Figure SI 10: Raw Data of Electromobility shift assay with data shown in Figure 2a Raw data of EMSA with varying concentrations of TALE_HEY2b and DNA bearing C, 5mC, or 5hmC at position 6 opposite an HD RVD of the TALE (only lanes with highest TALE concentrations 0.75 µm - 2.5 µm and visible differences in complex formation are shown). TALE DNA complex is marked with an arrow. O.T. = Off-Target (Sequence HEY2c, see Figure 1c of the manuscript) S16

SI Figure 11: Representative raw data of selectivity profiles of designed TALE repeats shown in Figure 6b S17

SI Figure 12 Principle of DNA Polymerase Accessibility Assay Principle of the DNA Polymerase accessibility assay: A synthetic oligonucleotide primer/template complex with 5-32 P-phosphate-labelled primer is incubated with a TALE that binds to a long stretch of the template strand in the direction 5-3 /N- to C- terminus (upper sequence, TALE target sequence underlined). To this complex, Klenow Fragment (KF) of E. coli DNA Polymerase I (5-3 -exo-) and dntp are added, the mixture is incubated and stopped by the addition of EDTA/formamide. The reaction mixture is resolved by denaturing PAGE and quantified by a phosphorimager. The binding range of KF(exo-) DNA polymerase is the last four nucleotides of the primer 3 -terminus as shown and thus overlaps with the binding range of the TALEs N-terminal part. Whereas no primer extension is observed for fully canonical templates, the presence of single 5mC (bold, red) in the TALE target range of the template strand leads to increased primer extension and thus indicates a local destabilization/opening of the N-terminal part of the TALE -DNA complex or its full dissociation. S18

References [1] G. Kubik, S. Batke, D. Summerer, J Am Chem Soc 2015, 137, 2-5. [2] T. Cermak, E. L. Doyle, M. Christian, L. Wang, Y. Zhang, C. Schmidt, J. A. Baller, N. V. Somia, A. J. Bogdanove, D. F. Voytas, Nucleic Acids Res 2011, 39, e82. [3] G. Kubik, M. J. Schmidt, J. E. Penner, D. Summerer, Angew Chem Int Ed Engl 2014, 53, 6002-6006. S19