Supporting Information. Trifluoroacetophenone-Linked Nucleotides and DNA for Studying of DNA-protein Interactions by 19 F NMR Spectroscopy
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1 Supporting Information Trifluoroacetophenone-Linked Nucleotides and DNA for Studying of DNA-protein Interactions by 19 F NMR Spectroscopy Agata Olszewska, Radek Pohl and Michal Hocek # * Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo namesti 2, Prague 6, Czech Republic # Dept. of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, Prague 2, Czech Republic Table of Contents 1. Additional results and figures SI 2. Copies of MALDI-TOF mass spectra SI 3. Copies of NMR spectra SI S1
2 1. Additional results and figures 1.1 Incorporation of dn TAP TP into DNA by PEX Table S1. List of oligonucleotides used or synthesized a Oligonucleotide Sequence prim A 5 -TCA AGA GAC ATG CCT-3 prim B 5 -CAT GGG CGG CAT GGG-3 prim C 5 -GGG TGG GTG GGT GGC TTT TGT-3 temp 19C 5 -C CCG CCC ATG CCG CCC ATG-3 temp 19A 5 -CCCTCCCATGCCGCCCATG-3 temp 19T 5 -CCCACCCATGCCGCCCATG-3 temp 19G 5 -AAACCCCATGCCGCCCATG-3 temp 30_1C 5 -ATA ATA AAC ATG TCT AGG CAT GTC TCT TGA-3 temp CTA GCA TGA GCT CAG TCC CAT GCC GCC CAT G CCC CTT TTT AAC AA A AGC CAC CCA CCC ACC C-3 temp 31_1U temp 30_2C temp 30_2A temp 30_2T temp 30_2G 5 -ATA ATA GAC ATG TCT AGG CAT GTC TCT TGA-3 5 -ACA ACA GAC AGT CTC AGG CAT GTC TCT TGA-3 5 -TTT TTA GGC ATG TCT AGG CAT GTC TCT TGA-3 5 -ATA ATA GAC ATG TCT AGG CAT GTC TCT TGA-3 19ON_1C TAP 5 -CAT GGG CGG CAT GGG CGG G-3 19ON_1A TAP 5 -CAT GGG CGG CAT GGG AGG G-3 19ON_1U TAP 5 -CAT GGG CGG CAT GGG UGG G-3 19ON_1G TAP 5 -CAT GGG CGG CAT GGG GTT T-3 30ON_1C TAP 5 -TCA AGA GAC ATG CCT AGA CAT GTT TAT TAT-3 30ON_2C TAP 5 -TCA AGA GAC ATG CCT AGA CAT GTC TAT TAT-3 31ON_1U TAP 5 -GGG TGG GTG GGT GGC TTT TGT UAA AAA GGG G-3 DNA_1U TAP 3 -GGG GAA AAA UTG TTT TCG GTG GGT GGG TGG G CCC CTT TTT AAC AA A AGC CAC CCA CCC ACC C-3 DNA_1C TAP 3 -AGT TCT CTG TAC GGA TCT GTA CAA ATA ATA-5 5 -TCA AGA GAC ATG CCT AGA CAT GTT TAT TAT-3 a primers are underline, p53 recognition sequences are red, nucleotides bearing modification are in bold. Table S2. List of oligonucleotides used in PCR study Oligonucleotide Sequence (5 3 ) Length prim FOR TAGGGGTTCCGCGCACATTTCCCCG 25-mer prim REV GGAGAGCGTTCACCGACAAACAACAG 26-mer prim FOR-L20 GACATCATGAGAGACATCGC 25-mer prim REV-LT25TH CAAGGACAAAATACCTGTATTCCTT 20-mer TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGA 339-mer temp Pveg CGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAA TAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTATTT GACAAAAATGGGCTCGTGTTGTACAATAAATGTGTCTAAGC TTGGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAA CGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGA GAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTC AGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCG temp FVL-A GACATCATGAGAGACATCGCCTCTGGGCTAATAGGACTACT TCTAATCTGTAAGAGCAGATCCCTGGACAGGCAAGGAATAC AGGTATTTTGTCCTTG 98-mer S2
3 Scheme 1. Model reaction of TAP modified monophosphate (dc TAP TP) with N-acetylserine Experimental procedure: dc TAP MP (25 mg, 0.05 mmol) and N-acetylserine (0.36 mg, 2.5 mmol, 50 equiv) were dissolved in triethylammonium acetate (TEAA) buffer (0.3 M, ph 8.4, 2.5 ml) or in acetone/0.1 M NaOH (v/v, 2:1) and the mixture was stirred for 6 days at 25 C. The reaction was monitored by TLC using C3H7OH/H2O/NH4OH (v/v/v, 11/2/7) as a mobile phase. We didn t observe any difference in the mobility of the mixture in comparison to the starting dc TAP MP. Products were then submitted for NMR and mass spectroscopy measurement. We did not observe any formation of the desired cross-linked product. All prepared modified dn TAP TP (da TAP TP, dc TAP TP, du TAP TP and dg TAP TP) were successfully incorporated into short (temp 19_C, temp 19_T, temp 19_A and temp 19_G ) or long DNA (temp 31 ) by primer extension (PEX) using KOD XL, Vent (exo-) or PWO DNA polymerase. Only full length products were observed on denaturing PAGE (Figure S1 and S2). S3
4 Figure S1. Primer extension using KOD XL/Vent(exo-)/PWO polymerases and 19 mer templates (temp 19_1C, temp 19_1T, temp 19_1A, temp 19_1G ), temp 19_1C (lanes 2-4), temp 19_1T (lanes 5-7), temp 19_1A (lanes 8-10) or temp 19_1G (lanes 11-13) with primer prim B. Lane 1 (P): Primer; lanes 2, 5, 8, 11 (C+/T+/A+/G+): natural dntps; lanes 3, 6, 9, 12 (C-/T-/A-/G-): negative control without dctp, dttp, datp or dgtp; lane 4 (C TAP ): dc TAP TP, dttp, dgtp, datp; lane 7 (U TAP ): du TAP TP, datp, dgtp, dctp; lane 10 (A TAP ): da TAP TP, dttp, dgtp, dctp; lane 13 (dg TAP ): dg TAP TP, dttp, dctp, datp. Figure S2. Primer extension using KOD XL/Vent(exo-)/PWO polymerases and 31 mer templates (temp 31 ) with primer prim B. Lane 1, 11, 21 (P): Primer; lanes 2, 12, 22 (+): natural dntps; lanes 3, 5, 7 9, 13, 15, 17, 19, 23, 25, 27, 29 (C-/T-/A-/G-): negative control without dctp, dttp, datp or dgtp; lanes 4, 14, 24 (C TAP ): dc TAP TP, dttp, dgtp, datp; lanes 6, 16, 26, (U TAP ): du TAP TP, datp, dgtp, dctp; lane 8, 18, 28 (A TAP ): da TAP TP, dttp, dgtp, dctp; lanes 10, 20, 30 (dg TAP ): dg TAP TP, dttp, dctp, datp. S4
5 Figure S3. Primer extension using KOD XL polymerases and 31 mer templates (temp 31_1U ) with primer prim C. Lane 1 (P): Primer; lane 2 (+): natural dntps; lane 3(-): negative control without dttp; lane 4 (U TAP ): du TAP TP, dgtp, datp. Figure S4. Primer extension using KOD XL polymerases and 30 mer templates (temp 30_2C, temp 30_2A, temp 30_2T, temp 30_2G ), temp 30_2C (lanes 2-4), temp 30_2A (lanes 5-7), temp 30_2T (lanes 8-10) or temp 30_2G (lanes 11-13) with primer prim A. Lane 1 (P): Primer; lanes 2, 5, 8, 11 (C+/T+/A+/G+): natural dntps; lanes 3, 6, 9, 12 (C-/T-/A-/G-): negative control without dctp, dttp, datp or dgtp; lane 4 (C TAP ): dc TAP TP, dttp, dgtp, datp; lane 7 (A TAP ): da TAP TP, dttp, dgtp, dctp; lane 10 (U TAP ): du TAP TP, datp, dgtp, dctp; lane 13 (dg TAP ): dg TAP TP, dttp, dctp, datp. S5
6 Figure S5. Agarose gel analysis of PCR products amplified by Pwo, Vent(exo-) or KOD XL DNA polymerases, temp FVL-A ; Lane 1 (L): ladder; lanes 2, 5, 8 (+): natural dntps; lanes 3, 6, 9 (C-/A-/T-/G-): negative controls without dctp, dttp or datp; Lane 4 (C TAP ): dc TAP TP, dttp, dgtp, datp; lane 7 (A TAP ): da TAP TP, dctp, dgtp, dttp; lane 10 (U TAP ): du TAP TP, datp, dctp, dgtp; 1.3 % (2%) agarose gel for 339-mer (98-mer) stained with GelRed. Figure S6. Agarose gel analysis of PCR products amplified by Pwo, Vent(exo-) or KOD XL DNA polymerase, temp Pveg ; Lane 1 (L): ladder; lanes 2, 5, 8, 11 (+): natural dntps; lanes 3, 6, 9, 12 (C-/A-/T-/G-): negative controls without dctp, dttp, datp or dgtp; Lane 4 (C TAP ): ): dc TAP TP, dttp, dgtp, datp; lane 7 (A TAP ): da TAP TP, dctp, dgtp, dttp; lane 10 (U TAP ): du TAP TP, datp, dctp, dgtp; 1.3 % (2%) agarose gel for 339-mer (98-mer) stained with GelRed. S6
7 Figure S7. Agarose gel analysis of PCR products amplified by Pwo, Vent(exo-) or KOD XL DNA polymerase, temp FVL-A and temp Pveg ; Lane 1 (L): ladder; lanes 2, 5, 8, 11 (+): natural dntps; lanes 3, 6, 9, 12 (C-/A-/T-/G-): negative controls without dctp, dttp, datp or dgtp; Lane 4 (C TAP ): ): dc TAP TP, dttp, dgtp, datp; lane 7 (A TAP ): da TAP TP, dctp, dgtp, dttp; lane 10 (U TAP ): du TAP TP, datp, dctp, dgtp; 1.3 % (2%) agarose gel for 339-mer (98-mer). Incubation of modified DNA with tumour suppressor protein p53 Modified DNAs (DNA_2C TAP ) were prepared by PEX and incubated with different ratios of p53 mutants in order to test its binding activity. The ability of GSTp53_C275S or GSTp53_C277S mutants of p53 to recognize modified DNA was monitored by 5% EMSA (Figure S8A ) confirmed that the DNA modifications do not prevent binding of p53. Then SDS PAGE was performed to see if covalent cross-links have been formed. (Figure S8B) does not show formation of covalent conjugates represented by the bands with lower mobility. Figure S8. A) 5% EMSA analysis of formation of complex between DNA_1C TAP and GSTp53_C275S or GSTp53_C277S mutants of GSTp53 protein (with increasing number of equivalents of the protein, see below the gels); B) 12.5 % SDS PAGE analysis of the potential S7
8 formation of covalently cross-linked product DNA_1C TAP + GSTp53_C275S or C277S ; (with increasing number of equivalents of the protein, see below the gels). Conditions: DNA (0.3 pmol), ph 7.6, 0 C/30 min (a) then 25 C/2 hours (b). Figure S9. A) 5% EMSA analysis of formation of complex between DNA_2X TAP and GSTp53_C277S mutant of GSTp53 protein; B) 12.5 % SDS PAGE analysis of the potential formation of covalently cross-linked product DNAC TAP + GSTp53_C277S. Lane 1: natural DNA; lanes 2, 4, 6, 8,11, 13, 15, 17: natural DNA with p53; lanes 3, 12: DNA_2C TAP + p53; Lanes 5, 14: DNA_2A TAP + p53; lane 7, 16 DNA_2U TAP + p53; lanes 9, 18: DNA_2G TAP + p53. Conditions: DNA_2X TAP (0.3 pmol), ph 7.6, 0 C/30 min (a) then 25 C/2 hours (b). Figure S10. 5% EMSA analysis of formation of complex between A) DNA_1C TAP and GSTp53CD B) DNA_1C TAP and BSA for 19 F NMR studies. lane 1: DNA_1C TAP, lane 2: DNA_1C TAP + GSTp53CD; lane 3: DNA_1C TAP ; lane 4: DNA_1C TAP + BSA. Conditions: DNA_1C TAP (0.7 nmol), GSTp53CD (1.19 nmol) BSA (1.19 nmol) ph 7.6, 0 C/30 min S8
9 Figure S11. 5% EMSA analysis of the formation of the complex between DNA_1C TAP and GSTp53CD for 19 F NMR studies A) DNA_1C TAP and GSTp53CD before 19 F NMR studies B) DNA_1C TAP and GSTp53CD after overnight 19 F NMR measurement C) DNA_1C TAP and GSTp53CD after protein denaturation. lane 1, 3, 5: DNA_1C TAP, lane 2, 4, 6: DNA_1C TAP + GSTp53CD. Conditions: DNA_1C TAP (0.7 nmol), GSTp53CD (1.75 nmol) ph 7.6, 0 C/30 min; for the denaturation 55 C/60 min. S9
10 Figure S F NMR spectra of DNA_1C TAP and GSTp53CD for 19 F NMR studies A) DNA_1C TAP B) DNA_1C TAP and GSTp53CD C) DNA_1C TAP and GSTp53CD after protein denaturation. Conditions: DNA_1C TAP (0.7 nmol), GSTp53CD (1.75 nmol) ph 7.6, 0 C/30 min; for the denaturation 55 C/60 min. Figure S13. Analysis of oligonucleotide annealing by electrophoresis in agarose gel; Lane 1 ssdna (31ON_1U TAP ); lane 2: dsdna. S10
11 2. Copies of MALDI-TOF mass spectra SI Figure S14. MALDI spectrum of 19ON_1C TAP. M (calcd) = Da, M (found) = Da [M+3H] -. S11
12 Figure S15. MALDI spectrum of 19ON_1G TAP. M (calcd) = Da, M (found) = Da [M+1H] -. Figure S16. MALDI spectrum of 19ON_1A TAP. M (calcd) = 6150 Da, M (found) = Da [M+H] -. S12
13 Figure S17. MALDI spectrum of 30ON_1C TAP. M (calcd) = Da, M (found) = Da [M+H] -. Figure S18. MALDI spectrum of 19ON_1U TAP. M (calcd) = Da, M (found) = Da [M+2H] -. S13
14 Figure S19. MALDI spectrum of 31ON_1U TAP. M (calcd) = Da, M (found) = 9958 Da [M+H] Copies of NMR spectra 1.dC TAP S14
15 S15
16 2.dC TAP MP S16
17 S17
18 3.dC TAP TP S18
19 S19
20 4. da TAP S20
21 S21
22 S22
23 5. da TAP MP S23
24 S24
25 6. da TAP TP S25
26 S26
27 7. du TAP S27
28 S28
29 S29
30 8. du TAP MP S30
31 S31
32 9. du TAP TP S32
33 S33
34 10. dg TAP S34
35 S35
36 S36
37 11. dg TAP MP S37
38 S38
39 12. dg TAP TP S39
40 S40
41 13. DNA_1C TAP A) Without C6F6 for external lock and reference signal (-163 ppm). B) With C6F6 in 1 mm coaxial capillary for external lock and reference signal (-163 ppm). S41
42 14. DNA_1C TAP + GSTp53CD A) Without C6F6 for external lock and reference signal (-163 ppm). DNA_1C TAP : GSTp53CD 1:1.7 B) With C6F6 in 1 mm coaxial capillary for external lock and reference signal (-163 ppm). DNA_1C TAP : GSTp53CD 1:2.5 S42
43 15. DNA_1C TAP after p53 denaturation With C6F6 in 1 mm coaxial capillary for external lock and reference signal (-163 ppm). 16. DNAC TAP + BSA S43
44 17. ssdna_u TAP 18. dsdna_u TAP S44
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