Supplementary Information

Transcription

1 Supplementry Informtion Polypurine reverse-hoogsteen (PPRH) oligonucleotides cn form triplexes with their trget sequences even under conditions where they fold into G-qudruplexes. Ann Solé, Emmnuelle Delgoutte, Crlos J. Ciudd, Véronique Noé, Ptrizi Alerti * Deprtement of Biochemistry nd Physiology, School of Phrmcy, University of Brcelon, Brcelon, Spin Structure et Instility of Genomes, Soronne Universités, Muséum ntionl d Histoire nturelle, Inserm U 54, CNRS UMR 796, Pris, Frnce * lerti@mnhn.fr

2 .2 5 Tt < 20 C NCl KCl Tt 33 C NCl + MgCl2 KCl + MgCl c µm 20 µm KCl + MgCl2 Figure S UV-melting profiles of HpE6 oligonucleotide Asornce t 260 nm s function of temperture of () 2µM HpE6 in 00 mm NCl (red) nd in 00 mm KCl (lue), () 2 µm HpE6 in 00 mm NCl + 0 mm MgCl2 (red) nd in 00 mm KCl + 0 mm MgCl2 (lue). (c) 2 µm (red) nd 20 µm (lue) HpE6 in 00 mm KCl + 0 mm MgCl2. The opticl pth length ws 0 mm for 2µM HpE6 nd mm for 20 µμ HpE6. Both cooling nd heting profiles re shown.

3 HpE6 HpE6rep s 295 nm HpE2 HpE2rep s 295 nm Figure S2 UV-melting profiles of HpE6(rep6) nd HpE2(rep2) oligonucleotides () Asornce t 260 nm s function of temperture of HpE6 (red) nd HpE6rep6 (lue). () Asornce t 295 nm s function of temperture of HpE2 (red) nd HpE2rep2 (lue). Mesurements were crried out t 2 µm strnd concentrtion for HpE6 nd HpE2 nd t.5 µm strnd concentrtion for HpE6rep6 nd HpE2rep2, in uffer contining 00 mm KCl nd 0 mm MgCl2. Both cooling nd heting profiles re shown.

4 C CD (mdeg) C wvelength (nm) Figure S3 CD spectr of HpE2 oligonucleotide in potssium nd mgnesium Circulr dichroism spectr of 2 µm HpE2 in 00 mm KCl + 0 mm MgCl2, t 5 C (lue) nd 90 C (red).

5 5 0.6 NCl Tt 33 C NCl + MgCl2 Tt 39 C µm Tt 39 C 20 µm Tt 45 C Figure S4 UV-melting profiles of HpE2 oligonucleotide in sodium () Asornce t 260 nm s function of temperture of 2 µm HpE2 in 00 mm NCl (red) nd in 00 mm NCl + 0 mm MgCl2 (lue). () Asornce t 260 nm s function of temperture of HpE2 in 00 mm NCl + 0 mm MgCl2, t 2 µm (lue) nd 20 µm (lck) strnd concentrtion. Both cooling nd heting profiles re shown.

6 ? Y6rep6? HpE6rep6 R6rep6 Y6rep6 Y6rep6 Figure S5 UV-melting profiles nd PAGE of the complex HpE6rep6 + Y6rep6 () Asornce t 260 nm s function of temperture of µm Y6rep6 + µm HpE6rep6 (red) nd of µm Y6rep6 + µm R6rep6 (lck), in 00 mm KCl + 0 mm MgCl2. Both cooling nd heting profiles re shown. () Non-denturing PAGE of : Lne : Y6rep6 25 µm; lne 2: HpE6rep6 25 µm + Y6rep6 25 µm; lne 3: Y6rep6 25 µm plus its complementry strnd R6rep6 25 µm; lne 4: HpE6rep6 25 µm + Y6rep6 25 µm + R6rep6 25 µm. Smples were prepred in uffer contining 00 mm KCl + 0 mm MgCl2, heted t 90 C for 2 min nd slowly cooled t 4 C. The gel nd the migrtion uffer contined 20 mm KCl + 0 mm MgCl2. Oligonucleotides were detected y UV-shdow. Identicl UV melting profiles nd PAGE ptterns were otined in 00 mm NCl + 0 mm MgCl2. The UV-melting profile of Y6rep6 + HpE6rep6 is similr to the one of the duplex formed y Y6rep6 nd its complementry strnd R6rep6; oth exhiit single melting trnsition t 73 C. This demonstrtes tht the HpE6rep6 forms duplex with Y6rep6, ut does not llow inferring triplex formtion y the HpE6 motif. In non-denturing PAGE, nneling of Y6rep6 in the presence of oth HpE6rep6 nd Rrep6 results in the formtion of two nds of similr intensities (lne 4), one corresponding to the duplex formed y Y6rep6 + R6rep6 (in lne 3), the other migrting s the complex formed y Y6rep6 + HpE6rep6 (in lne 2). The fct tht the two nds hve similr intensities indictes tht the structure formed y HpE6rep6 with its trget Y6rep6 is s stle s the duplex formed y Y6rep6 with its complementry strnd R6rep6.

7 2 3 Y2 * * Figure S6 EMSA of the complex HpE2 + Y2 in potssium nd mgnesium Electrophoretic moility shift ssy of the HpE2 system in KCl + MgCl2. Lne : rdioleled Y2 strnd (Y2*); lne 2: Y2* + HpE2; the mix ws heted t 95 C nd slowly nneled t 5 C; lne 3: HpE2 ws heted t 95 C nd slowly nneled t 5 C, then Y2* ws dded. EMSA ws crried out s descried in the Mteril nd Methods section. Anneling of HpE2 with Y2* from high to low temperture resulted in single thin nd (lne 2), supporting the formtion of single complex ( Wtson-Crick duplex with hnging third strnd or triplex?); while incution of Y2* with structured HpE2 (seprtely nneled lone in the presence of potssium) resulted in smer nd supporting the presence of multiple conformtionl sttes.

8 C/min 0.05 C/min das(260 nm)/dt Figure S7 UV-nneling profiles of the complex(es) formed y HpE2rep2 nd Y2rep2 in potssium nd mgnesium t different temperture scnning rtes () Asornce t 260 nm s function of temperture from 95 C to 5 C of Y2rep2 + HpE2rep2 t scnning rte of 0.2 C/min (lue) nd of 0.05 C/min (lck). () First derivtive of sornce s function of temperture of the nneling profiles shown in (). Ech oligonucleotide were t µm strnd concentrtion. Mesurements were run in uffer contining 00 mm KCl nd 0 mm MgCl2.