Table of Contents: Table S1. Total PQSs and PQS densities (per 100000 nucleotides) of sequences analyzed. Table S2. Most frequent PQSs harbouring already confirmed G4s in literature and the G4-iM Grinder database (version 2.502) per genome of T. cruzi analyzed. Table S3. Total PQSs with high G4-probability (that score ≥ 40), per genome of T. cruzi analyzed. Table S4. Most frequent PQSs with high G4-probability (that score ≥ 40), per genome of T. cruzi analyzed. Figure S1. Characterization of T. cruzi sequences (DCr, RCr, TCr and MCr) by NMM fluorescence assay. Figure S2. Characterization of T. cruzi sequences (DCr, RCr, TCr and MCr) by thermal difference spectra (TDS). Figure S3. Characterization of T. cruzi sequences (DCr, RCr, TCr and MCr) by isothermal difference spectra at 25 °C (IDS). Figure S4. CD melting curves of T. cruzi sequences (DCr, RCr, TCr and MCr) at 25 °C. Figure S5. CD spectra of RCr sequence in the presence of 100, 10 and 1 mM K+ concentration. Figure S6. Imino region of the proton-NMR spectra of RCr, TCr, MCr and DCr sequences. Figure S7. Imino region of the proton-NMR spectra of RCr sequence at different temperatures. Figure S8. Isomerisation of L3 to L4 followed by 1H NMR. Figure S9. Isomerisation of L5 to L6 followed by 1H NMR. Figure S10. Isomerisation of L7 to L8 followed by 1H NMR. Figure S11. Isomerisation of L11 to L12 followed by 1H NMR. Figure S12. Isomerisation of L13 to L14 followed by 1H NMR. Figure S13. FRET melting assay. FRET melting curves of A) F-MCr-T, B) F-TCr-T, C) F-21-T, D) F-MYC-T (all fluorescently labeled) DNA quadruplexes (0.2 μM) and E) F-dx-T DNA duplex (0.2 μM) in the absence and in the presence of the tested compounds L3 and L4 (10 μM). Figure S14. Competition FRET melting experiments run on A) F-21-T or B) F-RCr-T, for ligands L3 and L4 in the presence of increasing concentrations of ds26 competitor (0, 1, 3, 10 M). Figure S15. Imino region of the 1H-NMR spectra of A) RCr, B) TCr or C) MCr in aqueous buffer solution in the absence and presence of compound L3 or L4. Figure S16. CD melting curve of RCr quadruplex in the absence and presence of compound L4. Figure S17. CD melting curve of MCr quadruplex in the absence and presence of compound L3 and L4. Figure S18. UV spectra of L3 (A) or L4 (B) in the absence and presence of MCr or RCr DNA quadruplexes. Figure S19. UV spectra of L3 (A) or L4 (B) in the absence and presence of ds26 duplex DNA. 1H-NMR and 13C-NMR Spectra of the new compounds HPLC traces of compounds L3 and L4.

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