Encoding canonical DNA quadruplex structure

The remarkable architectural diversity of four-stranded DNA known as G-quadruplexes can be rationally designed.


This PDF file includes:
Assessment of folding of DNA sequences Characterization of structure Identification of topology NMR chemical shifts tables Structural statistics tables Fig. S1. Expansions of 1D NMR spectra of imino proton regions for DNA sequences folding into quadruplexes in this study. Fig. S2. NMR structure characterization of 2MFT. Fig. S3. NMR structure characterization of aromatic and anomeric regions of 2MW6. Fig. S4. NMR structure characterization of inosine substitutions for 2MW6. Fig. S5. Intraresidue aromatic-imino assignments for guanines in the stem of 2MW6. Fig. S6. Exchangeable proton assignments for the structure of 2M6W. Fig. S7. Nonexchangeable 1 H and 31 P assignments for 5J6U. Fig. S8. Exchangeable proton assignments for 5J6U. Fig. S9. Nonexchangeable 1 H assignments for 5J05. Fig. S10. Exchangeable proton assignments for 5J05. Fig. S11. Sequence-specific assignments for 5J4W. Fig. S12. Exchangeable proton assignments for 5J4W. Fig. S13. Nonexchangeable 1 H assignments for 5J4P. Fig. S14. Exchangeable proton assignments for 5J4P. Fig. S15. Nonexchangeable 1 H and 31 P assignments for 2M6V. Fig. S16. Exchangeable proton perturbations for the inosine substitutions on 2M6V. Fig. S17. Exchangeable proton assignments for 2M6V. Fig. S18. NMR experiments for characterization of the 4(−l w d+l n ) topology formed by the DNA sequences S069, S067, S036, and S080. Fig. S19. Solution NMR experiments for characterization of the 3(−l w d+l n ) topology formed by the DNA sequences S231, S090, S089, S088, and S093. Fig. S20. Solution NMR experiments for characterization of the 2(−l w d+l n ) topology formed by the DNA sequences S167, S171, and S172. Fig. S21. Use of riboguanosines to induce folding of the 3(−l w d+l n ) topology. Fig. S22. Exchangeable proton assignments for 3(−l w d+l n ) topology formed by S090. Table S1. Proton chemical shifts for the structure of 2MFT. Table S2. Proton and phosphorous chemical shifts for structure of 2M6W. Table S3. Proton and phosphorous chemical shifts for structure of 5J6U. Table S4. Proton chemical shifts for the structure of 5J05. Table S5. Proton chemical shifts for the structure of 5J4W. Table S6. Proton chemical shifts for the structure of 5J4P. Table S7. Proton and phosphorous chemical shifts for the structure of 2M6V. Table S8. NMR restraints and structural statistics for the structures of 2MFT. Table S9. NMR restraints and structural statistics for the structures of 2M6W. Table S10. NMR restraints and structural statistics for the structures of 5J6U. Table S11. NMR restraints and structural statistics for the structures of 5J05. Table S12. NMR restraints and structural statistics for the structures of 5J4W. Table S13. NMR restraints and structural statistics for the structures of 5J4P. Table S14. NMR restraints and structural statistics for the structures of 2M6V.

Assessment of folding of DNA sequences
Fig. S1. Expansions of 1D NMR spectra of imino proton regions for DNA sequences folding into quadruplexes in this study. Expansions of 1D NMR spectra of imino proton regions for DNA sequences at ~2 mM oligonucleotide concentrations in 80 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 6.8 solutions at at 5 o C. Shown are also schematic representations of the (-lwd+ln) topology they adopt with 2'-deoxyguanosines of the stem in syn (magenta) and anti (cyan) conformations, green for non-stem guanosines, and orange for adenines, and yellow for thymines.

Structural assignments for 2M6W in sodium
Fig. S3. NMR structure characterization of aromatic and anomeric regions of 2MW6. Nonexchangeable 1 H and 31 P assignments for the 4(-lwd+ln) adopted by the DNA sequence 2M6W in 16 mM NaCl, 4 mM NaH2PO4/Na2HPO4, pH 6.8, at 20 °C. In panel (A), expansions of 1 H-1 H NOESY spectra (20 o C) depicting anomeric-aromatic regions of the 1 H-1 H NOESY and showing labelled intraresidual H1′-H6/H8 and H6/H8-H2'/2'' NOE interactions. Sequential correlations are denoted with lines. The inset contains assignments for the characteristic sequential connectivities (SynG-AntiG-T-T-T-T) of the diagonal loop. Methyl-H8/H6 sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third Thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'-deoxyguanosines of the stem in syn (pink) and anti (cyan) conformations, green for non-stem guanosines, and yellow for thymines. In panel (B) sequential coupling correlations of the type H3'(i-1)-P(i)-H4'/H5'/H5'' in a[ 1 H-31 P] HSQC spectrum are shown. In panel (C) DQF COSY intraresidue correlations H1'-H2'/H2'' are shown. A complete list of proton assignments is shown in Table S2.

Structural assignments for 5J6U in sodium
Fig. S7. Nonexchangeable 1 H and 31 P assignments for 5J6U. Non-exchangeable 1 H and 31 P assignments for the 4(-lwd+ln) adopted by the DNA sequence 5J6U in 16 mM NaCl, 4 mM NaH2PO4/Na2HPO4, pH 6.8, at 20 °C. The spectrum on top shows expansions of 1 H-1 H NOESY spectra (20 o C) depicting anomeric-aromatic regions of the 1 H-1 H NOESY and showing labelled intraresidual H1′-H6/H8 and H6/H8-H2'/2'' NOE interactions. Sequential correlations are denoted with lines. The inset contains assignments for the characteristic sequential connectivities (SynG-AntiG-T-T-T-T) of the diagonal loop. Methyl-H8/H6 sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third Thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'deoxyguanosines of the stem in syn (pink) and anti (cyan) conformations, orange for adenines, and yellow for thymines. In the bottom spectrum sequential coupling correlations of the type H3'(i-1)-P(i)-H4'/H5'/H5'' in a [ 1 H-31 P] HSQC spectrum are shown. A complete list of proton assignments is shown in Table S3. ]. An expanded JR-NOESY spectrum (200 ms) in 16 mM NaCl, 4 mM NaH2PO4/Na2HPO4, pH 6.8 at 5 °C is shown, illustrating the dipolar connectivities between imino H1-H1 exchangeable protons. Peaks 1-18 are assigned as follows: (1) G8H1-G22H1, (2) G8H1-G18H1, G18H1-G24H1. These assignments allow for the formation of the hydrogen bond alignments shown in the chemical structures shown in (C).

Structural assignments for 5J05 in sodium
Fig. S9. Nonexchangeable 1 H assignments for 5J05. Non-exchangeable 1 H assignments for the 3(-lwd+ln) adopted by the DNA sequence 5J05 in 80 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 6.8. In panel (A), expansions of 1 H-1 H NOESY spectra (20 o C) depicting anomeric-aromatic regions of the 1 H-1 H NOESY and showing labelled intraresidual H1′-H6/H8 and H6/H8-H2'/2'' NOE interactions. Sequential correlations are denoted with lines. The inset contains assignments for the characteristic sequential connectivities (SynG-AntiG-T-T-T-T) of the diagonal loop. Methyl-H8/H6 sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third Thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'-deoxyguanosines of the stem in syn (pink) and anti (cyan) conformations, orange for adenines, and yellow for thymines. In panel (B) an expansion of JR [ 1 H-1 H] NOESY spectrum (200 ms mixing time) in 1 H2O at 5°C of the substitution of G2 for Inosine in DNA sequence 5J05. The spectrum illustrates the chemical shift of H8 upon substitution, which unambiguously proves the assignment of the sequentially linked G1 as a syn residue for 5J05. A complete list of proton assignments is shown in Table S4. spectrum (200 ms) in 80 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 6.8 is shown, illustrating the dipolar connectivities between imino H1-H8 imino H1-H1. Peaks 1-12 are assigned as follows: These assignments allow for the formation of the hydrogen bond alignments shown in the chemical structures below.

Structural assignments for 5J4W in sodium
Fig. S11. Sequence-specific assignments for 5J4W. Non-exchangeable 1 H assignments for the 2(-lwd+ln) adopted by the DNA sequence 5J4W in 80 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 6.8 and 20 o C. Expansions of 1 H-1 H NOESY spectra (20 o C) depicting anomeric-aromatic regions of the 1 H-1 H NOESY and showing labelled intraresidual H1′-H6/H8 and H6/H8-H2'/2'' NOE interactions. Sequential correlations are denoted with lines. The inset contains assignments for the characteristic sequential connectivities (SynG-AntiG-T-T-T-T) of the diagonal loop. Methyl-H8/H6 sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third Thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'-deoxyguanosines of the stem in syn (pink) and anti (cyan) conformations, and yellow for thymines. A complete list of proton assignments is shown in Table S5.

Structural assignments for 5J4P in sodium
Fig. S13. Nonexchangeable 1 H assignments for 5J4P. Non-exchangeable 1 H assignments for the 2(-lwd+ln) adopted by the DNA sequence 5J4P in 80 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 6.8 and 20 o C. Expansions of 1 H-1 H NOESY spectra depicting anomeric-aromatic regions of the 1 H-1 H NOESY and showing labelled intraresidual H1′-H6/H8 and H6/H8-H2'/2'' NOE interactions. Sequential correlations are denoted with lines. The inset contains assignments for the characteristic sequential connectivities (SynG-AntiG-T-T-T-T) of the diagonal loop. Methyl-H8/H6 sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third Thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'-deoxyguanosines of the stem in syn (pink) and anti (cyan) conformations, and yellow for thymines. A complete list of proton assignments is shown in Table S6.

Structural assignments for 2M6V in sodium
Fig. S15. Nonexchangeable 1 H and 31 P assignments for 2M6V. Non-exchangeable 1 H and 31 P assignments for the 2(-lwd+ln) adopted by the DNA sequence 2M6V in 20 mM NaCl, 4 mM NaH2PO4/Na2HPO4, pH 6.8, at 20 °C. The spectrum on top shows expansions of 1 H-1 H NOESY spectra (20 o C) depicting anomeric-aromatic regions of the 1 H-1 H NOESY and showing labelled intraresidual H1′-H6/H8 and H6/H8-H2'/2'' NOE interactions. Sequential correlations are denoted with lines. The inset contains assignments for the characteristic sequential connectivities (SynG-AntiG-T-T-T-T) of the diagonal loop. Methyl-H8/H6 sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third Thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'deoxyguanosines of the stem in syn (pink) and anti (cyan) conformations, orange for adenines, and yellow for thymines. In the bottom spectrum sequential coupling correlations of the type H3'(i-1)-P(i)-H4'/H5'/H5'' in a [ 1 H-31 P] HSQC spectrum are shown. A complete list of proton assignments is shown in Table S7.  sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third Thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'-deoxyguanosines of the stem in syn (magenta) and anti (cyan) conformations. Methyl-H8/H6 sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third Thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'-deoxyguanosines of the stem in syn (magenta) and anti (cyan) conformations.

Fig. S20. Solution NMR experiments for characterization of the 2(−lwd+ln) topology formed by
the DNA sequences S167, S171, and S172. Anomeric-aromatic regions of the 1 H-1 H NOESY (tm 250 ms, 20 o C) spectra of DNA sequences S167, S171, and S172 in 80 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 6.8. Intraresidual H1′-H6/H8 NOE interactions are labelled and sequential correlations are denoted with lines. The inset contains assignments for the characteristic sequential connectivities (SynG-AntiG-T-T-T-T) of the diagonal loop. Methyl-H8/H6 sections illustrate the characteristic connectivity between the aromatic H8 of the 5'-SynG residue of the stem and the methyl of the third thymine in the diagonal loop. Shown are also schematic representations of the topologies they adopt with 2'-deoxyguanosines of the stem in syn (magenta) and anti (cyan) conformations.

Fig. S22. Exchangeable proton assignments for 3(−lwd+ln) topology formed by S090.
Exchangeable proton assignments for S090 in 80 mM NaCl, 20 mM NaH2PO4/Na2HPO4, pH 6.8. An expanded [ 1 H-1 H] JR-NOESY spectrum (200 ms) of S090 at 5° C, depicts NOE connectivities between imino (H1) exchangeable protons and aromatic (H8) protons. Sequential connectivities and those with loop residues are not indicated. These assignments allow for the hydrogen bond alignments depicted in the chemical structures below defining the topology shown. Table S1. Proton chemical shifts for the structure of 2MFT. Exchangeable-proton chemical shifts were obtained at 5° C while nonexchangeable proton chemical shifts were obtained at 20° C. H5'/H5'' protons are not stereo-specifically assigned.  Table S3. Proton and phosphorous chemical shifts for structure of 5J6U. Proton and phosphorous (20 °C) chemical shifts for sequence 5J6U in 20 mM NaPi (pH 6.8) aqueous buffer. Exchangeable-proton chemical shifts were obtained at 5° C at 500 MHz, while nonexchangeable proton chemical shifts were obtained at 20° C at 900 MHz. H5'/H5'' protons are not stereo-specifically assigned.