Rotation of Guanine Amino Groups in G-Quadruplexes: A Probe for Local Structure and Ligand Binding  Michael Adrian, Fernaldo Richtia Winnerdy, Brahim Heddi, Anh Tuân Phan  Biophysical Journal  Volume 113, Issue 4, Pages 775-784 (August 2017) DOI: 10.1016/j.bpj.2017.05.053 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Guanine amino groups in G-quadruplexes. (A) A G-tetrad. Amino groups are colored in red. The amino-group rotation about the C-N bond is represented by an arrow. (B) The guanine base is drawn as a rectangle with one red-colored edge indicating the location of the amino group. (C) Schematic structure of the tetrameric G-quadruplex [d(TTAGGGGT)]4 (Platform G4). Only guanine residues are drawn for clarity. To see this figure in color, go online. Biophysical Journal 2017 113, 775-784DOI: (10.1016/j.bpj.2017.05.053) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 NMR spectra of Platform G4 and a modified sequence. (A) Chemical structure of a guanine (G) and an N2-methyl guanine (M). (B) NMR spectrum of Platform G4 (top) showing a broadened amino proton peak (marked by an asterisk) and NMR spectrum of a modified sequence with N2-methyl guanine substitution at position 5 (bottom) showing a sharp amino proton peak (marked by a red asterisk). Imino proton assignments are indicated. To see this figure in color, go online. Biophysical Journal 2017 113, 775-784DOI: (10.1016/j.bpj.2017.05.053) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Rotation of amino groups. (A) NOE spectroscopy spectrum (mixing time, 100 ms) of Platform G4 at 298 K. (B) One-dimensional traces at G5 imino proton extracted from NOE spectroscopy spectra of Platform G4 recorded at different temperatures (in Kelvin scale), as indicated. Amino proton peaks are marked by asterisks. The dashed red line represents the fitting curve of the two-site-exchange lineshape function. Proton peaks marked by gray dots were excluded during fitting. (C) Plot of amino group rotation rate of Platform G4 as a function of temperature. Rotation-rate values from ∼300 to ∼2000 Hz lie within the intermediate regime (indicated), in which the lineshape analysis method is unable to determine the exact values. To see this figure in color, go online. Biophysical Journal 2017 113, 775-784DOI: (10.1016/j.bpj.2017.05.053) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Stacking of G-quadruplexes affects amino-group rotation. (A) The schematic structures of Platform G4 (left) and Platform G4 Dimer (right) are shown. The G-tetrad at the 3′-3′ interface is highlighted (G7#), as is one of the inner G-tetrad next to it (G6#). (B) The amino-group rotation rates of G6 and G6#, as well as G7 and G7# at 298 K are shown in a bar chart. The data show a significant change for the amino-group rotation rates at the stacking interface, G7, as compared to that of the inner G-tetrad, G6. To see this figure in color, go online. Biophysical Journal 2017 113, 775-784DOI: (10.1016/j.bpj.2017.05.053) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Local arrangement of a basepair affects amino-group rotation. (A) The schematic of the CEB25 G-quadruplex. The G-tetrad of interest (consisting of G3, G7, G19, and G23) is indicated in cyan. Paired adenine (A2) and thymine (T18) residues are drawn in green and orange, respectively. Connecting loops are colored gray. Other residues are not drawn, for clarity. (B) Solution structure of the CEB25 G-quadruplex showing the A2•T18 basepair stacking onto the G3•G7•G19•G23 tetrad (PDB:2LPW) (33). Guanine, adenine, and thymine residues are colored cyan, green, and orange, respectively. The amino groups are highlighted in blue (nitrogen) and light gray (hydrogen). (C) Amino proton spectra and estimated rotation rates of G21 and G5, as well as G7 and G23. Observable amino proton peaks are marked by asterisks. To see this figure in color, go online. Biophysical Journal 2017 113, 775-784DOI: (10.1016/j.bpj.2017.05.053) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Ligand binding affects amino-group rotation. (A) The schematic of a telomeric G-quadruplex being targeted by the ligand L2H. Guanine residues at the binding interface (G3, G9, G17, and G21) are indicated in cyan. L2H is represented by a purple-colored ring. Connecting loops are colored gray. Other residues are not drawn, for clarity. (B) Solution structure of the complex showing L2H binding on the G3•G9•G17•G21 tetrad (PDB:2MB3) (51). Guanine residues are colored in cyan, the amino groups are highlighted in blue (nitrogen) and light gray (hydrogen). (C) Amino proton spectra and estimated rotation rates of G3, G9, and G17 located below the ligand and G5 and G15 in the free site are displayed. Observable amino proton peaks are marked by asterisks. To see this figure in color, go online. Biophysical Journal 2017 113, 775-784DOI: (10.1016/j.bpj.2017.05.053) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 An additional hydrogen bond affects amino group rotation. (A) The schematic of a left-handed G-quadruplex, Z-G4. Guanine residues of interest (G2, G5, G8, and G11) are indicated in cyan. (B) The details of the additional hydrogen bond that formed between G5 and T7 are represented in stick-figure format (52). Guanine and thymine residues are colored cyan and orange, respectively. The amino groups are highlighted in blue (nitrogen) and light gray (hydrogen), and the oxygen of the thymine sugar is highlighted in red. (C) Amino proton spectra of G5 and G8 (with the additional hydrogen bond) are compared to those of G9 and G18 (without the additional hydrogen bond). Observable amino proton peaks are marked with asterisks. To see this figure in color, go online. Biophysical Journal 2017 113, 775-784DOI: (10.1016/j.bpj.2017.05.053) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 8 An additional hydrogen bond affects amino-group rotation. (A) The schematic of the GGA8 G-quadruplex, which forms two heptads in the middle. (B) Stick figure of the middle heptad of a GGA8 G-quadruplex solution structure solved by NMR spectroscopy (31). Guanine and adenine residues are colored in cyan and green, respectively. The amino groups are highlighted in blue (nitrogen) and light gray (hydrogen). The figure shows an additional hydrogen bond formed between the amino proton of a guanine and a nitrogen of the neighboring adenine. (C) Comparison of NMR spectra between two guanine residues, G7 and G1, which have an additional hydrogen bond, located at the middle tetrad, and two other guanine residues, G11 and G23, located at the outer tetrad. Observable amino proton peaks are marked by asterisks. To see this figure in color, go online. Biophysical Journal 2017 113, 775-784DOI: (10.1016/j.bpj.2017.05.053) Copyright © 2017 Biophysical Society Terms and Conditions