SUPPLEMENTARY INFORMATION

NMR based structure reveals groove binding of mitoxantrone to two sites

of [d-(TTAGGGT)]4 having human telomeric DNA sequence leading to

thermal stabilization of G-quadruplex

Sweta Tripathi and Ritu Barthwal*

Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India

*Corresponding author: Phone: +91-1332-285807 Fax: +91-1332-273560

Email: ritubfbs@iitr.ac.in (Prof. Ritu Barthwal)

SUPPLEMENTARY TABLES

D/N τ1 (ns) τ2 (ns) α1 α2 χ2

0.0 0.14 - 100.0 - 1.140.5 0.45 0.26 44.6 55.4 1.161.0 0.47 0.23 30.0 70.0 1.301.5 0.44 0.21 23.1 76.9 1.202.0 0.44 0.21 18.3 81.7 1.203.0 0.47 0.15 11.8 88.2 1.194.0 0.59 0.19 10.8 89.2 1.11

Table SI 1: The fluorescence lifetime (τ) and the corresponding relative amplitude (α)

of 3 µM MTX in the absence and presence of [d-(TTAGGGT)]4 at different D/N ratios

at 25 ºC. χ2 is best chi square fitting values.

Protons

T1 T2 A3 G4

δb δf ∆δ δb δf ∆δ δb δf ∆δ δb δf ∆δ

H8/H6 7.49 7.44 0.05 7.35 7.35 0.00 8.46

8.44 0.02 7.94 7.99 -0.05

H1' 6.03 6.02 0.01 6.19 6.22 -0.03

6.29

6.29 0.00 6.04 6.05 -0.01

H2' 2.14 2.13 0.01 2.08 2.08 0.00 2.89

2.86 0.03 2.69 2.70 -0.01

H2'' 2.38 2.38 0.00 2.37 2.36 0.01 2.92

2.92 0.00 2.93 2.94 -0.01

H3' 4.63 4.78 5.12

5.12 0.00 5.08 5.07 0.01

H4' 4.05 4.03 0.02 4.09 4.09 0.00 4.44

4.44 0.00 4.48 4.46 0.02

H5' 3.69 3.67 -0.02

3.99 3.95 0.04 4.18

4.17 0.01 4.28 4.28 0.00

H5'' 3.69 3.70 -0.02

3.99 3.95 0.04 4.08

4.09 -0.01

4.28 4.28 0.00

H2/CH3

1.68 1.68 0.00 1.83 1.77 0.06 8.12

8.16 -0.04

NH2b

NH2nb

NH 11.57

11.63

-0.06

G5 G6 T7

δb δf ∆δ δb δf ∆δ δb δf ∆δ

H8/H6 7.75 7.79 -0.04

7.71 7.72 -0.01

7.42 7.41 0.01

H1' 6.05 6.06 -0.01

6.27 6.28 -0.01

6.11 6.11 0.00

H2' 2.63 2.64 -0.01

2.58 2.57 0.01 2.17 2.19 -0.02

H2'' 2.70 2.72 -0.02

2.69 2.68 0.01 2.23 2.23 0.00

H3' 5.06 5.09 -0.03

4.96 4.96 0.00 4.53 4.50 0.02

H4' 4.50 4.53 4.51 0.02 4.23 4.23 0.00

H5' 4.33 4.31 0.02 4.29 4.28 0.01 4.12 4.11 0.01

H5'' 4.33 4.31 0.02 4.29 4.28 0.01 4.12 4.11 0.01

H2/CH3

1.67 1.67 0.00

NH2b 9.25

NH2nb 5.91

NH 11.22 11.28 -0.06

10.98

11.04 -0.06

Dd +ve denotes downfield shift, Dd –ve denotes upfield shift

Table SI 2a: Chemical shift (ppm) of [d-(TTAGGGT)]4 protons in free (δf ) state

(D/N=0.0) and that bound to MTX (δb) at D/N=2.0 at 25 ºC along with change in

chemical shift on binding, that is ∆δ=δb(D/N=2.0)-δf (D/N=0.0).

MTXProtons

df(D/N=0.0) δb(D/N=1.0) δb(D/N=2.0) ∆δ=δb(D/N=1.0)-δf

(D/N=0.0) ∆δ=δb(D/N=2.0)-

δf (D/N=0.0) 11NH 9.68 9.58 9.58 -0.10 -0.106/7H 6.82 6.74 6.75 -0.08 -0.072/3H 6.71 6.19 6.18 -0.52 -0.53

1/4OH 13.23 12.74 12.74 -0.49 -0.4911CH2 3.53 3.27 3.27 -0.26 -0.2612CH2 3.24 3.14 3.13 -0.10 -0.1113CH2 3.21 3.13 3.12 -0.08 -0.0914CH2 3.88 3.78 3.77 -0.10 -0.11

Table SI 2b: Chemical shift (ppm) of MTX protons from NOESY spectra of free (df)

MTX (D/N=0.0) and MTX-[d-(TTAGGGT)]4 complex (db) at D/N=1.0 and 2.0 at 25 ºC

and change in chemical shift on binding at D/N=1.0 and 2.0.

S. No. 31P signalD/N=0

df D/N=0

db

D/N=0.5db

D/N=1.0db

D/N=1.5

31P signal*D/N=2.0

db

D/N=2.01 A3pG4 -0.27 -0.27 -0.34 -0.28 A3pG4 -0.301' - -0.29 - -0.23 A3pG4 -0.212 T1pT2,

T2pA3-0.55 -0.55 -0.57 -0.48 G6pT7 -0.46

3 G6pT7 -0.59 -0.60 -0.62 -0.53 T1pT2, T2pA3

-0.53

4 G5pG6 -0.62 -0.63 -0.68 -0.60 G5pG6 -0.555 G4pG5 -0.64 -0.67 -0.71 -0.65 G4pG5 -0.616 - - - - -0.647 - - - - -0.66

* Tentative Assignment

Table SI 3: Chemical shift of 31P resonances of the phosphate groups of [d-

(TTAGGGT)]4, in free [d-(TTAGGGT)]4 (D/N=0.0) and MTX-[d-(TTAGGGT)]4

complex at 35 ºC at different D/N ratios.

SUPPLEMENTARY FIGURES

Figure SI 1: (a,b) 1D Proton NMR spectrum of free G-quadruplex [d-(TTAGGGT)]4

showing non-exchangeable and exchangeable (imino) resonances at 25 ºC and (c) Proton

NMR spectra of 10 mM MTX in KBPES buffer.

Figure SI 2: Expansion of NOESY spectra of free G-quadruplex [d-(TTAGGGT)]4 at

25 ºC showing (a) NH-NH connectivity between adjacent imino protons, (b) Inter strand

guanine NH connectivities with its own and 5' flanking base protons in A3-G4-G5-G6

segment, (c) intra strand base H8/H6-H1'sequential connectivity and (d) Intra strand

base H8/H6-H2'H2''sequential connectivity in T1-T2-A3-G4-G5-G6-T7 segment.

Figure SI 3: Overlap of 1H-13C HSQC spectra at 45 ºC of free MTX (blue) and free [d-

(TTAGGGT)]4 (red), bound MTX (green) and bound [d-(TTAGGGT)]4 (black) in 2:1

MTX-[d-(TTAGGGT)]4 complex showing (a) aromatic protons and (b) aliphatic

protons.

Figure SI 4: 2D 1H-31P HMBC spectra of (a) free [d-(TTAGGGT)]4 (D/N=0.0) and (b)

MTX-[d-(TTAGGGT)]4 complex at D/N=2.0 at 35 ºC.

Figure SI 5: (a,b) Selected regions of NOESY spectra of mitoxantrone complexed to [d-

(TTAGGGT)]4 at D/N=1.0 showing drug-DNA inter molecular (*) NOE contacts at 25

ºC. The drug and DNA protons are shown in blue and black color, respectively.

Figure SI 6: Thermal melting profiles showing derivative of CD (θ) with respect to

temperature (dθ/dT) as a function of temperature for 20 µM free [d-(TTAGGGT)]4

(D/N=0) and its complex with mitoxantrone at D/N=1.0, 2.0 and 3.0 in KBPES buffer

(pH 7) containing KCl=100 mM, K2HPO4=10 mM, EDTA=1mM.