Study of STAT3 G-quadruplex folding patterns by CD

spectroscopy and molecular modeling

Sen Lin a, Ming Xu b,*, Gu Yuan a,*a Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of

Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing

100871, Chinab Key Laboratory of Molecular Cardiovascular Sciences of Ministry of Education, Institute of Vascular Medicine, Third Hospital, Peking

University, Beijing 100191, China

Received 22 August 2011

Available online 24 January 2012

Abstract

The G-quadruplexes formed from G-rich strands in the telomere and oncogene-promoter regions are regarded as new promising

targets in the cancer therapy. A G-quadruplex in the downstream flanking region of the signal transducer and activator of

transcription 3 (STAT3) gene was explored. Its folding patterns were proposed to be 3:2:2 and 3:3:1 loop isomers by the mutation

analysis by CD spectroscopy. The structures were constructed and refined by molecular modeling method.

# 2012 Gu Yuan. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: G-quadruplex; STAT3 gene; CD spectroscopy; Molecular modeling

STAT3, a key signal transducer and transcription activator, plays a very important role in many cell processes [1].

Its activity is required for optimal function of the electron transport chain in mitochondria, regulating processes of

neovascularization, cardiac growth and even providing protection against various cellular stresses [2,3]. There is a G-

rich sequence in the downstream flanking region of STAT3 gene (Gene ID: 6774 in NCBI database), it is transcribed

into the 30-UTR of STAT3 mRNA. This G-rich sequence has the potential to form a secondary DNA structure called G-

quadruplex. Stabilization and formation of the G-quadruplex may down-regulate the expression of STAT3 gene. So it

is important to explore the folding patterns of STAT3 G-quadruplex for the future study of developing this DNA

structure into a new drug target.

In this research, CD spectroscopy was used to probe the folding patterns of G-quadruplex in STAT3 gene through

the mutation analysis. Meanwhile, molecular modeling was carried out to construct and refine the theoretical

structures of the STAT3 G-quadruplex by AMBER 10 [4]. Our results not only reported a new G-quadruplex formed in

STAT3 gene, but also provided a novel method to explore the structures of the new-founded G-quadruplexes and gave

new clues for structure-based drug design.

www.elsevier.com/locate/cclet

Available online at www.sciencedirect.com

Chinese Chemical Letters 23 (2012) 329–331

* Corresponding authors.

E-mail addresses: xuminghi@bjmu.edu.cn (M. Xu), guyuan@pku.edu.cn (G. Yuan).

1001-8417/$ – see front matter # 2012 Gu Yuan. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2011.11.002

1. Experimental

The oligonucleotides were purchased from Sangon (Shanghai, China), and their sequences are listed in Table 1. The

CD spectra of DNA were measured by using a J-810 spectropolarimeter (JASCO Co., Ltd., Japan) with a 0.1 cm path-

length quartz cell at 25 8C. The concentration of oligonucleotide was 10 mmol/L in each sample. All the samples were

annealed at 90 8C for 10 min and then cooled to room temperature overnight.

The crystal structure of the telemetric DNA quadruplex-drug complex (PDB code 3CDM) was used as an initial

model and then treated by some necessary replacement and deletion to construct the STAT3 G-quadruplex structures.

Each system was neutralized by Na+ and solvated with a truncated octahedral box containing an 8 A buffer of TIP3P

water. Energy minimization was performed in these explicit solvents using the sander module of AMBER 10. First, the

system of G-quadruplex soaked in water was minimized for 1000 steps with holding the DNA fixed by

500 kcal mol�1 A�2. Then 2500 steps of minimization were done for the entire system with no restraints. This was

followed by molecular dynamics with 20 ps equilibration while restraining the solute by 10 kcal mol�1 A�2 and then

20 ns simulations for the whole system at 300 K. Trajectories with constant RMSD were averaged using the ptraj

module and the obtained structures were finally minimized in explicit system.

2. Results and discussion

There are only three guanine residues in the first run of the G-rich sequence (S1), which means that three G-quartets

could form in this G-quadruplex structure [5]. During the next three G-tracts, if a guanine base participates in the

formation of a G-quartet, the characteristic peak at 264 nm would be decreased obviously when the guanine was

mutated into a thymine. However, if the guanine locates at the loop region, the thymine mutation would result in a

smaller decrease of the characteristic peak at 264 nm. Fig. 1 shows that the mutation of G9 lead to a greater decline of

characteristic peak at 264 nm than the mutation of G6, which suggests that G9 belongs to the G-quartet and G6 belongs

to the loop region. According to the same analysis, G17 and G18 appear to be involved in the G-quartets. But in the

S. Lin et al. / Chinese Chemical Letters 23 (2012) 329–331330

Table 1

Oligonucleotides used in this work.

Name Sequence

S1 (STAT3) 50-GGGCTGGGGATGGGGAGGGGG-30

WT-G 50-TTGGGCTGGGGATGGGGAGGGGGTT-30

G6T 50-TTGGGCTTGGGATGGGGAGGGGGTT-30

G9T 50-TTGGGCTGGGTATGGGGAGGGGGTT-30

G12T 50-TTGGGCTGGGGATTGGGAGGGGGTT-30

G15T 50-TTGGGCTGGGGATGGGTAGGGGGTT-30

G17/18T 50-TTGGGCTGGGGATGGGGATTGGGTT-30

G20/21T 50-TTGGGCTGGGGATGGGGAGGGTTTT-30

240 260 280 300 320

-2.50x105

0.00

2.50x105

5.00x105

7.50x105

1.00x106

Mol

ar E

llipt

icity

(deg

cm

2 dm

ol-1)

Wavelength (nm)

WTG6TG9TG12TG15TG17/18TG20/21T

Fig. 1. CD spectra of the wild type and mutations of S1: WT-G, G6T and G9T, G12T and G15T, G17/18T and G20/21T. The concentration of

oligonucleotide was 10 mmol/L in each sample and 50 mmol/L Tris–HCl (pH = 7.4) was used as buffer solution.

third run of G-tract, the mutation of G12 caused the 264 nm peak to decrease by almost the same amount as the

mutation of G15 did. In addition, the chemical symmetries of these two possible patterns were similar. If G12 is in the

G-quartet, it would be a 3:2:2 loop isomer. If G15 is in the G-quartet, it would change into a 3:3:1 loop isomer. Based

on the results and analysis above, it is proposed that the guanine residues involved in G-quartet formation are G1–G3,

G7–G9, G12–G14 and G17–G19 or G1–G3, G7–G9, G13–G15 and G17–G19.

All the theoretical structures of 12 possible loop isomers of STAT3 G-quadruplex were constructed. Specially, the

loop isomers 3:2:2 and 3:3:1 proposed by CD analysis were further refined through 20 ns molecular dynamics

simulation using AMBER 10; and the obtained structures of the loop isomers 3:2:2 and 3:3:1 are presented in Fig. 2.

Three G-quartets formed and kept stable during the refinement.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 90913004 and 30821001), the

Natural Science Foundation of Beijing (No. 7082101), and Program for New Century Excellent Talents in University

and Beijing Talents Foundation. We also want to thank Beijing National Laboratory for Molecular Sciences for its

support.

References

[1] E. James Jr., Darnell, Science 277 (1997) 1630.

[2] K.Z. Gong, H. Zhang, J.H. Du, et al. Acta Pharmacol. Sin. 28 (2007) 153.

[3] K.D. Hilfiker, A. Hilfiker, H. Drexler, Pharmacol. Ther. 107 (2005) 131.

[4] N. Spackova, I. Berger, J. Sponer, J. Am. Chem. Soc. 121 (1999) 5519.

[5] H.H. Li, G. Yuan, Chin. Chem. Lett. 19 (2008) 1108.

S. Lin et al. / Chinese Chemical Letters 23 (2012) 329–331 331

Fig. 2. The structures of loop isomers 3:2:2 and 3:3:1 of STAT3 G-quadruplex obtained after 20 ns molecular dynamics by Amber 10. (a) 3:2:2, side-

view; (b) 3:2:2, top-view; (c) 3:3:1, side-view; (d) 3:3:1, top-view (presented by software PyMOL).