8-Oxo-7,8-dihydrodeoxyadenosine: The first example of a native DNA lesion that stabilizes human telomeric G-quadruplex DNA Manali Aggrawal, Hyun Joo, Wanbo Liu, Jerry Tsai, Liang Xue ⇑ Department of Chemistry, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA article info Article history: Received 5 April 2012 Available online 19 April 2012 Keywords: DNA lesions G-quadruplex DNA Thermal denaturation DNA conformations abstract Native DNA lesions in general destabilize DNA secondary structures such as duplex and G-quadruplex because they disrupt optimized interactions in DNA defined by nature. In this paper, we report the first example of a native DNA lesion (8-oxo-7,8-dihydrodeoxyadenosine, OxodA) that stabilizes human telo- meric G-quadruplex DNA. CD thermal denaturation studies explicitly displayed increased melting tem- peratures of telomeric G-quadruplex DNAs that contain OxodA(s) in different DNA loops, suggesting enhanced thermal stability. Conformation studies of G-quadruplex DNAs containing OxodA(s) in the loops using CD and native PAGE revealed that they adopt a similar antiparallel conformation in Na + but have much more versatile conformations in K + . According to computational calculations, the observed stabilization may result from the tight binding of K + into the pocket formed by the O8 of OxodA and its loop. The study reported here may provide better understanding of the effect of DNA lesions on G- quadruplex stability and conformation. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction G-quadruplex DNA structures are constructed by stacking planar Hoogsteen hydrogen-bonded G-quartets on top of each other [1]. Potential unimolecular quadruplex-forming sequences in Escherichia coli, other prokaryotic genomes, and the human genome have recently been identified via systematic surveys [2,3]. Amongst all the G-quadruplex-forming regions, telomeric DNA that consists of 5 0 -d(TTAGGG) repeating units at the ends of chromosomes has attracted most attention. Intramolecular G-quadruplexes formed in the telomeric regions were proven to inhibit the binding of telomerase, a reverse transcriptase that is ex- pressed in 80–85% tumor cells and is essential for the unrestricted growth of cancer cells [4]; therefore, they have become potential therapeutic targets for cancer intervention [5,6]. The conformation of telomeric G-quadruplexes under physiological conditions has been extensively studied because structure–function relationships may exist [7]. In our group, we are interested in investigating the effect of naturally occurring modified nucleotides (DNA lesions) on the structure of telomeric G-quadruplex DNA. The 3 0 overhang of single-stranded telomeric DNA in principle is prone to oxidation because it is more accessible to damaging agents such as reactive oxygen species (ROS) than duplex DNA. The resulting DNA lesions may regulate the conformation and stability of G-quadruplex DNA. However, to our knowledge, such studies are scarce in literature. 8- Oxo-7,8-dihydrodeoxyguanosine (OxodG) is the only native DNA lesion in G-quadruplex DNA that has been studied [8]. Another close example is an analog (dSpacer) mimicking abasic sites present in G-quadruplex DNA [9,10]. Both OxodG and dSpacer destabilize the corresponding G-quadruplex DNA regardless of their locations in the loops or G-quartets. In the present work, we report an unexpected stabilization of telomeric G-quadruplex DNA (1, Fig. 1) when we systematically replaced deoxyadenosine (dA) in the loops with its oxidized form (8-oxo-7,8-dihydrodeoxy- adenosine, OxodA). This is the first example of a native DNA lesion that augments the stability of G-quadruplex DNA. The observation reported here is quite significant and may reinstate a common be- lief on the role DNA lesions in the stabilization of G-quadruplex DNA. 2. Materials and methods All the chemicals and phosphoramidites for DNA synthesis were purchased from Glen Research. The DNA oligonucleotides were synthesized on an Applied Biosystems 392 DNA/RNA synthesizer, purified by polyacrylamide gel electrophoresis (PAGE), and charac- terized using a Shimadzu AXIMA-CFT MALDI-TOF mass spectrom- eter. UV spectra were collected on a Varian Cary 100 Bio UV–vis spectrophotometer. Circular dichroism spectra were recorded on a JASCO J-810 spectropolarimeter. T4 polynucleotide kinase was obtained from New England Biolabs. [c- 32 P]-ATP was purchased from MP Biochemicals. Quantification of 5 0 -[ 32 P]-labeled 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.04.059 ⇑ Corresponding author. Fax: +1 209 946 2607. E-mail address: lxue@pacific.edu (L. Xue). Biochemical and Biophysical Research Communications 421 (2012) 671–677 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc