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Structural Studies of a Stable Parallel-Stranded DNA Duplex

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Structural Studies of a Stable Parallel-Stranded DNA Duplex View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biophysical Journal Volume 75 September 1998 1163–1171 1163 Structural Studies of a Stable Parallel-Stranded DNA Duplex Incorporating Isoguanine:Cytosine and Isocytosine:Guanine Basepairs by Nuclear Magnetic Resonance Spectroscopy Xiang-Lei Yang,* Hiroshi Sugiyama,# Shuji Ikeda,§ Isao Saito,§ and Andrew H.-J. Wang* *Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 USA; #Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan; and §Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606-8501, Japan ABSTRACT Isoguanine (2-hydroxyladenine) is a product of oxidative damage to DNA and has been shown to cause mutation. It is also a potent inducer of parallel-stranded DNA duplex structure. The structure of the parallel-stranded DNA duplex (PS-duplex) 5Ј-d(TiGiCAiCiGiGAiCT) ϩ 5Ј-d(ACGTGCCTGA), containing the isoguanine (iG) and 5-methyl-isocytosine (iC) bases, has been determined by NMR refinement. All imino protons associated with the iG:C, G:iC, and A:T (except the two terminal A:T) basepairs are observed at 2°C, consistent with the formation of a stable duplex suggested by the earlier Tm measurements [Sugiyama, H., S. Ikeda, and I. Saito. 1996. J. Am. Chem. Soc. 118:9994–9995]. All basepairs are in the reverse Watson-Crick configuration. The structural characteristics of the refined PS-duplex are different from those of B-DNA. The PS duplex has two grooves with similar width (7.0 Å) and depth (7.7 Å), in contrast to the two distinct grooves (major groove width 11.7 Å, depth 8.5 Å, and minor groove width 5.7 Å, depth 7.5 Å) of B-DNA. The resonances of the amino protons of iG and C are clearly resolved and observable, but those of the G and iC are very broad and difficult to observe. Several intercalators with different complexities, including ethidium, daunorubicin, and nogalamycin, have been used to probe the flexibility of the backbone of the (iG, iC)-containing PS-duplex. All of them produce drug-induced UV/vis spectra identical to their respective spectra when bound to B-DNA, suggesting that those drugs bind to the (iG, iC)-containing PS-duplex using similar intercalation processes. The results may be useful in the design of intercalator-conjugated oligonucleotides for antisense applications. The study presented in this paper augments our understanding of a growing number of parallel- stranded DNA structures, including the G-quartet, the i-motif, and the unusual homo basepaired parallel-stranded double helix. Their possible relevance is discussed. INTRODUCTION The right-handed B-DNA is presumed to be the predomi- conformations. The telomere sequence (TTGGGG)n can nant conformation of DNA in vivo. However, other alter- adopt either parallel or anti-parallel quadruplex structures native stable conformations are also known. A-DNA, an- using novel G-G basepairs (Kang et al., 1992; Smith and other right-handed duplex conformation, is favored by a Feigon, 1992; Laughlan et al., 1994; Phillips et al., 1997). low-humidity environment. A more dramatic departure Under low pH condition, (C)n, part of the complementary from A- and B-form DNA is the left-handed Z-DNA, which sequence of (TTGGGG)n, adopts a novel parallel quadru- is favored by stretches of alternating purine-pyrimidine se- plex structure (called i-motif) with the cytosine bases inter- quences like (CG)n or (TG)n (Wang et al., 1979; Rich et al., calated with one another from the neighboring strands (Ge- 1984; Palecek, 1991). All three major duplex structures hring et al., 1993; Chen et al., 1994). consist of two anti-parallel stranded sugar-phosphate back- Therefore, it is clear that DNA adopts different confor- bones with Watson-Crick basepairs. mations using not only novel basepairs, but also different Interestingly, some repetitive sequences like (TT- chain polarities. Among the parallel DNA structures men- GGGG) , which is found in certain eukaryotic telomere n tioned above, all involve more than two strands of DNA. DNA (Blackburn, 1991, 1992; Sen and Gilbert, 1992); The question of whether a stable parallel DNA duplex exists (GGAAT) , which is found in centromere DNA (Grady et n has been addressed previously by Jovin and colleagues al., 1992); and (CCG) , which is found in the X-chromo- n (reviewed in Rippe and Jovin, 1992). A series of A, T- some (responsible for the fragile-X syndrome) (Kremer et containing DNA sequences was designed to form parallel- al., 1991; Oberle et al., 1991); have been implicated in various biological functions and are associated with specific stranded (PS) duplexes using the reversed Watson-Crick basepairs. The stability of those PS-duplexes is modest; for example, the Tm of a 21-mer PS-duplex is 15°C lower than that of the corresponding antiparallel duplex (Ramsing and Received for publication 10 April 1998 and in final form 2 June 1998. Jovin, 1988). Address reprint requests to Dr. Andrew Wang, Department of Cell and An important requirement for a PS-duplex is that the two Structural Biology, University of Illinois at Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-244-6637; Fax: glycosyl bonds within a basepair have to come from the 217-244-3181; E-mail: [email protected]. opposite direction because of the identical chain polarity. © 1998 by the Biophysical Society For the normal nucleic acid bases, this can be accomplished 0006-3495/98/09/1163/09 $2.00 using the reverse Watson-Crick basepair conformation. 1164 Biophysical Journal Volume 75 September 1998 However, A-T and G-C basepairs in a reverse Watson-Crick duced during the oxidative damage process is less than that conformation are not isostructural due to their hydrogen of 8-oxoguanine, it is still a significant product. It is known bonding restrictions. Therefore, it has not been easy to that both 2-hydroxyadenine (i.e., iG) and 8-oxoguanine are design a stable PS-duplex in which all four bases can be mutagens. The repair mechanism of the lesions involving incorporated in random order. these two oxidized bases is under intense study (Tchou and This problem has been overcome by using alternative Grollman, 1993; Kamiya and Kasai, 1996, 1997). Whereas nucleosides, 2Ј-deoxyisoguanosine (iG) and 2Ј-deoxy-5- the structural consequences due to the 8-oxoguanine incor- methyl-isocytosine (iC), which can form stable reverse poration have been studied extensively (Grollman et al., Watson-Crick basepairs with the normal 2Ј-deoxycytosine 1994; Lipscomb et al., 1995), little is known regarding the (C), and 2Ј-deoxyguanosine (G), respectively (Fig. 1). It structural effective of 2-hydroxyadenine (i.e., iG) in DNA. was noted by Seela and colleagues that iG is a potent Therefore it is desirable to gain insight to the properties inducer for the formation of parallel DNA duplex structure associated with the modified base 2-hydroxyadenine (Seela et al., 1996; Seela and Wei, 1997). Significantly, it (i.e., iG). has been shown by some of us that oligodeoxynucleotides Finally, the ability for (iG, iC)-containing DNA oli- containing iG and iC can form remarkably stable parallel- gomers to form specific stable parallel-stranded duplexes stranded duplexes with the complementary (G, C)-contain- may offer new opportunities for designing useful probes for ing DNA or RNA strands. The Tm of the PS-duplex is applications such as antisense or aptamer molecules. To comparable with that of the corresponding antiparallel du- date, only limited structural work on PS-duplexes has been plex even for a 10-mer (Sugiyama et al., 1996). More carried out (Germann et al., 1989; Zhou et al., 1993). Our recently, DNA molecules containing a string of iGs have understanding of the PS-duplexes remains mostly at the been demonstrated to form very stable parallel tetraplex level of stability from Tm measurements (Rippe and Jovin, structure (Seela et al., 1996; Roberts et al., 1997). 1992). Whether they have sequence-dependent conforma- Parenthetically, it may be useful to note that iG is a tions, as in B-DNA, or how they interact with other ligands product of oxidative damage to the adenine base in DNA. (proteins or small molecules) remains largely unexplored. The addition of a hydroxyl group at the C2-position of In this paper, we describe the structural analysis of the adenine resulting from the oxidative damage process pro- parallel-stranded DNA duplex 5Ј-d(TiGiCAiCiGiGAiCT) duces 2-hydroxyadenine, which is chemically identical to ϩ 5Ј-d(ACGTGCCTGA) by NMR refinement. In addition, isoguanine. While the amount of 2-hydroxyadenine pro- the flexibility of the backbone of the (iG, iC)-containing PS-duplex has been probed using several intercalators with different complexities, including ethidium, daunorubicin, and nogalamycin. MATERIALS AND METHODS Phosphoramidite derivatives of 5-methylisocytosine and isoguanine, 5Ј-O- DMT-N2-[(di-n-butylamino)methylene]-5-methyl-2Ј-deoxyisocytidine ␤-cyanoethyl-phosphoramidite and 5Ј-O-DMT-O2-(N,N-diphenylcar- bamoyl)-N6-[(di-n-butylamino)methylene]-2Ј-deoxyisoguanosine ␤-cya- noethyl-phosphoramidite were prepared as described previously (Sug- iyama et al., 1996). Calf intestine alkaline phosphatase (AP) (1000 U/␮l) and snake venom phosphodiesterase (s.v.PDE) (3 U/␮l) were purchased from Boehringer Mannheim (Tokyo). Deoxyoligonucleotides were pre- pared by the ␤-cyanoethyl phosphoramidite method on controlled pore glass supports (1 ␮mol) by ABI381A DNA synthesizer using normal protocol except for an extended coupling time of 5 min. After automated synthesis, oligonucleotides were cleaved from the supports and deprotected by heating the concentrated ammonia solutions at 50°C for 12 h. Oligo- nucleotides were purified by reverse phase HPLC using a CHEMCO- BOND 5-ODS-H column (10 mm ϫ 150 mm) with a flow rate of 3.0 ml/min and a linear gradient of acetonitrile in 50 mM ammonium formate.
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