J. Med. Chem. 2005, 48, 3471-3473
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Oxazine 170 Induces DNA:RNA:DNA Triplex Formation Guangtao Song,† Feifei Xing,† Xiaogang Qu,† Jonathan B. Chaires,§ and Jinsong Ren*,† Key Laboratory of Rare Earth Chemistry and Physics, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin 130022, China, and James Graham Brown Cancer Center, University of Louisville, 529 S. Jackson Street, Louisville, Kentucky 40202 Received February 11, 2005 Abstract: The oxazine dye (Oxazine 170) was found to induce formation of a hybrid triplex structure, poly rA:(poly dT)2, under solution conditions in which the triplex would not otherwise form. Formation of the hybrid triplex is driven by the structural selective binding of oxazine 170 to poly rA:(poly dT)2. Oxazine 170 serves as a lead compound for the design of new compounds that can modulate triplex formation.
Nucleic acid triple helices consisting of both DNA and RNA strands have attracted considerable attention because of their potential as tools in molecular biology as well as possible therapeutic agents.1 Stable DNA: RNA triplexes play key roles in many biological processes, including inhibition of RNAse, DNAse I, and RNA polymerase.2 Triplexes are, however, thermodynamically less stable than the corresponding duplexes, and only six of the eight possible hybrid (DNA:RNA: DNA) triplexes are stable under physiological conditions.3-5 The poor stability of these structures limits their use under physiological conditions. Ligands that stabilize otherwise unfavorable hybrid triplexes6-8 and which have a high discrimination between triplex and duplex open new avenues for the design of antisense, antigene, antiviral, and diagnostic agents. Much effort is under way to use small molecules to modulate the properties of triplex structures.9 Most of these studies, however, only assessed the impact of ligand binding on the properties of preformed triplexes. Only a few ligands have been shown to induce formation of hybrid triplex that would otherwise not form.6-8 The poly(rA)‚2poly(dT) triplex (Figure 1a), for example, forms only under extreme solution conditions (2.5 M Na+).10 Five ligands (berenil, DAPI, ethidium, netropsin,6,7 and neomycin8) were identified that facilitate the formation of the otherwise unstable poly(rA)‚2poly(dT) triplex. Such ligand-induced triplex formation is a kind of allostery driven by the preferential binding of the ligand to the triple helical form. The Hud laboratory recently demonstrated the remarkable ability of coralyne to disproportionate poly dA-poly dT into a triplex and a coralyne-poly dA complex.11 The disproportionation is driven by selective binding of coralyne to triplex and to poly dA, which we reported in our first description of the competition dialysis method.12 * To whom correspondence should be addressed. Phone: 86-4315262625. Fax: 86-431-5262656. E-mail:
[email protected]. † Chinese Academy of Sciences. § University of Louisville.
Figure 1. Structures of a dT-rA-dT triplet (a) and oxazine 170 (b).
Oxazine 170 (5,9-bis(diethylamino)-10-methylbenzo[R]phenoxazonium perchlorate) (Figure 1b) is a small crescent-shaped cationic molecule with four fused aromatic rings that mimics the shape of a base triplet and which also possesses the potential pharmacophore13 that is contained within compounds that are selective for DNA‚RNA hybrid structure. Its analog, Cresyl violet has been shown to stabilize triplex DNA.14 The nucleic acid binding properties of oxazine 170 have not been studied. We report here the remarkable ability of oxazine 170 to induce the formation of the poly(rA)‚2poly(dT) triplex, a consequence of its highly selective binding to triplex structures. The method of continuous variations15 was used to demonstrate the formation of a hybrid triplex only in the presence of oxazine 170. Inspection of the mixing curves in Figure 2A reveals that, in the absence of ligand, no inflection point is observed when duplex polyrA:poly dT is mixed with poly dT. The polyrA‚2polydT triplex does not form under the solution conditions employed (200 mM Na+, pH7.0). In the presence of oxazine 170, an inflection is clearly observed at 50% poly dT, indicating the formation of the polyrA‚2polyT triplex. Circular dichroism was used to characterize the interaction further. Figure 2B shows a strong induced CD spectrum for the ligand only in the presence of an equimolar mixture of duplex poly rA:poly dT and poly dT. Oxazine alone has no apparent CD signal over the wavelength range 220-800 nm, and there are only negligible CD bands evident in the presence of duplex poly rA:poly dT or single-stranded poly dT. These results strongly support the notion that oxazine 170 binds selectively to the hybrid triplex structure. The CD spectrum of polyrA‚2polydT-oxazine complex is distinctly different from the composite spectrum computed as the sum of the poly rA‚poly dToxazine and the poly dT-oxazine spectra (see Supporting Information). The ligand-induced formation of the hybrid triplex and the preferential binding of oxazine to the polyrA‚ 2polydT triplex was confirmed by thermal denaturation studies (Figure 3A). Without any ligand, only one transition that corresponds to the denaturation of the polyrA‚polydT duplex is evident.10,13,16 Upon addition of increasing molar ratios of oxazine, two transitions are evident in melting profiles. Under these ionic conditions, the Tm for the triplex transition is systematically increased from 25 °C up to a maximal shift of 50 °C. In marked contrast, melting of the duplex is largely unaffected. The magnitude of the Tm shift for the triplex, along with the lack of any shift for the duplex (Figure 3B), suggest that the triplex selectivity of oxazine 170
10.1021/jm050131g CCC: $30.25 © 2005 American Chemical Society Published on Web 04/19/2005
3472 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Figure 2. (A) Absorption mixing curves of polyrA:polydT and polydT in the absence and presence of oxazine (at a [total oxazine] to [nucleotide] ratio of 0.2) at 20 °C. A minimum at 50% of polyrA:polydT corresponds to the formation of the triplex polyrA:2polydT. (B) CD spectra of oxazine 170 (15 µM) in the presence of (75 µM) poly dT (O), poly rA: poly dT (4), or poly rA:2poly dT (b).
is much more stringent than that of other triplex stabilizer such as berenil and neomycin.6,8 Previous studies were unable to quantitatively evaluate the relative binding affinities and structural selectivity for different nucleic acids for those ligands that induced triplex formation because of the instability of the triplex form in the absence of bound ligands.3,4 Both berenil6 and neomycin8 were reported to be effective polyrA‚2polydT triplex stabilizers, using thermal denaturation methods. The interactions of berenil and neomycin with other structural forms were not studied in depth. We developed a competition dialysis method for rapidly screening the structural and sequence selectivity of ligand-nucleic acids interaction.12,13,16,17 In the assay, a test ligand of interest is dialyzed against an array of nucleic acid structures. At equilibrium, all structures are in contact with same free ligand solution, and the amount bound to each structure directly and quantitatively indicates the relative ligand binding affinity. Figure 4 shows the results of the competition dialysis assay using oxazine 170. Table S1 (Supporting Information) lists the 20 nucleic acid structures used in this study. Data are shown as a bar graph, in which the amount of oxazine dye bound to each nucleic acid sample is plotted. There is a wealth of information in Figure 4. Oxazine 170 binds preferentially to triplex DNA and G-tetraplex DNA, with poly rA‚2poly dT representing the next most preferred form. It shows less affinity or no binding to other nucleic acid structures. The most
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Figure 3. (A) UV melting curves of solution containing a 1:1 stoichiometric mixture of polydT 20 µM and polyrA:polydT 20µM in the presence of increasing molar ratios of added oxazine 170. (B) Plot of ∆Tm (the difference in the apparent Tm in the presence of oxazine relative to the melting of polyA: 2polydT). Filled circles are for the transition for dissociation of the third strand. ∆Tm3-2 (triplexfduplex+polydT) is calculated by assuming a Tm3-2 of 10 °C in the absence of oxazine 170 (no transition seen). Open circles are for the duplex melting transition.
Figure 4. Results of competition dialysis experiment using oxazine. The amount of oxazine bound to the various nucleic acid structures listed in the table S1 is shown as a bar graph. Full experimental details are provided as Supporting Information and in ref 16.
striking result to emerge is the strong preference of oxazine 170 for the poly rA‚2poly dT hybrid triplex over its duplex form. Since oxazine 170 shows no binding to poly dT and only weak binding to the hybrid duplex poly rA-poly dT, its apparent preferential binding to the poly rA-2poly dT mixture must arise from an induced structural change. Circular dichroism was used to confirm that the oxazine 170 in the dialysis experiment showed an induced CD spectrum identical to that shown in Figure 2B (Supporting Information). Binding to the polyrA‚2polydT triplex is nearly 3-fold greater than that to hybrid duplex and the mixture of 2polyrA + polydT that could form a DNA:RNA:RNA
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triplex. The lesser affinity toward the 2polyrA + polydT mixture is consistent with the inability of poly rA to bind to the polyrA‚polydT duplex in the presence of ligands. The apparent binding to 2polyrA + polydT mixture is the result of binding to duplex form. From the data obtained in Figure 4, apparent binding constants (Kapp) of 6.0, 2.1, and 2.2 × 104 M-1 may be calculated for the binding of oxazine to the poly rA‚2 poly dT triplex, to poly rA‚poly dT duplex, and to calf thymus DNA, respectively. To validate the Kapp values, a fluorescence titration study was done for the interaction of oxazine 170 with calf thymus. (Supporting Information). The measured binding constant of 2.6 × 104 M-1 is in excellent agreement with the competition dialysis data. Ligand-induced formation of new classes of triplexes potentially has important implications in the recognition of RNA‚DNA hybrid duplexes by cellular and viral proteins.6,7 The results of the experiment described here identify a small molecule with the ability to induce hybrid triplex formation. Oxazine 170 can serve as a lead compound for the design and synthesis of new types of triplex selective binders. We cannot yet specify the structural basis for the molecular recognition of hybrid triplex by the compound discovered here. The strand composition of the third strand and the target duplex, the mode of ligand binding, and the structural/conformational nature of the ligand-nucleic acids complex may all influence ligandinduced formation and stabilization of triplex. Further investigations of these points are under way in our lab. Acknowledgment. Supported by Funds for Distinguished Young Scholars of China and of Jilin province (20325101, 20225102), the Distinguished Talent Program from the Chinese Academy of Sciences, National Natural Science Foundation of China for key program (20331020), and National Cancer Institute Grant CA35635 (to J.B.C.) Supporting Information Available: Experimental details, table of structures used in the studies shown in Figure 4, figures showing UV and CD spectra, results of binding isotherm for the interaction of oxazine with CT DNA. This material is available free of charge via the Internet at http:// pubs.acs.org.
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