Binding Differences of Two Homochiral [Ru(bpy)2dppz]2+ Complexes

Communication pubs.acs.org/IC

Binding Differences of Two Homochiral [Ru(bpy)2dppz]2+ Complexes with poly(U)·poly(A)*poly(U) Triplex RNA Meng-Na Peng,† Zhi-Yuan Zhu,† and Li-Feng Tan*,‡ †

Key Laboratory of Environment-friendly Chemistry and Application in Ministry of Education and ‡College of Chemistry, Xiangtan University, Xiangtan 411105, China S Supporting Information *

methylenedioxyphenyldipyrido[3,2-a:2′,3′-c]phenazine), [Ru(bpy)2uip]2+ (bpy = 2,2′-bipyridine; uip = 2-(5-uracil)-1Himidazo[4,5-f ][1,10]phenanthroline), and [Ru(phen)2uip]2+ has been reported by our group.4 [Ru(phen)2mdpz]2+ could not only stabilize the triplex but also display the characteristic of the molecular light switch via intercalative binding. Triplex stabilization by intercalating [Ru(phen)2uip]2+ was more marked, while the effect of [Ru(bpy)2uip]2+ was similar to that of berberine and palmatine,5 which stabilized the third strand with little effect on the template duplex. In addition, the interactions between the chiral transitionmetal complexes and nucleic acids in recent years have aroused intense interest because of the enantioselective binding properties.6 In particular, ruthenium(II) polypyridyl complexes containing planar aromatic ligands have many spectroscopic features, such as strong visible absorbance and strong fluorescence emission, which provide a convenient handle for monitoring the DNA binding process and have the potential to discriminate between right- and left-handed duplex DNA.7 Previous reports indicated that the Δ isomer of [Ru(bpy)2dppz]2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) binds more tightly than the Λ isomer to right-handed B-form DNA, resulting in the Δ isomer exhibiting a higher integrated luminescence intensity upon binding to DNA.8 However, the Λ and Δ enantiomers of [Ru(phen)2dppz]2+ are found to show luminescence in the presence of DNA to which they bound very strongly (K ≈ 108 M−1) but without noticeable enantioselectivity.9 Interestingly, dialysis experiments indicated that the Λ enantiomer of [Ru(bpy)2hpip]2+ (hpip = 2-(4-hydroxy-phenyl)imidazo[4,5-f ][1,10]phenanthroline) bound DNA more rapidly than the Δ enantiomer did because of their different intercalative binding geometries.10 Recently, the crystal structures of Λ[Ru(phen)2dppz]2+ and Δ-[Ru(bpy)2dppz]2+ as well as rac[Ru(phen)2dppz]2+ with oligonucleotide demonstrate that each enantiomer is intercalated from the minor groove of the duplex and there is some sequence specificity in the orientation of the dppz chromophore,11 while evidence for the minor groove binding of Δ-[Ru(phen)2dppz]2+ reflects that this enantiomer could bind in the minor groove of DNA with five different binding modes, which offers a new hypothesis for interpretation of the solution data.12 Although recent significant efforts have been made to gain insight into the structural properties of chiral metal complexes binding with DNA, to the best of our knowledge, however, none

ABSTRACT: The first investigation of chiral ruthenium(II) complexes Δ- and Λ-[Ru(bpy)2dppz]2+ and triplex RNA poly(U)·poly(A)*poly(U) was carried out, which showed that Δ enantiomer displayed significant ability in stabilizing model triplex RNA.

T

riplex RNA has created a resurgent interest because of its potential applications in molecular biology, diagnostics, and therapeutics.1 However, stabilization of the third strand is usually weaker than that of the template duplex because of the electrostatic repulsion between three polyanionic strands, which hinders its practical use under physiological conditions.1a Altering the third-strand stability by small-molecule binding could have potential biotechnological applications. Many efforts in recent years have found that a handful of groove binders with diverse structural traits are able to increase the third-strand stability.2 However, the present research is mainly focused on aminoglycosides and alkaloids, and there is currently little information concerning metal complexes altering the third-strand stabilization.3,4 Recently, stabilization of the poly(U)·poly(A)*poly(U) triplex (Figure 1, where · denotes the Watson−Crick base pairing and * denotes the Hoogsteen base pairing) by racemic ruthenium(II) complexes [Ru(phen)2mdpz]2+ (phen = 1,10-phenanthroline; mdpz = 7,7′-

Figure 1. Chemical structures of Δ-[Ru(bpy)2dppz]2+ (Δ-1), Λ[Ru(bpy)2dppz]2+ (Λ-1), and the base-pairing scheme in poly(U)· poly(A)*poly(U). © 2017 American Chemical Society

Received: March 16, 2017 Published: June 21, 2017 7312

DOI: 10.1021/acs.inorgchem.7b00670 Inorg. Chem. 2017, 56, 7312−7315

Communication

Inorganic Chemistry

We next investigated the two enantiomers’ effect on the triplex viscosity to further elucidate their binding modes. Figure 3 indicates that the viscosity of the triplex RNA solution increases upon complexation with Λ-1, while a different changing

were based on triplex RNA. Given that recent reports demonstrate that the “light-switch” complex [Ru(bpy)2dppz]2+ is able to thermodynamically destabilize noncanonical nucleic acid structures and its Λ and Δ enantiomers (Figure 1) must be considered as individual species when they interact with doublestranded DNA,13 we are intrigued to see how the two enantiomers would bind to the poly(U)·poly(A)*poly(U) triplex RNA, especially altering the triplex stabilization. Enantiomers of Λ-[Ru(bpy)2dppz]Cl2 (Λ-1) and Δ-[Ru(bpy)2dppz]Cl2 (Δ-1) were prepared and purified as previously described.11 Successful resolution and enantiomeric purity were established by circular dichroism (CD; Figure S1) and matrixassisted laser desorption ionization time-of-flight mass spectroscopy (Figure S2). Formation of the poly(U)·poly(A)*poly(U) triplex was prepared as reported earlier,4a and its concentration was determined optically using molar extinction coefficients, ε (M−1 cm−1), reported in the literature.14 To determine the binding affinities of Λ- and Δ-1 for the poly(U)·poly(A)*poly(U) triplex, a series of the triplex titrations were primarily carried out. Figure S3 shows the overall changes in the absorption spectra of Λ- and Δ-1, and the quantitative data are given in Table S1. Binding of each enantiomer to the triplex resulted in considerable hypochromism of the metal-to-ligand charge-transfer (443 nm for Λ-1 and 444 nm for Δ-1) transitions and the π−π* intraligand transitions (371 nm) of the intercalating ligands in the visible region of the ruthenium(II) species, while no obvious red shifts were observed here. Using these data, their intrinsic binding constants (Kb) were determined by the nonlinear fitting of the binding equation.15 Surprisingly, at all wavelength numbers, the binding constant for Δ-1 is significantly larger than that for Λ-1. A greater binding for Δ-1 reflected a stronger intermolecular interaction involving effective overlap of the π-electron clouds of Δ-1 with the base triplets,4b,5,16 which was further verified by spectrofluorimetric studies. Λ- and Δ-1 shows negligible luminescence in aqueous solutions (Figure 2), similar to rac-[Ru(bpy)2dppz]2+ without

Figure 3. Viscometric complex Λ- and Δ-1 titrations of poly(U)· poly(A)*poly(U) (153 μM) in buffer at 20 °C, where Ru stands for Λand Δ-1, respectively. The solution conditions are the same as those described in the legend of Figure 2.

trend is observed for Δ-1, which clearly shows that the two enantiomers may exhibit different binding behaviors. Upon the addition of Δ-1, the triplex RNA viscosities displayed initial decreases and subsequent increases. The initial decreases might be assigned to the conformational change of the triplex RNA induced by binding Δ-1.4a Such a conformational change arose from the kinking or bending of the triplex RNA at the metal complex binding site, thereby reducing its effective molecular length.19 Also, no matter what, the subsequent increases in the solution viscosities are indicative of intercalation of the ligand between the triplex RNA base pairs.19a Therefore, the binding modes of the two enantiomers with the triplex RNA are intercalations,5 but the binding sites might be different.4a Notably, the final change of the triplex RNA viscosity in the presence of Λ-1 is somewhat less than that of Δ-1. This might be an indication that the enhanced rigidity of the triplex RNA strand by Λ-1 is relatively smaller than that by Δ-1, leading to the triplex RNA being less sensitive to the structural perturbations arising from Λ-1 binding.4b The slightly smaller structural perturbations of the triplex RNA with Λ-1 are further verified by CD titrations (Figure S4). The CD spectrum of the triplex RNA without enantiomers (Figure S4, red lines) was characterized by a large positive band at about 266 nm and an adjacent weak negative band at about 240 nm followed by a small positive band at about 220 nm.20 The changes at 266 nm in the presence of Λ- and Δ-1 display no obvious differences, while the changes at the 285−320 nm range are clearly different. A new negative peak located at about 295 nm and a new positive peak at about 307 nm upon the binding of Λ-1 are observed. In the case of Δ-1, however, only a new negative peak located at about 303 nm exists but with significant higher intensity, suggesting that the binding sites of Λand Δ-1 binding the triplex RNA might be different. In addition, the CD spectra changes at the 285−320 nm range with significantly higher intensity in the presence of Δ-1 reveal that the triplex RNA structure perturbed by Δ-1 is more obvious than that by Λ-1. Thermal melting experiments were employed to determine triplex stabilization by Λ- and Δ-1, which also could detect the binding specificity.5,21 The melting profile of free poly(U)· poly(A)*poly(U) (Figure S5) is clearly biphasic, as is typically observed when this triplex is formed.4a,20 The lower melting temperature (Tm1; Table 1) at 37.0 °C represents the transformation of the triplex to a poly(U)·poly(A) duplex and a single strand of poly(U) by dissociation of the Hoogsteen base-

Figure 2. Representative fluorescence emission spectra of Λ-1 (A) and Δ-1 (B) treated with poly(U)·poly(A)*poly(U) in a phosphate buffer. [Λ-1] = [Δ-1] = 2.0 μM; for Λ- and Δ-1, [UAU] = 0−82.0 and 0−103.7 μM, respectively, where UAU stands for poly(U)·poly(A)*poly(U). The arrows show the intensity change upon increasing triplex concentration.

DNA.17 The addition of the triplex to each enantiomer resulted in a “light-switch” effect. Eventually, their luminescence enhanced about 21 and 32 times that of the initial intensity for Λ- and Δ-1, respectively, suggesting that Δ-1 binding with the triplex is more marked.4b,18 The result also indicated that the location of the bound Λ- and Δ-1 in a hydrophobic environment was similar to that of an intercalated state4,5 and Δ-1 was protected by the triplex more efficiently than Λ-1. 7313

DOI: 10.1021/acs.inorgchem.7b00670 Inorg. Chem. 2017, 56, 7312−7315

Communication

Inorganic Chemistry

(bpy)2dppz]2+ should be considered as individual species with their own distinct properties of stabilizing the third strand observed in the poly(U)·poly(A)*poly(U) case. Therefore, further systematic studies are very necessary.

Table 1. Melting Temperature (°C) for the poly(U)· poly(A)*poly(U) Triplex in the Absence and Presence of Λand Δ-1, Respectively compd UAU UAU + Λ-1

UAU + Δ-1

[Ru]/ [UAU]

Tm1 (°C)

Tm2 (°C)

0 0.10 0.20 0.30 0.10 0.20 0.30

37.0 36.5 37.0 38.0 43.9 44.9 44.5

46.0 49.9 52.9 54.9 50.9 54.6 58.0

ΔTm1 (°C) −0.5 0 1.0 6.9 7.9 7.5

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ΔTm2 (°C)

ASSOCIATED CONTENT

S Supporting Information *

3.9 6.9 8.9 4.9 8.6 12.0

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b00670. Equations used to calculate the binding constants, Table S1, and Figures S1−S5 (PDF)

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paired poly(U) strand from the major groove of the template duplex. The higher temperature (Tm2; Table 1) at 46.0 °C corresponds to the dissociation of the duplex, poly(U)·poly(A), into single strands.20 Binding of Λ- or Δ-1 resulted in Tm1 and Tm2 different changes (Figure 4 and Table 1). Tm2 increased to

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 731 58293997. ORCID

Li-Feng Tan: 0000-0002-8110-2281 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for this work was provided by the National Natural Science Foundation of China (Grants 21671165 and 21371146) and Hunan Provincial Natural Science Foundation of China (Grant 2016JJ2121).

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REFERENCES

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Figure 4. Melting curves at 260 nm of poly(U)·poly(A)*poly(U) (32.1 μM) and its complexation with Λ-1 (A) and Δ-1 (B) at different [Ru]/ [UAU] ratios. Solution conditions are the same as those described in the legend of Figure 2, and [Na+] = 35 mM.

54.9 °C upon the addition of Λ-1 at a mixing ratio of 0.3, while Tm1 increased to only 38.0 °C under the same condition, indicating that the intercalating Λ-1 preferred to stabilize the Watson−Crick base-pairing duplex strand rather than stabilize the third strand. However, in the presence of Δ-1 at the same mixing ratio of 0.3, Tm1 and Tm2 occurred at 44.5 and 58.0 °C, respectively, suggesting the effects of the intercalating Δ-1 stabilizing the triplex were more marked. This observation reflects that Δ-1 may stay bind to not only the Watson−Crick base-paired duplex but also the Hoogsteen paired poly(U) third strand, while Λ-1 may only intercalate between the Watson−Crick base-paired duplex. Thus, we suspect that rac-[Ru(bpy)2dppz]2+ is similar to a nonspecific metallointercalator for this triplex. Notably, the triplex stabilization effects of Λ- and Δ-1 clearly differed from those observed for ethidium and proflavine, where these binders exerted stabilization effects on the template duplex and definitely destabilized the third strand.3,22 Furthermore, the behaviors of the two enantiomers also differed from those of berberine, coralyne, and [Ru(bpy)2pip]2+,5,23 which only stabilized the third strand. These reveal that the effects of small molecules on triplex stabilization are very complicated and sensitive to their structural features. In conclusion, our observations suggest that the third-strand stabilizing effect of Δ-[Ru(bpy)2dppz]2+ obviously differs from that of Λ-[Ru(bpy)2dppz]2+ when they interact with the chiral environment of triple-helical poly(U)·poly(A)*poly(U). However, it remains to be seen whether the Λ- and Δ-[Ru7314

DOI: 10.1021/acs.inorgchem.7b00670 Inorg. Chem. 2017, 56, 7312−7315

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Inorganic Chemistry telomeric G-quadruplex DNA. Nucleic Acids Res. 2008, 36, 5695−5703. (c) Zhao, C. Q.; Wu, L.; Ren, J. S.; Xu, Y.; Qu, X. G. Targeting human telomeric higher-order DNA: dimeric G-quadruplex units serve as preferred binding site. J. Am. Chem. Soc. 2013, 135, 18786−18789. (d) Johnstone, T. C.; Lippard, S. J. The chiral potential of phenanthriplatin and its influence on guanine binding. J. Am. Chem. Soc. 2014, 136, 2126−2134. (7) (a) Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Neither Δ-nor Λ-tris(phenanthroline) ruthenium(II) binds to DNA by classical intercalation. Biochemistry 1992, 31, 9319−9324. (b) Gill, M. R.; Thomas, J. A. Ruthenium(II) polypyridyl complexes and DNAfrom structural probes to cellular imaging and therapeutics. Chem. Soc. Rev. 2012, 41, 3179−3192. (c) Komor, A. C.; Barton, J. K. The path for metal complexes to a DNA target. Chem. Commun. 2013, 49, 3617−3630. (8) Lim, M. H.; Song, H.; Olmon, E. D.; Dervan, E. E.; Barton, J. K. Sensitivity of [Ru(bpy)2dppz]2+ luminescence to DNA defects. Inorg. Chem. 2009, 48, 5392−5397. (9) Hiort, C.; Lincoln, P.; Norden, B. DNA Binding of Δ- and Λ[Ru(phen)2DPPZ]2+. J. Am. Chem. Soc. 1993, 115, 3448−3454. (10) Liu, J. G.; Ye, B. H.; Zhang, Q. L.; Zou, X. H.; Zhen, Q. X.; Tian, X.; Ji, L. N. Enantiomeric ruthenium(II) complexes binding to DNA: binding modes and enantioselectivity. JBIC, J. Biol. Inorg. Chem. 2000, 5, 119−128. (11) (a) Song, H.; Kaiser, J. T.; Barton, J. K. Crystal structure of Δ[Ru(bpy)2dppz]2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation. Nat. Chem. 2012, 4, 615−620. (b) Niyazi, H.; Hall, J. P.; O’sullivan, K.; Winter, G.; Sorensen, T.; Kelly, J. M.; Cardin, C. J. Crystal structures of Λ-[Ru(phen)2dppz]2+ with oligonucleotides containing TA/TA and AT/AT steps show two intercalation modes. Nat. Chem. 2012, 4, 621−628. (c) Hall, J. P.; Cook, D.; Morte, S. R.; McIntyre, P.; Buchner, K.; Beer, H.; Cardin, D. J.; Brazier, J. A.; Winter, G.; Kelly, J. M.; Cardin, C. J. X-ray crystal structure of rac-[Ru(phen)2dppz]2+ with d(ATGCAT)2 shows enantiomer orientations and water ordering. J. Am. Chem. Soc. 2013, 135, 12652− 12659. (12) Hall, J. P.; Keane, P. M.; Beer, H.; Buchner, K.; Winter, G.; Sorensen, T. L.; Cardin, D. J.; Brazier, J. A.; Cardin, C. J. Delta chirality ruthenium ″light-switch″ complexes can bind in the minor groove of DNA with five different binding modes. Nucleic Acids Res. 2016, 44, 9472−9482. (13) (a) McConnell, A. J.; Song, H.; Barton, J. K. Luminescence of [Ru(bpy)2(dppz)]2+ bound to RNA mismatches. Inorg. Chem. 2013, 52, 10131−10136. (b) McKinley, A. W.; Andersson, J.; Lincoln, P.; Tuite, E. M. DNA sequence and ancillary ligand modulate the biexponential emission decay of intercalated [Ru(L)2dppz]2+ enantiomers. Chem. Eur. J. 2012, 18, 15142−15150. (14) Ray, A.; Kumar, G. S.; Das, S.; Maiti, M. Spectroscopic studies on the interaction of aristololactam-β-D-glucoside with DNA and RNA double and triple helices: a comparative study. Biochemistry 1999, 38, 6239−6247. (15) Carter, M. T.; Rodriguez, M.; Bard, A. J. Voltammetric studies of the interaction of metal chelates with DNA. 2. tris-chelated complexes of cobalt(III) and iron(II) with 1, 10-Phenanthroline and 2, 2′-bipyridine. J. Am. Chem. Soc. 1989, 111, 8901−8911. (16) Fleisher, M. B.; Waterman, K. C.; Turro, N. J.; Barton, J. K. Lightinduced cleavage of DNA by metal complexes. Inorg. Chem. 1986, 25, 3549−3551. (17) Friedman, A. E.; Chambron, J. C.; Sauvage, J. P.; Turro, N. J.; Barton, J. K. Molecular ‘‘light switch” for DNA: Ru(bpy)2(dppz)2+. J. Am. Chem. Soc. 1990, 112, 4960−4962. (18) Zhang, H.; Liu, X. W.; He, X. J.; Liu, Y.; Tan, L. F. Experimental and density functional theory(DFT) studies on the interactions of Ru(II) polypyridyl complexes with the RAN triplex poly(U).poly (A)*poly(U). Metallomics 2014, 6, 2148−2156. (19) (a) Lerman, L. S. Structural considerations in the interaction of DNA and acridines. J. Mol. Biol. 1961, 3, 18−30. (b) Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Tris(phenanthroline) ruthenium(II) enantiomer interactions with DNA: mode and specificity of binding. Biochemistry 1993, 32, 2573−2584.

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DOI: 10.1021/acs.inorgchem.7b00670 Inorg. Chem. 2017, 56, 7312−7315