Insight into achirality and chirality effects in interactions of an racemic ruthenium(II) polypyridyl complex and its Δ- and Λ-enantiomers with an RNA triplex

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Abstract

RNA triplexes have a variety of potential applications in molecular biology, diagnostics and therapeutics, while low stabilization of the third strand hinders their practical utilities under physiological conditions. In this regard, achieving the third-strand stabilization by binding small molecules is a promising strategy. Chirality is one of the basic properties of nature. To clarify achirality and chirality effects on the binding and stabilizing effects of RNA triplexes by small molecules, we report for the first time the RNA interactions of an racemic ruthenium(II) polypyridyl complex [Ru(bpy)2(11-CN-dppz)]2+ (rac-Ru1) and its two enantiomers Δ/Λ-[Ru(bpy)2(11-CN-dppz)]2+ (Δ/Λ-Ru1) with an RNA triplex poly(U-A*U) (where “-” represents Watson-Crick base pairing, and “*” denotes Hoogsteen base pairing, respectively) in this work. Research shows that although rac-Ru1 and its two enantiomers Δ/Λ-Ru1 bind to the RNA triplex through the same mode of intercalation, the binding affinity for enantiomer Δ-Ru1 is much higher than that for rac-Ru1 and enantiomer Λ-Ru1. However, compared to enantiomer Λ-Ru1, the binding affinity for rac-Ru1 does not show much of an advantage, which is slightly greater than that for the former. Thermal denaturation measurements reveal both rac-Ru1 and Δ-Ru1 to have a preference for stabilizing the third strand rather than the template duplex of the RNA triplex, while Λ-Ru1 stabilizes the RNA triplex without significant selectivity. Besides, the third-strand stabilizing effects by rac-Ru1 and Δ-Ru1 are not markedly different from each other, but more marked than that by Λ-Ru1. This work shows that the binding properties of the racemic Ru(II) polypyridyl complex with the RNA triplex are not simply an average of its two enantiomers, indicating potentially complicated binding events.

Introduction

RNA triplexes are important tertiary structure motifs in RNA [1], which have important roles in many biological processes, such as antigen strategy, post-transcriptional RNA processing and gene regulation [2]. However, stabilization of the Hoogsteen base-paired strand (the third strand) is much weaker than that of the Watson-Crick base-paired duplex (the template duplex) of an RNA triplex due to unfavorable charge repulsion between the three negatively charged polyanionic strands [3], which hinders their practical utilities under physiological conditions [4]. In order for the complicated structures to function steadily in biological processes, it is necessary to stabilize third-strand of an RNA triplex [5]. Combining with the RNA triplexes is one of the effective methods to alter the stability of the third-strand of the triplexes. Whereas, it is surprised to find that, in comparison with RNA triplexes, investigations of the stabilization of DNA triplexes by small molecules binding at present are well established. Previous studies have shown that small molecules as intercalating agents can both stabilize and destabilize RNA triplexes [6]. For example, the study of flavonoids has shown that it stabilizes strongly the third-strand of the RNA triplex by intercalation binding [7]. Similarly, the binding of berberine-analogs to RNA triplex poly(U-A*U) show high binding affinity, and the melting temperature of the third-strand in the RNA triplexes are significantly increased [8], whereas ethidium, proxanthin and the platinum(II) proxanthin complexes tend to destabilize the triplex [9].

While most of these studies are limited to organic small molecules and much lesser on metal complexes, while such attention in recent years has been paid to the design of the configuration of the inert octahedron transition metal complexes [10]. Many researchers have designed and synthesized transition metal complexes with d6\d8\d10 electronic structure [11]. By changing the structure of the ligand, the binding stability and recognition of the ligand with the triplexes can also be changed, and most of the complexes which have favourable photophysical or photochemical activity have great practical applications in detection [12]. For instance, complexes [Ru(bpy)2(7-CH3-dppz)]2+ (bpy = 2,2′-bipyridine, 7-CH3-dppz = 7-methyldipyrido[3,2-a:2′,3′-c]- phenazine) [13] and [Ru(bpy)2(dppz)]2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) [14] not only stabilize RNA triplex but also embody the characteristic of the molecular “light switch” by intercalative binding. Moreover, for [Ru(phen)2(7-F-dppz)]2+ (phen = 1,10-phenanthrolline, 7-F-dppz = 7-fluorodipyrido[3,2-a:2′,3′-c]phenazine) [15] or [Ru(phen)2ttbd]2+ (ttbd = 4-(6-propenyl-pyrido[3,2-a]phenzain-10-yl-benzene-1,2-diamine) [16], both of which are conducive to stabilize the Hoogsteen base-paired third-strand poly(U) and serve as pH-controlled reversible visual light-switches for triplex.

The enantioselective binding between chiral transition metal complexes and DNA have aroused intense interest in recent years [17]. In particular, the strong visible absorbance and fluorescence emission of chiral ruthenium(II) polypyridyl complexes provide a simple method for detecting the nucleic acid binding processes, and it is possible to distinguish between left- and right-handed DNA [18]. In addition, an RNA triplex is itself a chiral molecule, being a right-handed helical structure in its common A-form [1]. Surprisingly, before we began to study the interaction of chiral small molecules with RNA triplexes [14], [19], [20], [21], [22], there was no research in this area. Our recent studies have preliminarily demonstrated that the chiral properties of Ru(II) complexes have a significant role in the binding and stabilization of RNA triplexes. For example, for complex [Ru(bpy)2(dppz)]2+, its Δ-enantiomer stabilized the template duplex and the third strand of poly(U-A*U) with no obvious selectivity, while its Λ-enantiomer preferred to stabilize the template duplex [14]. However, for complex [Ru(bpy)2(6-F-dppz)]2+ [22], its two Δ/Λ-enantiomers stabilized the template duplex of triplex poly(U-A*U) more effectively than the third strand, indicating that the two enantiomers preferred to bind with the template duplex rather than the third strand to some extent. Therefore, for racemic small molecules, in addition to their own interactions with RNA triplexes, the binding of their enantiomers with RNA triplexes should also considered.

To competitively determine chiral and achiral effects on the third-strand stabilization by small molecules, two new chiral Ru(II) complexes Λ-[Ru(bpy)2(11-CN-dppz)]2+ (Λ-Ru1; 11-CN-dppz = 11-cyanodipyrido-[3,2-a,2′,3′-c]-phenazine) and Δ-[Ru(bpy)2(11-CN-dppz)]2+ (Δ-Ru1) as well as their corresponding racemic complex rac-[Ru(bpy)2(11-CN-dppz)]2+ (rac-Ru1) (Fig. 1) have been synthesized and characterized in this work. Binding properties of the three complexes to the RNA triplex poly(U-A*U) have been studied by various biophysical methods. To our knowledge, this work is the first example of competitive insight into chirality and achirality effects in intercalations of an racemic compound and its enantiomers with an RNA triplex.

Section snippets

Materials

All commercially available chemicals were used directly without additional purification. Compounds 1,10-phenanthroline-5,6-dione [23], cis-[Ru(bpy)2Cl2]·2H2O [24], cis-[Ru(bpy)2(py)2]Cl2 and Λ/Δ-[Ru(bpy)2(py)2][O,O′-dibenzoyl-L/D-tartrate]·12H2O (py = pyridine) [25], [26] were prepared and characterized according to the literatures. Polynucleotide samples of single-stranded poly(U) and double-stranded poly(A-U) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) and were used as

Synthesis and characterization

The synthetic routes of ligand 11-CN-dppz and its three complexes rac-Ru1, Λ-Ru1 and Δ-Ru1 are shown in Scheme S1. According the methods established by Dickeson and Summers [25], ligand 11-CN-dppz was prepared by condensation reaction using 1,10-phenanthroline-5,6-dione and 3,4-diaminobenzonitrile in the presence of ethanol, giving the ligand with high yield. Complex rac-Ru1 was synthesized by treating ligand 11-CN-dppz with the equal molar quantities of precursor cis-[Ru(bpy)2Cl2]·2H2O in

Conclusions

In summary, in the present study the comparative studies of the racemic ruthenium(II) polypyridyl complex [Ru(bpy)2(11-CN-dppz)]2+ (rac-Ru1) and its two enantiomers Λ-Ru1 and Δ-Ru1 with the RNA triplex poly(U-A*U) have been studied by various biophysical methods. The obtained results suggest that the binding interactions of the racemic complex [Ru(bpy)2(11-CN-dppz)]2+ with the RNA triplex poly(U-A*U) are not simply an average of its two enantiomers, reflecting potentially complicated binding

CRediT authorship contribution statement

Hui Wang: Validation.: Formal analysis.: Investigation.: Data curation, Writing- Original draft preparation.: Writing - Review & Editing Xiaohua Liu: Resources Lifeng Tan: formulation or evolution of overarching research goals and aims.: Writing - Review & Editing.: Supervision.: Project administration.: Funding acquisition.

Uncited reference

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Declaration of competing interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Acknowledgements

We would like to thank the National Natural Science Foundation of China (21671165) for financial supports.

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