Thermodynamics of the formation of Ag(I)-mediated azole base pairs in DNA duplexes

https://doi.org/10.1016/j.jinorgbio.2016.03.003Get rights and content

Highlights

  • The thermodynamics of Ag(I)-mediated base pair formation were probed by calorimetry.

  • Imidazole–Ag(I)–imidazole base pairs are of superior stability.

  • Cooperative formation of metal-mediated base pairs may take place.

  • The Ag(I)-binding affinity of single imidazole:imidazole pairs is not sequence-dependent.

Abstract

Isothermal titration calorimetry was applied to determine the thermodynamic parameters for the specific binding of Ag(I) ions to a series of DNA duplexes comprising Im:Im or Tr:Tr mispairs to form metal-mediated Im–Ag(I)–Im or Tr–Ag(I)–Tr base pairs (Im = imidazole nucleoside; Tr = 1.2,4-triazole nucleoside). A total of seven different duplexes are discussed, incorporating one to three artificial base pairs in neighboring or non-neighboring positions. The association constant related to the formation of Tr–Ag(I)–Tr base pairs is estimated to be < 103 M 1. In contrast, Im–Ag(I)–Im base pairs are much more stable. The intrinsic association constant for their formation is in the order of 106 M 1 and is therefore larger than that for the formation of T–Hg(II)–T and C–Ag(I)–C base pairs consisting of natural nucleobases. Two neighboring Im–Ag(I)–Im base pairs form cooperatively, whereas two remotely located Im–Ag(I)–Im base pairs form non-cooperatively. In general, the specific binding of Ag(I) to Im:Im-containing duplexes is enthalpically driven, with a significant additional entropic contribution in most cases.

Graphical abstract

The specific binding of Ag(I) ions to DNA duplexes containing imidazole:imidazole or triazole:triazole mispairs in various sequence contexts was probed by isothermal titration calorimetry, yielding a quantitative insight into the thermodynamics of the formation of these artificial Ag(I)-mediated base pairs.

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Introduction

It is well-established that the structural integrity of nucleic acid tertiary structures is strongly affected by the presence of biologically relevant metal ions [1]. In addition, nucleic acids can be modified in a way that they bind transition metal ions in a site-specific manner. The resulting metal-modified nucleic acids are highly relevant in the generation of self-assembling function-bearing objects on the nanoscale [2]. One prominent method for the site-specific introduction of metal ions into nucleic acids is the use of metal-mediated base pairs [3], [4], [5], [6]. In these artificial base pairs, the complementary nucleobases are held together via coordinate bonds to a central metal ion rather than via hydrogen bonds. While a few metal-mediated base pairs are known with natural nucleobases (Fig. 1a, b) [7], most base pairs of this type reported to date comprise artificial nucleosides. These include imidazole (Fig. 1c) [8], [9], [10], [11], 1,2,4-triazole (Fig. 1d) [12], [13], hydroxypyridone [14], [15], and salen [14], [16], [17], to name just a few. More recently, dinuclear Ag(I)-mediated base pairs have been reported, too [18], [19], [20], [21], [22], [23]. The use of metal-mediated base pairs can be easily extended to parallel-stranded DNA [24], [25] and to other nucleic acids and nucleic acid derivatives such as RNA [26], GNA (glycol nucleic acid) [27], and PNA (peptide nucleic acid) [28], [29]. The introduction of metal-mediated base pairs opens several opportunities for fascinating applications, including enhanced charge-transfer through DNA [30], expansion of the genetic code [31], discrimination of the natural nucleobases for a potential specific recognition of biologically relevant short RNAs [32], [33], [34], and numerous sensing applications [35].

To understand better the formation of metal-mediated base pairs, several crystal and solution structures have been reported [9], [10], [31], [36], [37], [38]. Moreover, the thermodynamics of the formation of the metal-mediated T–Hg(II)–T and C–Ag(I)–C base pairs, i.e. those based on the canonical pyrimidine nucleobases, have been thoroughly investigated. For T–Hg(II)–T, association constants Ka for the specific binding of Hg(II) to a single T:T mismatch within a DNA duplex were determined to be in the order of 5 · 105 M 1[39]. Moreover, cooperative formation of two T–Hg(II)–T base pairs was reported, irrespective of whether the underlying T:T mispairs are directly adjacent to one another or whether they are separated by one canonical A:T base pair [40]. The Ka for the binding of the second Hg(II) was reported to be about 7–15 times larger than the Ka of the first binding event [40]. Similarly, the association constant for the specific binding of Ag(I) to a C:C mismatch to yield a C–Ag(I)–C base pair was reported to be in the order of 4 · 105 M 1[41].

In contrast, much less is known on the thermodynamics of the formation of metal-mediated base pairs involving artificial nucleobases. In fact, the sole study in this respect is our recent communication on the cooperative formation of adjacent Im–Ag(I)–Im base pairs [11]. The Ka for the specific binding of Ag(I) to one Im:Im mispair within a DNA duplex is in the order of 3 · 106 M 1. In the case of two adjacent Im:Im mispairs, the Ka of the second binding event is about 25-fold larger than that of the first one. Hence, cooperative formation of neighboring Im–Ag(I)–Im base pairs is evident.

In this study, we extend that initial investigation of the thermodynamics of the formation of the Im–Ag(I)–Im base pair to other sequence contexts. In particular, we were interested whether cooperativity of the base pair formation can be observed also for duplexes in which two metal-mediated base pairs are separated from each other by several natural base pairs. Moreover, as 1,2,4-triazole (Tr) has previously been reported as another artificial nucleobase for metal-mediated base pairing [12], [42], this study also aimed at the first determination of the thermodynamic parameters of the formation of Tr–Ag(I)–Tr base pairs within DNA duplexes.

Section snippets

Synthetic procedures

The phosphoramidites of the artificial imidazole and 1,2,4-triazole nucleosides were prepared according to published procedures [10], [12]. All other phosphoramidites were purchased from Glen Research. The oligonucleotides were synthesized and purified as described previously [19]. The desalted oligonucleotides were characterized by MALDI-TOF mass spectrometry (ODN1Tr: calcd. for [M + H]+: 7887 Da; found: 7879 Da; ODN2Tr: calcd. for [M + H]+: 7916 Da; found: 7910 Da; ODN3Tr: calcd. for [M + H]+: 7829 Da;

DNA sequences under investigation

To investigate in detail the thermodynamics of the formation of Ag(I)-mediated azole base pairs within a DNA double helix, several slightly different 26mer duplexes were investigated. Duplex I comprises one central Im:Im (IIm) or Tr:Tr (ITr) mispair. Duplexes II and III contain two artificial azole mispairs. In the case of II, these are directly next to one another, whereas they are separated by seven canonical base pairs in duplex III. Duplex IVIm holds three neighboring Im:Im mispairs.

Summary and conclusions

This comprehensive study on the thermodynamics of the specific binding of Ag(I) to a variety of DNA oligonucleotide duplexes with Im:Im or Tr:Tr mispairs to form Im–Ag(I)–Im or Tr–Ag(I)–Tr metal-mediated base pairs had led to several fascinating insights. While the association constant Ka for the formation of a Tr–Ag(I)–Tr base pair is comparatively low, with an estimated upper limit of 103 M 1, the association constant for the formation of an Im–Ag(I)–Im base pair is in the order of at least 106

Abbreviations

    A

    adenine

    C

    cytosine

    G

    guanine

    GNA

    glycol nucleic acid

    ITC

    isothermal titration calorimetry

    PNA

    peptide nucleic acid

    RT

    room temperature

    T

    thymine

Acknowledgements

Financial support from the Deutsche Forschungsgemeinschaft (SFB 858, GRK 2027) is gratefully acknowledged.

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    Dedicated to Professor Bernhard Lippert on the occasion of his 70th birthday.

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