Elsevier

Biochimie

Volume 93, Issue 8, August 2011, Pages 1341-1350
Biochimie

Research paper
Interaction of minor groove ligands with G-quadruplexes: Thermodynamic contributions of the number of quartets, T–U substitutions, and conformation

https://doi.org/10.1016/j.biochi.2011.06.001Get rights and content

Abstract

In the presence of specific metal ions, DNA oligonucleotides containing guanine repeat sequences can adopt G-quadruplex structures. In this work, we used a combination of spectroscopic and calorimetric techniques to investigate the conformation and unfolding thermodynamics of the K+-form of five G-quadruplexes with sequences: d(G2T2G2TGTG2T2G2), G2, d(G3T2G3TGTG3T2G3), G3, their analogs where T is replaced with U, G2-U and G3-U, and r(G2U2G2UGUG2U2G2), rG2. These G-quadruplexes show CD spectra characteristic of the “chair” conformation (G2 and G2-U), or “basket” conformation (rG2); or a mixture of these two conformers (G3 and G3-U). Thermodynamic profiles show that the favorable folding of each G-quadruplex results from the typical compensation of a favorable enthalpy and unfavorable entropy contributions. G-quadruplex stability increase in the following order (in ΔG°20): rG2 (−1.3 kcal/mol) < G2 < G2-U <G3-U (chair) < G3 (chair) <G3-U (basket) < G3 (basket) (−8.6 kcal/mol), due to favorable enthalpy contribution from the stacking of G-quartets.

We used ITC to determine thermodynamic binding profiles for the interaction of the minor groove ligands, netropsin and distamycin, with each G-quadruplex. Both ligands bind with high exothermic enthalpies (∼−10.8 kcal/mol), 1:1 stoichiometries, and weak affinities (∼8 × 104 M−1). The similarity of the binding thermodynamic profiles, together with the absence of induced Cotton effects, indicates a surface or outside binding mode. We speculate that the top and bottom surfaces of the G-quadruplex comprise the potential MGBL binding sites, where the ligand lies on the surface forming van der Waals interactions with the guanines of the G-quartets and loop nucleotides.

Highlights

► Quadruplex conformation: “chair” (G2 & G2-U), “basket” (rG2); and both (G3 & G3-U). ► DSC profiles showed the following order of stability: rG2 < G2 < G2-U < G3-U < G3. ► Netropsin and distamycin yielded exothermic binding enthalpies of ∼−10.8 kcal/mol. ► Isotherms yielded affinities of ∼8 × 104 and 1:1 stoichiometries. ► Thermodynamic binding profiles suggest binding to the terminal G-quartets and loops.

Introduction

DNA is most often regarded as a duplex molecule in which the two self-complementary strands are held together by Watson–Crick base pairs. However, purine rich DNA sequences containing runs of guanines can form four-stranded structures called G-quadruplexes. Such sequences are found at the ends of chromosomes in the so-called telomeric regions and in transcriptional regulatory regions of several important oncogenes [1]. G-quartets are made up of guanine bases in DNA and RNA that associate via Hoogsteen hydrogen bonds to form planar G-quartets [2]. Guanine rich sequences can then form inter- or intramolecular G-quadruplexes. These G-quartets stack and form a platform connected by intermediate loops sequences of several nucleotides. Loops play a key role in the overall folding and stability of G-quadruplexes [3], [4], [5], [6], [7]; therefore, the length and sequence of these loops can either stabilize or destabilize a G-quadruplex. This is due to the strength of several types of molecular interactions of which the most important ones are hydrogen bonding, base–base stacking within the loops and stacking of the loops onto the G-quartets [8], [9], [10].

The conformation of a nucleic acid G-quadruplex is often investigated by circular dichroism spectroscopy, numerous publications have reported the spectral characteristics of folded G-quadruplexes in terms of the antiparallel or parallel arrangements of guanines [11], [12], [13], [14]. However, it is the population of syn/anti geometries of guanosine glycosidic torsional angles that determines their CD spectra [11]. Since this geometry is not directly related to the strand orientation, CD spectroscopy can provide only indicative information on strand orientation [15], [16]. On the other hand, the measured changes of CD peak positions and ratios of their magnitudes do reflect topological changes of the measured spectra of G-quadruplexes. The appearance of different G-quadruplex conformations and thus the ratio between CD peaks often depends highly on the nature of cations present in the solution (Na+, K+) [17], [18], [19], [20].

The G-rich DNA sequences with potential to form quadruplexes have been found in a number of important biological processes. For instance the defects in proteins that have high affinity for quadruplex DNA can lead to errors in replication, transcription, and recombination as well as to increase in the rates of aging and tumor formation [21]. Telomeric G-quadruplex structures occurring in the single-stranded overhangs at the ends of chromosomes seem to inhibit telomerase [22], [23], [24], [25]. There are also reports on G-quadruplexes forming oligonucleotides that act as inhibitors of HIV integrase [26] and thrombin [27]. Molecules have also been discovered that can bind to quadruplex DNA and inhibit proliferation in cancer cells presumably by means of telomerase inhibition [28], [29], [30]. These observations have attracted a lot of scientists to study the use of DNA quadruplexes as drugs and as therapeutic targets [1], [31]. To increase the efficiency of existing drugs and to develop new ones, a better understanding of interactions between ligands and DNA at the molecular level is needed. It is now well established that this cannot be accomplished by structural or computational studies alone but an insight into the binding thermodynamic properties of these systems is a suitable alternative approach [32], [33], [34], [35], [36], [37], [38], [39]. In the absence of high resolution structural studies, thermodynamics can help to determine the type of binding mode by which ligands bind non-covalently to DNA. There are four different ways that ligands bind to DNA: intercalation, minor groove binding, partial insertion and outside or surface binding [33], [38]. The binding of the so-called “minor groove binding ligands” (MGBL) to B-DNA is of great interest to researchers and has been extensively studied for several decades now [39], [40], [41], [42], [43], [44]. Whereas intercalation between adjacent base pairs is a common phenomenon in duplex DNA, in the case of quadruplexes there seems to be no binding of chromophores between G-quartets [45]. Early NMR and X-ray studies on structurally simple tetramolecular quadruplex–ligand complexes concurred in establishing that the chromophores stack onto the terminal G-quartets of quadruplexes [46]. One example is the crystal structure of the 1:1 complex of a disubstituted acridine ligand with the dimeric Oxytricha nova telomeric quadruplex showing this G-quartet end-stacking binding mode [47], [48]. However, this may not be the only way ligands can interact with G-quadruplexes, as indicated below. G-quadruplexes have already been extensively studied by our laboratory. One such investigation is of the thrombin aptamer d(G2T2G2TGTG2T2G2), that inhibits thrombin-catalyzed fibrin clot formation [9], [49], [50], [51], [52], [53], [54]. One focus of the studies was to modify the sequence of the loops to determine their energetic, ion and water binding contributions. Very recent investigations shows that distamycin and its derivatives interacts with the grooves of the [d(TG4T)]4 quadruplex [55], [56]. In light of these results, we have decided to investigate whether the MGBL netropsin and distamycin (Scheme 1) can bind to the thrombin aptamer and what is the nature of their binding. We are also interested on how additional G-quartets, the substitutions of thymine with uracil, and conformational changes are influencing the binding of MGBL.

Section snippets

Materials

The oligonucleotides (ODNs) and their designations: d(G2T2G2TGTG2T2G2), G2; d(G2U2G2UGUG2U2G2), G2-U; d(G3T2G3TGTG3T2G3), G3; d(G3U2G3UGUG3U2G3), G3-U; r(G2U2G2UGUG2U2G2), rG2 were synthesized by the Core Synthetic Facility of the Eppley Research Institute at UNMC, HPLC purified, and desalted by column chromatography using G-10 Sephadex exclusion chromatography. The concentrations of the oligomer solutions were determined at 260 nm and 25 °C using a Perkin Elmer Lambda-10 spectrophotometer and

CD spectra of G-quadruplexes

We started off our investigation by determining the conformation of each G-quadruplex, their sequences are shown in Scheme 1. This conformation was obtained from simple inspection of their CD spectra at low temperatures. To make sure each oligonucleotide forms the proper G-quadruplex fold, they were subjected to a temperature treatment. This treatment consisted of an initial heating to 95 °C, cooled at a rate of 1 °C per minute to 60 °C, hold for 15 min and cooled again to 5 °C [9]. The CD

Conclusions

We used a combination of spectroscopic and calorimetric techniques to investigate the conformation and unfolding thermodynamics of the K+-form of five G-quadruplexes, based on the sequence of the thrombin aptamer (G2). These G-quadruplexes show CD spectra characteristic of the “chair” conformation (G2 and G2-U), “basket” conformation (rG2); or a mixture “chair & basket” (G3 and G3-U). Thermodynamic folding profiles indicate the following order of stability: G3 (basket) > G3-U (basket) > G3

Acknowledgments

This work was supported by Grants MCB-0315746 and MCB-0616005 from the National Science Foundation, and a Shared Instrumentation Grant 1S10RR027205 from the National Institutes of Health. Partial financial support (to I.P.) from the Slovenian Research Agency (P1 0201) is greatly appreciated.

References (67)

  • S. Mazur et al.

    A thermodynamic and structural analysis of DNA minor-groove complex formation

    J. Mol. Biol.

    (2000)
  • I. Haq et al.

    Specific binding of hoechst 33258 to the d(CGCAAATTTGCG)2 duplex: calorimetric and spectroscopic studies

    J. Mol. Biol.

    (1997)
  • J. Lah et al.

    Energetic diversity of DNA minor groove recognition by small molecules displayed through some model ligand–DNA systems

    J. Mol. Biol.

    (2004)
  • S. Neidle

    The structures of quadruplex nucleic acids and their drug complexes

    Curr. Opin. Struct. Biol.

    (2009)
  • S.M. Haider et al.

    Crystal structure of the potassium form of an oxytricha nova G-quadruplex

    J. Mol. Biol.

    (2002)
  • S.M. Haider et al.

    Structure of a G-quadruplex–ligand complex

    J. Mol. Biol.

    (2003)
  • B. Pagano et al.

    Targeting DNA quadruplexes with distamycin A and its derivatives: an ITC and NMR study

    Biochemie

    (2008)
  • R.H. Shafer et al.

    Biological aspects of DNA/RNA quadruplexes

    Biopolymers

    (2001)
  • W.I. Sundquist et al.

    Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops

    Nature

    (1989)
  • R.F. Macaya et al.

    Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution

    Proc. Natl. Acad. Sci. USA

    (1993)
  • M.A. Keniry et al.

    The contribution of thymine–thymine interactions to the stability of folded dimeric quadruplexes

    Nucleic Acids Res.

    (1997)
  • C.M. Olsen et al.

    Unfolding of G-quadruplexes: energetic, and ion and water contributions of G-quartet stacking

    J. Phys. Chem. B.

    (2006)
  • P. Hazel et al.

    Loop-length-dependent folding of G-quadruplexes

    J. Am. Chem. Soc.

    (2004)
  • M. Vorlíčková et al.

    Guanine tetraplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats

    Nucleic Acids Res.

    (2005)
  • M. Lu et al.

    Thermodynamics of G-tetraplex formation by telomeric DNAs

    Biochemistry

    (1993)
  • I.N. Rujan et al.

    Vertebrate telomere repeat DNAs favor external loop propeller quadruplex structures in the presence of high concentrations of potassium

    Nucleic Acids Res.

    (2005)
  • J. Kypr et al.

    Circular dichroism spectroscopy and conformational properties of DNA

    Nucleic Acids Res.

    (2009)
  • S. Masiero et al.

    A non-empirical chromophoric interpretation of CD spectra of DNA G-quadruplex structures

    Org. Biomol. Chem.

    (2010)
  • G.R. Bishop et al.

    Characterization of DNA structures by circular dichroism

  • T.I. Gaynutdinov et al.

    Structural polymorphism of intramolecular quadruplex of human telomeric DNA: effect of cations, quadruplex-binding drugs and flanking sequences

    Nucleic Acids Res.

    (2008)
  • C.-C. Chang et al.

    Investigation of spectral conversion of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titration by a G-quadruplex recognizer BMVC molecule

    Nucleic Acids Res.

    (2007)
  • K.N. Luu et al.

    Structure of the human telomere in K+ solution: an intramolecular (3+1) G-quadruplex scaffold

    J. Am. Chem. Soc.

    (2006)
  • V. Đapić et al.

    Biophysical and biological properties of quadruplex oligodeoxyribonucleotides

    Nucleic Acids Res.

    (2003)
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