Selective G-quadruplex DNA recognition by a new class of designed cyanines

Abstract

A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.

Figure 1

Trimethine cyanine analogs used in the current study. The modifications performed on the parent cyanine (24) are highlighted in different colors (compounds 25–32). The detailed synthetic procedure is described in Section 4.1. All compounds have one positive charge on the cyanine system and most have charged alkyl amine substituents. The synthesis and characterization of the related pentamethine cyanine analogs (compounds 1–23) are previously reported [18].

Figure 2

SPR sensorgrams and the steady-state binding plots for the parent cyanine (24, panel a) and the brominated analog (31, panel b) with Tel22 and cMYC19 quadruplexes and a control duplex (AATT). The injected concentration range for 24 is 10 nM–10 µM and for 31 is 10 nM–1 µM. The binding plots were obtained by fitting the steady-state response values (RU) as a function of free ligand concentration (C_free) and fit to a two-site binding model. The estimated equilibrium binding affinities (K₁ and K₂) are reported in Table 2.

Figure 3

CD spectra of the parent cyanine (24, panel a) and the brominated analog (31, panel b) with Tel22 and cMYC19 quadruplex sequences. The ligands were titrated into the quadruplex solutions (5 µM) until no further change in ICD signals were obtained. The insets show the mole ratio of drug:DNA.

Figure 4

Imino proton spectra of MYC22 quadruplex titrated with the parent cyanine 24 (a) and the brominated analogue 31 (b). Ligands were added to the quadruplex at the ratio indicated on the plot. Selected imino protons of 3ʹ-end (9, red) middle (8, blue) and 5ʹ-end (16, green) tetrads are marked.

Figure 5

(a) Schematic structure of the c-myc quadruplex MYC22 adopted from [30]. For clarity the imino protons of the guanine tetrads are color coded. Red: 3ʹ-end tetrad, blue: center tetrad, and green: 5ʹ-end tetrad. Imino proton titration curves of 3ʹ-end, middle and 5ʹ-end tetrad for the parent cyanine (24, b) and the brominated analog (31, c).

Figure 6

Fluorescence emission spectra of the parent cyanine (24, panel a) and the brominated analog (31, panel b) with Tel22 and cMYC19 quadruplex sequences. The arrow indicates increasing concentrations of DNA titrated into the ligand solution (1 µM) until no further change in the fluorescence emission signal was detected.

Scheme 1

Synthetic route for the preparation of trimethine cyanine G-quadruplex binding agents.