Selective recognition of pyrimidine-pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators

Abstract

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine-pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes.

Chart 1.

Structures of macrocyclic ligands and acyclic control compounds used in this study. The charges correspond to the presumable protonation sites at pH 6.0. All ligands were handled as hydrochloride salts.

Chart 2.

Sequences of duplex oligodeoxynucleotides used in this study. X, Y = A, C, G or T; 2 = 2-aminopurine; M = N⁶-methyladenine.

Figure 1.

Ligand-induced changes of melting temperature (ΔT_m) of the fully matched (12-TA: black) and mismatch-containing duplexes (12-TG: orange, 12-TC: cyan, 12-TT: magenta bars) at ligand-to-duplex ratios of q = 1 (horizontally hatched bars), q = 2 (filled bars) and q = 3 (cross-hatched bars, only for 12-TA duplex); [12-TX] = 3 µM; estimated error in T_m determination is ±1.0°C.

Figure 2.

Ligand-induced changes of melting temperature (ΔT_m) of fully matched and mismatch-containing duplexes 17-TX (for sequence see Chart 2); [17-TX] = 6 µM; estimated error in T_m determination is ±0.5°C. For assignment of data sets see Figure 1 caption.

Figure 3.

ESI-MS spectra of duplexes 14-TX in the presence of macrocycles BisA (A), BisA-NH₂ (B) and BisNP (C). [14-TX] = [ligand] = 5 µM, except for 14-TA and BisA-NH₂ (B, lower spectrum), where [ligand] = 12 µM. The free duplexes are labeled as [TX]^6– and the 1 : 1 complexes as [TX + ligand]^6–. The diamonds indicate peaks corresponding to the triply charged single strands, the circles indicate triply charged 1 : 1 complexes with single strands when detected, and the arrows indicate 2 : 1 complexes [TX + 2 L]^6– when detected.

Figure 4.

FID titrations (excitation at 520 nm and emission at 615 nm) of 17-TT (filled symbols) and 17-TA (empty symbols) (c = 100 nM each) in the presence of ethidium bromide (c = 333 nM) with BisA (squares), BisA-NH₂ (diamonds) or BisNP (circles). The solid lines represent the analytical fits for a 1:1 binding model.

Figure 5.

Relative fluorescence intensity decrease (excitation at 520 nm and emission at 615 nm) upon addition of BisNP (c = 120 nM) to all sixteen 17-YX duplexes (100 nM each) in the presence of ethidium bromide (1 µM).

Figure 6.

Competition of BisNP (green) and M.TaqI (blue) for a TT-mismatch in DNA. (A) Principle of the competitive binding assay: (I) binding of M.TaqI to a DNA duplex with 2-aminopurine (2) at the target position leads to base flipping and a fluorescence increase (yellow) of 2-aminopurine; (II) in the presence of a DNA duplex with a TT-mismatch at the target position, the 2-aminopurine fluorescence increase is retarded; (III) adding BisNP to the two DNA duplexes leads to a partial reversal of the fluorescence increase retardation. (B) Corresponding fluorescence titration curves (excitation at 320 nm and emission at 384 nm), with I: 16-2T (100 nM), II: 16-2T and 17-TT (100 nM each), III: 16-2T and 17-TT (100 nM each) in the presence of BisNP (2 µM). Solid lines: curve fittings according to a one equilibrium (I) or a two equilibria (II, III) binding model for M.TaqI.

Figure 7.

Comparison between ligand-induced melting temperature increase of duplex DNA (left, q = 2) and binding affinity constants determined by ESI-MS (center) and by FID experiments (right) for binding of BisA, BisA-NH₂ and BisNP to TT mismatch-containing DNA duplexes 12-TT (T_m), 14-TT (ESI-MS) or 17-TT (FID).

Figure 8.

Comparison between BisNP induced shift of melting temperature of 12-TX (left, q = 2), binding constants of BisNP to 14-TX determined by ESI-MS (center) and background-corrected decrease of ethidium bromide fluorescence upon addition of 1.2 eq. BisNP to 17-TX (right).