N-methylmesoporphyrin IX fluorescence as a reporter of strand orientation in guanine quadruplexes

Abstract

Guanine quadruplexes (GQ) are four-stranded DNA structures formed by guanine-rich DNA sequences. The formation of GQs inhibits cancer cell growth, although the detection of GQs in vivo has proven difficult, in part because of their structural diversity. The development of GQ-selective fluorescent reporters would enhance our ability to quantify the number and location of GQs, ultimately advancing biological studies of quadruplex relevance and function. N-methylmesoporphyrin IX (NMM) interacts selectively with parallel-stranded GQs; in addition, its fluorescence is sensitive to the presence of DNA, making this ligand a possible candidate for a quadruplex probe. In the present study, we investigated the effect of DNA secondary structure on NMM fluorescence. We found that NMM fluorescence increases by about 60-fold in the presence of parallel-stranded GQs and by about 40-fold in the presence of hybrid GQs. Antiparallel GQs lead to lower than 10-fold increases in NMM fluorescence. Single-stranded DNA, duplex, or i-motif, induce no change in NMM fluorescence. We conclude that NMM shows promise as a 'turn-on' fluorescent probe for detecting quadruplex structures, as well as for differentiating them on the basis of strand orientation.

Keywords: N-methylmesoporphyrin IX; fluorescent probe; guanine quadruplex; human telomeric DNA; selectivity.

Figure 1

Structures of GQ and NMM. (A) Structure of a G‐tetrad. (B) Schematic representations of mixed‐hybrid monomolecular GQ (left), antiparallel bimolecular GQ (middle) and tetramolecular parallel‐stranded GQ (right). (C) Structure of NMM. Note that commercially available NMM is a mixture of four regioisomers that differ in the position of the N‐Me group (only one isomer is shown); each isomer forms a pair of enantiomers with the N‐Me group pointing up or down.

Figure 2

Normalized fluorescence data for titration of (A) 1.0 μm NMM with G4 and G8 in TB buffer and (B) 0.1 μm NMM with G₄T₄G₄ in 5K buffer at 25 °C. Data for G4 and G8 are the average of three individual titrations. Solid lines represent global fits to a 1 : 1 binding model and dashed lines represent the 95% confidence interval. Binding constants were determined to be (1.4 ± 0.2) × 10⁶, (1.7 ± 0.2) × 10⁶ and (1.26 ± 0.07) × 10⁷ L·mol⁻¹ for G4, G8 and G₄T₄G₄, respectively. Note, the data for G₄T₄G₄ could be satisfactory fit to a 1 NMM : 2 GQ binding model with K_a of (5.4 ± 0.5) × 10⁷ L·mol⁻¹.

Figure 3

Summary of steady‐state fluorescence data for NMM incubated with a 10‐fold molar excess of indicated sequences. Fluorescence enhancement is reported relative to the fluorescence of NMM alone. Error bars are 1 SD (confidence interval of 68.2%).