Highly fluorescent guanosine mimics for folding and energy transfer studies

Abstract

Guanosines with substituents at the 8-position can provide useful fluorescent probes that effectively mimic guanine residues even in highly demanding model systems such as polymorphic G-quadruplexes and duplex DNA. Here, we report the synthesis and photophysical properties of a small family of 8-substituted-2'-deoxyguanosines that have been incorporated into the human telomeric repeat sequence using phosphoramidite chemistry. These include 8-(2-pyridyl)-2'-deoxyguanosine (2PyG), 8-(2-phenylethenyl)-2'-deoxyguanosine (StG) and 8-[2-(pyrid-4-yl)-ethenyl]-2'-deoxyguanosine (4PVG). On DNA folding and stability, 8-substituted guanosines can exhibit context-dependent effects but were better tolerated by G-quadruplex and duplex structures than pyrimidine mismatches. In contrast to previously reported fluorescent guanine analogs, 8-substituted guanosines exhibit similar or even higher quantum yields upon their incorporation into nucleic acids (Φ = 0.02-0.45). We have used these highly emissive probes to quantify energy transfer efficiencies from unmodified DNA nucleobases to 8-substituted guanosines. The resulting DNA-to-probe energy transfer efficiencies (η(t)) are highly structure selective, with η(t)(duplex) < η(t)(single-strand) < η(t)(G-quadruplex). These trends were independent of the exact structural features and thermal stabilities of the G-quadruplexes or duplexes containing them. The combination of efficient energy transfer, high probe quantum yield, and high molar extinction coefficient of the DNA provides a highly sensitive and reliable readout of G-quadruplex formation even in highly diluted sample solutions of 0.25 nM.

Figure 1.

(A) Structures and numbering of 8-substituted-2′-deoxyguanosines and an ion-containing G-tetrad (gray). (B) Wavelengths of maximal absorbance (λ_abs) and emission (λ_em) in water, and quantum yield (Φ) of each nucleoside in acetonitrile, water or D₂O.

Scheme 1.

Synthesis of the 8-(substituted)-2′-deoxyguanosine phosphoramidites 16 and 17.

Figure 2.

Excitation spectra of 2 µM of StG (A), 4PVG (B) or 2PyG (C) in the presence of 100 mM KCl (K⁺), 100 mM NaCl (Na⁺) or 100 mM LiCl (Li⁺). Excitation spectra of hTeloG23(StG) (D), hTeloG23(4PVG) (E) and hTeloG23(2PyG) (F) were collected using emission at 450, 475 and 415 nm, respectively. Emission spectra of hTeloG23(StG) (G), hTeloG23(4PVG) (H) and hTeloG23(2PyG) (I) were collected using excitation at 260 nm. Double-stranded samples ‘DS’ included 1.1 equivalents of the complementary strand and were prepared in 100 mM NaCl. All samples contained 2 µM of DNA or 8-substituted-2′-deoxyguanosine nucleoside (2–4) in an aqueous 10 mM cacodylate buffer (pH 7.4).

Figure 3.

CD spectra of single-stranded hTeloG9 and hTeloG23 derivatives in 100 mM LiCl (A), 100 mM KCl (B and C) or 100 mM NaCl (D). Double-stranded DNA samples ‘DS’ included 1.1 equivalents of the complementary strand and were prepared in 100 mM NaCl (E and F). All samples contained 2 µM of DNA in an aqueous 10 mM cacodylate buffer (pH 7.4).