Development of fluorescence oligonucleotide probes based on cytosine- and guanine-rich sequences

Abstract

The properties of cytosine- and guanine-rich oligonucleotides contributed to employing them as sensing elements in various biosensors. In this paper, we report our current development of fluorescence oligonucleotide probes based on i-motif or G-quadruplex forming oligonucleotides for cellular measurements or bioimaging applications. Additionally, we also focus on the spectral properties of the new fluorescent silver nanoclusters based system (ChONC12-AgNCs) that is able to anchor at the Langmuir monolayer interface, which is mimicking the surface of living cells membrane.

Figure 1

The spectra of tC-MB-520 probe (1 μM) prefolded in different pH buffers: (A) CD spectra with insert showing the dependence of CD signals at 286 nm against the pH values; (B) UV–Vis absorption spectra with insert showing absorption changes in UV region.

Figure 2

The maximum fluorescence intensity of molecular beacons in different pH solutions: (A) tC-MB-520 probe (0.25 μM); (B) MB-520 reference probe (0.25 μM).

Figure 3

Confocal microscopy images of HeLa cells transfected with 50 nM tC-MB-520 using Lipofectamine 2000: (A) Atto520 fluorescence, marked red; (B) tC/Atto520 fluorescence, marked blue (C) bright field image (D) overlay of all images. Fluorescence emission filters: (1) for red color, 520–640 nm; excitation wavelength: 514 nm; (2) for blue color, 520–640 nm; excitation wavelength: 405 nm. Scale bars: 20 μm.

Figure 4

Confocal microscopy images of HeLa cells treated with 50 nM Ch(F-TBA-T) for 3.5 h (A): (a) FAM fluorescence, marked green; (b) FRET image, marked red; (c) TAMRA image, marked red; (d) overlay of all images. Fluorescence emission filters: (1) for FAM, 510–540 nm; excitation wavelength: 480 nm; (2) for FRET, 595–630 nm; excitation wavelength: 480 nm; (3) for TAMRA, 595–630 nm; excitation wavelength: 560 nm. Scale bars: 50 μm. (B) Scheme of using the FRET process to generate a fluorescent signal by the Ch(F-TBA-T) probe; (C) fluorescence intensity ratio (F₅₈₃/F₅₂₀) for Ch(F-TBA-T) (C) plotted against K⁺ concentration in the presence 150 mM Na⁺ at 25.0 °C (circles) and 36.6 °C (triangles).

Figure 5

Absorption spectra (A) and CD spectra (B) of the ChONC12-AgNCs. The solutions contained 2 µM ChONC12, Tris–CH₃COOH buffer (10 mM, pH = 7.5), [Ag⁺] = [BH₄] = 24 µM.

Figure 6

Upper panel: Excitation and emission spectra of ChONC12Ag-NCs (A) and the effect of time on the emission stability of ChONC12-AgNCs for 1 h to 21 days after the reduction of silver (B). Conditions: 2 µM DNA, Tris–CH₃COOH buffer solution (10 mM, pH 7.5), [Ag⁺] = [BH₄⁻] = 24 µM; λ_ex = 475 nm/λ_em = 560 nm, λ_ex = 560 nm/λ_em = 610 nm. Lower panel: Absorbance (C) and emission (D) spectra of ChONC12-AgNCs nanoclusters obtained at different molar ratio C/Ag⁺. Conditions: 2 μM DNA, 10 mM Tris-CH3COOH (pH = 7.5), λ_ex = 475 nm and λ_ex = 560 nm.

Scheme 1

Scheme showing the synthesis of ChONC12-AgNCs incorporated into the Langmuir monolayer.

Figure 7

The π-A isotherms recorded for ChONC12 (black line), DODAB monolayer (red line) and ChONC12-AgNCs/DODAB complex (green line). Conditions: 50 µl (1 × 10^–4 M) ChONC12, 5 µl DODAB (1 × 10^–3 M), [Ag⁺] = 6 µl (1 × 10^–2 M), [BH₄^-] = 120 µl (1 × 10^–2 M); subphase contained 10 mM buffer Tris–CH₃COOH (pH = 7.5).

Figure 8

Excitation and emission spectra of ChONC12-AgNCs/DODAB system recorded at the air/water interface. Conditions: 50 µl (1 × 10^–4 M) ChONC12, 5 µl DODAB (1 × 10^–3 M), [Ag⁺] = 6 µl (1 × 10^–2 M), [BH₄⁻] = 120 µl (1 × 10^–2 M); subphase contained 10 mM buffer Tris-CH₃COOH (pH = 7.5), λ_ex = 475 nm/λ_em = 560 nm, λ_ex = 560 nm/λ_em = 620 nm.

Figure 9

Confocal fluorescence imaging of HeLa cells loaded with 1 μM ChONC12-AgNCs for 2 h (A) or control HeLa cells treated with AgNO₃ for 30 min (B). Fluorescence emission filter: 510–550 nm; excitation wavelength: 500 nm. Scale bars: 50 μm.

Scheme 2

Schematic diagram of the preparation of DNA-Ag NCs.