The purpose of the present work was to design, synthesize and spectrally characterize cholesterol-anchored fluorescent oligonucleotide probes (Ch(F-TBA-T), Ch(py-TBA-py)), based on G-quadruplexes, which were able to incorporate into a lipid structure (Langmuir monolayer, living cell membrane). The probes, based on the thrombin-binding aptamer (TBA) sequence, were labeled with fluorescent dyes which enabled simultaneous monitoring of the formation of G-quadruplex structures and visualization of probe incorporation into the cellular membrane. The combinations of fluorophores used included fluorescence resonance energy transfer (FRET) and excimer emission approaches. The structural changes of the probes upon binding with K+ or Na+ ions were monitored with fluorescence techniques. These systems showed a very high binding preference for K+ over Na+ ions. The use of confocal fluorescence microscopy indicated successful anchoring of the cholesterol-bearing fluorescent probes to the living cell membrane. These structurally simple cholesterol-based fluorescent probes have good potential for opening up new and exciting opportunities in the field of biosensors; e.g., in vivo detection of K+ ions.

on DNA. Finally, the obtained solution was further stored in the dark at 4 °C for 2 h to prepare stable DNA-AgNCs. The schematic diagram of the preparation of DNA-AgNCs is presented on Scheme 2. Spectroscopy measurements of fluorescent DNA-AgNCs were performed at 25 °C using 2 µM solutions of a ChONC12 oligonucleotide in 10 mM Tris-CH 3 COOH buffer (pH 7.5) as follows. Absorption spectra of fluorescent DNA-AgNCs. UV-Vis spectra of the DNA-AgNCs were recorded in the spectral range of 200-800 nm by means of Jasco V-750 spectrophotometer (Jasco, Tokyo, Japan). CD spectra of fluorescent DNA-AgNCs. CD measurements were carried out on a Jasco J-820 Spectropolarimeter (Jasco, Tokyo, Japan) with connected a temperature controller (PTC-423L). The CD spectra were obtained by taking the average of three scans in the range of 350-200 nm, with a scan rate of 200 nm/min. Fluorescence spectra. Fluorescence measurements were performed on a Jasco spectrofluorimeter FP-8200 (Jasco, Tokyo, Japan) with 10 nm excitation and 10 nm emission slits and were carried out using 0.4 × 1 cm quartz cuvettes containing 1 ml of solution. Excitation and emission spectra of DNA-AgNCs were recorded in the 200-750 nm range with λ ex = 475 nm/λ em = 560 nm, λ ex = 560 nm/λ em = 610 nm. Sample solution containing 2 μM of fluorescent oligonucleotide in 10 mM Tris-CH 3 COOH buffer (pH 7.5) was equilibrated in a quartz cell at 25 °C for 10 min. Measurements of π-A isotherms and fluorescence spectra at the monolayer interface. To obtain the surface pressure-area (π-A) isotherms for the DODAB (dimethyldioctadecylammonium bromide) monolayer and DODAB/ AgNCs complex at the air-water interface we used the computer-contolled balance system (Langmuir trough small, KSV NIMA, Espoo, Finland) as in Ref. 42. The procedure for creating a monolayer using silver nanoclusters consisted of mixing a DODAB surfactant (5 µL, 1 × 10-3 M in CHCl 3) with a ChONC12 oligonucleotide (5 µL, 1 × 10-3 M in water) (1:1 ratio) and AgNO 3 (6 µl, 1 × 10-2 M in water) and such a mixture was applied to the subphase (after 60 min). At a later stage, freshly prepared NaBH 4 (to reduce Ag + ions) was added behind the barriers. The compression as well as fluorescence measurements of monolayer-silver nanoclusters were carried out analogues as in Ref. 42. Fluorescence emission spectra for ChONC12-AgNCs were recorded in the 400-750 nm range, with the excitation wavelength of 460 nm and 550 nm (λ em = 550 nm/λ em = 610 nm).

In this study, we examined properties of silver nanoclusters, which are AgNCs stabilized by DNA oligonucleotide scaffold containing G-quadruplex-forming sequences: human telomeric (Tel22) or thrombin-binding aptamer (TBA). Thus, we obtained two fluorescent probes abbreviated as Tel22C12-AgNCs and TBAC12-AgNCs, which were characterized using absorption, circular dichroism and fluorescence spectroscopy. Both probes emit green and red fluorescence. The presence of silver nanoclusters did not destabilize the formed G-quadruplexes. The structural changes of probes upon binding K+ or Na+ ions cause quenching in their red emission. Green emission was slightly quenched only in the case of Tel22C12-AgNCs; on the contrary, for TBAC12-AgNC’s green emission, we observed an increasing fluorescence signal. Moreover, the Tel22C12-AgNCs system shows not only a higher binding preference for K+ over Na+, but it was able to monitor small changes in K+ concentrations in the buffer mimicking extracellular conditions (high content of Na+ ions). These results suggest that Tel22C12-AgNCs exhibit the potential to monitor transmembrane potassium transport.

No abstract

Silver nanoclusters (AgNCs) generated on DNA templates belong to a new class of fluorescent tags showing excellent brightness, photostability and biocompatibility. Thus, AgNCs-DNA has been applied in various applications, from the detection of DNA/RNA and environmental monitoring to bioimaging and cancer therapy. In this work, we report fluorescent AgNCs synthesized using two 1,3-diaza-2-oxophenothiazine (tC)-modified oligonucleotides that contain RET-related sequence CCCCGCCCCGCCCCGCCCCA. The communication compares the absorption and emission properties of the obtained systems with silver nanoclusters synthesized on the unmodified oligonucleotide. First, we showed the optimal conditions for AgNCs-DNA synthesis on three DNA templates: (1) RET20 with the sequence 5′-CCC CGC CCC GCC CCG CCC CA-3′; (2) RET19tC with the sequence 5′-CCC CGC CCC GCC CCG CCC tCA-3′; and (3) RET14tC with the sequence 5′-CCC CGC CCC GCC CtCG CCC CA-3′. Next, the silver nanoclusters were characterized by UV/Vis absorption, fluorescence and circular dichroism spectroscopy. Silver nanoclusters RET19tC-AgNCs and RET14tC-AgNCs indicated several times higher fluorescence intensities in the long-wave emission spectra as compared to RET20-AgNCs. Moreover, silver nanoclusters on tC-modified oligonucleotides showed higher stability over time. The possibility of using the silver nanoclusters RET19tC-AgNCs for monitoring pH changes will be also tested.