Abstract
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.
Highlights
The structure and function of biological membranes are largely controlled by their lipid composition, and by cholesterol, which represents up to 40% of the lipids in plasma membranes [1,2]
The model cholesterol-anchored fluorescent oligonucleotide probes for potassium sensing at the cell membrane interface are illustrated in Scheme 1
The amphiphilic lipid−DNA probe consists of a hydrophobic cholesterol moiety, hydrophilic DNA strand, and fluorophores attached to DNA (Scheme 1A)
Summary
The structure and function of biological membranes are largely controlled by their lipid composition, and by cholesterol, which represents up to 40% of the lipids in plasma membranes [1,2]. Membrane of Living Cell for Fluorescence Imaging Experiment. HeLa were seeded at a density of 1.2 × 105 cells per well in 4-chamber glass-bottom cell culture dishes (Grenier Bio-One, Kremsmünster, Austria) and cultured in RPMI 1640 antibiotic-free medium (Sigma, Kawasaki, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), and 1%. The Ch(F-TBA-T) probe was added to the culture medium to obtain the final concentration, nM or 200 nM. The positive control was HeLa cells treated with the F-TBA-T probe (50 nM) added to the culture medium 24 h before visualization. In experiments using Hoechst 33342 (Sigma, Kawasaki, Japan), the dye was applied to medium in a final concentration of 5 μg/mL and images were taken after 15 min
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