Cholesterol-Bearing Fluorescent G-Quadruplex Potassium Probes for Anchoring at the Langmuir Monolayer and Cell Membrane

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.

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

“…The ChONC12-AgNCs probe was designed and studied in order to verify its potential as a fluorescent tag that can be integrate with G-quadruplex DNA to create FRET working system able to anchor into the cellular membrane and monitor changes in potassium concentration. Previously, we proved the successful incorporation of cholesterol-modified fluorescent probes based on G-quadruplexes to the outer cell membrane of living HeLa cells 42 . So far, we have optimizied the synthesis of ChONC12-AgNCs in buffer as well as in cell lysate 19 .…”

“…The ability of cholesterol-modified ssDNA for spontaneous anchoring into the hydrophobic interior of lipid membranes was proven by Patolsky et al 52 . In our group, we developed cholesterol-anchored fluorescent probes based on G-quadruplexes for spontaneous anchoring into the hydrophobic interior of living cell membrane 42 . The Ch(F-TBA-T) probe was labeled with carboxyfluorescein (FAM) and carboxytetramethylrhodamine (TAMRA) dyes ( Table 1, Fig.…”

“…4A). What is important, the analogous probe without cholesterol moiety was not only unable to anchor to the cell membrane, but also did not penetrate inside the cells 42 . These results revealed the important role of the cholesterol group in the localization of G-quadruplex probes on the cell membrane.…”

“…The cholesterol-linked oligonucleotide (ChONC12) was soluble in all physiologically relevant solutions and could interact with the Langmuir monolayer via hydrophobic interaction between the cholesterol moiety and the membrane 42,67 , or via electrostatic interaction between the positively charged DODAB head groups and the negatively charged phosphate groups of DNA [68][69][70] . Figure 7 shows π-A isotherms recorded for ChONC12 oligonucleotide (black line), the DODAB monolayer (red line) and ChONC12-AgNCs/DODAB complex (green line) on the subphase containing 10 mM Tris-CH 3 COOH buffer.…”

“…Cell culturing. HeLa cells culturing was performed as we have done previously 17,42 . Thus, HeLa cells were seeded at a density of 1.2 × 10 5 cells per well in 4-chamber glass-bottom cell culture dishes (Grenier Bio-One, Kremsmünster, Austria) and cultured in RPMI 1,640 medium (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), and 1% RPMI 1,640 vitamin solution (Sigma) and 1% antibiotic antimycotic solution (Sigma) at 37 °C under a 5% CO 2 atmosphere.…”