Fluorescence Imaging of Extracellular Potassium Ion Using Potassium Sensing Oligonucleotide

Abstract

Potassium-sensing oligonucleotide, PSO, a conjugate of a quadruplex structure-forming oligonucleotide with a peptide incorporating a Förster Resonance Energy Transfer (FRET) chromophore pair, has been developed for fluorescent detection of potassium ion (K⁺) in aqueous medium. PSO 1 could be introduced into cells for real-time imaging of cytoplasmic K⁺ concentrations. To perform fluorescent imaging of K⁺ on the cell surface, we synthesized twelve PSO derivatives with different types of peptide types and lengths, and oligonucleotide sequences including thrombin-binding aptamer (TBA) sequences with FAM and TAMRA as a FRET chromophore pair, and evaluated their performance. 1 was shown to respond selectively to K⁺, not to most ions present in vivo, and to show reciprocal fluorescence changes in response to K⁺ concentration. For the peptide chains and oligonucleotide sequences examined in this study, the PSO derivatives had K _d values for K⁺ in the range of 5-30 mM. All PSO derivatives showed high K⁺ selectivity even in the presence of excess Na⁺. The PSO derivatives were successfully localized to the cell surface by biotinylated concanavalin A (ConA) or sulfo-NHS-biotin via streptavidin (StAv). Fluorescence imaging of extracellular K⁺ upon addition of apoptosis inducers was successfully achieved by 1 localized to the cell surface.

Keywords: G-quadruplex; cell surface; fluorometric imaging; potassium ion; potassium ion efflux; potassium sensing oligonucleotide; sodium ion.

FIGURE 1

(A) Example of potassium-sensing oligonucleotide (PSO) 1 consisting of oligonucleotide-carrying TBA sequence, peptide linker with biotin, and FRET chromophore pair of FAM and TAMRA. (B) PSO localized on a cell surface through sugar chain, concanavalin A (ConA), and avidin (left) or biotinylated membrane protein, avidin (right). Black circle: biotin; red circle: FRET chromophore pair.

FIGURE 2

(A) Fluorescence spectra of 0.2 μM 1, 0.3 μM StAv in the absence (red) or presence (blue) of 150 mM KCl, 20 mM Tris-HCl (pH 7.4) without (A) or with 145 mM NaCl (B).

FIGURE 3

ΔRatio of 0.2 μM 1 in the absence (−) or presence (+) of 0.3 μM streptavidin (StAv) in 20 mM Tris-HCl (pH 7.4) under several cations; 150 mM KCl, 145 mM NaCl, 2 mM MgCl₂, 2 mM CaCl₂, 20 mM CH₃COONH₄, or 150 mM LiCl. Ex: 495 nm.

FIGURE 4

(A) Extracell imaging by 1-StAv-ConA, (B) fluorescence ratio of F_red/F_green (F_red: F.I. of 550–640 nm,/F_green: F.I. of 495–540 nm) after adding KCl, λ _ex = 488 nm. (C) Extracell imaging by 1-StAv-sulfo-NHS-biotin, (D) fluorescence ratio of F_red/F_green after adding KCl, λ _ex = 488 nm. (↑) indicates the change of DMEM medium.

FIGURE 5

(A) Cell surface imaging by 1-StAv-ConA after adding 10 μM Amphotericin B, 10 μM Bumetanide, and 10 μM Ouabain/DMEM at 11 min (↑), (B) fluorescence ratio of F_red/F_green (F_red: F.I. of 575–595 nm,/F_green: F.I. of 508–528 nm), λ _ex = 488 nm in all cells. (C) Extracell imaging by 1-StAv-sulfo-NHS-biotin and (D) fluorescence ratio of F_red/F_green (F_red: F.I. of 554–620 nm, F_green: F.I. of 500–554 nm), λ _ex = 488 nm after adding 26 μM Amphotericin B at 11 min (↑). The three cells shown in (C) were surrounded by ROI (region of interests), and their fluorescence ratios were shown in (D).