Synthesis of Fluorescent Potassium Ion-Sensing Probes Based on a Thrombin-Binding DNA Aptamer-Peptide Conjugate.

Abstract

This unit provides a procedure for synthesis of the potassium-sensing peptide-oligodeoxyribonucleotide conjugate PSO-5 for visualizing potassium ions (K(+) ) in living cells. It is constructed by combining an oligodeoxyribonucleotide carrying a thrombin-binding DNA aptamer (TBA) sequence with an uncharged peptide carrying biotin and the fluorescence tags fluorescein (FAM) and tetramethylrhodamine (TAMRA). The PSO-5 and biotin-modified nuclear export signal peptide are conjugated through streptavidin, and this sensing molecule is introduced into the cell where it is localized in the cytoplasm. The TBA part of PSO-5 shows a conformational change from a random coil to a tetraplex structure induced by K(+) and a change in the fluorescence resonance energy transfer (FRET) efficiency between FAM and TAMRA arising from its conformational change, enabling fluorometric detection of changes in K(+) concentration.