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Chloride plays a major role in cellular homeostasis by regulating the lumenal pH of intracellular organelles. We have described a pH-independent, fluorescent chloride reporter called Clensor that has successfully measured resting chloride in organelles of living cells. Here, we describe the rational design of Clensor . Clensor integrates a chloride sensitive fluorophore called 10,10'-bis[3-carboxypropyl]-9,9'-biacridinium dinitrate (BAC) with the programmability, modularity and targetability available to nucleic acid scaffolds. We show that simple conjugation of BAC to a DNA backbone fails to yield a viable chloride-sensitive reporter. Fluorescence intensity and lifetime investigations on a series of BAC-functionalized structural variants yielded molecular insights that guided the rational design and successful realization of the chloride sensitive fluorescent reporter, Clensor . This study provides some general design principles that would aid the realization of diverse ion-sensitive nucleic acid reporters based on the sensing strategy of Clensor .

Rational design of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter