The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

Lysosomes are multi-functional, sub-cellular organelles with roles in plasma membrane repair, autophagy, pathogen degradation and nutrient sensing. Dysfunctional lysosomes underlie Alzheimers, Parkinsons and rare lysosomal storage diseases but their contributions to these pathophysiologies are unclear. Live imaging has revealed lysosome sub-populations with different physical characteristics including dynamics, morphology or cellular localization. Here we chemically resolve lysosome sub-populations using a DNA-based combination reporter that quantitatively images pH and chloride simultaneously in the same lysosome while retaining single lysosome information in live cells. We call this technology two-ion measurement or 2-IM. 2-IM of lysosomes in primary skin fibroblasts derived from normal individuals show two major lysosome populations, one of which is lost in primary cells derived from Niemann-Pick disease patients. When patient cells are treated with relevant therapeutic, the second population re-emerges. Chemically resolving lysosomes by 2-IM could enable decoding the mechanistic underpinnings of lysosomal diseases, monitoring disease progression or evaluating therapeutic efficacy.

It is extremely challenging to quantitate lumenal Ca 2+ in acidic Ca 2+ stores of the cell because all Ca 2+ indicators are pH sensitive, and Ca 2+ transport coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca 2+ simultaneously, it functions as a pHcorrectable, Ca 2+ reporter. By targeting CalipHluor to the endolysosomal pathway we mapped lumenal Ca 2+ changes during endosomal maturation and found a surge in lumenal Ca 2+ specifically in lysosomes. Using lysosomal proteomics and genetic analysis we found that catp-6, a C. elegans homolog of ATP13A2, was responsible for lysosomal Ca 2+ accumulation-the first example of a lysosome-specific Ca 2+ importer in animals. By enabling the facile quantification of compartmentalized Ca 2+ , CalipHluor can expand our understanding of subcellular Ca 2+ importers.

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The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid--based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid--based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand.