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

Prakash, Ved; Saha, Sonali; Chakraborty, Kasturi; Krishnan, Yamuna

doi:10.1039/c5sc04002g

Cited by 23 publications

(23 citation statements)

References 64 publications

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“…In the I-switch intrastrand i-motif formation is used to bring about a pH-dependent conformational change, that leverages fluorescence resonance energy transfer (FRET) to create a ratiometric fluorescent pH reporter. (Figure 1; figure supplementary 2) The DNA-based chloride sensor, Clensor, is composed of three modules: a sensing module, a normalizing module and a targeting module (Figure 1a) (Saha et al, 2015) , (Prakash et al, 2016). The sensing module is a 12 base long peptide nucleic acid (PNA) oligomer conjugated to a fluorescent, chloride-sensitive molecule 10,10΄-Bis[3-carboxypropyl]-9,9΄-biacridinium dinitrate (BAC), (Figure 1a) (Sonawane et al, 2002).…”

Section: Resultsmentioning

confidence: 99%

High lumenal chloride in the lysosome is critical for lysosome function

Krishnan

Chakraborty

Leung

2017

Preprint

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Lysosomes are organelles responsible for the breakdown and recycling of cellular machinery. Dysfunctional lysosomes give rise to lysosomal storage disorders as well as common neurodegenerative diseases. Here, we use a DNA-based, fluorescent chloride reporter to measure lysosomal chloride in Caenorhabditis elegans as well as murine and human cell culture models of lysosomal diseases. We find that the lysosome is highly enriched in chloride, and that chloride reduction correlates directly with a loss in the degradative function of the lysosome. In nematodes and mammalian cell culture models of diverse lysosomal disorders, where previously only lysosomal pH dysregulation has been described, massive reduction of lumenal chloride is observed that is ~10 4 -fold greater than the accompanying pH change. Reducing chloride within the lysosome impacts Ca 2+ release from the lysosome and impedes the activity of specific lysosomal enzymes indicating a broader role for chloride in lysosomal function.

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Section: Resultsmentioning

confidence: 99%

High lumenal chloride in the lysosome is critical for lysosome function

Krishnan

Chakraborty

Leung

2017

Preprint

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“…Unusual chloride ion concentrations in the lysosome can lead to lysosomal storage disorders. A DNA nanodevice called Clensor can report changes in chloride ion concentrations in a pH‐independent manner . Clensor consists of three parts: a sensing module containing a 12‐base long PNA moiety labeled with a chloride‐sensitive dye, 10,10′‐bis[3‐carboxypropyl]‐9,9′‐biacridinium dinitrate (BAC), a normalizing module containing a 38‐base long Alexa 647‐labeled DNA sequence, and a targeting module containing a 26‐base long DNA.…”

Section: Aptamer‐based Probesmentioning

confidence: 99%

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

2019

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The chemical composition of cells at the molecular level determines their growth, differentiation, structure, and function. Probing this composition is powerful because it provides invaluable insight into chemical processes inside cells and in certain cases allows disease diagnosis based on molecular profiles. However, many techniques analyze fixed cells or lysates of bulk populations, in which information about dynamics and cellular heterogeneity is lost. Recently, nucleic‐acid‐based probes have emerged as a promising platform for the detection of a wide variety of intracellular analytes in live cells with single‐cell resolution. Recent advances in this field are described and common strategies for probe design, types of targets that can be identified, current limitations, and future directions are discussed.

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“…To improve this, our sensor Clensor uses a double-stranded DNA backbone to achieve quantitative Cl − mapping across the entire physiological regime of Cl − by displaying BAC on one strand and a normalizing Alexa Fluor 647 (AF647) fluorophore on the other in a precise 1:1 stoichiometry (Fig. 3C) (Prakash et al, 2016;Saha et al, 2015). The DNA portion also acts as a negatively charged ligand for scavenger receptors, which, after binding, traffic the sensor along the endolysosomal pathway, after which it can be directed to other organelles by using aptamers (Dan et al, 2019;Modi et al, 2009Modi et al, , 2013Modi et al, , 2014Narayanaswamy et al, 2019;Thekkan et al, 2018).…”

Section: Second Generationthe Development Of Ratiometric Quantificationmentioning

confidence: 99%

What biologists want from their chloride reporters – a conversation between chemists and biologists

Zajac

Chakraborty

Saha

et al. 2020

Journal of Cell Science

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Impaired chloride transport affects diverse processes ranging from neuron excitability to water secretion, which underlie epilepsy and cystic fibrosis, respectively. The ability to image chloride fluxes with fluorescent probes has been essential for the investigation of the roles of chloride channels and transporters in health and disease. Therefore, developing effective fluorescent chloride reporters is critical to characterizing chloride transporters and discovering new ones. However, each chloride channel or transporter has a unique functional context that demands a suite of chloride probes with appropriate sensing characteristics. This Review seeks to juxtapose the biology of chloride transport with the chemistries underlying chloride sensors by exploring the various biological roles of chloride and highlighting the insights delivered by studies using chloride reporters. We then delineate the evolution of small-molecule sensors and genetically encoded chloride reporters. Finally, we analyze discussions with chloride biologists to identify the advantages and limitations of sensors in each biological context, as well as to recognize the key design challenges that must be overcome for developing the next generation of chloride sensors.

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

Abstract: This study describes the rational design of a DNA-based chloride reporter.

Cited by 23 publications

References 64 publications

High lumenal chloride in the lysosome is critical for lysosome function

High lumenal chloride in the lysosome is critical for lysosome function

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

What biologists want from their chloride reporters – a conversation between chemists and biologists

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