A DNA nanomachine chemically resolves lysosomes in live cells

Abstract

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.

Programmable low-cost DNA-based platform for viral RNA detection

“…DNA-PAINT in super-resolution microscopy 51 and DNA scaffolds for NMR 52 and cryo-EM 53 ) and biosensing (e.g. detection of lysosomal disorders 54 and mapping cellular endocytic pathways 55 ). Our DNA nanoswitches take a reductionist approach, resulting in assays that are robust and sensitive, yet simple to adapt and don't require multiple steps or expensive equipment.…”

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…Recently, combining the advantages of I‐switch and Clensor, a probe that can detect pH and chloride ion concentration simultaneously was developed . Using this probe termed as ChloropHore, subpopulations of lysosomes could be chemically resolved based on their chloride and proton content.…”

“…For instance, Sticky‐Flares have been used to show for the first time that β‐actin mRNA colocalizes with mitochondria in HeLa cells . Furthermore, ChloropHore has been used for simultaneous tracking of pH and chloride ion concentrations, providing fundamental insight into the relationship between these analytes and Niemann–Pick disease . Microscopy is the primary technique used in studying spatiotemporal behavior of analytes.…”

Dynamic DNA Structures

“…This switch was used as a fluorescent reporter to measure and map intracellular pH changes in living cells with spatial and temporal resolution. They later developed a series of DNA nanomachines that can sense and measure important physiological ions (e.g., Ca 2+ , Cl −1 ) in intracellular compartments . In addition to ions, biological molecules in living cells such as ATP may be probed as well.…”