DNA-based fluorescent probes of NOS2 activity in live brains.

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.

Controlled release of bioactive signaling molecules.

Membrane-initiated steroid signaling (MISS) involves rapid second messenger based intracellular signaling without coupling to transcription or translation. MISS activates important cellular signaling cascades such as mitogen-activated protein kinase (MAPK) or adenylate cyclase pathways. Despite its vital role in signaling, the downstream second messengers involved in MISS and their temporal dynamics remain elusive. A technology which can offer pristine spatiotemporal control over the release of the steroid initiator could pave the way to understand these rapid and ultrasensitive signaling processes. Toward this, we describe a DNA-nanocapsule based technology to chemically release steroids and study MISS in endothelial cells. Here we discuss the synthesis and cellular protocols for investigators who seek to utilize DNA-nanocapsules for the chemically triggered release of small molecules.

A DNA-based fluorescent probe maps NOS3 activity with subcellular spatial resolution.

Nitric oxide synthase 3 (NOS3) produces the gasotransmitter nitric oxide (NO), which drives critical cellular signaling pathways by S-nitrosylating target proteins. Endogenous NOS3 resides at two distinct subcellular locations: the plasma membrane and the trans-Golgi network (TGN). However, NO generation arising from the activities of both these pools of NOS3 and its relative contribution to physiology or disease is not yet resolvable. We describe a fluorescent DNA-based probe technology, NOckout, that can be targeted either to the plasma membrane or the TGN, where it can quantitatively map the activities of endogenous NOS3 at these locations in live cells. We found that, although NOS3 at the Golgi is tenfold less active than at the plasma membrane, its activity is essential for the structural integrity of the Golgi. The newfound ability to spatially map NOS3 activity provides a platform to discover selective regulators of the distinct pools of NOS3.

DNA nanodevices map enzymatic activity in organelles.

Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in subcellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, subcellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in Caenorhabditis elegans and identified that exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.

Chemical control over membrane-initiated steroid signaling with a DNA nanocapsule.

Membrane-initiated steroid signaling (MISS) is a recently discovered aspect of steroidal control over cell function that has proved highly challenging to study due to its rapidity and ultrasensitivity to the steroid trigger [Chow RWY, Handelsman DJ, Ng MKC (2010) Endocrinology 151:2411-2422]. Fundamental aspects underlying MISS, such as receptor binding, kinetics of ion-channel opening, and production of downstream effector molecules remain obscure because a pristine molecular technology that could trigger the release of signaling steroids was not available. We have recently described a prototype DNA nanocapsule which can be programmed to release small molecules upon photoirradiation [Veetil AT, et al. (2017) Nat Nanotechnol 12:1183-1189]. Here we show that this DNA-based molecular technology can now be programmed to chemically trigger MISS, significantly expanding its applicability to systems that are refractory to photoirradiation.

Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules.

Achieving triggered release of small molecules with spatial and temporal precision at designated cells within an organism remains a challenge. By combining a cell-targetable, icosahedral DNA-nanocapsule loaded with photoresponsive polymers, we show cytosolic delivery of small molecules with the spatial resolution of single endosomes in specific cells in Caenorhabditis elegans. Our technology can report on the extent of small molecules released after photoactivation as well as pinpoint the location at which uncaging of the molecules occurred. We apply this technology to release dehydroepiandrosterone (DHEA), a neurosteroid that promotes neurogenesis and neuron survival, and determined the timescale of neuronal activation by DHEA, using light-induced release of DHEA from targeted DNA nanocapsules. Importantly, sequestration inside the DNA capsule prevents photocaged DHEA from activating neurons prematurely. Our methodology can in principle be generalized to diverse neurostimulatory molecules.

Nucleic Acid-Based Nanodevices in Biological Imaging.

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.