A DNA nanomachine that maps spatial and temporal pH changes inside living cells

DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upon stimulation by external triggers. Previously, the performance of DNA devices has been limited to in vitro applications. Here we report the construction of a DNA nanomachine called the I-switch, which is triggered by protons and functions as a pH sensor based on fluorescence resonance energy transfer (FRET) inside living cells. It is an efficient reporter of pH from pH 5.5 to 6.8, with a high dynamic range between pH 5.8 and 7. To demonstrate its ability to function inside living cells we use the I-switch to map spatial and temporal pH changes associated with endosome maturation. The performance of our DNA nanodevices inside living systems illustrates the potential of DNA scaffolds responsive to more complex triggers in sensing, diagnostics and targeted therapies in living systems

Structural DNA nanotechnology: from bases to bricks, from structure to function.

ABSTRACT The two fields of structural DNA nanotechnology and functional nucleic acids have been independently coevolving, with the former seeking to arrange and bring about movement of nucleic acid modules precisely and with control in space and the latter producing modules with incredible diversity in effective recognition and function. Here, we track the key developments in structural DNA nanotechnology that reveal a current trend that is seeing the integration of functional nucleic acid modules into their architectures to access a range of new functions. This contribution will seek to provide a perspective for the field of structural DNA nanotechnology where the integration of such functional modules on precisely controlled architectures can uncover phenomena of interest to physical chemists. D NA has proven to be a powerful material for construction on the nanoscale on the basis of the following properties: (i) the availability of automated synthetic methods and continually dropping costs, (ii) chemical robustness that confers stability on the resultant architectures and their subsequent ability to be functional under a variety of environmental conditions, (iii) the uniformly rodlike nature of the DNA double helix irrespective of its primary sequence, (iv) the specificity of Watson-Crick base pairing, which functions as an easily engineerable, site-specific, molecular-scale glue applicable to any DNA double helix, (v) the periodic nature of the DNA double helix and the predictable nature of sequence-specific thermal stability, both of which predispose it to computational methods to design and fabricate superarchitectures, (vi) the availability of well-characterized biochemical and molecular biological methods to cut, copy, and covalently link B-DNA double helices sequencespecifically, which allows manipulation of the construction material, (vii) the modular nature of the DNA scaffold that allows fabrication of architectures that are complex in terms of both structure and function when multiple modules are appended to each other, and (viii) single-stranded DNA sequences, called functional nucleic acids, which can fold and offer three-dimensional cavities suited to bind with great specificity a range of molecular entities with diverse function. In 1982, Ned Seeman proposed that DNA, which until then had been thought of as a linear polymer, could be used to make branched architectures by using stable artificial junctions with helical DNA limbs radiating from a central node. 1 These structures were analogous to metastable naturally occurring DNA motifs, such as the replication fork and Holliday junction. "It appears to be possible to generate covalently joined...networks of nucleic acids which are periodic in connectivity and perhaps in space." 1 This marked the origin of structural DNA nanotechnology that seeks to create defined architectures on the nanoscale using sequences of DNA that self-assemble into rigid rods that are, in turn, connected to form superarchitectures of precise dimensions. In 1999, it was shown that DNA could switch between two forms (the B-form and the Z-form), and this motion could be transduced along a DNA architecture, making it undergo a twisting motion. 2 Thus began a complementary aspect of structural DNA nanotechnology, of bringing about defined molecular-scale movements of DNA architectures triggered by the addition of input stimuli that are chemical, photonic, thermal, or electrical in nature. Functional nucleic acids are obtained from a test tube evolution method called SELEX independently conceptualized by the Szostak and Gold groups. 3,4 It uses molecular biology tools to pick out from a library of ∼10 15 different DNA (or RNA) sequences, a subset of sequences based on a given selection criterion and amplify them. 5 When subjected to the same selection criterion repeatedly with progressively higher stringencies, it is possible to progressively enrich from the library, a pool of DNA (or RNA) sequences with a specific functionality. If the selection criterion is the recognition of a target molecule, then selected single-stranded DNA (ssDNA) sequences are capable of binding to the target with high specificity and affinity. Thus, SELEX has yielded DNA sequences that can bind a huge variety of chemical entities ranging from small molecules to proteins, peptides, transition-stat

The non-adrenergic imidazoline-1 receptor protein nischarin is a key regulator of astrocyte glutamate uptake

Astrocytic GLT-1 is the main glutamate transporter involved in glutamate buffering in the brain, pivotal for glutamate removal at excitatory synapses to terminate neurotransmission and for preventing excitotoxicity. We show here that the surface expression and function of GLT-1 can be rapidly modulated through the interaction of its N-terminus with the nonadrenergic imidazoline-1 receptor protein, Nischarin. The phox domain of Nischarin is critical for interaction and internalization of surface GLT-1. Using live super-resolution imaging, we found that glutamate accelerated Nischarin-GLT-1 internalization into endosomal structures. The surface GLT-1 level increased in Nischarin knockout astrocytes, and this correlated with a significant increase in transporter uptake current. In addition, Nischarin knockout in astrocytes is neuroprotective against glutamate excitotoxicity. These data provide new molecular insights into regulation of GLT-1 surface level and function and suggest new drug targets for the treatment of neurological disorders

The PNA–DNA hybrid I-motif: implications for sugar–sugar contacts in i-motif tetramerization

We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands from an equimolar mixture of a C-rich DNA and analogous PNA sequence. Nano-electrospray ionization mass spectrometry confirmed the formation of a tetrameric species, composed of PNA–DNA heteroduplexes. Thermal denaturation and CD experiments revealed that the structure was held together by C-H(+)-C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H(+)-C base pairs per heteroduplex. The imino protons are protected from D(2)O exchange suggesting intercalation of the heteroduplexes as seen in DNA(4) i-motifs. FRET established the relative DNA and PNA strand polarities in the hybrid. The DNA strands were arranged antiparallel with respect to one another. The same topology was observed for PNA strands. Fluorescence quenching revealed that both PNA–DNA parallel heteroduplexes are intercalated, such that both DNA strands occupy one of the narrow grooves. H1′–H1′ NOEs show that both heteroduplexes are fully intercalated and that both DNA strands are disposed towards a narrow groove, invoking sugar–sugar interactions as seen in DNA(4) i-motifs. The hybrid i-motif shows enhanced thermal stability, intermediate pH dependence and forms at relatively low concentrations making it an ideal nanoscale structural element for pH-based molecular switches. It also serves as a good model system to assess the contribution of sugar–sugar contacts in i-motif tetramerization

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes

Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways

International audienceDNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a 'handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the 'handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications

Neuronal control of lipid metabolism by STR‐2 G protein‐coupled receptor promotes longevity in Caenorhabditis elegans

The G protein-coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. Here, we show that C. elegans longevity is regulated by a chemosensory GPCR STR-2, expressed in AWC and ASI amphid sensory neurons. STR-2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR-2 regulates expression of delta-9 desaturases, fat-5, fat-6 and fat-7, and of diacylglycerol acyltransferase dgat-2. Rescue of the STR-2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat-5, dgat-2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wild-type levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR-2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.</p