Parameterizing the Binding Properties of XNA Aptamers Isolated from a Low Stringency Selection.

Machine learning offers a guided approach to aptamer discovery, but more information is needed to develop algorithms that can intelligently identify high-performing aptamers to a broad array of targets. Critical to this effort is the need to experimentally parameterize the difference between low and high affinity binders to a given target. Although classical selection experiments help define the upper limit by converging on a small number of tight binding sequences, very little is known about the lower limit of binding that defines the boundary between binders and nonbinders. Here, we apply a quantitative approach to explore the diversity of aptamers isolated from two identical in vitro selections performed under low stringency conditions. Starting from a library of 1 trillion unique threose nucleic acid (TNA) sequences, 7 rounds of selection were performed to enrich binders to a known aptagenic target. High density sequencing of each round of selection followed by a detailed kinetic analysis of 136 TNA aptamers yielded a narrow range of equilibrium dissociation constants (KD = ∼ 1-15 nM) that were consistent between two experimental replicates. These findings offer insights into the lower limit of binding that may be expected for aptamers generated against aptagenic targets and could provide useful constraints for evaluating the results of experimental and computational approaches.

Os(II/III) complex supports pH-insensitive electrochemical DNA-based sensing with superior operational stability than the benchmark methylene blue reporter.

DNA-based electrochemical sensors use redox reporters to transduce affinity events into electrical currents. Ideally, such reporters must be electrochemically reversible, chemically stable for thousands of redox cycles, and tolerant to changing chemical environments. Here we report the first use of an Os(II/III) complex in DNA-based sensors, which undergoes pH-insensitive electron transfer with 35% better operational stability relative to the benchmark methylene blue, making it a promising reporter for continuous molecular monitoring applications where pH fluctuates with time.

Lymphangioleiomyomatosis: a metastatic lung disease.

Lymphangioleiomyomatosis (LAM) is a rare disease affecting women, caused by somatic mutations in the TSC1 or TSC2 genes, and driven by estrogen. Similar to many cancers, it is metastatic, primarily to the lung. Despite its monogenetic nature, like many cancers, LAM is a heterogeneous disease. The cellular constituents of LAM are very diverse, including mesenchymal, epithelial, endothelial, and immune cells. LAM is characterized by dysregulation of many cell signaling pathways, distinct populations of LAM cells, and a rich microenvironment, in which the immune system appears to play an important role. This review delineates the heterogeneity of LAM and focuses on the metastatic features of LAM, the deregulated signaling mechanisms and the tumor microenvironment. Understanding the tumor-host interaction in LAM may provide insights into the development of new therapeutic strategies, which could be combinatorial or superlative to Sirolimus, the current U.S. Food and Drug Administration-approved treatment.

Human Cyclophilin B Nuclease Activity Revealed via Nucleic Acid-Based Electrochemical Sensors.

Human cyclophilin B (CypB) is oversecreted by pancreatic cancer cells, making it a potential biomarker for early-stage disease diagnosis. Our group is motivated to develop aptamer-based assays to measure CypB levels in biofluids. However, human cyclophilins have been postulated to have collateral nuclease activity, which could impede the use of aptamers for CypB detection. To establish if CypB can hydrolyze electrode-bound nucleic acids, we used ultrasensitive electrochemical sensors to measure CypB's hydrolytic activity. Our sensors use ssDNA and dsDNA in the biologically predominant d-DNA form, and in the nuclease resistant l-DNA form. Challenging such sensors with CypB and control proteins, we unequivocally demonstrate that CypB can cleave nucleic acids. To our knowledge, this is the first study to use electrochemical biosensors to reveal the hydrolytic activity of a protein that is not known to be a nuclease. Future development of CypB bioassays will require the use of nuclease-resistant aptamer sequences.

A STING agonist preconditions against ischaemic stroke via an adaptive antiviral Type 1 interferon response.

Converging lines of inquiry have highlighted the importance of the Type I antiviral response not only in defending against viruses but also in preconditioning the brain against ischaemic stroke. Despite this understanding, treatments that foster brain resilience by driving antiviral interferon responses have yet to be developed for human use. Studies from our laboratory showed that tilorone, the first human antiviral immunomodulatory agent to be developed, robustly preconditioned against stroke in mice and rats. Tilorone is a DNA intercalator; therefore, we hypothesized that it stabilizes cytosolic DNA (released from the mitochondria or the nucleus), thereby activating cyclic GMP-AMP synthase, a homeostatic DNA sensor, and its downstream pathway. This pathway involves st imulator of in terferon g enes (STING), tank-binding kinase 1 (TBK1), and i nterferon r egulatory p rotein-3 and culminates in a protective Type I interferon response. We tested this hypothesis by examining the ability of structurally diverse small-molecule agonists of STING to protect against oxygen/glucose deprivation in vitro in mouse cortical cultures and in vivo against transient ischaemia in mice. The STING agonists significantly reduced cell death both in vitro and in vivo but failed to do so in STING knockout mice. As expected, STING agonist-induced protection was associated with the induction of interferon related genes and the effects could be abrogated in vitro by a TBK1 inhibitor. Taken together, these findings in mice identify STING as a therapeutic target for preconditioning the brain against ischaemic stroke in vitro and in vivo. Moreover, they suggest that clinically approved STING agonists such as Ganciclovir or α-Mangostin are candidate drugs that could be tested in humans as a prophylactic treatment to alleviate brain injury associated with ischaemic stroke.

Integration of chemically modified nucleotides with DNA strand displacement reactions for applications in living systems.

Watson-Crick base pairing rules provide a powerful approach for engineering DNA-based nanodevices with programmable and predictable behaviors. In particular, DNA strand displacement reactions have enabled the development of an impressive repertoire of molecular devices with complex functionalities. By relying on DNA to function, dynamic strand displacement devices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation in living systems has been a slow process due to several persistent challenges, including nuclease degradation. To circumvent these issues, researchers are increasingly turning to chemically modified nucleotides as a means to increase device performance and reliability within harsh biological environments. In this review, we summarize recent progress toward the integration of chemically modified nucleotides with DNA strand displacement reactions, highlighting key successes in the development of robust systems and devices that operate in living cells and in vivo. We discuss the advantages and disadvantages of commonly employed modifications as they pertain to DNA strand displacement, as well as considerations that must be taken into account when applying modified oligonucleotide to living cells. Finally, we explore how chemically modified nucleotides fit into the broader goal of bringing dynamic DNA nanotechnology into the cell, and the challenges that remain. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > Biosensing.

Kinetics of heterochiral strand displacement from PNA-DNA heteroduplexes.

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as 'heterochiral' strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction - strand displacement from PNA-DNA heteroduplexes - remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA-DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA-DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.

Nuclease Hydrolysis Does Not Drive the Rapid Signaling Decay of DNA Aptamer-Based Electrochemical Sensors in Biological Fluids.

Electrochemical aptamer-based (E-AB) sensors are a technology capable of real-time monitoring of drug concentrations directly in the body. These sensors achieve their selectivity from surface-attached aptamers, which alter their conformation upon target binding, thereby causing a change in electron transfer kinetics between aptamer-bound redox reporters and the electrode surface. Because, in theory, aptamers can be selected for nearly any target of interest, E-AB sensors have far-reaching potential for diagnostic and biomedical applications. However, a remaining critical weakness in the platform lies in the time-dependent, spontaneous degradation of the bioelectronic interface. This progressive degradation-seen in part as a continuous drop in faradaic current from aptamer-attached redox reporters-limits the in vivo operational life of E-AB sensors to less than 12 h, prohibiting their long-term application for continuous molecular monitoring in humans. In this work, we study the effects of nuclease action on the signaling lifetime of E-AB sensors, to determine whether the progressive signal loss is caused by hydrolysis of DNA aptamers and thus the loss of signaling moieties from the sensor surface. We continuously interrogate sensors deployed in several undiluted biological fluids at 37 °C and inject nuclease to reach physiologically relevant concentrations. By employing both naturally occurring d-DNA and the nuclease-resistant enantiomer l-DNA, we determine that within the current lifespan of state-of-the-art E-AB sensors, nuclease hydrolysis is not the dominant cause of sensor signal loss under the conditions we tested. Instead, signal loss is driven primarily by the loss of monolayer elements-both blocking alkanethiol and aptamer monolayers-from the electrode surface. While use of l-DNA aptamers may extend the E-AB operational life in the long term, the critical issue of passive monolayer loss must be addressed before those effects can be seen.

Mirror-Image Oligonucleotides: History and Emerging Applications.

As chiral molecules, naturally occurring d-oligonucleotides have enantiomers, l-DNA and l-RNA, which are comprised of l-(deoxy)ribose sugars. These mirror-image oligonucleotides have the same physical and chemical properties as that of their native d-counterparts, yet are highly orthogonal to the stereospecific environment of biology. Consequently, l-oligonucleotides are resistant to nuclease degradation and many of the off-target interactions that plague traditional d-oligonucleotide-based technologies; thus making them ideal for biomedical applications. Despite a flurry of interest during the early 1990s, the inability of d- and l-oligonucleotides to form contiguous Watson-Crick base pairs with each other has ultimately led to the perception that l-oligonucleotides have only limited utility. Recently, however, scientists have begun to uncover novel strategies to harness the bio-orthogonality of l-oligonucleotides, while overcoming (and even exploiting) their inability to Watson-Crick base pair with the natural polymer. Herein, a brief history of l-oligonucleotide research is presented and emerging l-oligonucleotide-based technologies, as well as their applications in research and therapy, are presented.

Ferroptosis in Neurons and Cancer Cells Is Similar But Differentially Regulated by Histone Deacetylase Inhibitors.

Ferroptotic death is a mechanism for tumor suppression by pharmacological inhibitors that target the Xc- transporter (cystine/glutamate antiporter) in a host of non-CNS and CNS tumors. Inhibition of this transporter leads to reduction of cystine uptake, cyst(e)ine deprivation, subsequent depletion of the versatile antioxidant glutathione, and reactive lipid species-dependent death. Accordingly, pharmacological inhibitors of the Xc- transporter can also induce neuronal cell death raising concerns about toxicity in the CNS and PNS if these agents are used for chemotherapy. Here, we show that ferroptotic death induced by the canonical ferroptosis inducer erastin is similar in HT1080 fibrosarcoma cells and primary cortical neurons although cell death is mediated more potently in cancer cells. Reducing the toxicity of ferroptosis inducers will require, among other things, the identification of agents that protect neurons from ferroptosis but exacerbate it in tumor cells. Although we show that a number of agents known to block ferroptosis in primary mouse neurons also inhibit ferroptosis in fibrosarcoma cells, class I histone deacetylase (HDAC) inhibitors selectively protect neurons while augmenting ferroptosis in cancer cells. Our results further suggest that cell death pathways induced by erastin in these two cell types are statistically identical to each other and identical to oxidative glutamate toxicity in neurons, where death is also mediated via inhibition of Xc- cystine transport. Together, these studies identify HDACs inhibitors as a novel class of agents to augment tumor suppression by ferroptosis induction and to minimize neuronal toxicity that could manifest as peripheral neuropathy or chemo brain.

Heterochiral Nucleic Acid Circuits.

The programmability of DNA/RNA-based molecular circuits provides numerous opportunities in the field of synthetic biology. However, the stability of nucleic acids remains a major concern when performing complex computations in biological environments. Our solution to this problem is L-(deoxy)ribose nucleic acids (L-DNA/RNA), which are mirror images (i.e. enantiomers) of natural D-nucleotides. L-oligonucleotides have the same physical and chemical properties as their natural counterparts, yet they are completely invisible to the stereospecific environment of biology. We recently reported a novel strand-displacement methodology for transferring sequence information between oligonucleotide enantiomers (which are incapable of base pairing with each other), enabling bio-orthogonal L-DNA/RNA circuits to be easily interfaced with living systems. In this perspective, we summarize these so-called "heterochiral" circuits, provide a viewpoint on their potential applications in synthetic biology, and discuss key problems that must be solved before achieving the ultimate goal of engineering complex and reliable functionality.

Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway.

The Cancer Genome Atlas (TCGA) data indicate that high MDM2 expression correlates with all subtypes of breast cancer. Overexpression of MDM2 drives breast oncogenesis in the presence of wild-type or mutant p53 (mtp53). Importantly, estrogen-receptor positive (ER+) breast cancers overexpress MDM2 and estrogen mediates this expression. We previously demonstrated that this estrogen-MDM2 axis activates the proliferation of breast cancer cell lines T47D (mtp53 L194F) and MCF7 (wild-type p53) in a manner independent of increased degradation of wild-type p53 (ie, p53-independently). Herein we present data supporting the role of the estrogen-MDM2 axis in regulating cell proliferation and mammary tissue architecture of MCF7 and T47D cells in a p53-independent manner. Inducible shRNA mediated MDM2 knockdown inhibited colony formation in soft agar, decreased mass size and induced lumen formation in matrigel and also significantly reduced mitosis as seen by decreased phospho-histone H3 positive cells. The knockdown of MDM2 in both cell lines decreased Rb phosphorylation and the level of E2F1 protein. This signaling was through the estrogen receptor because fulvestrant (a selective estrogen receptor degrader) decreased MDM2 protein levels and decreased phosphorylation of Rb. Taken together these data indicate that in some ER+ breast cancers the estrogen-MDM2-Rb-E2F1 axis is a central hub for estrogen-mediated p53-independent signal transduction. This is the first indication that estrogen signaling utilizes the estrogen-MDM2 axis to provoke phosphorylation of Rb and increase E2F1 while promoting abnormal mammary architecture.

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo.

BACKGROUND: Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. RESULTS: Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3ß ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3ß activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. CONCLUSIONS: Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3ß to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.

A p53-independent role of Mdm2 in estrogen-mediated activation of breast cancer cell proliferation.

INTRODUCTION: Estrogen receptor positive breast cancers often have high levels of Mdm2. We investigated if estrogen signaling in such breast cancers occurred through an Mdm2 mediated pathway with subsequent inactivation of p53. METHODS: We examined the effect of long-term 17ß-estradiol (E2) treatment (five days) on the p53-Mdm2 pathway in estrogen receptor alpha (ERα) positive breast cancer cell lines that contain wild-type p53 (MCF-7 and ZR75-1). We assessed the influence of estrogen by examining cell proliferation changes, activation of transcription of p53 target genes, p53-chromatin interactions and cell cycle profile changes. To determine the effects of Mdm2 and p53 knockdown on the estrogen-mediated proliferation signals we generated MCF-7 cell lines with inducible shRNA for mdm2 or p53 and monitored their influence on estrogen-mediated outcomes. To further address the p53-independent effect of Mdm2 in ERα positive breast cancer we generated cell lines with inducible shRNA to mdm2 using the mutant p53 expressing cell line T-47D. RESULTS: Estrogen increased the Mdm2 protein level in MCF-7 cells without decreasing the p53 protein level. After estrogen treatment of MCF-7 cells, down-regulation of basal transcription of p53 target genes puma and p21 was observed. Estrogen treatment also down-regulated etoposide activated transcription of puma, but not p21. Mdm2 knockdown in MCF-7 cells increased p21 mRNA and protein, decreased cell growth in 3D matrigel and also decreased estrogen-induced cell proliferation in 2D culture. In contrast, knockdown of p53 had no effect on estrogen-induced cell proliferation. In T-47D cells with mutant p53, the knockdown of Mdm2 decreased estrogen-mediated cell proliferation but did not increase p21 protein. CONCLUSIONS: Estrogen-induced breast cancer cell proliferation required a p53-independent role of Mdm2. The combined influence of genetic and environmental factors on the tumor promoting effects of estrogen implicated Mdm2 as a strong contributor to the bypass of cell cycle checkpoints. The novel finding that p53 was not the key target of Mdm2 in the estrogen activation of cell proliferation could have great benefit for future Mdm2-targeted breast cancer therapies.