Protein-to-DNA Converter with High Signal Gain.

DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

Design strategies for countering the effect of fluorophore-quencher labelling on DNA hairpin thermodynamics.

We report the impact of fluorophore-quencher labelling on the thermodynamics of hairpin opening by testing five fluorophores and two quenchers labelled at the end and/or internal positions. Two counter strategies were introduced, i.e. label the hairpin probe at an internal position or append an external hairpin stem on the trigger strand to promote coaxial stacking hybridization. The observations remained valid for complex hairpin opening operations such as hybridization chain reaction.

Protein-DNA Conjugates with a Discrete Number of Oligonucleotide Strands for Highly Reproducible Protein Quantification by the DNA Proximity Assay.

Protein-oligonucleotide conjugates are increasingly used as detection probes in biological applications such as proximity sensing and spatial biology. The preparation of high-quality conjugate probes as starting reagents is critical for achieving good and consistent performance, which we demonstrate via the DNA proximity assay (DPA) for the one-pot quantification of protein targets. We first established a complete conjugation and anion-exchange chromatography purification workflow to reproducibly obtain pure subpopulations of protein probes carrying a discrete number of oligonucleotide strands. A systematic study using the purified conjugate sub-populations confirmed that the order of conjugate (number of oligonucleotides per protein) and its purity (the absence of the unconjugated antibody) were important for ensuring optimal and reproducible assay performance. The streamlined workflow was then successfully used to conjugate a pair of universal DPA initiator oligonucleotides onto a wide range of binders including antibodies, nanobodies, and antigens which enabled the versatile detection of different types of proteins such as cytokines, total antibodies, and specific antibody isotypes. The good assay robustness (the inter-assay coefficient of variation lower than 5%) and linear calibration curve was achieved across all targets with just a single mix-and-incubate reaction step and a short reaction time of 30 min. We anticipate the streamlined protein-oligonucleotide probe preparation workflow developed in this work to have broad utility across applications leveraging the specificity of protein bio-recognition with the programmability of DNA hybridization.

Harnessing the Immunogenic Potential of Gold Nanoparticle-Based Platforms as a Therapeutic Strategy in Breast Cancer Immunotherapy: A Mini Review.

Breast cancer remains the most common malignancy among women worldwide. Although the implementation of mammography has dramatically increased the early detection rate, conventional treatments like chemotherapy, radiation therapy, and surgery, have significantly improved the prognosis for breast cancer patients. However, about a third of treated breast cancer patients are known to suffer from disease recurrences and progression to metastasis. Immunotherapy has recently gained traction due to its ability to establish long-term immune surveillance, and response for the prevention of disease recurrence and extension of patient survival. Current research findings have revealed that gold nanoparticles can enhance the safety and efficacy of cancer immunotherapy, through their unique intrinsic properties of good biocompatibility, durability, convenient surface modification, as well as enhanced permeability and retention effect. Gold nanoparticles are also able to induce innate immune responses through the process of immunogenic cell death, which can lead to the establishment of lasting adaptive immunity. As such gold nanoparticles are considered as good candidates for next generation immunotherapeutic strategies. This mini review gives an overview of gold nanoparticles and their potential applications in breast cancer immunotherapeutic strategies.

Dean migration of unfocused micron sized particles in low aspect ratio spiral microchannels.

We present an analysis of the microfluidic Dean migration of 2.5 µm particles, which do not meet focus criterion, in tall and low aspect ratio microchannels. We demonstrate the use of such low aspect ratio and tall spirals (h > 50 µm) for isolating high concentration (> 106 particles or cells/mL) micron sized particles without an initial off-chip dilution step. We specifically show the need for a sheath fluid for isolation and systematically analyze the particle stream profile (i.e. thickness and distance from the channel wall) as a function of downstream channel length and curvature ratio, with changes in the fluid velocity and the flow rate ratio of particles to sheath fluid (FRR). We also show that the width of the particle stream can control the particle migration and that a threshold stream width and Dean drag is necessary to initiate the particle stream migration from the channel wall. We then propose a design guide based on the selection of optimum curvatures, flow velocities and the FRRs required for achieving a narrow particle stream through a particular outlet. Finally, we use the design guide to demonstrate the isolation of bacteria from bladder epithelial cells.

Dynamically elongated associative toehold for tuning DNA circuit kinetics and thermodynamics.

Associative toehold is a powerful concept enabling efficient combinatorial computation in DNA circuit. A longer association length boosts circuit kinetics and equilibrium signal but results in higher leak rate. We reconcile this trade-off by using a hairpin lock design to dynamically elongate the effective associative toehold length in response to the input target. Design guidelines were established to achieve robust elongation without incurring additional leakages. Three hairpin initiators with different combinations of elongated associative toehold (4 → 6 nt, 5 → 8 nt and 6 → 9 nt) were shortlisted from the design framework for further discussion. The circuit performance improved in terms of reaction kinetics, equilibrium signal generated and limit of detection. Overall, the elongated associative toehold served as a built-in function to stabilize and favour the forward, desired reaction when triggered.

Design of Split Proximity Circuit as a Plug-and-Play Translator for Point Mutation Discrimination.

Point mutations are a common form of genetic variation and have been identified as important disease biomarkers. Conventional methods for analyzing point mutations, e.g., polymerase chain reaction (PCR), are based on differences in thermal stability of the DNA duplex, which require extensive optimization of the reaction condition and nontrivial design of sequence-selective primers. This motivated the design of molecular translators to convert molecular inputs into generic output sequences, which allows for the target recognition and signal generation regions to be designed independently. In this work, we propose a translator design based on the concept of split proximity circuit (SPC) to achieve both high sequence selectivity and assay robustness using a universal reaction condition, i.e., room temperature and constant ionic concentration. We discussed the design aspects of the SPC recognition regions and demonstrated its plug-and-play capability to discriminate different point mutations for both DNA (seven G6PD mutations) and RNA (let-7 microRNA family members) targets while retaining the same signal generation region. Despite its simple design and nonstringent assay condition requirements, the SPC retained good analytical performance to detect subnanomolar target concentration within a reasonable time of an hour.

Dynamic Stabilization of DNA Assembly by Using Pyrrole-Imidazole Polyamide.

We used N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamide as an exogenous agent to modulate the formation of DNA assemblies at specific double-stranded sequences. The concept was demonstrated on the hybridization chain reaction that forms linear DNA. Through a series of melting curve analyses, we demonstrated that the binding of Py-Im polyamide positively influenced both the HCR initiation and elongation steps. In particular, Py-Im polyamide was found to drastically stabilize the DNA duplex such that its thermal stability approached that of an equivalent hairpin structure. Also, the polyamide served as an anchor between hairpin pairs in the HCR assembly, thus improving the originally weak interstrand stability. We hope that these proof-of-concept results can inspire future use of Py-Im polyamide as a molecular tool to modulate the formation of DNA assemblies.

The role of spacer sequence in modulating turn-on fluorescence of DNA-templated silver nanoclusters.

Guanine activation of fluorescence in DNA templated silver nanoclusters (AgNCs) is an interesting physical phenomenon which has yet to be fully understood to date. While the individual role of cytosine and guanine has been established, there is still a knowledge gap on how the AgNC-DNA system switches from dark to bright state. Here, we present evidence on the universal role of the DNA spacer sequence in physically separating two Ag+-binding cytosine sites to maintain the dark state while holding them together for structural re-organization by the guanine-rich strand to activate the bright state. The extent of turn-on signal could be modulated by adjusting the spacer length and composition. The ATATA spacer sequence was found to have negligible dark state fluorescence and a turn-on effect of 2440-fold, which was almost five times of the highest factor reported to date.

Real-time monitoring of the Trojan-horse effect of silver nanoparticles by using a genetically encoded fluorescent cell sensor.

Silver nanoparticles (AgNPs) are widely incorporated into commercial products due to their antimicrobial properties. As a consequence, concerns about the adverse effects induced by AgNPs to humans and the environment need to be carefully examined. The existing literature reveals that AgNPs exhibit certain toxic effects, but it remains to be proved whether AgNPs or the ionic silver (Ag+) released from AgNPs are the main toxic species. Here, a genetically encoded fluorescent protein sensor with high affinity to Ag+ was developed. The resulting sensor, MT2a-FRET, was found to be ratiometric, sensitive and selective toward only Ag+ but inert against AgNPs. This makes this sensor a potential useful tool for monitoring the real-time intracellular dissolutions of AgNPs. Our data supported that AgNPs display the "Trojan-horse" mechanism, where AgNPs are internalized by cells and undergo dissolution intracellularly. We further found that cells exhibited a detoxification ability to remove active Ag+ from cells in 48 hours.

Localized Visualization and Autonomous Detection of Cell Surface Receptor Clusters Using DNA Proximity Circuit.

Cell surface receptors play an important role in mediating cell communication and are used as disease biomarkers and therapeutic targets. We present a one-pot molecular toolbox, which we term the split proximity circuit (SPC), for the autonomous detection and visualization of cell surface receptor clusters. Detection was powered by antibody recognition and a series of autonomous DNA hybridization to achieve localized, enzyme-free signal amplification. The system under study was the human epidermal growth factor receptor (HER) family, that is, HER2:HER2 homodimer and HER2:HER3 heterodimer, both in cell lysate and in situ on fixed whole cells. The detection and imaging of receptors were carried out using standard microplate scans and confocal microscopy, respectively. The circuit operated specifically with minimal leakages and successfully captured the receptor expression profiles on three cell types without any intermediate washing steps.

Gold nanoparticles induce serum amyloid A 1-Toll-like receptor 2 mediated NF-kB signaling in lung cells in vitro.

Gold nanoparticles (AuNPs) have emerging applications in biomedicine and the industry. Exposure to AuNPs has previously been shown to alter the transcriptional activity of nuclear factor kappa B (NF-kB), which is known to mediate physiological and pathological processes. This study seeks to provide mechanistic insights into AuNP-induced NF-kB activation in Small Airway Epithelial Cells (SAECs) in vitro. Increased NF-kB transcriptional activity (quantified by the luciferase reporter assay) was observed in AuNP-treated SAECs. Transcriptomic analysis revealed differential expression of 42 genes, which regulate functional processes that include cellular response to stimulus, chemicals and stress as well as immune response. Notably, the gene expression of serum amyloid A1 (SAA1), an acute phase protein and Toll-like receptor 2 (TLR2) were found to be up-regulated. As TLR2 is known to be a functional receptor of SAA1, a co-immunoprecipitation assay was performed. SAA1 was observed to be co-immunoprecipitated with the TLR2 protein and this protein-protein interaction was further supported by in silico computer based protein modeling. The present study suggests that AuNPs may potentially induce SAA1-TLR2-mediated NF-kB transcription factor activation in lung epithelial cells, highlighting that nano-bio interactions could result in biological effects that may affect cells.

miR-128 Regulates Genes Associated with Inflammation and Fibrosis of Rat Kidney Cells In Vitro.

microRNAs (miRNAs) regulate diverse cellular functions and signaling pathways via inhibiting the expression of their target genes. Given that miR-128 mediates mitogen-activated protein kinase signaling and production of reactive oxygen species and pro-inflammatory chemokines in various types of cells and tissues, and that miR-128 is differentially expressed in aged and diseased kidneys, we hypothesized that miR-128 may play key roles in kidney inflammation. Hence, in this study, we evaluated the biological effects of miR-128 in normal rat kidney (NRK) cells in vitro. Our results revealed that overexpression of miR-128 enhanced expression of genes associated with inflammation, pro-inflammatory cytokines and fibrosis in NRK cells. The recent reports showing that expression of miR-128 is increased in liver and lung fibrosis, together with the findings in this study, suggest that miR-128 may be a pro-fibrotic miRNA that regulates fibrosis in various tissues. Anat Rec, 301:913-921, 2018. © 2017 Wiley Periodicals, Inc.

Gold Nanoplate-Based 3D Hierarchical Microparticles: A Single Particle with High Surface-Enhanced Raman Scattering Enhancement.

Formation of intended nano- and microstructures with regular building blocks has attracted much attention because of their potential applications in the fields of optics, electronics, and catalysis. Herein, we report a novel strategy to spontaneously grow three-dimensional (3D) hierarchical cabbagelike microparticles (CLMPs) constructed by individual Au nanoplates. By reducing gold precursor to gold atoms, N-(3-amidino)-aniline (NAAN) itself was oxidized to form poly(N-(3-amidino)-aniline) (PNAAN), which specifically binds on Au(111) facet as a capping agent and which leads to the formation of gold nanoplates. Because of the incomplete coverage of Au(111) facet, new gold nanoplate growth sites were spontaneously generated from the crystal plane of existing Au nanoplates for the growth of other nanoplates. This process continued until the nanoplate density reached its maximum range, eventually resulting in CLMPs with well-controlled structures. This opens a new avenue to utilize the imperfection during nanoparticle (NP) growth for the construction of microstructures. The individual CLMP shows excellent surface-enhanced Raman scattering (SERS) performance with high enhancement factor (EF) and good reproducibility as it integrates the SERS enhancement effects of individual Au nanoplate and the nanogaps formed by the uniform and hierarchical structures.

Formation and Self-assembly of Gold Nanoplates through an Interfacial Reaction for Surface-Enhanced Raman Scattering.

3D hierarchical architectures assembled from individual particles have attracted great interest because they displayed novel properties from the individual building blocks as well as their complex structures. Here we present a new strategy to form 3D hierarchical gold (Au) nanostructures via an interfacial reduction reaction. An aniline (ANI) derivative, N-(3-amidino)-aniline (NAAN), and HAuCl4 were separately dissolved in toluene and water to form an organic/water interface. Au nanoplates formed at the interface and subsequently moved to the aqueous phase. As a capping agent for the nanoplate formation, the oxidized NAAN, i.e., poly(N-(3-amidino)-aniline) (PNAAN), also facilitated the self-assembly of Au nanoplates into 3D hierarchical Au nanoflowers (AuNFs) through π-π stacking. The individual AuNF exhibited good surface-enhanced Raman scattering (SERS) response both in enhancement factor and reproducibility because it integrates the SERS enhancement effects of individual Au nanoplates and their hierarchical structures. This is the first report depicting the one-pot formation and self-assembly of Au nanoplates into 3D organized hierarchical nanostructures through the molecular interaction of conducting polymer.

Engineering a robust DNA split proximity circuit with minimized circuit leakage.

DNA circuit is a versatile and highly-programmable toolbox which can potentially be used for the autonomous sensing of dynamic events, such as biomolecular interactions. However, the experimental implementation of in silico circuit designs has been hindered by the problem of circuit leakage. Here, we systematically analyzed the sources and characteristics of various types of leakage in a split proximity circuit which was engineered to spatially probe for target sites held within close proximity. Direct evidence that 3'-truncated oligonucleotides were the major impurity contributing to circuit leakage was presented. More importantly, a unique strategy of translocating a single nucleotide between domains, termed 'inter-domain bridging', was introduced to eliminate toehold-independent leakages while enhancing the strand displacement kinetics across a three-way junction. We also analyzed the dynamics of intermediate complexes involved in the circuit computation in order to define the working range of domain lengths for the reporter toehold and association region respectively. The final circuit design was successfully implemented on a model streptavidin-biotin system and demonstrated to be robust against both circuit leakage and biological interferences. We anticipate that this simple signal transduction strategy can be used to probe for diverse biomolecular interactions when used in conjunction with specific target recognition moieties.

Detection of G-Quadruplex Formation via Light Scattering of Defined Gold Nanoassemblies Modulated by Molecular Hairpins.

G-quadruplexes are of great scientific interest, as these unique DNA structures play key regulatory roles in cell replication, such as safeguarding against uncontrolled cellular divisions. The quadruplexes have also been applied for detecting DNA and protein biomarkers via methods like fluorescence resonance energy transfer (FRET) and gold nanoparticle (AuNP) aggregation. As an alternative and complementary platform to the established molecular techniques for the study of quadruplexes, we have developed a strategy coupling poly-G (PG)-mediated quadruplex formation with AuNP assembly detectable via dynamic light scattering (DLS). The presence of quadruplex-forming sequences also uniquely modifies the AuNP nanoassembly readout on DLS. In addition, molecular hairpins co-attached onto the AuNP together with PG successfully modulated the quadruplex-induced nanoassembly. Through molecular beacon-based fluorescence restoration and light scattering signal changes, the open/closed conformations of the hairpins are leveraged to tune the size of the quadruplex-mediated nanoassembly.

Rational design of hybridization chain reaction monomers for robust signal amplification.

We established four-point guidelines for the sequence design of hairpin monomers in hybridization chain reaction (HCR). This enabled greater flexibility to customize specific hairpin sequences for use with the readout platform of interest. Using shorter hairpin stem length, a one-pot signal amplification system was demonstrated by incorporating distance-sensitive Förster resonance energy transfer (FRET) readout.

Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis.

Silver nanoparticles (AgNPs), one of the most popular nanomaterials, are commonly used in consumer products and biomedical devices, despite their potential toxicity. Recently, AgNP exposure was reported to be associated with male reproductive toxicity in mammalian models. However, there is still a limited understanding of the effects of AgNPs on spermatogenesis. The fruit fly Drosophila testis is an excellent in vivo model to elucidate the mechanisms underlying AgNP-induced defects in spermatogenesis, as germ lineages can be easily identified and imaged. In this study, we evaluated AgNP-mediated toxicity on spermatogenesis by feeding Drosophila with AgNPs at various concentrations. We first observed a dose-dependent uptake of AgNPs in vivo. Concomitantly, AgNP exposure caused a significant decrease in the viability and delay in the development of Drosophila in a dose-dependent manner. Furthermore, AgNP-treated male flies showed a reduction in fecundity, and the resulting testes contained a decreased number of germline stem cells (GSCs) compared to controls. Interestingly, testes exposed to AgNPs exhibited a dramatic increase in reactive oxygen species levels and showed precocious GSC differentiation. Taken together, our study suggests that AgNP exposure may increase ROS levels in the Drosophila testis, leading to a reduction of GSC number by promoting premature GSC differentiation.

Inflammatory Changes in Lung Tissues Associated with Altered Inflammation-Related MicroRNA Expression after Intravenous Administration of Gold Nanoparticles in Vivo.

Potential adverse effects of gold nanoparticles (AuNPs) are gaining attention due to their wide industrial, consumer, and biomedical applications. This may give rise to possible health risks from direct exposure to the NPs. Excessive inflammatory response is known to be one of the main effects induced by NPs. In this study, inflammatory and miRNA expression changes in lung tissues were evaluated in rats following intravenous administration of AuNPs. AuNPs (20 nm) at a mass concentration of 256 µg/mL were intravenously injected into 6-8 week old male Wistar rats at single doses of 0.025, 0.05, 0.1, and 0.2 mg/kg and sacrificed at 1 week, 1 month, and 2 months, respectively. The biodistribution of AuNPs in the lungs of the rats was determined by inductively coupled plasma mass spectrometry. There were no apparent changes observed in the body weight of the experimental rats. Histopathological examination revealed the presence of infiltrating lymphocytes in lung interstitial tissues and enhanced IL-1α immunostaining in the lung tissues. Out of 84 rat microRNAs (miRNAs) analyzed, the expression of three miRNAs in rat lungs were dysregulated by more than 2-fold in the 0.1 and 0.2 mg/kg AuNP-treated rats 1 week after exposure. In particular, miR-327 was significantly down-regulated in both groups of treated rats. Taken together, it would seem that miRNAs may regulate inflammatory changes in the lungs after exposure to AuNPs in vivo.