Hanyong Peng

Binding‐Induced DNA Nanomachines Triggered by Proteins and Nucleic Acids

We introduce the concept and operation of a binding-induced DNA nanomachine that can be activated by proteins and nucleic acids. This new type of nanomachine harnesses specific target binding to trigger assembly of separate DNA components that are otherwise unable to spontaneously assemble. Three-dimensional DNA tracks of high density are constructed on gold nanoparticles functionalized with hundreds of single-stranded oligonucleotides and tens of an affinity ligand. A DNA swing arm, free in solution, is linked to a second affinity ligand. Binding of a target molecule to the two ligands brings the swing arm to AuNP and initiates autonomous, stepwise movement of the swing arm around the AuNP surface. The movement of the swing arm, powered by enzymatic cleavage of conjugated oligonucleotides, cleaves hundreds of oligonucleotides in response to a single binding event. We demonstrate three nanomachines that are specifically activated by streptavidin, platelet-derived growth factor, and the Smallpox gene. Substituting the ligands enables the nanomachine to respond to other molecules. The new nanomachines have several unique and advantageous features over DNA nanomachines that rely on DNA self-assembly.

A microRNA-initiated DNAzyme motor operating in living cells

Synthetic DNA motors have great potential to mimic natural protein motors in cells but the operation of synthetic DNA motors in living cells remains challenging and has not been demonstrated. Here we report a DNAzyme motor that operates in living cells in response to a specific intracellular target. The whole motor system is constructed on a 20 nm gold nanoparticle (AuNP) decorated with hundreds of substrate strands serving as DNA tracks and dozens of DNAzyme molecules each silenced by a locking strand. Intracellular interaction of a target molecule with the motor system initiates the autonomous walking of the motor on the AuNP. An example DNAzyme motor responsive to a specific microRNA enables amplified detection of the specific microRNA in individual cancer cells. Activated by specific intracellular targets, these self-powered DNAzyme motors will have diverse applications in the control and modulation of biological functions.

Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing ☆

The occurrence of a large number of diverse arsenic species in the environment and in biological systems makes it important to compare their relative toxicity. The toxicity of arsenic species has been examined in various cell lines using different assays, making comparison difficult. We report real-time cell sensing of two human cell lines to examine the cytotoxicity of fourteen arsenic species: arsenite (AsIII), monomethylarsonous acid (MMAIII) originating from the oxide and iodide forms, dimethylarsinous acid (DMAIII), dimethylarsinic glutathione (DMAGIII), phenylarsine oxide (PAOIII), arsenate (AsV), monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV), monomethyltrithioarsonate (MMTTAV), dimethylmonothioarsinate (DMMTAV), dimethyldithioarsinate (DMDTAV), 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox), and 4-aminobenzenearsenic acid (p-arsanilic acid, p-ASA). Cellular responses were measured in real time for 72hr in human lung (A549) and bladder (T24) cells. IC50 values for the arsenicals were determined continuously over the exposure time, giving rise to IC50 histograms and unique cell response profiles. Arsenic accumulation and speciation were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS). On the basis of the 24-hr IC50 values, the relative cytotoxicity of the tested arsenicals was in the following decreasing order: PAOIII≫MMAIII≥DMAIII≥DMAGIII≈DMMTAV≥AsIII≫MMTTAV>AsV>DMDTAV>DMAV>MMAV≥Rox≥p-ASA. Stepwise shapes of cell response profiles for DMAIII, DMAGIII, and DMMTAV coincided with the conversion of these arsenicals to the less toxic pentavalent DMAV. Dynamic monitoring of real-time cellular responses to fourteen arsenicals provided useful information for comparison of their relative cytotoxicity.

Arsenic Species in Chicken Breast: Temporal Variations of Metabolites, Elimination Kinetics, and Residual Concentrations.

Background: Chicken meat has the highest per capita consumption among all meat types in North America. The practice of feeding 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox) to chickens lasted for more than 60 years. However, the fate of Rox and arsenic metabolites remaining in chicken are poorly understood. Objectives: We aimed to determine the elimination of Rox and metabolites from chickens and quantify the remaining arsenic species in chicken meat, providing necessary information for meaningful exposure assessment. Methods: We have conducted a 35-day feeding experiment involving 1,600 chickens, of which half were control and the other half were fed a Rox-supplemented diet for the first 28 days and then a Rox-free diet for the final 7 days. We quantified the concentrations of individual arsenic species in the breast meat of 229 chickens. Results: Rox, arsenobetaine, arsenite, monomethylarsonic acid, dimethylarsinic acid, and a new arsenic metabolite, were detected in breast meat from chickens fed Rox. The concentrations of arsenic species, except arsenobetaine, were significantly higher in the Rox-fed than in the control chickens. The half-lives of elimination of these arsenic species were 0.4–1 day. Seven days after termination of Rox feeding, the concentrations of arsenite (3.1 μg/kg), Rox (0.4 μg/kg), and a new arsenic metabolite (0.8 μg/kg) were significantly higher in the Rox-fed chickens than in the control. Conclusion: Feeding of Rox to chickens increased the concentrations of five arsenic species in breast meat. Although most arsenic species were excreted rapidly when the feeding of Rox stopped, arsenic species remaining in the Rox-fed chickens were higher than the background levels. Citation: Liu Q, Peng H, Lu X, Zuidhof MJ, Li XF, Le XC. 2016. Arsenic species in chicken breast: temporal variations of metabolites, elimination kinetics, and residual concentrations. Environ Health Perspect 124:1174–1181; http://dx.doi.org/10.1289/ehp.1510530

Enzyme-assisted extraction and liquid chromatography mass spectrometry for the determination of arsenic species in chicken meat

Chicken is the most consumed meat in North America. Concentrations of arsenic in chicken range from μg kg(-1) to mg kg(-1). However, little is known about the speciation of arsenic in chicken meat. The objective of this research was to develop a method enabling determination of arsenic species in chicken breast muscle. We report here enzyme-enhanced extraction of arsenic species from chicken meat, separation using anion exchange chromatography (HPLC), and simultaneous detection with both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESIMS). We compared the extraction of arsenic species using several proteolytic enzymes: bromelain, papain, pepsin, proteinase K, and trypsin. With the use of papain-assisted extraction, 10 arsenic species were extracted and detected, as compared to 8 detectable arsenic species in the water/methanol extract. The overall extraction efficiency was also improved using a combination of ultrasonication and papain digestion, as compared to the conventional water/methanol extraction. Detection limits were in the range of 1.0-1.8 μg arsenic per kg chicken breast meat (dry weight) for seven arsenic species: arsenobetaine (AsB), inorganic arsenite (As(III)), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), inorganic arsenate (As(V)), 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone), and N-acetyl-4-hydroxy-m-arsanilic acid (NAHAA). Analysis of breast meat samples from six chickens receiving feed containing Roxarsone showed the presence of (mean±standard deviation μg kg(-1)) AsB (107±4), As(III) (113±7), As(V) (7±2), MMA (51±5), DMA (64±6), Roxarsone (18±1), and four unidentified arsenic species (approximate concentration 1-10 μg kg(-1)).

Arsenic Metabolites, Including N-Acetyl-4-hydroxy-m-arsanilic Acid, in Chicken Litter from a Roxarsone-Feeding Study Involving 1600 Chickens

The poultry industry has used organoarsenicals, such as 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, ROX), to prevent disease and to promote growth. Although previous studies have analyzed arsenic species in chicken litter after composting or after application to agricultural lands, it is not clear what arsenic species were excreted by chickens before biotransformation of arsenic species during composting. We describe here the identification and quantitation of arsenic species in chicken litter repeatedly collected on days 14, 24, 28, 30, and 35 of a Roxarsone-feeding study involving 1600 chickens of two strains. High performance liquid chromatography separation with simultaneous detection by both inductively coupled plasma mass spectrometry and electrospray ionization tandem mass spectrometry provided complementary information necessary for the identification and quantitation of arsenic species. A new metabolite, N-acetyl-4-hydroxy-m-arsanilic acid (N-AHAA), was identified, and it accounted for 3-12% of total arsenic. Speciation analyses of litter samples collected from ROX-fed chickens on days 14, 24, 28, 30, and 35 showed the presence of N-AHAA, 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), inorganic arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), and ROX. 3-AHPAA accounted for 3-19% of the total arsenic. Inorganic arsenicals (the sum of As(III) and As(V)) comprised 2-6% (mean 3.5%) of total arsenic. Our results on the detection of inorganic arsenicals, methylarsenicals, 3-AHPAA, and N-AHAA in the chicken litter support recent findings that ROX is actually metabolized by the chicken or its gut microbiome. The presence of the toxic metabolites in chicken litter is environmentally relevant as chicken litter is commonly used as fertilizer.

Methylated and thiolated arsenic species for environmental and health research — A review on synthesis and characterization

Hundreds of millions of people around the world are exposed to elevated concentrations of inorganic and organic arsenic compounds, increasing the risk of a wide range of health effects. Studies of the environmental fate and human health effects of arsenic require authentic arsenic compounds. We summarize here the synthesis and characterization of more than a dozen methylated and thiolated arsenic compounds that are not commercially available. We discuss the methods of synthesis for the following 14 trivalent (III) and pentavalent (V) arsenic compounds: monomethylarsonous acid (MMAIII), dicysteinylmethyldithioarsenite (MMAIII(Cys)2), monomethylarsonic acid (MMAV), monomethylmonothioarsonic acid (MMMTAV) or monothio-MMAV, monomethyldithioarsonic acid (MMDTAV) or dithio-MMAV, monomethyltrithioarsonate (MMTTAV) or trithio-MMAV, dimethylarsinous acid (DMAIII), dimethylarsino-glutathione (DMAIII(SG)), dimethylarsinic acid (DMAV), dimethylmonothioarsinic acid (DMMTAV) or monothio-DMAV, dimethyldithioarsinic acid (DMDTAV) or dithio-DMAV, trimethylarsine oxide (TMAOV), arsenobetaine (AsB), and an arsenicin-A model compound. We have reviewed and compared the available methods, synthesized the arsenic compounds in our laboratories, and provided characterization information. On the basis of reaction yield, ease of synthesis and purification of product, safety considerations, and our experience, we recommend a method for the synthesis of each of these arsenic compounds.

An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes

Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (10(16) and 10(14)) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind preferentially to the M11 M-type of S. pyogenes. Estimated binding dissociation constants (Kd) were in the low nanomolar range for the M11 specific sequences; for example, sequence E-CA20 had a Kd of 7±1 nM. These affinities are comparable to those of a monoclonal antibody. The improved bacterial cell-SELEX technique is successful in generating aptamers selective for S. pyogenes and some of its M-types. These aptamers are potentially useful for detecting S. pyogenes, achieving binding profiles of the various M-types, and developing new M-typing technologies for non-specialized laboratories or point-of-care testing.

A Target-Triggered DNAzyme Motor Enabling Homogeneous,Amplified Detection of Proteins

We report here the concept of a self-powered, target-triggered DNA motor constructed by engineering a DNAzyme to adapt into binding-induced DNA assembly. An affinity ligand was attached to the DNAzyme motor via a DNA spacer, and a second affinity ligand was conjugated to the gold nanoparticle (AuNP) that was also decorated with hundreds of substrate strands serving as a high-density, three-dimensional track for the DNAzyme motor. Binding of a target molecule to the two ligands induced hybridization between the DNAzyme and its substrate on the AuNP, which are otherwise unable to spontaneously hybridize. The hybridization of DNAzyme with the substrate initiates the cleavage of the substrate and the autonomous movement of the DNAzyme along the AuNP. Each moving step restores the fluorescence of a dye molecule, enabling monitoring of the operation of the DNAzyme motor in real time. A simple addition or depletion of the cofactor Mg2+ allows for fine control of the DNAzyme motor. The motor can translate a single binding event into cleavage of hundreds of substrates, enabling amplified detection of proteins at room temperature without the need for separation.

p‐Azidophenylarsenoxide: An Arsenical “Bait” for the In Situ Capture and Identification of Cellular Arsenic‐Binding Proteins

Identification of arsenic-binding proteins is important for understanding arsenic health effects and for developing arsenic-based therapeutics. We report here a strategy for the capture and identification of arsenic-binding proteins in living cells. We designed an azide-labeled arsenical, p-azidophenylarsenoxide (PAzPAO), to serve bio-orthogonal functions: the trivalent arsenical group binds to cellular proteins in situ, and the azide group facilitates click chemistry with dibenzylcyclooctyne. The selective and efficient capture of arsenic-binding proteins enables subsequent enrichment and identification by shotgun proteomics. Applications of the technique are demonstrated using the A549 human lung carcinoma cells and two in vitro model systems. The technique enables the capture and identification of 48 arsenic-binding proteins in A549 cells incubated with PAzPAO. Among the identified proteins are a series of antioxidant proteins (e.g., thioredoxin, peroxiredoxin, peroxide reductase, glutathione reductase, and protein disulfide isomerase) and glyceraldehyde-3-phosphate dehydrogenase. Identification of these functional proteins, along with studies of arsenic binding and enzymatic inhibition, points to these proteins as potential molecular targets that play important roles in arsenic-induced health effects and in cancer treatment.