DNA-conjugated Gold Research Articles

Hollow Polyethyleneimine Nanoparticles with Drug Loaded DNA for Chemotherapeutic Applications.

The next generation of anticancer agents are emerging from rationally designed nanostructured materials. This work involved the synthesis and characterization of novel hollow DNA-conjugated gold nanoparticles (DNA-AuNPs) for controlled drug delivery. Polyethyleneimine (PEI) was bound to AuNPs, forming polymer-shell nanoparticles. Dissolution of the gold core via iodine formed hollow core polymeric nanoparticles (HCPNPs) and a high density (85 molecules/particle) of DNA intercalated with daunorubicin was conjugated. Particles were spherical with an average diameter of 105.7±17.3 nm and zeta potential of 20.4±3.54 mV. We hypothesize the DNA backbone electrostatically condensed to the primary amines on the surface of the particle toroidally, weaving itself within the polymer shell. During the DNA intercalation process, increasing the ionic concentration and decreasing the amine/phosphate ratio 10-fold increased drug intercalation 64 % and 61 %, respectively, allowing us to determine the optimal method of particle synthesis. As intercalation sites increased with increasing DNA strand length, drug loading increased. An average of 874±40.1 daunorubicin molecules were loaded per HCPNP. HCPNPs with drug intercalated DNA have strong potential to be clinically efficacious drug delivery vehicles due to the versatility of DNA and high drug loading capacities.

A microchamber-free and enzyme-free digital assay based on ultrabright fluorescent microspheres

Digital analysis is an effective single-molecule detection method and has attracted extensive attention in the field of bioassays. However, most digital assays require microchambers for signal compartmentalization. Herein, we developed a microchamber-free and enzyme-free digital assay by labeling paramagnetic beads directly with ultrabright fluorescent microspheres. In this assay, a DNA sandwich analysis was firstly performed on the bead to label a fluorescent microsphere. Then, the beads were loaded on the glass slide to form a monolayer film for signal readout. The whole analysis process does not require the participation of enzymes and the preparation of microchambers, which greatly simplifies the experimental steps and saves the costs. Furthermore, by introducing non-enzymatic hybridization chain reaction (HCR) and biotinylated DNA-conjugated gold nanoparticles (Au NPs-Bio), the capture efficiency and analytical sensitivity were improved. As a proof of concept, single-stranded DNA (ssDNA) of SARS-CoV-2 fragment was chosen as a model, and a detection limit of 1.5 fM was achieved. Spiked and recovery experiments on human serum and saliva samples validated the good performance of the proposed digital assay in real biological samples. The proposed assay provides a facile way of signal generation and readout for digital analysis.

Aggregation of DNA-Grafted Nanoparticles in Water: The Critical Role of Sequence-Dependent Conformation of DNA Coating.

Aggregation behavior of nanoparticles in water, particularly in the presence of surface coating, is a fundamental behavior to understand as it closely relates to the material function, reactivity, and toxicity. Engineered DNA, an important type of surface coating, is essentially different from conventional polymeric coatings with homogeneous repeating units. The heterogeneous nature of DNA with numerous combinations of the four nucleotides poses a challenge to understand the aggregation of nanoparticles coated with them. In this study, we use both experimental and computational approaches to study how DNA properties influence the aggregation of DNA-conjugated gold nanoparticles (DNA-AuNPs). Aggregation kinetics under different pH values and in three salts (NaCl, CaCl2, and MgCl2) were investigated. Overall, DNA-AuNPs demonstrated similar aggregation behavior of electrosterically stabilized nanoparticles, where the surface coating layer thickness played a determining role. The underlying interactions between DNA-AuNPs were analyzed by an extended DLVO model, and the excluded volume repulsion, which varied as a function of layer thickness, was the dominant stabilizing repulsion. Interestingly, the DNA sequence was found to cause different layer thicknesses and thus profoundly different aggregation behaviors in the presence of cations. The interactions between DNA strands with different sequences and surrounding counterions were investigated via molecular dynamics simulations. The simulation results were consistent with experimental observations and supported the sequence-dependent conformational change of DNA coating. Our study provides new molecular-level insights into aggregation of nanoparticles with heterogeneous surface coating such as DNA.

Facile construction of targeted pH-responsive DNA-conjugated gold nanoparticles for synergistic photothermal-chemotherapy

Recently, stimuli-responsive DNA nanostructure-based nanodevices have been applied for cancer therapy. In this study, pH-responsive i-motif DNA was modified on gold nanoparticles (AuNPs) via a facile, time-saving freeze-thaw method and utilized to construct stimuli-responsive drug nanocarriers. When the environment pH changes from 7.4 to 5.0, the i-motif DNA would be folded into four-stranded (C-quadruplex) that could be characterized by circular dichroism, and the characteristic of acid stimulate was verified by fluorescence resonance energy transfer (FRET). To enhance specifical cellular uptake, MUC1 aptamer was employed as the targeting moiety. Doxorubicin (Dox) is an anticancer drug that can be efficiently intercalated into GC base pairs of DNA nanostructure to form drug-loaded nanovehicles (Dox@AuNP-MUC1). Additionally, owing to the excellent photothermal conversion efficiency of AuNPs, the synergistic effect between chemotherapy and PTT can be readily achieved by 808 nm near-infrared (NIR) irradiation, which exhibits specifically and efficiently anticancer efficiency. Hence, this multifunctional drug carrier shows the potential for synergistic photothermal-chemotherapy.

A syringe-aided apta-nanosensing method for colorimetric determination of acetamiprid

A syringe-aided apta-nanosensing method is reported for the colorimetric determination of acetamiprid. The method employs double-stranded (ds) DNA-conjugated gold nanoparticle@magnetic agarose beads, i.e., dsDNA-AuNP@MABs as peroxidase-mimicking composite probes, in which the aptamer is indirectly attached to the AuNP surface through its hybridization with complementary DNA (cDNA). Upon contact with the acetamiprid target, the probes can give perceptible color change due to the possible conformation switch from dsDNA’s brush-like to cDNA’s ‘pancake’ regime. An “air-spaced pumping” procedure using a syringe equipped with ring magnets as the operation platform was proposed to facilitate the magnetic separation of the sensing probes. Therefore, the analytical steps can be easily accomplished in a syringe, including probe loading, acetamiprid capture and magnetic separation from crude samples, chromogenic reagent loading and colorimetric visualization. Acetamiprid concentration down to 3.3 ppb can be easily identified by the naked eye. The final solution also can be transferred for quantitative measurement. Under spectrometer, the ratio of the absorbance at 652 nm in the presence and absence of acetamiprid (A/A0) is linearly related to the acetamiprid concentration in the 0.4–4.5 ppb range. The limit of detection is calculated to be 0.24 ppb. Moreover, satisfactory recoveries ranging from 90.90 to 91.82% with relative standard deviations of ≤2.96% were obtained in analyzing real spiked samples.

Stabilization of DNA by sodium and magnesium ions during the synthesis of DNA-bridged gold nanoparticles

Nanostructures synthesized using DNA-conjugated gold nanoparticles have a wide range of applications in the field of biosensorics. The stability of the DNA duplex plays a critical role as it determines the final geometry of these nanostructures. The main way to control DNA stability is to maintain a high ionic strength of the buffer solution; at the same time, high salt concentrations lead to an aggregation of nanoparticles. In this study, by means of the instrumentality of DNA-bridged seeds using tris(hydroxymethyl)aminomethane as a soft reducing agent the dumbbell-like gold nanoparticles up to 35 nm were synthesized with a high concentration of sodium ions of up to 100 mM and magnesium ions up to 1 mM. We also examined at the atomic level the details of the effect of the gold nanoparticle surface, as well as Na+ and Mg2+ ions, on the stability of nucleotide pairs located in close proximity to the grafting site.

Biodistribution of Graphene Oxide Determined through Postadministration Labeling with DNA-Conjugated Gold Nanoparticles and ICPMS.

Recent research has revealed the use of graphene oxide (GO) and its derivatives as a potential biomaterial because of their attractive physicochemical characteristics and functional properties. However, if GO and related derivatives are to become useful materials for biomedical applications, it will be necessary to evaluate their biodistribution for health and safety considerations. To obtain a more accurate biodistribution for GO, we (i) developed a postadministration labeling strategy employing DNA-conjugated gold nanoparticles (DNA-AuNPs) to selectively label administered GO in Solvable-treated tissue samples and (ii) constructed an automatic sample pretreatment scheme (using a C18-packed minicolumn) to effectively separate the DNA-AuNP-labeled GO from the unbound DNA-AuNPs and the dissolved tissue matrices, thereby enabling ultrasensitive, interference-free quantification of GO through measurement (inductively coupled plasma mass spectrometry) of the Au signal intensities. The DNA-AuNPs can bind to GO in a concentration- and time-dependent manner. After optimizing the labeling conditions (DNA length, incubation pH, DNA-AuNP concentration, and incubation time) and the separation scheme (sample loading flow rate, rinsing volume, and eluent composition), we found that A20R20-AuNPs (R20: random DNA sequence including A, T, C, and G) had the strongest binding affinity for labeling of the administered GO (dissociation constant: 36.0 fM) and that the method's detection limit reached 9.3 ag L-1 with a calibration curve having a working range from 10-1 to 1010 fg L-1. Moreover, this approach revealed that the intravenously administered GO accumulated predominantly in the liver and spleen at 1 and 12 h post administration, with apparent discrepancies in the concentrations measured using pre- and postadministration labeling strategies.

An aptasensor strip-based colorimetric determination methodfor kanamycin using cellulose acetate nanofibers decorated DNA-gold nanoparticle bioconjugates.

The preparation ofportable colorimetric biosensor strips is describedby combining aptamer-immobilized electrospun nanofiber membranes (A-NFMs) with signal probes (DNA-conjugated gold nanoparticles (AuNPs)) for determination of kanamycin (KMC) as a model analyte. The A-NFMs were decorated with complementary single-stranded DNA (cDNA) of KMC aptamer-conjugated AuNPs (cDNA@Au) to get the colorimetric biosensor strips. The constructed biosensor strips showed a significant absorbance decreasing band at 510nm which induce a visual color change from pink to white when exposed to KMC, with a low detection limit of 2.5nM (at S/N = 3). The effect is due to disassembling of cDNA@Au from NFMs in the presence of KMC because the aptamer has a higher affinity to KMC than its complementary DNA, which resulted in replacing cDNA@Au with KMC. Satisfactory performance was observed in real sample (drinking water and milk) analysis with a recovery of 98.9-102.2%. The constructed colorimetric biosensor test strips hold great application promise for food safety control. Graphical abstract Schematic representation of biosensor strips for kanamycin detection prepared with the cDNA@Au immobilized aptamer-based cellulose acetate nanofibers.

Facile Construction of i-Motif DNA-Conjugated Gold Nanostars as Near-Infrared and pH Dual-Responsive Targeted Drug Delivery Systems for Combined Cancer Therapy.

Stimuli-responsive DNA-based nanostructures have emerged as promising vehicles for intelligent drug delivery. In this study, i-motif DNA-conjugated gold nanostars (GNSs) were fabricated in a facile manner as stimuli-responsive drug delivery systems (denoted as A-GNS/DNA/DOX) for the treatment of cancer via combined chemo-photothermal therapy. The i-motif DNA is sensitive to the environmental pH and can switch from a single-stranded structure to a C-tetrad (i-motif) structure as the environmental pH decreases from neutral (∼7.4) to acidic (<6.0). The loaded drug can then be released along with the conformational changes. To enhance cellular uptake and improve cancer cell selectivity, the aptamer AS1411, which recognizes nucleolins, was employed as a targeting moiety. The A-GNS/DNA/DOX nanocomposites were found to be highly capable of photothermal conversion and exhibited photostability under near-infrared (NIR) irradiation, and the pH and NIR irradiation effectively triggered the drug-release behaviors. In addition, the A-GNS/DNA/DOX nanocomposites exhibited good biocompatibility. The targeting recognition enabled the A-GNS/DNA/DOX to exhibit higher cellular uptake and therapeutic efficiency than the GNS/DNA/DOX. Notably, under NIR irradiation, a synergistic effect between chemotherapy and photothermal therapy can be achieved with the proposed delivery system, which exhibits much higher therapeutic efficiency both in monolayer cancer cells and tumor spheroids as compared with a single therapeutic method. This study highlights the potential of GNS/DNA nanoassemblies for intelligent anticancer drug delivery and combined cancer therapy.

DNA-Conjugated Gold Nanoparticles as High-Mass Probes in Imaging Mass Cytometry.

We employ imaging mass cytometry (IMC) to investigate in vitro uptake and cellular distribution of DNA-functionalized gold nanoparticles (AuNPs). IMC enables the multiparametric imaging of cell components and allows for the detection of AuNPs in cells with >100 times higher sensitivity than conventional confocal fluorescence imaging, as each nanoparticle contains thousands of atoms for signal amplification. Changes in the accumulation of nanoparticles in cells due to oligonucleotide sequence-dependent interactions are exploited to examine a model biomarker for hypoxia-microRNA-210. We find that AuNPs functionalized with microRNA-210-targeting sequence accumulate in hypoxic cells 3 to 4-fold compared to normoxic cells. The work examines the potential use of DNA-AuNP as high-mass probes for the analysis of nonabundant nucleic acids.

Polymer Pen Lithography-Fabricated DNA Arrays for Highly Sensitive and Selective Detection of Unamplified Ganoderma Boninense DNA.

There is an increasing demand for lithography methods to enable the fabrication of diagnostic devices for the biomedical and agri-food sectors. In this regard, scanning probe lithography methods have emerged as a possible approach for this purpose, as they are not only convenient, robust and accessible, but also enable the deposition of “soft” materials such as complex organic molecules and biomolecules. In this report, the use of polymer pen lithography for the fabrication of DNA oligonucleotide arrays is described, together with the application of the arrays for the sensitive and selective detection of Ganoderma boninense, a fungal pathogen of the oil palm. When used in a sandwich assay format with DNA-conjugated gold nanoparticles, this system is able to generate a visually observable result in the presence of the target DNA. This assay is able to detect as little as 30 ng of Ganoderma-derived DNA without any pre-amplification and without the need for specialist laboratory equipment or training.

Universal mRNA Translation Enhancement with Gold Nanoparticles Conjugated to Oligonucleotides with a Poly(T) Sequence.

DNA-conjugated gold nanoparticles (AuNPs) have been shown to enhance the translation of mRNA. However, the specific sequence on the DNA dictates the specific mRNA to be enhanced. This study describes poly(thymine)-functionalized AuNPs (AuNP-p(T)DNA) capable of enhancing the translation of any mRNA template that is incorporated into pcDNA6 vector with bovine growth hormone (BGH) polyadenylation signal (P(A)). We demonstrated this by incorporating four genes: green fluorescence protein (GFP), general control nonderepressible 5 (GCN5), cAMP-responsive element binding protein 1 (CREB1), and X-box-binding protein 1-spliced (XBP-1S) separately into pcDNA6 vector with BGH P(A) before their expression in HeLa lysate. The addition of AuNP-p(T)DNA to HeLa lysate containing GFP, GCN5, CREB1, and XBP-1S mRNA increased protein synthesis 1.80, 1.99, 1.95, and 2.20 times, respectively. Similar translation enhancement was also observed in a multiplex reaction containing the mRNA of three genes together in the lysate. Complementary p(T)DNA hybridization to the poly(A) tail of the mRNA was critical as the removal of either p(T)DNA or BGH P(A) in XBP-1S mRNA or the replacement of p(T)DNA with p(A)DNA reduced the translation back to baseline level. Finally, an optimum length of 25 nucleotides for the DNA oligomer and a AuNP-p(T)DNA:mRNA ratio of 0.658 achieved a 3.08-fold translation enhancement. The AuNP-p(T)DNA nanoconstruct could be incorporated into commercial cell-free protein synthesis kits as a universal translation enhancer.

Sequence-Modulated Interactions between Single Multivalent DNA-Conjugated Gold Nanoparticles.

DNA-conjugated gold nanoparticle (AuNP) is an attractive building block to construct elegant plasmonic nanomaterials by self-assembly but the complicated interaction between multivalent nanoconjugates governing the assembly process and the properties of assembled structures remains poorly understood. Herein, with an in situ kinetic single-particle imaging method, we report the dynamic interaction between single multivalent DNA-conjugated AuNPs quantitatively depends on the nucleic acid sequence in nanoconjugates. From the binding dynamics analysis, it was found that the binding of nanoconjugates with DNA length longer than nine bases is kinetically irreversible and the binding rate is dependent on both the sequence length and GC content, enabling us to predict the rational modulation of binding rates of individual building blocks for stepwise assembly. Moreover, the reversibility for the multivalent interaction between single nanoconjugates at constant temperature can be reinstated by adopting the DNA sequence with single-nucleotide mismatch and the lifetime for nanoconjugates at bound state can be tailored by changing the mismatch positions in DNA strands, providing new opportunity to create active nanostructures with controlled dynamic properties. All these findings provide new insights for understanding the multivalent interaction during the assembly process at the single-nanoconjugate level and predicting the programmable self-assembly of engineered nanoconjugates for the fabrication of dynamic nanomaterials.

DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors

Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

Model-Guided Interface Probe Arrangement for Sensitive Protein Detection.

A wide range of analytical techniques in bioanalysis relies on surface-based biomolecular detection, which requires the confinement of probes onto a heterogeneous surface to react with targets. Probe arrangement on the interface is critical for target recognition and determines assay performance. Much effort has been devoted to screen the optimized probe arrangement according to experimental tests. Such a data-driven posteriori pattern faces low efficiency, ambiguous orientation, and possible deviated tested ranges from the best case. Herein, we demonstrate that a model can effectively guide probe arrangement onto the interface to facilitate probe-target recognition, embodied by the assay of human telomerase activity with DNA-conjugated gold nanoparticles (AuNPs). Both theoretical calculation and experimental results indicate that telomerase activity is maximized on the AuNP surface under guidance of the model. The detection limit is at least 1 order of magnitude lower than that of AuNP bearing densely packed DNA, comparable to that of the telomeric repeat amplification protocol (TRAP). The model-guided interface probe arrangement is proved to be highly useful in regulating interface recognition and may offer a new paradigm to promote surface-based biomolecular detection.

PH and near-infrared light dual-stimuli responsive drug delivery using DNA-conjugated gold nanorods for effective treatment of multidrug resistant cancer cells

A thiolated pH-responsive DNA conjugated gold nanorod (GNR) was developed as a multifunctional nanocarrier for targeted, pH-and near infrared (NIR) radiation dual-stimuli triggered drug delivery. It was further passivated by a thiolated poly(ethylene glycol)-biotin to improve its cancer targeting ability by specific binding to cancer cell over-expressed biotin receptors. Doxorubicin (DOX), a widely used clinical anticancer drug, was conveniently loaded into nanocarrier by intercalating inside the double-stranded pH-responsive DNAs on the GNR surface to complete the construction of the multifunctional nanomedicine. The nanomedicine can rapidly and effectively release its DOX payload triggered by an acidic pH environment (pH~5) and/or applying an 808nm NIR laser radiation. Compared to free DOX, the biotin-modified nanomedicine displayed greatly increased cell uptake and significantly reduced drug efflux by model multidrug resistant (MDR) breast cancer cell lines (MCF-7/ADR). The application of NIR radiation further increased the DOX release and facilitated its nuclear accumulation. As a result, this new DNA-GNR based multifunctional nanomedicine exerted greatly increased potency (~67 fold) against the MDR cancer cells over free DOX.

Biomedical Applications of DNA-Conjugated Gold Nanoparticles.

Gold nanoparticles (AuNPs) are useful for diagnostic and biomedical applications, mainly because of their ease in preparation and conjugation, biocompatibility, and size-dependent optical properties. However, bare AuNPs do not possess specificity for targets. AuNPs conjugated with DNA aptamers offer specificity for various analytes, such as proteins and small molecules/ions. Although DNA aptamers themselves have therapeutic and target-recognizing properties, they are susceptible to degradation in vivo. When DNA aptamers are conjugated to AuNPs, their stability and cell uptake efficiency both increase, making aptamer-AuNPs suitable for biomedical applications. Additionally, drugs can be efficiently conjugated with DNA aptamer-AuNPs to further enhance their therapeutic efficiency. This review focuses on the applications of DNA aptamer-based AuNPs in several biomedical areas, including anticoagulation, anticancer, antibacterial, and antiviral applications.

Characteristics of DNA-AuNP networks on cell membranes and real-time movies for viral infection.

This data article provides complementary data for the article entitled “DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding” Li et al. (2016) [1]. The experimental methods for the preparation and characterization of DNA-conjugated nanoparticle networks on cell membranes were described. Confocal fluorescence images, agarose gel electrophoresis images and hydrodynamic diameter of DNA-conjugated gold nanoparticle (DNA-AuNP) networks were presented. In addition, we have prepared QDs-labeled RSV (QDs-RSV) to real-time monitor the RSV infection on HEp-2 cells in the absence and presence of DNA-AuNP networks. Finally, the cell viability of HEp-2 cells coated by six types of DNA-nanoparticle networks was determined after RSV infection.

DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

Hybrid Sensor Using Gold Nanoparticles and Conjugated Polyelectrolytes for Studying Sequence Rule in Protein–DNA Interactions

Protein-DNA interactions play center roles in many biological processes. Studying sequence specific protein-DNA interactions and revealing sequence rules require sensitive and quantitative methodologies that are capable of capturing subtle affinity difference with high accuracy and in a high throughput manner. In this study, double stranded DNA-conjugated gold nanoparticles (dsDNA-AuNPs) and water-soluble conjugated polyelectrolytes (CPEs) are used as cooperative sensing elements to construct a suit of hybrid sensors for detecting protein-DNA interactions, exploiting the differential Förster resonance energy transfer (FRET) with and without protein binding. Through a proper selection of CPEs in terms of charge properties relative to the charge of dsDNA-AuNPs and emission wavelengths relative to the AuNP extinction peak, the hybrid sensors can be constructed into "light-on", "light-off", and "two-way" models. Protein binding can be detected by fluorescence recovery, fluorescence quenching, or both ways, respectively. The "two-way" sensor allows for detection of proteins of any charge properties or unknown charge properties. With estrogen receptor (ERα and ERβ), their consensus DNA (5'-GGTCAnnnTGACC-5') element, and all 15 possible singly mutated elements (i.e., 3 possible base substitutions at each of 1 to 5 positions from left to right of the 5' end half site, GGTCA), we have demonstrated the accuracy of the hybrids sensors for determination of binding affinity constant, binding stoichiometry, and site- and nucleotide-specific binding energy matrix. The in vitro binding energy determined by the hybrid sensors correlates very well with the energy matrix computed from in vivo genome-wide ERα binding data using Thermodynamic Modeling of ChIP-Seq (rank correlation coefficient 0.98). The high degree of correlation of the in vitro energy matrix versus the in vivo matrix renders the new method a highly reliable alternative for understanding in vivo protein binding in the whole genome.