Dendrimer-like DNA Research Articles

An amplifiable ratiometric fluorescent aptasensor for aflatoxin B1 detection based on dendrimer-like DNA nanostructures coupled with catalytic hairpin self-assembly

Herein, an amplifiable ratiometric fluorescent aptasensor was constructed based on dendrimer-like DNA nanostructures coupled with catalytic hairpin self-assembly for the specific detection of aflatoxin B1 (AFB1) in food. Compared with the single-signal mode, the ratio-signal mode based on FCy3/FCy5 has a wider linear range and a higher R2 for the detection of AFB1. Moreover, for the spiking experiment of peanut, soybean and wine samples, taking the average of the recovery obtained by three standard curves of FCy3, FCy5, FCy3/FCy5 as the reference value, the ratio-signal-based aptasensor showed higher accuracy than the single-signal-based aptasensor. In addition, the presence of a dendrimer-like scaffold can not only improve the stability of the sensor but also enhance FRET efficiency. In the whole detection process, the specific signal difference with dendrimer-like DNA nanostructures was 5.7 times of that without dendrimer-like DNA nanostructures. Besides, catalytic hairpin self-assembly, an enzyme-free amplification strategy, was introduced to achieve signal amplification by the complementary chain of the aptamer (c-DNA) cycle. Furthermore, the sensor can detect various mycotoxins in food by different aptamer recognition.

Tailored protein-conjugated DNA nanoplatform for synergistic cancer therapy.

Multidrug resistance (MDR) to chemotherapeutic drugs and targeted drug delivery are recurring issues in clinical cancer treatment. Here, a multifunctional fusion protein-DNA conjugate was designed as a co-delivery vehicle for anticancer peptides and chemotherapeutic drugs to combat both drug-resistant and drug-sensitive tumor cells. The fusion protein was constructed by fusing a PsTag polypeptide, a matrix metalloproteinase 2 (MMP2)-degradable domain, and the mitochondria-targeted pro-apoptotic peptide KLAKLAKKLAKLAK. Doxorubicin was efficiently loaded into the fusion protein pre-conjugated dendrimer-like DNA nanostructure. With the incorporation of enhanced stability, tumor targeting, and controlled-release elements, the tailored nanostructure can selectively enter tumor cells and synergistically exert antitumor activity with no significant adverse effects. Thus, these protein-conjugated DNA nanocarriers could be a potential co-delivery system for protein/peptide and chemotherapeutic drugs delivery in synergistic cancer therapy.

A DNAzyme-catalyzed label-free aptasensor based on multifunctional dendrimer-like DNA assembly for sensitive detection of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is an important malign tumor marker. In this study, a simple, label-free and antibody-free aptasensor was fabricated based on a multifunctional dendrimer-like DNA nanoassembly. The DNA nanoassembly was embedded with multiple G-quadruplex DNAzyme motifs and a hanging CEA aptamer motif. It was prepared from short DNA sequences by autonomous-assembly. The aptasensor was prepared simply by self-assembly of a capture DNA (cpDNA) on a gold electrode, followed by hybridization with a CEA aptamer (AptGAC-P). CEA as a model target was detected through competitive binding of CEA with AptGAC-P, exposing cpDNA to bind with the DNA nanoassembly. The detection process only contains 2 incubation steps. The high load of G-quadruplex DNAzyme motifs and their catalytic activity resulted in an amplified and label-free differential pulse voltammetry (DPV) electrochemical signal. The peak current correlated linearly with the CEA concentration, with a linear range of 2–45 ng mL−1, and an LOD value of 0.24 ng mL−1. The aptasensor showed high specificity and reproducibility, and retained 96.5% of detection signal intensities after 31 days of storage. The recovery rates for spiked CEA in human serum were within 100 ± 5%, and the coincidence rates for clinical human serum samples with ELISA kits were 80.7–111%. Conceivably, possessing simplicity, sensitivity, reproducibility, storage stability, and accuracy, the aptasensor should be a very prominent and applicable tool for clinical CEA detection and cancer diagnosis, and is promisingly applicable as a platform for detecting other targets of interests.

Structure and stimuli-responsiveness of all-DNA dendrimers: theory and experiment.

We present a comprehensive theoretical and experimental study of the solution phase properties of a DNA-based family of nanoparticles - dendrimer-like DNA molecules (DL-DNA). These charged DNA dendrimers are novel macromolecular aggregates, which hold high promise in targeted self-assembly of soft matter systems in the bulk and at interfaces. To describe the behaviour of this family of dendrimers (with generations ranging from G1 to G7), we use a theoretical model in which base-pairs of a single DL-DNA molecule are modeled by charged monomers, whose interactions are chosen to mimic the equilibrium properties of DNA correctly. Experimental results on the sizes and conformations of DL-DNA are based on static and dynamic light scattering; and molecular dynamics simulations are employed to model the equilibrium properties of DL-DNA, which compare favorably to the findings from experiments while at the same time providing a host of additional information and insight into the molecular structure of the nanostructures. We also examine the salt-responsiveness of these macromolecules, finding that despite the strong screening of electrostatic interactions brought about by the added salt, the macromolecules shrink only slightly, their size robustness stemming from the high bending rigidity of the DNA-segments. The study of these charged dendrimer systems is an important field of research in the area of soft matter due to their potential role for various interdisciplinary applications, ranging from molecular cages and carriers for drug delivery in a living organism to the development of dendrimer- and dendron-based ultra-thin films in the area of nanotechnology. These findings are essential to determine if DL-DNA is a viable candidate for the experimental realization of cluster crystals in the bulk, a novel form of solid with multiple site occupancy.

Self-Assembled DNA Dendrimer Nanoparticle for Efficient Delivery of Immunostimulatory CpG Motifs.

Dendrimer-like DNA nanostructures have attractive properties such as mechanical stability, highly branched nanostructure, customized sizes, and biocompatibility. In this study, we construct programmable DNA dendrimeric nanoparticles as efficient vehicles to deliver immunostimulatory cytosine-phosphate-guanosine (CpG) sequences for activation of the immune response. DNA dendrimers decorated with CpG-containing hairpin-loops triggered stronger immune response characterized by pro-inflammatory cytokines production, in contrast to DNA dendrimers loading linear CpG. After further modification with TAT peptide, a typical cell-penetrating peptide, on the surface of the nanocarriers, CpG loops-loaded DNA dendrimers showed the enhanced cell internalization and cytokines production. The TAT-DNA dendrimer-CpG loops constructs did not affect the viability of immune cells and no detectable cytotoxicity was observed. Our results demonstrate that the DNA dendrimers can serve as designable and safe vehicles for delivery of immune modulators and anticancer drugs.

Target-catalyzed autonomous assembly of dendrimer-like DNA nanostructures for enzyme-free and signal amplified colorimetric nucleic acids detection

Self-assembly of DNA nanostructures is of great importance in nanomedicine, nanotechnology and biosensing. Herein, a novel target-catalyzed autonomous assembly pathway for the formation of dendrimer-like DNA nanostructures that only employing target DNA and three hairpin DNA probes was proposed. We use the sticky-ended Y shape DNA (Y-DNA) as the assembly monomer and it was synthesized by the catalyzed hairpin assembly (CHA) instead of the DNA strand annealing method. The formed Y-DNA was equipped with three ssDNA sticky ends and two of them were predesigned to be complementary to the third one, then the dendrimer-like DNA nanostructures can be obtained via an autonomous assembly among these sticky-ended Y-DNAs. The resulting nanostructure has been successfully applied to develop an enzyme-free and signal amplified gold nanoparticle (AuNP)-based colorimetric nucleic acids assay.

SNAP Display - an In Vitro Method for the Selection of Protein Binders

Display technologies are powerful tools that are widely used for selecting protein binders against different targets by mimicking natural selection. Such in vitro display methods have been pivotal in the isolation and evolution of antibodies with affinities greater than those produced by the natural immune response. Here we describe SNAP display, which covalently links genotype with phenotype by the action of the SNAP-tag in an emulsion microdroplet. Successful model selections suggest that SNAP display, has potential as a robust display platform suitable for selecting protein binders. A multivalent format of SNAP display using dendrimer-like DNA has also been established to probe avidity effects in multivalent protein display selections. This review describes the principles of SNAP display and extrapolates its scope for in vitro selections.

SNAP Dendrimers: Multivalent Protein Display on Dendrimer‐Like DNA for Directed Evolution

Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered.

Three‐Dimensional Structure and Thermal Stability Studies of DNA Nanostructures by Energy Transfer Spectroscopy

Structural changes and stability of DNA nanoarchitectures including Y-shaped DNA (see picture), dendrimer-like DNA, and DNA hydrogels are investigated. The results demonstrate the feasibility and flexibility of FRET and NSET (Förster resonance/ nanometal surface- energy transfer) in determining difficult-to-obtain 3D structures and characterizing the thermal responses of DNA nanoarchitectures in real time.

Bio-bar-code dendrimer-like DNA as signal amplifier for cancerous cells assay using ruthenium nanoparticle-based ultrasensitive chemiluminescence detection

Bio-bar-code dendrimer-like DNA (bbc-DL-DNA) is employed as a label for the amplification assay of cancer cells in combination with the newly explored chemiluminescence (CL) system of luminol-H(2)O(2)-Ru(3+) and specificity of structure-switching aptamers selected by cell-based SELEX.

The assembly of a short linear natural cytosine-phosphate-guanine DNA into dendritic structures and its effect on immunostimulatory activity

DNA containing unmethylated CpG dinucleotides, or CpG motifs, (CpG DNA) has been explored as a therapeutic agent, owing to its potent immunostimulatory activity. A previous study showing that Y-shaped (Y-) CpG DNA has a high immunostimulatory activity compared with single- or double stranded CpG DNA suggests the possibility that CpG DNA in a more complicated structure is a stronger activator of the immune system. In the present study, dendrimer-like DNA (DL-DNA) was prepared by ligating Y-DNA monomers. The DL-DNA of the second or third generation with 12 or 24 highly potent CpG motifs in one unit, respectively, were designed and successfully prepared for the first time. These DL-DNAs induced greater amounts of tumor necrosis factor-α and interleukin-6 from RAW264.7 macrophage-like cells than did a mixture of Y-DNA with the same sequences as the corresponding DL-DNA. DL-DNA was more efficiently taken up by RAW264.7 cells than Y-DNA, but the increase was lower than that exhibited by the levels of cytokine release. These results suggest that the dendritic structure formation is a potential approach to increasing the immunostimulatory activity of CpG DNA without any modifications of the chemical structure of the natural phosphodiester DNA.

Highly Efficient Method for Constructing a Single-Stranded Comb-Like Oligonucleotide via Reversible Photocrosslinking

Abstract Branched oligodeoxyribonucleotides (ODNs) are key molecules in constructing nano-scale structures such as DNA lattices, dendrimer-like DNA as well as DNA devices including DNA walkers, DNA memory, and DNA computer. In this paper, two types of highly efficient methods were used to construct the comb-like DNA by using template-directed reversible DNA photocrosslinking via 5-(2-carbamoylvinyl)-2′-deoxyuridine (cvU). The efficiency and thermodynamic stability of three types of reversible DNA photocrosslinked, linear ODN, single-branched ODN (5′-end) and single-branched ODN (mid) reported previously, were also compared. The results show that the most efficient one of the three is a single-branched ODN (5′-end), despite its low thermodynamic stability. In addition, we also showed that this reversible photocrosslinking method is applicable to nanotechnology by constructing comb-like DNA. This method should have great potential in nanotechnology by serving as a platform for fabrication and synthesis, because their sizes and structures can be changed through the alteration of building blocks. We believe that this method is versatile and may find a myriad of applications in the nano-biotechnology field.

Enzyme-catalysed assembly of DNA hydrogel

DNA is a remarkable polymer that can be manipulated by a large number of molecular tools including enzymes. A variety of geometric objects, periodic arrays and nanoscale devices have been constructed. Previously we synthesized dendrimer-like DNA and DNA nanobarcodes from branched DNA via ligases. Here we report the construction of a hydrogel entirely from branched DNA that are three-dimensional and can be crosslinked in nature. These DNA hydrogels were biocompatible, biodegradable, inexpensive to fabricate and easily moulded into desired shapes and sizes. The distinct difference of the DNA hydrogel to other bio-inspired hydrogels (including peptide-based, alginate-based and DNA (linear)-polyacrylamide hydrogels) is that the crosslinking is realized via efficient, ligase-mediated reactions. The advantage is that the gelling processes are achieved under physiological conditions and the encapsulations are accomplished in situ-drugs including proteins and even live mammalian cells can be encapsulated in the liquid phase eliminating the drug-loading step and also avoiding denaturing conditions. Fine tuning of these hydrogels is easily accomplished by adjusting the initial concentrations and types of branched DNA monomers, thus allowing the hydrogels to be tailored for specific applications such as controlled drug delivery, tissue engineering, 3D cell culture, cell transplant therapy and other biomedical applications.

Dendrimer-like DNA-based fluorescence nanobarcodes

A major challenge in clinical diagnostics and environmental analysis is the difficulty in rapid and sensitive detection of multiple target molecules simultaneously (i.e., multiplexed detections). Our group has designed and synthesized a dendrimer-like DNA (DL-DNA) that is multivalent and anisotropic; using this unique DNA structure, we have developed a fluorescence-tagged nanobarcode system for multiplex detection. This nanobarcode system allows the rapid and sensitive detection of multiple pathogens simultaneously using the ratios of two different fluorescent dyes, green and red, with which different DL-DNAs are labeled. The key step of our nanobarcode model lies in the monodisperse preparation of DL-DNA. Two methods, solution phase and solid phase, are presented here. With slight modifications, this platform technology can also be extended to the multiplexed detection of RNA and proteins. This protocol can be completed in 2-5 d.

Detection and identification of nucleic acid engineered fluorescent labels in submicrometrefluidic channels

Nucleic acid engineers have created nanoscale fluorescent labels that are uniquelyidentifiable by the number of conjugated fluorophores, and with binding characteristics thatpermit recognition of individual specific biomolecules. The viability of this technology foruse in multi-analyte homogeneous assays depends on the ability to optically detectindividual labels, and distinguish the fluorescence emission of each label. We describe theuse of fluidic channels with submicrometre dimensions to rapidly detect individual labels insolution. Labels with small differences in fluorophore composition were differentiatedwith varying degrees of accuracy. Labels were synthesized at the molecular levelfrom dendrimer-like DNA, with the identity encoded into the number of AlexaFluor 488 and BODIPY 630/650 fluorophores conjugated with the structure. Toexplore the decoding resolution limit, labels with a single fluorophore of eachcolour were detected, and were found to be distinguishable as a group, but notindividually, from labels with one additional red fluorophore. Labels with one green andthree red fluorophores were individually distinguishable with greater than 80%accuracy from labels with one red and three green fluorophores. Photon countinghistograms were analysed to differentiate the various labels, and fluorescencecorrelation spectroscopy was used to measure their mobilities. Fluidic channelswere fabricated in fused silica with a 500 nm square cross section, resulting ina focal volume of approximately 500 al. Because the entire channel width wasilluminated, every fluorescent molecule in solution passing through the channel wasuniformly excited and analyzed. Flow control enabled a balance of rapid dataacquisition and efficient fluorescence collection with these nanoscale systems.

Diffusion of Single Star-Branched Dendrimer-like DNA

We report a single-molecule fluorescence study on the diffusion of star-branched polymer dendrimer-like DNA (DL-DNA). The DL-DNA molecules were synthesized from ligating Y-shaped DNA to the fourth generation. It was found through single particle tracking that the diffusion coefficient of DL-DNA changes in a nonmonotonic fashion with its increased concentration possibly due to arm arrest and arm retraction. The diffusion of DL-DNA in linear lambda DNA solution displayed a monotonic concentration dependence 1 order of magnitude greater than the diffusion of DL-DNA in DL-DNA solution. This difference is attributed to the different conformation of DL-DNA and lambda DNA and the entanglement of lambda DNA with a large radius of gyration. Our diffusion study facilitates DL-DNA transportation for drug delivery.