Framework Nucleic Acids-Based VEGF Signaling Activating System for Angiogenesis: A Dual Stimulation Strategy.

Angiogenesis is crucial for tissue engineering, wound healing, and regenerative medicine. Nanomaterials constructed based on specific goals can be employed to activate endogenous growth factor-related signaling. In this study, based on the conventional single-stranded DNA self-assembly into tetrahedral framework nucleic acids (tFNAs), the Apt02 nucleic acid aptamer and dimethyloxallyl glycine (DMOG) small molecule are integrated into a complex via a template-based click chemistry reaction and toehold-mediated strand displacement reaction. Thus, being able to simulate the VEGF (vascular endothelial growth factor) function and stabilize HIF (hypoxia-inducible factor), a functional whole is constructed and applied to angiogenesis. Cellular studies demonstrate that the tFNAs-Apt02 complex (TAC) has a conspicuous affinity to human umbilical vein endothelial cells (HUVECs). Further incubation with DMOG yields the tFNAs-Apt02-DMOG complex (TACD), which promotes VEGF secretion, in vitro blood vessel formation, sprouting, and migration of HUVECs. Additionally, TACD enhances angiogenesis by upregulating the VEGF/VEGFR and HIF signaling pathways. Moreover, in a diabetic mouse skin defect repair process, TACD increases blood vessel formation and collagen deposition, therefore accelerating wound healing. The novel strategy simulating VEGF and stabilizing HIF promotes blood-vessel formation in vivo and in vitro and has the potential for broad applications in the vascularization field.

Utilizing peptide-anchored DNA templates for novel programmable nanoparticle assemblies in biological macromolecules: A review.

Biological macromolecules such as proteins and DNA are known to self-assemble into various structural moieties with distinct functions. While nucleic acids are the structural building blocks, peptides exemplify diversity as tailorable biochemical units. Thus, combining the scaffold properties of the biomacromolecule DNA and the functionality of peptides could evolve into a powerful method to obtain tailorable nano assemblies. In this review, we discuss the assembly of non-DNA-coated colloidal NPs on DNA/peptide templates using functional anchors. We begin with strategies for directly attaching metallic NPs to DNA templates to ascertain the functional role of DNA as a scaffold. Followed by methods to assemble peptides onto DNA templates to emphasize the functional versatility of biologically abundant DNA-binding peptides. Next, we focus on studies corroborating peptide self-assembling into macromolecular templates onto which NPs can attach to emphasize the properties of NP-binding peptides. Finally, we discuss the assembly of NPs on a DNA template with a focus on the bifunctional DNA-binding peptides with NP-binding affinity (peptide anchors). This review aims to highlight the immense potential of combining the functional power of DNA scaffolds and tailorable functionalities of peptides for NP assembly and the need to utilize them effectively to obtain tailorable hierarchical NP assemblies.

Primer Exchange Reaction Coupled with DNA-Templated Silver Nanoclusters for Label-Free and Sensitive Detection of MicroRNA.

MicroRNAs (MiRNAs) play pivotal roles in regulating gene expression, and serve as crucial biomarkers for diagnosis of a variety of disease. However, label-free and sensitive miRNA detection remains a huge challenge due to the low abundance. Herein, we developed an approach through integrating primer exchange reaction (PER) with DNA-templated silver nanoclusters (AgNCs) for label-free and sensitive miRNA detection. In this method, PER was used to amplify miRNA signals and produce single-strand DNA (ssDNA) sequences. The produced ssDNA sequences mediated DNA-templated AgNCs based signal generation by unfolding the designed hairpin probe (HP). The generated AgNCs signal was correlated with the dosage of target miRNA. Eventually, the established approach exhibited a low detection of limit of 47 fM with a great dynamic range of more than five orders of magnitude. In addition, the method was also utilized to detect the miRNA-31 expression in collected clinical samples from pancreatitis patients and demonstrated that miRNA-31 was upregulated in patients, showing a great promising of the method in clinical application.

DNA Mold-Based Fabrication of Palladium Nanostructures.

DNA origami molds allow a shape-controlled growth of metallic nanoparticles. So far, this approach is limited to gold and silver. Here, the fabrication of linear palladium nanostructures with controlled lengths and patterns is demonstrated. To obtain nucleation centers for a seeded growth, a synthesis procedure of palladium nanoparticles (PdNPs) using Bis(p-sulfonatophenyl)phenylphosphine (BSPP) both as reductant and stabilizer is developed to establish an efficient functionalization protocol of the particles with single-stranded DNA. Attaching the functionalized particles to complementary DNA strands inside DNA mold cavities supports subsequently a highly specific seeded palladium deposition. This provides rod-like PdNPs with diameters of 20-35 nm of grainy morphology. Using an annealing procedure and a post-reduction step with hydrogen, homogeneous palladium nanostructures can be obtained. With the adaptation of the procedure to palladium the capabilities of the mold-based tool-box are expanded. In the future, this may allow a facile adaptation of the mold approach to less noble metals including magnetic materials such as Ni and Co.

A synthetic DNA template for fast manufacturing of versatile single epitope mRNA.

A flexible, affordable, and rapid vaccine platform is necessary to unlock the potential of personalized cancer vaccines in order to achieve full clinical efficiency. mRNA cancer vaccine manufacture relies on the rigid sequence design of multiepitope constructs produced by laborious bacterial cloning and time-consuming plasmid preparation. Here, we introduce a synthetic DNA template (SDT) assembly process, which allows cost- and time-efficient manufacturing of single (neo)epitope mRNA. We benchmarked SDT-derived mRNA against mRNA derived from a plasmid DNA template (PDT), showing that monocyte-derived dendritic cells (moDCs) electroporated with SDT-mRNA or PDT-mRNA, encoding HLA-I- or HLA-II-restricted (neo)epitopes, equally activated T cells that were modified to express the cognate T cell receptors. Furthermore, we validated the SDT-mRNA platform for neoepitope immunogenicity screening using the characterized HLA-A2-restricted neoepitope DHX40B and four new candidate HLA-A2-restricted melanoma neoepitopes. Finally, we compared SDT-mRNA with PDT-mRNA for vaccine development purposes. moDCs electroporated with mRNA encoding the HLA-A2-restricted, mutated Melan-A/Mart-1 epitope together with TriMix mRNA-generated high levels of functional Melan-A/Mart-1-specific CD8+ T cells. In conclusion, SDT single epitope mRNA can be manufactured in a more flexible, cost-efficient, and time-efficient way compared with PDT-mRNA, allowing prompt neoepitope immunogenicity screening, and might be exploited for the development of personalized cancer vaccines.

Effects of DNA template preparation on variability in cell-free protein production.

DNA templates for protein production remain an unexplored source of variability in the performance of cell-free expression (CFE) systems. To characterize this variability, we investigated the effects of two common DNA extraction methodologies, a postprocessing step and manual versus automated preparation on protein production using CFE. We assess the concentration of the DNA template, the quality of the DNA template in terms of physical damage and the quality of the DNA solution in terms of purity resulting from eight DNA preparation workflows. We measure the variance in protein titer and rate of protein production in CFE reactions associated with the biological replicate of the DNA template, the technical replicate DNA solution prepared with the same workflow and the measurement replicate of nominally identical CFE reactions. We offer practical guidance for preparing and characterizing DNA templates to achieve acceptable variability in CFE performance.

Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production.

Performance variability is a common challenge in cell-free protein production and hinders a wider adoption of these systems for both research and biomanufacturing. While the inherent stochasticity and complexity of biology likely contributes to variability, other systematic factors may also play a role, including the source and preparation of the cell extract, the composition of the supplemental reaction buffer, the facility at which experiments are conducted, and the human operator (Cole et al. ACS Synth Biol 8:2080-2091, 2019). Variability in protein production could also arise from differences in the DNA template-specifically the amount of functional DNA added to a cell-free reaction and the quality of the DNA preparation in terms of contaminants and strand breakage. Here, we present protocols and suggest best practices optimized for DNA template preparation and quantitation for cell-free systems toward reducing variability in cell-free protein production.

Label-free and sensitive MiRNA detection based on turn-on fluorescence of DNA-templated silver nanoclusters coupled with duplex-specific nuclease-assisted signal amplification.

A novel strategy for microRNAs (miRNAs) detection has been developed utilizing duplex-specific nuclease-assisted signal amplification (DSNSA) and guanine-rich DNA-enhanced fluorescence of DNA-templated silver nanoclusters (AgNCs). The combination between target miRNA, DSNSA, and AgNCs is achieved by the unique design of DNA sequences. Target miRNA opens the hairpin structure of the Hairpin DNA probe (HP) by hybridizing with the HP and initiates the duplex-specific nuclease-assisted signal amplification (DSNSA) reaction. The DSNSA reaction generates the release of the guanine-rich DNA sequence, which can turn on the fluorescence of the dark AgNCs by hybridizing with the DNA template of the dark AgNCs. The fluorescence intensity of AgNCs corresponds to the dosage of the target miRNA. This is measured at 630 nm by exciting at 560 nm. The constructed method exhibits a low detection limit (~8.3 fmol), a great dynamic range of more than three orders of magnitude, and excellent selectivity. Moreover, it has a good performance for miR-21 detection in complex biological samples. A novel strategy for microRNAs (miRNAs) detection has been developed utilizing duplex-specific nuclease-assisted signal amplification (DSNSA) and guanine-rich DNA-enhanced fluorescence of DNA-templated silver nanoclusters (AgNCs).

Schottky junction devices by using bio-molecule DNA template-based one dimensional CdS-nanostructures.

Creating a well-defined nanostructure through de-oxyribo nucleic acid (DNA)-nanotechnology, and specifically the development of metal/inorganic semiconductor junctions on DNA-assembled nanostructures, is an emerging research area. Herein, we investigate the electrical properties of biomolecule DNA-template based one-dimensional nanowires (NWs)-CdS/Au and without-template based nanoparticles (NPs)-CdS/Au devices grown on the Indium Tin Oxide (ITO) glass substrates. More importantly, the NWs-CdS/Au device displays a dramatic augmentation of current flow and also a striking change in threshold voltage (~55 mV) in comparison to NPs (~190 mV) and reported bulk-CdS/Au (~680 mV) devices. Albeit the manifestation of non-linear/asymmetric current-voltage (I-V) characteristic establishes the CdS/Au junction as Schottky device, but captivatingly, the large ideality factor of about 24 found in NWs-CdS/Au device could be due to the DNA-assembled based organic process CdS-semiconductor. Capacitance-voltage (C-V) measurements of the NWs-CdS/Au divulge a remarkable hump-like feature at lower frequency owing to the frequency dispersion effect. In contrast, the effect appears to be enfeebled with increasing frequency. We conjecture that the density of surface/interface traps materialises at the interface of nanostructures-CdS/metal-Au results in the changes in underlying electrical properties. The observation of significant differences in the electrical properties of DNA-assembled NWs-based Schottky junctions could possibly be helpful for the fabrication of more sophisticated and higher multispecificity biosensors for medical applications.

Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology.

Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics.

Dynamic metallization of spherical DNA via conformational transition into gold nanostructures with controlled sizes and shapes.

Despite the reversible condensation properties of DNA, DNA metallization during controlled conformational transitions has been rarely investigated. We perform dynamic metallization of spherically condensed DNA nanoparticles (DNA NPs) via a globule-to-coil transition. A positively charged new Au3+ reagent is prepared via ligand-exchange of conventional complex Au3+ ions, which was used to synthesize spherically condensed DNA NPs simply based on the fundamental electrostatic and coordinative interactions between DNA and Au3+ions. Interestingly, the size of the Au3+-condensed DNA NPs (Au3+-DNA NPs) and the type of reducing agents lead to the formation of different Au nanostructures with unprecedented morphologies (cracked NPs, bowl-shaped NPs, and small NPs), owing to the controlled conformational changes in the Au3+-DNA NPs during metallization. The condensed DNA NPs play significant roles for Au nanostructures as (1) the dynamic template for the synthesis, (2) the reservoir and supply of Au3+ for the growth, and (3) the surface stabilizer. The synthesized Au nanostructures are remarkably stable against high ionic strength and exhibit catalytic activities and excellent SERS properties. This is the first study on the morphological control and concomitant dynamic metallization of spherically condensed DNA, proposing new synthetic routes for bioinorganic nanomaterials.

A novel electrochemical aptasensor for ochratoxin a sensing in spiked food using strand-displacement polymerase reaction.

Herein, an aptasensor is presented for electrochemical determination of ochratoxin A (OTA) based on nontarget-triggered production of rolling circular amplification (RCA). The surface of gold electrode is modified with thiolated complementary strand of aptamer (CS) as both capture probe and primer and OTA aptamer (Apt) as both sensing molecule and padlock probe (PLP). Following the addition of OTA, Apt/OTA conjugate is formed and detached from the electrode surface. Therefore, no RCA is produced after incubation of the modified electrode with T4 DNA ligase and phi29 DNA polymerase and a sharp current signal occurs. The analytical response ranged from 30 pM to 120 nM with detection limit of 5 pM. The designed aptasensor showed superior analytical performance in comparison with other approaches for OTA detection. Also, the approach exhibited good performance for OTA determination in spiked grape juice samples. The technique presented in this study, can be applied to develop sensors for detecting different toxins by replacing the relevant aptamers and complementary strands.

Label-Free Analysis of H5N1 Virus Based on Three-Segment Branched DNA-Templated Fluorescent Silver Nanoclusters.

Since H5N1 virus is a highly infectious pathogen that causes outbreaks of avian influenza, developing a sensitive and rapid diagnostic platform to sense it becomes significant. Here, a novel label-free fluorescence sensing platform based on DNA-templated silver nanoclusters (DNA-Ag NCs) is developed to detect the H5N1 gene sequence representing H5N1 virus. The three-segment-branched DNA structure with closed cytosine-rich loop is designed as an effective template to produce fluorescent Ag NCs, which is different with the previous design of cytosine-rich loop formed by hairpin-like single-stranded DNA or double-stranded DNA. The proposed fluorescence detection approach gives a wide linear range (500 pM-2 µM) and a low detection limit (500 pM) to sense H5N1 gene sequence. Furthermore, selective analysis of target DNA shows that our constructed analytical strategy has a high selectivity to H5N1 gene sequence. It is regarded as a promising method for highly sensitive and selective sensing of H5N1 virus.

A simplified method to produce mRNAs and functional proteins from synthetic double-stranded DNA templates.

We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning.

Construction of manganese oxide nanowire-like cluster arrays on a DNA template: Application to detection of hydrogen peroxide.

Improved electron transfer properties and catalytic activity of manganese oxide (MnOx) was demonstrated following its electrochemical deposition on a deoxyribonucleic acid (DNA) modified glassy carbon electrode. The MnOx showed different morphologies, electrocatalytic properties and electrochemical kinetics. Scanning electron microscopy showed that electrodeposition of MnOx on a bare glassy carbon electrode led to the formation of irregular-shapes while a nanowire cluster (NWC) was formed on a GCE/DNA due to the DNA serving as a template. Electrochemical impedance spectroscopy (EIS) revealed lower charge transfer resistance of the MnOxNWC compared with MnOx. A new mechanism is presented for the electrodeposition of MnOx on the surface of a GC/DNA electrode. An electrochemical biosensor was fabricated based on depositing MnOx onto a glassy carbon /DNA electrode (GCE/DNA/MnOxNWC) and was used to detect hydrogen peroxide (H2O2). The MnOx nanowire cluster and DNA exhibited significant electrocatalytic activity for simultaneous electrocatalytic oxidation at two oxidation potentials (0.6 V and 0.98 V vs Ag/AgCl) and one reduction potential (-0.5 V vs Ag/AgCl) for H2O2 at pH 6.0. A new mechanism for the detection of H2O2 is presented. Excellent electrocatalytic activity, stability and facility for simultaneous detection of H2O2 at different of applied potentials are proposed advantages of the proposed electrochemical biosensor.

DNA-Templated Fluorescent Nanoclusters for Metal Ions Detection.

DNA-templated fluorescent nanoclusters (NCs) have attracted increasing research interest on account of their prominent features, such as DNA sequence-dependent fluorescence, easy functionalization, wide availability, water solubility, and excellent biocompatibility. Coupling DNA templates with complementary DNA, aptamers, G-quadruplex, and so on has generated a large number of sensors. Additionally, the preparation and applications of DNA-templated fluorescent NCs in these sensing have been widely studied. This review firstly focuses on the properties of DNA-templated fluorescent NCs, and the synthesis of DNA-templated fluorescent NCs with different metals is then discussed. In the third part, we mainly introduce the applications of DNA-templated fluorescent NCs for sensing metal ions. At last, we further discuss the future perspectives of DNA-templated fluorescent NCs in the synthesis and sensing metal ions in the environmental and biological fields.

DNA template-mediated click chemistry-based portable signal-on sensor for ochratoxin A detection.

A novel signal-on portable sensing system has been developed for OTA detection using personal glucose meter (PGM) as signal transducer. In the study, we explore the potential of using a short dsDNA as template to trigger the "click" ligation of two DNA strands, further improve the stability of DNA strand on the magnetic beads (MBs) surface, and thereby reduce the background signal. Compared with no "click" ligation, the background signal decreases 7.5 times. Both the sensitivity and selectivity are greatly promoted. A high sensitivity with OTA detection down to 72 pg/mL is achieved, which is comparable with several existing detectors, such as fluorescence-based detectors and electrochemical detectors. The feasibility of the strategy in real samples is well verified and evaluated by detecting OTA in feed samples, indicating the potential application in the food safety field.

Enzyme-free fluorescent detection of microcystin-LR using hairpin DNA-templated copper nanoclusters as signal indicator.

Microcystin-LR (MC-LR) is a potent hepatotoxin that is often associated with blooms of cyanobacteria. The analysis of trace MC-LR plays important role in environmental and health fields. Herein, we developed a low-cost and enzyme-free detection method of MC-LR by using hairpin DNA-templated copper nanoclusters (hpDNA-CuNCs) as fluorescent probe. The hpDNA-template was designed and fabricated by a MC-LR aptamer loop and a double strand stem, which can specifically recognize target MC-LR with strong affinity. The AT-rich and complementary double strand stem serves as a template for the formation of CuNCs. The formed fluorescent sensing probe of hpDNA-CuNCs exhibits maximum emission wavelength at 575 nm. Upon the addition of target MC-LR into the hpDNA-CuNCs, we observed fluorescence was quenched considerably due to the high affinity between MC-LR and hpDNA aptamer strand loop, which indicated a conformational change of hairpin probe from the stem-loop DNA structure to single-stranded DNA. Then, the change of fluorescence intensity can be used to monitor the concentration of MC-LR from 0.005 to 1200 µg L-1 with a detection limit of 0.003 ng L-1. Compared with the previous reports, this method does not require complex DNA sequence design, fluorescence dye label and sophisticated experimental techniques. Moreover, the target MC-LR in real water samples has been detected.

Structural Influence on the Post-Clustering Stability of DNA/AgNCs Fluorescence.

DNA-encapsulated Silver Nanoclusters (DNA/AgNCs) based sensors have gained increasing attention in past years due to their diverse applications in bioimaging, biosensing, and enzymatic assays. Given the potential of DNA/AgNCs for practical applications, the systematic studies of the fluorescent stability over an extended period is necessary. However, the correlation between nucleic acid properties and the long-term stability of DNA/AgNCs is less known. With locking-to-unlocking sensors, in which the secondary structure of DNA template is standardized, we investigated the correlation between the DNA structure and the fluorescence stability of AgNCs. Post-synthesis of DNA/AgNCs, the fluorescence, and structures of templates were monitored over three weeks. By combining the fluorescence spectroscopy with the in-gel fluorescent assay, we found that AgNCs encapsulated by dimer-structured DNA/AgNCs templates were more stable than those of hairpin-structured DNA/AgNCs templates. While the orange fluorescence from the dimer templates increased over three weeks, the red fluorescence from the hairpin templates was diminished by >80% within two days at room temperature. Further tests revealed that hairpin-encapsulated red-emissive AgNCs is more sensitive to oxidation by atmospheric oxygen compared to dimer encapsulated orange AgNCs. Our observations may provide an important clue in encapsulating photophysically more stable AgNCs by tuning the DNA secondary structures. The proposed strategy here can be essential for pragmatic applications of DNA/AgNCs templates.

Fabrication of Metal Nanostructures with Programmable Length and Patterns Using a Modular DNA Platform.

Recently introduced DNA nanomolds allow the shape-controlled growth of metallic nanoparticles. Here we demonstrate that this approach can be used to fabricate longer linear metal nanostructures of controlled lengths and patterns. To this end, we establish a set of different interfaces that enable mold interactions with high affinity and specificity. These interfaces enable and control the modular assembly of mold monomers into larger mold superstructure with programmable dimension in which each mold monomer remains uniquely addressable. Preloading the molds with nanoparticle seeds subsequently allows the growth of linear gold nanostructures whose lengths are controlled by the DNA structure. Exploiting the addressability of individual mold monomers furthermore allows achievement of site-specific metallization, that is, to create defined metal patterns. We think that the introduced approach provides a useful basis to fabricate nanomaterials with complex shapes and material composition in a fully programmable and modular fashion.