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The process of coordinating between the same or multiple types of cells to jointly execute various instructions in a controlled and carefully regulated environment is a very appealing field. In order to provide clearer insight into the role of cell-cell interactions and the cellular communication of this process in their local communities, several interdisciplinary approaches have been employed to enhance the core understanding of this phenomenon. DNA nanostructures have emerged in recent years as one of the most promising tools in exploring cell-cell communication and interactions due to their programmability and addressability. Herein, this review is dedicated to offering a new perspective on using DNA nanostructures to explore the progress of cell-cell communication. After briefly outlining the anchoring strategy of DNA nanostructures on cell membranes and the subsequent dynamic regulation of DNA nanostructures, this paper highlights the significant contribution of DNA nanostructures in monitoring cell-cell communication and regulating its interactions. Finally, we provide a quick overview of the current challenges and potential directions for the application of DNA nanostructures in cellular communication and interactions.

Liquid coacervate microdroplets have been widely explored as membrane-free compartment protocells for cargo delivery in therapeutic applications. In this study, coacervate protocells were developed as gene carriers for transfection of nitric oxide synthase (NOS) and overproduction of nitric oxide (NO) for killing of cancer cells. The coacervate microdroplet protocells were formed via the liquid-liquid phase separation of oppositely charged diethylaminoethyl-dextran/polyacrylic acids. The coacervate microdroplet protocells were found to facilitate gene transfection, which was demonstrated by cell imaging of the internalized coacervate microdroplets containing plasmids of enhanced green fluorescent protein. Due to their high transfection capability, the coacervate protocells were subsequently utilized for the delivery of NOS plasmids (pNOS). The cellular internalization of pNOS-containing coacervate carriers was found to result in high NOS expression coupled with NO overproduction, which then induced cell apoptosis and decreased cell viability. The cell apoptosis is associated with NO-mediated mitochondrial damage. The enhanced gene transfection was attributed to coacervate microdroplets' unique high sequestration capability and liquid-like fluidity. Overall, the incorporation of genes in coacervate microdroplets was demonstrated as a viable and novel strategy for the development of cargo biocarriers for biomedical applications.

A self-assembled DNA nanostructure based on a DNA nanocreeper and multiplexed fluorescence supersandwich was designed for the sensitive and specific detection of tumour cells. This nanostructure could improve the binding affinity of current aptamers and trigger signal amplification, which provide potential for the discrimination of low abundant target cells in liquid biopsy.

Aptamer-tailored DNA supersandwich assemblies are proposed as artificial receptors anchored onto a cell membrane, enabling catalase delivery with an 8-fold enhancement in efficiency. The catalase transfection led to inhibition of intracellular ROS, while the cell viability was also remarkably improved even when exposed to H2O2 and lipopolysaccharide environments.

Accurate measurement of intracellular temperature is of great significance in biology and medicine. With use of DNA nanotechnology and inspiration by nature's examples of "protective and reversible responses" exoskeletons, a scallop-inspired DNA nanomachine (SDN) is desgined as a ratiometric nanothermometer for intracellular temperature sensing. The SDN is composed of a rigid DNA tetrahedron, where a thermal-sensitive molecular beacon (MB) is embedded in one edge of the DNA tetrahedron. Relying on the thermal-sensitive MB and fluorescence resonance energy transfer (FRET) signaling mechanism, the "On" to "Off" signal is reversibly responding to "below" and "over" the melting temperature. Mimicking the functional anatomy of a scallop, the SDN exhibits high cellular permeability and resistance to enzymatic degradation, good reversibility, and tunable response range. Furthermore, FRET ratiometric signal that allows the simultaneous recording of two emission intensities at different wavelengths can provide a feasible approach for precise detection, minimizing the effect of system fluctuations.

We report self-assembled nanocentipedes as multivalent vehicles for the delivery of immunostimulatory CpG ODNs. The multivalent vehicles could be internalized by RAW264.7 cells and stimulate the secretion of large amounts of cytokines, successively inducing effective apoptosis of cancer cells.

DNA nanostructures have emerged as powerful and versatile building blocks for the construction of programmable nanoscale structures and functional sensors for biomarker detection, disease diagnostics, and therapy. Here we integrated multiple sensing modules into a single DNA three-dimensional (3D) nanoarchitecture with a triangular-prism (TP) structure for ratiometric and multiplexed biomolecule detection on a single microbead. In our design, the complementary hybridization of three clip sequences formed TP nanoassemblies in which the six single-strand regions in the top and bottom faces act as binding sites for different sensing modules, including an anchor module, reference sequence module, and capture sequence module. The multifunctional modular TP nanostructures were thus exploited for ratiometric and multiplexed biomolecule detection on microbeads. Microbead imaging demonstrated that, after ratiometric self-calibration analysis, the imaging deviations resulting from uneven fluorescence intensity distribution and differing probe concentrations were greatly reduced. The rigid nanostructure also conferred the TP as a framework for geometric positioning of different capture sequences. The inclusion of multiple targets led to the formation of sandwich hybridization structures that gave a readily detectable optical response at different fluorescence channels and distinct fingerprint-like pattern arrays. This approach allowed us to discriminate multiplexed biomolecule targets in a simple and efficient fashion. In this module-designed strategy, the diversity of the controlled DNA assembly coupled with the geometrically well-defined rigid nanostructures of the TP assembly provides a flexible and reliable biosensing approach that shows great promise for biomedical applications.

Herein, we proposed an enzyme-free strategy for the amplified detection of DNA by combining the efficient fluorescence enhancement capability of a β-cyclodextrin-tethered cationic polymer (cationic polyβ-CD) to pyrene with the amplification capability of target DNA triggered hybridization chain reaction (HCR). Cationic polyβ-CD with positive charge was synthesized. Two hairpin probes, H1 and H2, were employed in the system and the pyrene-labelled H2 was chosen as the signal unit. The pyrene attached on the sticky end of H2 was flexible and there was strong electrostatic interaction between cationic polyβ-CD and negatively-charged H2, so pyrene could easily enter the cavity of CD that is tethered on the cationic polymer, accompanied by significant fluorescence enhancement. Once target DNA was introduced, HCR was triggered to form a rigid long dsDNA polymer with pyrene attached on it. The pyrene was hardly able to enter the cavity of cationic polyβ-CD because of steric hindrance, leading to a weak fluorescent signal. Owing to the efficient pyrene fluorescence enhancement of cationic polyβ-CD and the amplified capability of HCR, an enzyme-free sensitive detection of target DNA was achieved with a detection limit of 0.1 nM and high selectivity.

A DNA-protein hybrid hydrogel was constructed based on a programmable assembly approach, which served as a biomimetic physiologic matrix for efficient enzyme encapsulation. A dsDNA building block tailored with precise biotin residues was fabricated based on supersandwich hybridization, and then the addition of streptavidin triggered the formation of the DNA-protein hybrid hydrogel. The biocompatible hydrogel, which formed a flower-like porous structure that was 6.7 ± 2.1 μm in size, served as a reservoir system for enzyme encapsulation. Alcohol oxidase (AOx), which served as a representative enzyme, was encapsulated in the hybrid hydrogel using a synchronous assembly approach. The enzyme-encapsulated hydrogel was utilized to extend the duration time for ethanol removal in serum plasma and the enzyme retained 78% activity after incubation with human serum for 24 h. The DNA-protein hybrid hydrogel can mediate the intact immobilization on a streptavidin-modified and positively charged substrate, which is very beneficial to solid-phase biosensing applications. The hydrogel-encapsulated enzyme exhibited improved stability in the presence of various denaturants. For example, the encapsulated enzyme retained 60% activity after incubation at 55 °C for 30 min. The encapsulated enzyme also retains its total activity after five freeze-thaw cycles and even suspended in solution containing organic solvents.

The chemistry of singlet O2 toward the guanine base of DNA is highly relevant to DNA lesion, mutation, cell death, and pathological conditions. This oxidative damage is initiated by the formation of a transient endoperoxide through the Diels-Alder cycloaddition of singlet O2 to the guanine imidazole ring. However, no endoperoxide formation was directly detected in native guanine or guanosine, even at -100 °C. Herein, gas-phase ion-molecule scattering mass spectrometry was utilized to capture unstable endoperoxides in the collisions of hydrated guanine ions (protonated or deprotonated) with singlet O2 at ambient temperature. Corroborated by results from potential energy surface exploration, kinetic modeling, and dynamics simulations, various aspects of endoperoxide formation and transformation (including its dependence on guanine ionization and hydration states, as well as on collision energy) were determined. This work has pieced together reaction mechanisms, kinetics, and dynamics data concerning the early stage of singlet O2 induced guanine oxidation, which is missing from conventional condensed-phase studies., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The detection of nucleic acids is fundamental for studying their functions and for the development of biological studies and medical diagnostics. Herein, we report a new strategy for nucleic acid amplified detection by combining target-catalyzed dynamic assembly with host-guest interaction between β-cyclodextrin polymer (β-CDP) and pyrene. In this strategy, a metastable pyrene-labeled hairpin DNA probe (probe H1) and a metastable unlabeled hairpin DNA probe (probe H2) were elaborately designed as the assembly components, which were kinetically handicapped from cross-opening in the absence of target DNA. In this state, pyrene labled at the 5'-termini of single-stranded stem of probe H1 would be easily trapped into the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to significant fluorescence enhancement. Once the dynamic assembly was catalyzed by target DNA, a hybridized DNA duplex H1-H2 would be created continuously. In this state, it is difficult for pyrene to enter the cavity of β-CDP due to steric hindrance and weak-binding interaction, leading to a weak fluorescent signal. Thus, target DNA could be detected by this simple mix-and-detect amplification method without the need of expensive and perishable protein enzymes. As low as 10 pM of the target DNA was detected by this assay, which was comparable to that of some reported enzyme-dependent amplification methods. Meanwhile, the proposed method was further successfully applied to detect DNA in cell lysate samples, showing great potential for target detection from complex fluids. In addition, as a novel transformation of dynamic DNA assembly technology into enzyme-free signal-amplification analytical application, the proposed strategy has shown great potential for applications in a wide range of fields, such as aptamer-based non-nucleic acid target sensing, biomedicine and bioimaging., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A multiple amplification strategy has been developed for nucleic acid detection based on host-guest interaction between the β-cyclodextrin polymer (β-CDP) and pyrene. Briefly, the detection system consists of three parts: the polymerase and nicking enzyme-assisted isothermal strand displacement amplification (SDA) activated by a target DNA or microRNA; the exonuclease III-aided cyclic enzymatic amplification (CEA); and the fluorescence enhancement effect based on host-guest interaction between β-CDP and pyrene. This strategy showed a good positive linear correlation with target DNA concentrations in the range from 75 fM to 1 pM with a detection limit of 41 fM. Significantly, our amplification platform was further validated and evaluated successfully by assaying miRNA-21 in human serum. The proposed assay has great potential as a nucleic acid quantification method for use in biomedical research, clinical analysis and disease diagnostics.

We developed a novel homogeneous fluorescence analysis based on a novel competitive host-guest interaction (CHGI) mechanism between β-cyclodextrin polymer (polyβ CD) and pyrene-labeled probe for biochemical assay. Pyrene labeling with oligonucleotide strands can be recruited and reside in lipophilic cavities of polyβ CD. This altered lipophilic microenvironment provides favored polarity for enhanced quantum efficiencies and extraordinarily increases the luminescence intensity of pyrene. However, with addition of complementary DNA, the pyrene-labeled probe formed double-strand DNA to hinder pyrene from entering the cavities of polyβ CD. The release of pyrene from polyβ CD, which are followed by fluorescence extinguishing, will provide the clear signal turn-off in the presence of target DNA. We also introduced Exodeoxyribonuclease I (Exo I) and Exodeoxyribonuclease III (Exo III) to improve the sensitivity of this system, and the following product of cleavage reaction, pyrene-nucleotide, could more easily host-guest interact with polyβ CD and emit stronger fluorescence than pyrene-labeled probe. In addition, the successful detection of adenosine is also demonstrated by using the similar sensing scheme. Although this scheme might be easily interfered by some biomolecules in the real test sample, it holds promising potential for detecting a broad range of other types of aptamer-binding chemicals and biomolecules.

We present a novel strategy for construction of a conformational switch of a molecular beacon based on the combination of nucleic acid (DNA) self-assembly and reversible host-guest inclusion interaction. With the functionalized probe, the nucleic acid hybridization process can be easily controlled with a photoactivated and thermal regeneration mode.

A label-free and non-enzymatic amplification fluorescent method for detection of DNA has been developed by using hybridization chain reaction (HCR) and dsDNA-templated copper nanoparticles (CuNPs). First, the biotinylated capture DNA probes were immobilized on the streptavidin-modified beads through the interaction of biotin and streptavidin. Then, target DNA hybridized with the capture DNA probes, which formed a hybridized DNA with sticky end. The sticky end triggered the HCR process and formation of dsDNA polymers while two hairpin probes coexisted. Subsequently, the dsDNA polymers were employed as template for synthesis of CuNPs with excellent fluorescent properties, which provided a label-free, non-enzymatic signal response. Meanwhile, the fluorescence sensing depended on the target DNA triggered HCR, which render this method a high selectivity against single-base mismatch sequences. The concept and methodology developed in this work show great promise in the quantitative detection of DNA in biological and medical applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel, high sensitive, and specific DNA assay based on gold nanoparticle (AuNP) colorimetric detection and hybridization chain reaction (HCR) amplification has been demonstrated in this article. Two hairpin auxiliary probes were designed with single-stranded DNA (ssDNA) sticky ends which stabilize AuNPs and effectively prevent them from salt-induced aggregation. The target DNA hybridized with the hairpin auxiliary probes and triggered the formation of extended double-stranded DNA (dsDNA) polymers through HCR. As a result, the formed dsDNA polymers provide less stabilization without ssDNA sticky ends, and AuNPs undergo aggregation when salt concentration is increased. Subsequently, a pale purple-to-blue color variation is observed in the colloid solution. The system is simple in design and convenient in operation. The novel strategy eliminates the need for enzymatic reactions, separation processes, chemical modifications, and sophisticated instrumentation. The detection and discrimination process can be seen with the naked eye. The detection limit of this method is lower than or at least comparable to previous AuNP-based methods. Importantly, the protocol offers high selectivity for the determination between perfectly matched target oligonucleotides and targets with single base-pair mismatches.

Torque teno sus virus 1 (TTSuV1) has a non-enveloped, single-stranded, negative-sense circular DNA genome, and it is widely distributed in pigs. Open reading frame 1 (ORF1) of TTSuV1 can be transcribed into mRNA and then translated into protein; however, its promoter has not yet been identified. We used a dual-luciferase reporter system, involving pGL3-Basic and pRL-TK, to identify the promoter of TTSuV1 ORF1. Our results revealed that the sequence between nucleotides 196 and 525 promoted the transcription of the firefly luciferase gene. The core sequence of the promoter was between nucleotides 250 and 400. A comparison of the identified TTSuV1 ORF1 promoter with that from cytomegalovirus (CMV) suggested that the two promoters were similar in strength. Our findings provide new information regarding the molecular biology of TTSuV1 and have revealed a new promoter that can be used in plasmids for numerous applications. [ABSTRACT FROM AUTHOR]

A signal amplification method has been developed for determination of human papillomavirus-16 DNA (HPV-16 DNA). It consists of a combination of using (a) two-color emitting fluorescent DNA-templated silver nanoclusters (DNA-AgNCs); (b) hairpin-blocked DNAzyme probe (H-Dz), and (c) catalytic hairpin assembly. In the absence of the model target HPV-16 DNA, the H-Dz forms a hairpin structure through intramolecular hybridization. It inhibits the catalytic activity of the DNAzyme. However, in the presence of HPV-16 DNA, it will hybridize with the DNAzyme probe which leads to the opening of the hairpin and the formation of an active secondary structure in the catalytic core. This produces an "active" DNAzyme which can cut H-Dz under the catalytic action of Mg(II) ions. Then, the DNA fragment and HPV-16 DNA are separated due to the weak hybridization. The former triggers a downstream catalytic hairpin assembly (CHA). This changes the color of the fluorescence of DNA-AgNCs from red (630 nm) to yellow (570 nm). The HPV-16 DNA will hybridize with another H-Dz to trigger the next round of activation cycle to cut H-Dz. In this way, recycling amplification results in the generation of a strong signal that allows human HPV-16 DNA to be detected with a 5.7 pM detection limit. [ABSTRACT FROM AUTHOR]

Highlights: [•] The advantages of DNA hairpin-based fluorescent probes (DHFPs) are discussed. [•] The various signal-labeled DHFP types and properties are listed. [•] The signal-transduction mechanisms are demonstrated by representative examples. [•] Some recent major bioanalytical applications of DHFPs are introduced. [Copyright &y& Elsevier]

Abstract: A simple and distinctive assay for quantitating target DNA was developed based on personal uric acid meter (PUAM) and xanthine oxidase modified DNA (xanthine oxidase-DNA). Capture DNA was assembled on the Au film by the formation of Au S bond. When target DNA was added, it was complementary to capture DNA at one-half of the segment and complementary to the xanthine oxidase-DNA at the other half-segment, resulting in the formation of a stable duplex complex. As a result, the xanthine oxidase was dragged to the Au film. Since xanthine oxidase could catalyze hydrolysis of xanthine to uric acid, the concentration of target DNA in the sample could be quantified by monitoring uric acid using PUAM. Since the signal was amplified based on the enzymatic turnovers, this enzyme-based assay made it possible that 10pM p53 DNA or 4pM HBV DNA could be detected using PUAM. This assay also showed excellent selectivity toward mismatches, and demonstrated its applicability for the target detection in human serum. Furthermore, this protocol showed a new strategy to detect non-uric acid targets using PUAM. [Copyright &y& Elsevier]