Enhanced Targeted Gene Transduction: AAV2 Vectors Conjugated to Multiple Aptamers via Reducible Disulfide Linkages.

Enhanced targeted gene transduction by AAV2 vectors is achieved by linking the vector to multiple sgc8 aptamers, which are selective for cell membrane protein PTK7. Aptamer molecules are conjugated to multiple sites on a DNA dendrimer (G-sgc8), which is then linked to AAV2 via a dithiobis(succinimidyl propionate) cross-linker containing a disulfide group, which can facilitate the release of AAV2 vectors by reaction with the reduced form of intracellular glutathione. The G-sgc8-AAV2 vectors showed a 21-fold enhancement in binding affinity and an enhanced ability to protect sgc8 aptamers against nuclease degradation to cells expressing PTK7 compared to single aptamer-AAV2 conjugates. The transduction efficiency was tested by loading AAV2 with the gene for green fluorescent protein. Therefore, this modified recombinant vector is an attractive and promising tool for targeted biomedical applications.

Constructing Smart Protocells with Built-In DNA Computational Core to Eliminate Exogenous Challenge.

A DNA reaction network is like a biological algorithm that can respond to "molecular input signals", such as biological molecules, while the artificial cell is like a microrobot whose function is powered by the encapsulated DNA reaction network. In this work, we describe the feasibility of using a DNA reaction network as the computational core of a protocell, which will perform an artificial immune response in a concise way to eliminate a mimicked pathogenic challenge. Such a DNA reaction network (RN)-powered protocell can realize the connection of logical computation and biological recognition due to the natural programmability and biological properties of DNA. Thus, the biological input molecules can be easily involved in the molecular computation and the computation process can be spatially isolated and protected by artificial bilayer membrane. We believe the strategy proposed in the current paper, i.e., using DNA RN to power artificial cells, will lay the groundwork for understanding the basic design principles of DNA algorithm-based nanodevices which will, in turn, inspire the construction of artificial cells, or protocells, that will find a place in future biomedical research.

Fabrication and evaluation of molecularly imprinted magnetic nanoparticles for selective recognition and magnetic separation of lysozyme in human urine.

In this study, lysozyme-based magnetic molecularly imprinted polymers (Lyz-MMIPs) for selective recognition and magnetic separation of lysozyme in human urine were prepared via surface imprinting technology. The morphology and structural properties of the resultant Lyz-MMIPs were characterized by using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), thermogravimetry (TG), X-ray diffraction (XRD) and a vibrating sample magnetometer (VSM). The results showed that the Lyz-MMIPs exhibited a uniform core-shell structure and favorable magnetic properties with a saturation magnetization of 14.8 emu g-1. To obtain the best selectivity and binding performance, the pH value of adsorption solution was investigated in detail. Under the optimized conditions, the Lyz-MMIPs possessed high binding and specific recognition capacity, as well as fast binding kinetics and phase separation rates. Moreover, the saturation binding capacity of Lyz-MMIPs reached 124.3 mg g-1, which was nearly 3.2 times that of lysozyme-based magnetic non-imprinted polymers (Lyz-MNIPs). In addition, the selectivity and reusability experiments showed that the Lyz-MMIPs displayed significant selectivity and favorable reusability. Furthermore, the Lyz-MMIPs were successfully applied for the determination and separation of lysozyme in human urine with satisfactory recovery rates. Above all, the synthetic process was quite simple and this strategy may provide a versatile approach for the fabrication of well-defined molecularly imprinted polymers on magnetic nanoparticles for the analysis of complicated matrixes.

Molecular Recognition-Based DNA Nanoassemblies on the Surfaces of Nanosized Exosomes.

Exosomes are membrane-enclosed extracellular vesicles derived from cells, carrying biomolecules that include proteins and nucleic acids for intercellular communication. Owning to their advantages of size, structure, stability, and biocompatibility, exosomes have been used widely as natural nanocarriers for intracellular delivery of theranostic agents. Meanwhile, surface modifications needed to endow exosomes with additional functionalities remain challenging by their small size and the complexity of their membrane surfaces. Current methods have used genetic engineering and chemical conjugation, but these strategies require complex manipulations and have only limited applications. Herein, we present an aptamer-based DNA nanoassemblies on exosome surfaces. This in situ assembly method is based on molecular recognition between DNA aptamers and their exosome surface markers, as well as DNA hybridization chain reaction initiated by an aptamer-chimeric trigger. It further demonstrated selective assembly on target cell-derived exosomes, but not exosomes derived from nontarget cells. The present work shows that DNA nanostructures can successfully be assembled on a nanosized organelle. This approach is useful for exosome modification and functionalization, which is expected to have broad biomedical and bioanalytical applications.

Biostable L-DNAzyme for Sensing of Metal Ions in Biological Systems.

DNAzymes, an important type of metal ion-dependent functional nucleic acid, are widely applied in bioanalysis and biomedicine. However, the use of DNAzymes in practical applications has been impeded by the intrinsic drawbacks of natural nucleic acids, such as interferences from nuclease digestion and protein binding, as well as undesired intermolecular interactions with other nucleic acids. On the basis of reciprocal chiral substrate specificity, the enantiomer of D-DNAzyme, L-DNAzyme, could initiate catalytic cleavage activity with the same achiral metal ion as a cofactor. Meanwhile, by using the advantage of nonbiological L-DNAzyme, which is not subject to the interferences of biological matrixes, as recognition units, a facile and stable L-DNAzyme sensor was proposed for sensing metal ions in complex biological samples and live cells.

Rationally designed molecular beacons for bioanalytical and biomedical applications.

Nucleic acids hold promise as biomolecules for future applications in biomedicine and biotechnology. Their well-defined structures and compositions afford unique chemical properties and biological functions. Moreover, the specificity of hydrogen-bonded Watson-Crick interactions allows the construction of nucleic acid sequences with multiple functions. In particular, the development of nucleic acid probes as essential molecular engineering tools will make a significant contribution to advancements in biosensing, bioimaging and therapy. The molecular beacon (MB), first conceptualized by Tyagi and Kramer in 1996, is an excellent example of a double-stranded nucleic acid (dsDNA) probe. Although inactive in the absence of a target, dsDNA probes can report the presence of a specific target through hybridization or a specific recognition-triggered change in conformation. MB probes are typically fluorescently labeled oligonucleotides that range from 25 to 35 nucleotides (nt) in length, and their structure can be divided into three components: stem, loop and reporter. The intrinsic merit of MBs depends on predictable design, reproducibility of synthesis, simplicity of modification, and built-in signal transduction. Using resonance energy transfer (RET) for signal transduction, MBs are further endowed with increased sensitivity, rapid response and universality, making them ideal for chemical sensing, environmental monitoring and biological imaging, in contrast to other nucleic acid probes. Furthermore, integrating MBs with targeting ligands or molecular drugs can substantially support their in vivo applications in theranositics. In this review, we survey advances in bioanalytical and biomedical applications of rationally designed MBs, as they have evolved through the collaborative efforts of many researchers. We first discuss improvements to the three components of MBs: stem, loop and reporter. The current applications of MBs in biosensing, bioimaging and therapy will then be described. In particular, we emphasize recent progress in constructing MB-based biosensors in homogeneous solution or on solid surfaces. We expect that such rationally designed and functionalized MBs will open up new and exciting avenues for biological and medical research and applications.

A cell-targeted, size-photocontrollable, nuclear-uptake nanodrug delivery system for drug-resistant cancer therapy.

The development of multidrug resistance (MDR) has become an increasingly serious problem in cancer therapy. The cell-membrane overexpression of P-glycoprotein (P-gp), which can actively efflux various anticancer drugs from the cell, is a major mechanism of MDR. Nuclear-uptake nanodrug delivery systems, which enable intranuclear release of anticancer drugs, are expected to address this challenge by bypassing P-gp. However, before entering the nucleus, the nanocarrier must pass through the cell membrane, necessitating coordination between intracellular and intranuclear delivery. To accommodate this requirement, we have used DNA self-assembly to develop a nuclear-uptake nanodrug system carried by a cell-targeted near-infrared (NIR)-responsive nanotruck for drug-resistant cancer therapy. Via DNA hybridization, small drug-loaded gold nanoparticles (termed nanodrugs) can self-assemble onto the side face of a silver-gold nanorod (NR, termed nanotruck) whose end faces were modified with a cell type-specific internalizing aptamer. By using this size-photocontrollable nanodrug delivery system, anticancer drugs can be efficiently accumulated in the nuclei to effectively kill the cancer cells.

Ionic Functionalization of Hydrophobic Colloidal Nanoparticles To Form Ionic Nanoparticles with Enzymelike Properties.

Inorganic colloidal nanoparticles (NPs) stabilized by a layer of hydrophobic surfactant on their surfaces have poor solubility in the aqueous phase, thus limiting their application as biosensors under physiological conditions. Here we report a simple model to ionize various types of hydrophobic colloidal NPs, including FePt, cubic Fe3O4, Pd, CdSe, and NaYF4 (Yb 30%, Er 2%, Nd 1%) NPs, to multicharged (positive and negative) NPs via ligand exchange. Surfaces of neutral hydrophobic NPs were converted to multicharged ions, thus making them soluble in water. Furthermore, peroxidase-like activity was observed for ionic FePt, Fe3O4, Pd, and CdSe NPs, of which FePt and CdSe catalyzed the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the blue-colored product in the absence of H2O2, while Pd and Fe3O4 catalyzed the oxidization of TMB in the presence of H2O2. With the benefit of the ionic functionalization protocols described herein, colloidal NPs should gain wider use as biomarkers, nanozymes, and biosensors.

Self-assembly of DNA nanohydrogels with controllable size and stimuli-responsive property for targeted gene regulation therapy.

Here, we report the synthesis and characterization of size-controllable and stimuli-responsive DNA nanohydrogels as effective targeted gene delivery vectors. DNA nanohydrogels were created through a self-assembly process using three kinds of building units, respectively termed Y-shaped monomer A with three sticky ends (YMA), Y-shaped monomer B with one sticky end (YMB), and DNA linker (LK) with two sticky ends. Hybridization at the sticky ends of monomers and LK leads to nanohydrogel formation. DNA nanohydrogels are size-controllable by varying the ratio of YMA to YMB. By incorporating different functional elements, such as aptamers, disulfide linkages, and therapeutic genes into different building units, the synthesized aptamer-based nanohydrogels (Y-gel-Apt) can be used for targeted and stimuli-responsive gene therapy. Y-gel-Apt strongly inhibited cell proliferation and migration in target A549 cells, but not in control cells. By taking advantage of facile modular design and assembly, efficient cellular uptake, and superior biocompatibility, this Y-gel-Apt holds great promise as a candidate for targeted gene or drug delivery and cancer therapy.

A Nonenzymatic Hairpin DNA Cascade Reaction Provides High Signal Gain of mRNA Imaging inside Live Cells.

Enzyme-free signal amplification has enabled sensitive in vitro detection of biomolecules such as proteins and nucleic acids. However, monitoring targets of interest in live cells via enzyme-free amplification is still challenging, especially for analytes with low concentrations. To the best of our knowledge, this paper reports the first attempt to perform mRNA imaging inside live cells, using a nonenzymatic hairpin DNA cascade reaction for high signal gain, termed a hairpin DNA cascade amplifier (HDCA). In conventional nucleic acid probes, such as linear hybridization probes, mRNA target signaling occurs in an equivalent reaction ratio (1:1), whereas, in HDCA, one mRNA target is able to yield multiple signal outputs (1:m), thus achieving the goal of signal amplification for low-expression mRNA targets. Moreover, the recycled mRNA target in the HDCA serves as a catalyst for the assembly of multiple DNA duplexes, generating the fluorescent signal of reduced MnSOD mRNA expression, thus indicating amplified intracellular imaging. This programmable cascade reaction presents a simple and modular amplification mechanism for intracellular biomarkers of interest, providing a significant boost to the search for clues leading to the accurate identification and effective treatment of cancers.

DLISA: A DNAzyme-Based ELISA for Protein Enzyme-Free Immunoassay of Multiple Analytes.

A DNAzyme-based ELISA, termed DLISA, was developed as a novel protein enzyme-free, triply amplified platform, combining a catalytic and molecular beacon (CAMB) system with a cation exchange reaction for ultrasensitive multiplex fluorescent immunosorbent assay. Classical ELISA, which employs protein enzymes as biocatalysts to afford amplified signals, suffers from poor stability caused by the irreversible denaturation of these enzymes under harsh conditions, such as heat and acidity. Compared with proteins, nucleic acids are more stable and adaptable, and they can be easily produced using a commercial DNA synthesizer. Moreover, the catalytic and cleavage activities of DNAzyme can be achieved in solution; thus, no enzyme immobilization is needed for detection. Taken together, these attributes suggest that a DNAzyme-based ELISA detection approach will be more robust than current ELISA assays. Importantly, the proposed triply amplified DLISA immunoassay method shows ultrasensitive detection of such targets as human IgG with a detection limit of 2 fg/mL (3 × 10(-17) M), which is well within the range of many important disease biomarkers. DLISA can also be used to construct a sensing array for simultaneous multiplexed detection. With these merits, this high-throughput, stable, simple, sensitive, and low-cost multiplex fluorescence immunoassay shows promise for applications in clinical diagnosis.

Cell membrane-anchored biosensors for real-time monitoring of the cellular microenvironment.

Cell membrane-anchored biochemical sensors that allow real-time monitoring of the interactions of cells with their microenvironment would be powerful tools for studying the mechanisms underlying various biological processes, such as cell metabolism and signaling. Despite the significance of these techniques, unfortunately, their development has lagged far behind due to the lack of a desirable membrane engineering method. Here, we propose a simple, efficient, biocompatible, and universal strategy for one-step self-construction of cell-surface sensors using diacyllipid-DNA conjugates as the building and sensing elements. The sensors exploit the high membrane-insertion capacity of a diacyllipid tail and good sensing performance of the DNA probes. Based on this strategy, we have engineered specific DNAzymes on the cell membrane for metal ion assay in the extracellular microspace. The immobilized DNAzyme showed excellent performance for reporting and semiquantifying both exogenous and cell-extruded target metal ions in real time. This membrane-anchored sensor could also be used for multiple target detection by having different DNA probes inserted, providing potentially useful tools for versatile applications in cell biology, biomedical research, drug discovery, and tissue engineering.

Facile surface functionalization of hydrophobic magnetic nanoparticles.

Nonpolar phase synthesized hydrophobic nanocrystals show attractive properties and have demonstrated prominent potential in biomedical applications. However, the preparation of biocompatible nanocrystals is made difficult by the presence of hydrophobic surfactant stabilizer on their surfaces. To address this limitation, we have developed a facile, high efficiency, single-phase and low-cost method to convert hydrophobic magnetic nanoparticles (MNPs) to an aqueous phase using tetrahydrofuran, NaOH and 3,4-dihydroxyhydrocinnamic acid without any complicated organic synthesis. The as-transferred hydrophilic MNPs are water-soluble over a wide pH range (pH = 3-12), and the solubility is pH-controllable. Furthermore, the as-transferred MNPs with carboxylate can be readily adapted with further surface functionalization, varying from small molecule dyes to oligonucleotides and enzymes. Finally, the strategy developed here can easily be extended to other types of hydrophobic nanoparticles to facilitate biomedical applications of nanomaterials.

Multicolor fluorescent biosensor for multiplexed detection of DNA.

Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

Gold-Coated Fe3O4 Nanoroses with Five Unique Functions for Cancer Cell Targeting, Imaging and Therapy.

The development of nanomaterials that combine diagnostic and therapeutic functions within a single nanoplatform is extremely important for molecular medicine. Molecular imaging with simultaneous diagnosis and therapy will provide the multimodality needed for accurate diagnosis and targeted therapy. Here, we demonstrate gold-coated iron oxide (Fe3O4@Au) nanoroses with five distinct functions, which integrate aptamer-based targeting, magnetic resonance imaging (MRI), optical imaging, photothermal therapy and chemotherapy into one single probe. The inner Fe3O4 core functions as an MRI agent, while the photothermal effect is achieved through near-infrared absorption by the gold shell, causing a rapid rise in temperature and also resulting in a facilitated release of the anticancer drug doxorubicin carried by the nanoroses. Where the doxorubicin is released is monitored by its fluorescent. Aptamers immobilized on the surfaces of the nanoroses enable efficient and selective drug delivery, imaging and photothermal effect with high specificity. The five-function-embedded nanoroses show great advantages in multimodality.

Nucleic acid based logical systems.

Researchers increasingly visualize a significant role for artificial biochemical logical systems in biological engineering, much like digital logic circuits in electrical engineering. Those logical systems could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expression in vivo. Nucleic acids (NA), as carriers of genetic information with well-regulated and predictable structures, are promising materials for the design and engineering of biochemical circuits. A number of logical devices based on nucleic acids (NA) have been designed to handle various processes for technological or biotechnological purposes. This article focuses on the most recent and important developments in NA-based logical devices and their evolution from in vitro, through cellular, even towards in vivo biological applications.

A targeted, self-delivered, and photocontrolled molecular beacon for mRNA detection in living cells.

The spatiotemporal dynamics of specific mRNA molecules are difficult to image and detect inside living cells, and this has been a significant challenge for the chemical and biomedical communities. To solve this problem, we have developed a targeted, self-delivered, and photocontrolled aptamer-based molecular beacon (MB) for intracellular mRNA analysis. An internalizing aptamer connected via a double-stranded DNA structure was used as a carrier probe (CP) for cell-specific delivery of the MB designed to signal target mRNA. A light activation strategy was employed by inserting two photolabile groups in the CP sequence, enabling control over the MB's intracellular function. After the probe was guided to the target cell via specific binding of aptamer AS1411 to nucleolin on the cell membrane, light illumination released the MB for mRNA monitoring. Consequently, the MB is able to perform live-cell mRNA imaging with precise spatiotemporal control, while the CP acts as both a tracer for intracellular distribution of the MB before photoinitiation and an internal reference for mRNA ratiometric detection.

Building a multifunctional aptamer-based DNA nanoassembly for targeted cancer therapy.

The ability to self-assemble one-dimensional DNA building blocks into two- and three-dimensional nanostructures via DNA/RNA nanotechnology has led to broad applications in bioimaging, basic biological mechanism studies, disease diagnosis, and drug delivery. However, the cellular uptake of most nucleic acid nanostructures is dependent on passive delivery or the enhanced permeability and retention effect, which may not be suitable for certain types of cancers, especially for treatment in vivo. To meet this need, we have constructed a multifunctional aptamer-based DNA nanoassembly (AptNA) for targeted cancer therapy. In particular, we first designed various Y-shaped functional DNA domains through predesigned base pair hybridization, including targeting aptamers, intercalated anticancer drugs, and therapeutic antisense oligonucleotides. Then these functional DNA domains were linked to an X-shaped DNA core connector, termed a building unit, through the complementary sequences in the arms of functional domains and connector. Finally, hundreds (~100-200) of these basic building units with 5'-modification of acrydite groups were further photo-cross-linked into a multifunctional and programmable aptamer-based nanoassembly structure able to take advantage of facile modular design and assembly, high programmability, excellent biostability and biocompatibility, as well as selective recognition and transportation. With these properties, AptNAs were demonstrated to have specific cytotoxic effect against leukemia cells. Moreover, the incorporation of therapeutic antisense oligonucleotides resulted in the inhibition of P-gp expression (a drug efflux pump to increase excretion of anticancer drugs) as well as a decrease in drug resistance. Therefore, these multifunctional and programmable aptamer-based DNA nanoassemblies show promise as candidates for targeted drug delivery and cancer therapy.

Responsive DNA-based hydrogels and their applications.

The term hydrogel describes a type of soft and wet material formed by cross-linked hydrophilic polymers. The distinct feature of hydrogels is their ability to absorb a large amount of water and swell. The properties of a hydrogel are usually determined by the chemical properties of their constituent polymer(s). However, a group of hydrogels, called "smart hydrogels," changes properties in response to environmental changes or external stimuli. Recently, DNA or DNA-inspired responsive hydrogels have attracted considerable attention in construction of smart hydrogels because of the intrinsic advantages of DNA. As a biological polymer, DNA is hydrophilic, biocompatible, and highly programmable by Watson-Crick base pairing. DNA can form a hydrogel by itself under certain conditions, and it can also be incorporated into synthetic polymers to form DNA-hybrid hydrogels. Functional DNAs, such as aptamers and DNAzymes, provide additional molecular recognition capabilities and versatility. In this Review, DNA-based hydrogels are discussed in terms of their stimulus response, as well as their applications.

A logical molecular circuit for programmable and autonomous regulation of protein activity using DNA aptamer-protein interactions.

Researchers increasingly envision an important role for artificial biochemical circuits in biological engineering, much like electrical circuits in electrical engineering. Similar to electrical circuits, which control electromechanical devices, biochemical circuits could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expressions in vivo. (1) As a consequence of their relative robustness and potential applicability for controlling a wide range of in vitro chemistries, synthetic cell-free biochemical circuits promise to be useful in manipulating the functions of biological molecules. Here, we describe the first logical circuit based on DNA-protein interactions with accurate threshold control, enabling autonomous, self-sustained and programmable manipulation of protein activity in vitro. Similar circuits made previously were based primarily on DNA hybridization and strand displacement reactions. This new design uses the diverse nucleic acid interactions with proteins. The circuit can precisely sense the local enzymatic environment, such as the concentration of thrombin, and when it is excessively high, a coagulation inhibitor is automatically released by a concentration-adjusted circuit module. To demonstrate the programmable and autonomous modulation, a molecular circuit with different threshold concentrations of thrombin was tested as a proof of principle. In the future, owing to tunable regulation, design modularity and target specificity, this prototype could lead to the development of novel DNA biochemical circuits to control the delivery of aptamer-based drugs in smart and personalized medicine, providing a more efficient and safer therapeutic strategy.