Aptamer-Directed Protein-Specific Multiple Modifications of Membrane Glycoproteins on Living Cells.

Understanding how a cell membrane protein functions on living cells remains a challenge for cell biology. Specific placement of functional molecules on specific proteins in their native environment would allow comprehensive study of proteins' dynamic functions. Existing methods cannot facilely achieve multiple modifications on specific membrane proteins. In this report, we describe an aptamer-induced, protein-specific bio-orthogonal modification technology for precise nongenetic immobilization of multiple small functional molecules on target membrane glycoproteins by combining metabolic technology and aptamer targeting. In brief, DNA probes were designed by modifying aptamers, which bind to target proteins on the surfaces of living cells pretreated with N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz). The cyclooctynes tagged of DNA probes will approach the azide groups to trigger the bio-orthogonal reactions. After UV irradiation and hybridization with cDNA (complementary DNA), the aptamers can be removed, and the process can be repeated to achieve multiple modifications for multicolor imaging and cell surface nanoengineering on specific proteins.

Circular Bispecific Aptamer-Mediated Artificial Intercellular Recognition for Targeted T Cell Immunotherapy.

Adoptive T cell immunotherapy, such as chimeric antigen receptor (CAR) T cell therapy, has proven to be highly efficient in the treatment of hematologic malignancies. However, it is challenged by complicated ex vivo engineering, systemic side effects, and low expression of tumor-specific antigen, especially in solid tumors. In this paper, we present a "recognition-then-activation" strategy, which first assists naïve T cells to recognize and adhere to cancer cells and then activates the accumulated T cell in situ to specifically kill cancer cells. In this way, we could unleash the antitumor power of the T cell without complicated and time-consuming cell engineering. To this end, circular bispecific aptamers (cb-aptamers), a class of chemically cyclized aptamers with improved stability and molecular recognition ability which can simultaneously bind to two different types of cells, were first constructed to form artificial intercellular recognition between naïve T cells and tumor cells. After T cell accumulation in the tumor mediated by cb-aptamers, T cells in the tumor site were subsequently activated in situvia commercial CD3/CD28 T cell activator beads to induce tumor-specific killing. Furthermore, by simply choosing different anticancer aptamers, the application of this "recognition-then-activation" strategy can be expanded for targeted treatment of various types of cancer. This may represent a simple T cell immunotherapy that is useful for the treatment of multiple cancers.

A programmable polymer library that enables the construction of stimuli-responsive nanocarriers containing logic gates.

Stimuli-responsive biomaterials that contain logic gates hold great potential for detecting and responding to pathological markers as part of clinical therapies. However, a major barrier is the lack of a generalized system that can be used to easily assemble different ligand-responsive units to form programmable nanodevices for advanced biocomputation. Here we develop a programmable polymer library by including responsive units in building blocks with similar structure and reactivity. Using these polymers, we have developed a series of smart nanocarriers with hierarchical structures containing logic gates linked to self-immolative motifs. Designed with disease biomarkers as inputs, our logic devices showed site-specific release of multiple therapeutics (including kinase inhibitors, drugs and short interfering RNA) in vitro and in vivo. We expect that this 'plug and play' platform will be expanded towards smart biomaterial engineering for therapeutic delivery, precision medicine, tissue engineering and stem cell therapy.

An Aptamer-Nanotrain Assembled from Six-Letter DNA Delivers Doxorubicin Selectively to Liver Cancer Cells.

Expanding the number of nucleotides in DNA increases the information density of functional DNA molecules, creating nanoassemblies that cannot be invaded by natural DNA/RNA in complex biological systems. Here, we show how six-letter GACTZP DNA contributes this property in two parts of a nanoassembly: 1) in an aptamer evolved from a six-letter DNA library to selectively bind liver cancer cells; and 2) in a six-letter self-assembling GACTZP nanotrain that carries the drug doxorubicin. The aptamer-nanotrain assembly, charged with doxorubicin, selectively kills liver cancer cells in culture, as the selectivity of the aptamer binding directs doxorubicin into the aptamer-targeted cells. The assembly does not kill untransformed cells that the aptamer does not bind. This architecture, built with an expanded genetic alphabet, is reminiscent of antibodies conjugated to drugs, which presumably act by this mechanism as well, but with the antibody replaced by an aptamer.

Aptamer Displacement Reaction from Live-Cell Surfaces and Its Applications.

The DNA strand displacement reaction has had sustained scientific interest in building complicated nucleic acid-based networks. However, extending the fundamental mechanism to more diverse biomolecules in a complex environment remains challenging. Aptamers bind with targeted biomolecules with high affinity and selectivity, thus offering a promising route to link the powers of nucleic acid with diverse cues. Here, we describe three methods that allow facile and efficient displacement reaction of aptamers from the living cell surface using complement DNA (cDNA), toehold-labeled cDNA (tcDNA), and single-stranded binding protein (SSB). The kinetics of the DNA strand displacement reaction is severely affected by complex physicochemical properties of the natural membrane. Toehold-mediated and SSB-mediated aptamer displacement exhibited significantly enhanced kinetics, and they completely removed the aptamer quickly to avoid a false signal caused by aptamer internalization. Because of its simplicity, aptamer displacement enabled detection of membrane protein post-translation and improved selection efficiency of cell-SELEX.

Generalized Preparation of Two-Dimensional Quasi-nanosheets via Self-assembly of Nanoparticles.

Two-dimensional (2D) nanomaterials are attracting increasing research interest because of their unique properties and promising applications. Here, we report a facile method to manipulate the assembly of nanoparticles (NPs) to fabricate free-standing 2D quasi-nanosheets. The as-generated 2D products are composed of few-layer NPs; that is, their thicknesses are only tens of nanometers but lateral dimensions could be up to several micrometers. Therefore, the novel structure was denoted as 2D "quasi-nanosheets (QNS)". Specifically, several types of building blocks could be assembled into 2D unary, binary, ternary, and even quaternary QNS by a universal procedure. The entire assembly process is carried out in solution and mediated simply by tuning the concentration of ligands surrounding the NPs. In contrast to traditional assembly techniques, even without any substrate or template, these QNS showed exceptionally high stability. They can remain intact for several days without any disassembly regardless of the solvent environment (e.g., water, ethanol, methanol, and hexane). In general, our method has effectively tackled several limitations associated with traditional assembly techniques and allows more freedom in manipulating assembly of NPs, which may hold great potential for future fabrication of 2D devices with rich functionalities.

Facile approach to prepare HSA-templated MnO2 nanosheets as oxidase mimic for colorimetric detection of glutathione.

In this work, a simple, rapid, and highly sensitive colorimetric assay for the determination of glutathione (GSH) was developed. It employs human serum albumin (HSA)-templated MnO2 nanosheets as an artificial oxidase. HSA-templated MnO2 nanosheets can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to a blue oxTMB product with a significant increase in absorbance at 652 nm in the absence of H2O2. When GSH is introduced, the MnO2 nanosheets are reduced to Mn2+ ions, thereby inhibiting the formation of oxTMB. Based on these findings, a simple colorimetric assay was developed for the detection GSH in the range of 10 nM to 5 µM with a low detection limit of 5.6 nM. Importantly, the proposed method was successfully used for quantitative determination of GSH in biological fluids, such as human serum samples.

Free-Floating 2D Nanosheets with a Superlattice Assembled from Fe3O4 Nanoparticles for Peroxidase-Mimicking Activity.

The organization of nanoparticles (NPs) with controlled chemical composition and size distribution into well-defined sheets will find many practical applications, but the chemistry remains problematic. Therefore, we report a facile method to assemble NPs to free-floating two-dimensional (2D) nanosheets with a superlattice and thicknesses reaching 22.8 nm. The ligand oleic acid is critical in the formation of nanosheets. As assembled, these free-floating 2D nanosheets remain intact in both polar and nonpolar solvents, e.g., deionized water, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, toluene, hexane, and chloroform, without any disassembly. Compared to Fe3O4 NP building blocks, these 2D nanosheets show more favorable catalytic properties and enhanced catalytic reactivity, which can be exploited to mimic natural enzymes. Our work is expected to open up a new avenue for synthesizing free-floating 2D supersheets by NP assembly, leading to a new generation of materials with enriched functions and broader applications.

Aptamer-mediated selective delivery of a cytotoxic cationic NHC-Au(i) complex to cancer cells.

A novel cationic NHC-Au(i) complex was synthesized and studied for its antitumor activity. For all the cell lines tested, cationic NHC-Au(i) complex 2 shows much higher cytotoxicity than its neutral analogue 1. To achieve selective cancer cell targeting, complex 2 was covalently conjugated to aptamer AS1411, a DNA aptamer with strong binding affinity for nucleolin. The successful conjugation was confirmed by HPLC, gel electrophoresis, fluorescence spectroscopy and UV-Vis absorption. Conjugate AS1411-2 was then examined for its specific targeting and binding ability towards cancer cells over human normal cells using flow cytometry analysis and confocal microscopy. The cytotoxicity of AS1411-2 was then estimated by MTS assay. It was found that AS1411-2 exhibits higher activity than complex 2 towards targeted cells. Importantly, AS1411-2 exhibits much lower cytotoxicity towards healthy normal cell lines. Concurrently, the control groups without the AS1411 aptamer or without the NHC-Au(i) complex do have significant impact on cancer cell viability.

Recognition-then-Reaction Enables Site-Selective Bioconjugation to Proteins on Live-Cell Surfaces.

Site-selective protein modification is a key step in facilitating protein functionalization and manipulation. To accomplish this, genetically engineered proteins were previously required, but the procedure was laborious, complex, and technically challenging. Herein we report the development of aptamer-based recognition-then-reaction to guide site-selective protein/DNA conjugation in a single step with outstanding selectivity and efficiency. As models, several proteins, including human thrombin, PDGF-BB, Avidin, and His-tagged recombinant protein, were studied, and the results showed excellent selectivity under mild reaction conditions. Taking advantage of aptamers as recognition elements with extraordinary selectivity and affinity, this simple preparation method can tag a protein in a complex milieu. Thus, with the aptamer obtained from cell-SELEX, real-time modification of live-cell membrane proteins can be achieved in one step without any pre-treatment.

Three Dimensional Multipod Superstructure based on Cu(OH)2 as a Highly Efficient Nanozyme.

A highly efficient nanozyme system, termed hollow multipod Cu(OH)2 superstructure (HMPS), has been developed via direct conversion from irregular nanoparticles. The HMPS displayed body size around 150 nm and branch lengths in the range of 150~250 nm. Based on the excellent catalytic property of HMPS, we developed a simple and highly sensitive colorimetric assay to detect urine glucose, and the results are in good agreement with hospital examination reports.

N-Heterocyclic Carbene-Gold(I) Complexes Conjugated to a Leukemia-Specific DNA Aptamer for Targeted Drug Delivery.

This report describes the synthesis and characterization of novel N-heterocyclic carbene (NHC)-gold(I) complexes and their bioconjugation to the CCRF-CEM-leukemia-specific aptamer sgc8c. Successful bioconjugation was confirmed by the use of fluorescent tags on both the NHC-Au(I) complex and the aptamer. Cell-viability assays indicated that the NHC-Au(I) -aptamer conjugate was more cytotoxic than the NHC-gold complex alone. A combination of flow cytometry, confocal microscopy, and cell-viability assays provided clear evidence that the NHC-Au(I) -aptamer conjugate was selective for targeted CCRF-CEM leukemia cells.

A Facile Process for the Preparation of Three-Dimensional Hollow Zn(OH)2 Nanoflowers at Room Temperature.

A facile strategy has been developed to synthesize double-shelled Zn(OH)2 nanoflowers (DNFs) at room temperature. The nanoflowers were generated via conversion of Cu2 O nanoparticles (NPs) using ZnCl2 and Na2 S2 O3 by a simple process. Outward diffusion of the Cu(2+) , produced by an oxidation process on the surface of NPs, and the inward diffusion of Zn(2+) by coordination and migration, eventually lead to a hollow cavity in the inner NPs with a double-shelled 3D hollow flower shapes. The thickness of the inner and outer shells is estimated to be about 20 nm, and the thickness of nanopetals is about 7 nm. The nanoflowers have large surface areas and excellent adsorption properties. As a proof of potential applications, the DNFs exhibited an excellent ability to remove organic molecules from aqueous solutions.

Fabrication of Ultrathin Zn(OH)2 Nanosheets as Drug Carriers.

Ultrathin two-dimensional (2D) porous Zn(OH)2 nanosheets (PNs) have been fabricated by using one-dimensional Cu nanowires as backbones. PNs have thicknesses of about 3.8 nm and pore sizes of 4~10 nm. To form "smart" porous nanosheets, DNA aptamers were covalently conjugated on the surface of PNs. These ultrathin nanosheets show good biocompatibility, efficient cellular uptake, and promising pH-stimulated drug release.

Single Nanoparticle to 3D Supercage: Framing for an Artificial Enzyme System.

A facile strategy has been developed to fabricate Cu(OH)2 supercages (SCs) as an artificial enzyme system with intrinsic peroxidase-mimic activities (PMA). SCs with high catalytic activity and excellent recyclability were generated via direct conversion of amorphous Cu(OH)2 nanoparticles (NPs) at room temperature. More specifically, the process that takes a single nanoparticle to a 3D supercage involves two basic steps. First, with addition of a copper-ammonia complex, the Cu(2+) ions that are located on the surface of amorphous Cu(OH)2 NPs would evolve into a fine lamellar structure by coordination and migration and eventually convert to 1D nanoribbons around the NPs. Second, accompanied by the migration of Cu(2+), a hollow cavity is generated in the inner NPs, such that a single nanoparticle eventually becomes a nanoribbon-assembled 3D hollow cage. These Cu(OH)2 SCs were then engineered as an artificial enzymatic system with higher efficiency for intrinsic PMA than the peroxidase activity of a natural enzyme, horseradish peroxidase.

N-heterocyclic carbene gold(I) and silver(I) complexes bearing functional groups for bio-conjugation.

This work describes several synthetic approaches to append organic functional groups to gold and silver N-heterocyclic carbene (NHC) complexes suitable for applications in biomolecule conjugation. Carboxylate appended NHC ligands (3) lead to unstable Au(I) complexes that convert into bis-NHC species (4). A benzyl protected carboxylate NHC-Au(I) complex 2 was synthesized but deprotection to produce the carboxylic acid functionality could not be achieved. A small library of new alkyne functionalized NHC proligands were synthesized and used for subsequent silver and gold metalation reactions. The alkyne appended NHC gold complex 13 readily reacts with benzyl azide in a copper catalyzed azide-alkyne cycloaddition reaction to form the triazole appended NHC gold complex 14. Cell cytotoxicity studies were performed on DLD-1 (colorectal adenocarcinoma), Hep-G2 (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma), CCRF-CEM (human T-Cell leukemia), and HEK (human embryonic kidney). Complete spectroscopic characterization of the ligands and complexes was achieved using (1)H and (13)C NMR, gHMBC, ESI-MS, and combustion analysis.

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.