Liqin Zhang

In vitro selection with artificial expanded genetic information systems

Significance Many chemicals are valuable because they bind to other molecules. Chemical theory cannot directly design “binders.” However, we might recreate in the laboratory the Darwinian processes that nature uses to create binders. This in vitro evolution uses nucleic acids as binders, libraries of DNA/RNA to survive a selection challenge before they can have “children” (systematic evolution of ligands by exponential enrichment, SELEX). Unfortunately, with only four nucleotides, natural DNA/RNA often yields only poor binders, perhaps because they are built from only four building blocks. Synthetic biology has increased the number of DNA/RNA building blocks, with tools to sequence, PCR amplify, and clone artificially expanded genetic information systems (AEGISs). We report here the first example of a SELEX using AEGIS, producing a molecule that binds to cancer cells. Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson–Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS–SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS–SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS–SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS–SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.

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.

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.

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.

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.

Aptasensor with Expanded Nucleotide Using DNA Nanotetrahedra for Electrochemical Detection of Cancerous Exosomes

Exosomes are extracellular vesicles (50-100 nm) circulating in biofluids as intercellular signal transmitters. Although the potential of cancerous exosomes as tumor biomarkers is promising, sensitive and rapid detection of exosomes remains challenging. Herein, we combined the strengths of advanced aptamer technology, DNA-based nanostructure, and portable electrochemical devices to develop a nanotetrahedron (NTH)-assisted aptasensor for direct capture and detection of hepatocellular exosomes. The oriented immobilization of aptamers significantly improved the accessibility of an artificial nucleobase-containing aptamer to suspended exosomes, and the NTH-assisted aptasensor could detect exosomes with 100-fold higher sensitivity when compared to the single-stranded aptamer-functionalized aptasensor. The present study provides a proof-of-concept for sensitive and efficient quantification of tumor-derived exosomes. We thus expect the NTH-assisted electrochemical aptasensor to become a powerful tool for comprehensive exosome studies.

Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution

Laboratory in vitro evolution (LIVE) might deliver DNA aptamers that bind proteins expressed on the surface of cells. In this work, we used cell engineering to place glypican 3 (GPC3), a possible marker for liver cancer theranostics, on the surface of a liver cell line. Libraries were then built from a six-letter genetic alphabet containing the standard nucleobases and two added nucleobases (2-amino-8H-imidazo[1,2-a][1,3,5]triazin-4-one and 6-amino-5-nitropyridin-2-one), Watson-Crick complements from an artificially expanded genetic information system (AEGIS). With counterselection against non-engineered cells, eight AEGIS-containing aptamers were recovered. Five bound selectively to GPC3-overexpressing cells. This selection-counterselection scheme had acceptable statistics, notwithstanding the possibility that cells engineered to overexpress GPC3 might also express different off-target proteins. This is the first example of such a combination.

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.

Self-Assembled DNA Immunonanoflowers as Multivalent CpG Nanoagents.

Synthetic unmethylated cytosine–guanine (CpG) oligodeoxynucleotides are immunostimulatory motifs that have shown promise as vaccines or adjuvants for diseases such as cancers and infectious diseases. In the present work, novel immuno-nanoflowers (NFs), self-assembled from long DNA integrated with tandem CpG through rolling circle replication, were developed for efficient CpG delivery and protection from nuclease degradation. In a model of macrophage-like cells, the CpG NFs proved to be potent immunostimulators by triggering the proliferation of these immune cells, which, in turn, secreted immunostimulatory cytokines, including tumor necrosis factor α, interleukin-6, and interleukin-10. These results demonstrate the ability of CpG NFs to induce cancer cell apoptosis and necrosis.