Modular Weaving DNAzyme in Skeleton of DNA Nanocages for Photoactivatable Catalytic Activity Regulation.

DNAzymes exhibit tremendous application potentials in the field of biosensing and gene regulation due to its unique catalytic function. However, spatiotemporally controlled regulation of DNAzyme activity remains a daunting challenge, which may cause nonspecific signal leakage or gene silencing of the catalytic systems. Here, we report a photochemical approach via modular weaving active DNAzyme into the skeleton of tetrahedral DNA nanocages (TDN) for light-triggered on-demand liberation of DNAzyme and thus conditional control of gene regulation activity. We demonstrate that the direct encoding of DNAzyme in TDN could improve the biostability of DNAzyme and ensure the delivery efficiency, comparing with the conventional surface anchoring strategy. Furthermore, the molecular weaving of the DNA nanostructures allows remote control of DNAzyme-mediated gene regulation with high spatiotemporal precision of light. In addition, we demonstrate that the approach is applicable for controlled regulation of the gene editing functions of other functional nucleic acids.

Modular Engineering of DNAzyme-Based Sensors for Spatioselective Imaging of Metal Ions in Mitochondria.

DNAzyme-based sensors remain at the forefront of metal-ion imaging efforts, but most lack the subcellular precision necessary to their applications in specific organelles. Here, we seek to overcome this limitation by presenting a DNAzyme-based biosensor technology for spatiotemporally controlled imaging of metal ions in mitochondria. A DNA nanodevice was constructed by integrating an optically activatable DNAzyme sensor and an upconversion nanoparticle with an organelle-targeting signal. We exemplify that this approach allows for mitochondria-specific imaging of Zn2+ in living cells in a near-infrared light-controlled manner. Based on this, the system is used for the monitoring of mitochondrial Zn2+ during drug treatment in a cellular model of ischemia insult. Furthermore, the DNA nanodevice is employed to assess dynamic Zn2+ change and pharmacological interventions in an injury cell model of Zn2+ toxicity. This method paves the way for engineering of DNAzyme sensors to investigate the pathophysiological roles of metal ions at the subcellular level.

Near-infrared-emitting upconverting BiVO4 nanoprobes for in vivo fluorescent imaging.

Near-infrared (NIR) emitting BiVO4:Yb3+,Tm3+ nanoparticles are synthesized by a new solvothermal strategy using solvents of oleic acid and methanol. The obtained BiVO4:Yb3+,Tm3+ samples show an average particle size of ≈164 nm and exhibit an asymmetry monoclinic crystal structure of BiVO4. At NIR excitation of 980 nm, the BiVO4:Yb3+,Tm3+ sample exhibits a nearly single NIR emission at ≈796 nm with extremely weak blue emissions from Tm3+ ions. These high-energy visible emissions are absorbed by the semiconducting host of BiVO4 that possesses a bandgap of ≈2.2 eV. Therefore, the NIR excitation to a single intense NIR emission fluorescent BiVO4 materials could be a potential ideal probe for deep-tissue high-resolution bioimaging. To validate the ability of BiVO4 materials for bio-applications, we conduct the cytotoxicity experiments. The results show that the cytotoxicity of HeLa cells is negligible at a concentration of 0.2 mg/ml of BiVO4:Yb3+,Tm3+ , and the cell viability approaches 90% at a high dosage of 0.5 mg/ml. The Daphnia magna and Zebrafish treated with nanoparticles (0.5 mg/ml) display bright NIR emission without any background, indicating the excellent in vivo fluorescent imaging capacity of BiVO4:Yb3+,Tm3+ nanoparticles. Our findings offer an environment-friendly strategy to synthesize BiVO4 UCL nanophosphors and provide a promising new class of fluorescent probes for biological applications.

Defective Ag-In-S/ZnS quantum dots: an oxygen-derived free radical scavenger for mitigating macrophage inflammation.

Oxidative stress plays an important role in the development of inflammatory diseases including allergy, heart disease, diabetes and cancer. Nanomaterial-mediated antioxidant therapy is regarded as a promising strategy to treat oxidative stress-mediated inflammation. Herein, defective Ag-In-S/ZnS quantum dots (AIS/ZnS QDs) with oxygen-derived radical-scavenging capabilities are developed. Owing to their intrinsic defects and abundant surface functional groups, these quantum dots exhibit excellent oxygen-derived free radical removal efficiency in vitro. In macrophages, AIS/ZnS QDs can eliminate intracellular excessive ROS stimulated by either H2O2 or lipopolysaccharide (LPS), thus can effectively protect macrophages against ROS-induced oxidative injury. Moreover, in the model of LPS-triggered macrophage inflammation, they exhibit benign anti-inflammatory ability by inhibiting the expression of related proinflammatory cytokines (e.g., TNF-α and IL-6). These findings indicate that AIS/ZnS QDs hold great potential for the treatment of ROS-related inflammatory disorders.

Enzyme-triggered DNA nanomimosa: A ratiometric nanoprobe for RNase H activity sensing in living cells.

Since ribonuclease H (RNase H) exhibits its importance in a variety of cellular processes. It is necessary to establish strategy for RNase H detection. In this work, we are enlightened by mimosa, a natural plant which can fold in response to stimuli, to construct a DNA tetrahedron-based nanoprobe, termed DNA nanomimosa, to sensing RNase H activity based on fluorescent resonance energy transfer (FRET). The DNA nanomimosa was self-assembled from four DNA chains and one RNA chain. One of the four DNA chains contains a FRET-paired fluorophores-labeled hairpin DNA structures which is unfolded by the RNA chain through hybridization. Without RNase H, the RNA chain separate the two FRET-paired fluorophores in hairpin DNA structure, giving a feeble FRET signal. However, the presence of RNase H can selectively digest the RNA strand in RNA/unfolded-hairpin DNA section, resulting in the hairpin DNA configuration changed from "unfolded" state to "folded" state and further turn on the FRET signal. The DNA nanomimosa can be applied to achieve the determination of RNase H activity by recording the emission intensity of donor and acceptor fluorophores. This strategy shows a low detection limit by 0.017 U/mL, good specificity, and distinct advantages like the self-delivery ability, good biocompatibility, and the capacity to minimize the effects of fluctuations. This design provides a potential application in ribonuclease research and could be expanded for other biomedical research and clinical diagnostics.

Polydopamine nanodots-based cost-effective nanoprobe for glucose detection and intracellular imaging.

The cellular glucose detection remains a vital topic, which could provide some essential information about the glucose-based pathological and physiological processes. In this study, a smart polydopamine nanodots-based cost-effective fluorescence turn-on nanoprobe (denoted as PDA-Ag-GOx) for intracellular glucose detection is established. Silver nanoparticles (AgNPs) are directly formed in one step by the reduction of fluorescent polydopamine nanodots (PDADs) which have much phenolic hydroxyls on the surface. The fluorescence of PDADs could be quenched by AgNPs through surface plasmon-enhanced energy transfer (SPEET) from donor PDADs to acceptor AgNPs. Glucose oxidase (GOx) is modified on the PDA-Ag NPs by covalent bond. In the presence of glucose, GOx could catalyze glucose to produce H2O2 and gluconic acid. The generated acid and H2O2 would degrade AgNPs into Ag+, the PDADs release and restore its fluorescence. The proposed nanoprobe has some advantages, such as cost-effective, easy preparation, and excellent selectivity toward glucose, which could be successfully utilized to intracellular glucose imaging.

A dual-functional biomimetic-mineralized nanoplatform for glucose detection and therapy with cancer cells in vitro.

Glucose detection is a crucial topic in the diagnosis of numerous diseases, such as hypoglycemia or diabetes mellitus. Research indicates that people with diabetes mellitus are at a higher risk of developing various types of cancer. A nanoplatform that combines both diabetes diagnosis and cancer therapy might be regarded as a more effective way to solve the above-mentioned problem. However, none of the known sensors has a smart strategy that can work as a fluorescent glucose sensor and a cancer therapeutic platform simultaneously. Here, we developed a pH responsive biomimetic-mineralized nanoplatform (denoted as CaCO3-PDA@DOX-GOx) for glucose detection in serum samples and applied it to treat the tumor cells combined chemotherapy with the starvation therapy in vitro. Doxorubicin (DOX) and glucose oxidase (GOx) were loaded through the mesoporous CaCO3-PDA nanoparticles (m-CaCO3-PDA NPs). The fluorescence of DOX is quenched as a result of fluorescence resonance energy transfer (FRET) caused by the broad absorption of m-CaCO3-PDA NPs. The nanoplatform would recover fluorescence under lower pH values due to the catalytic reaction of GOx with glucose or tumor microenvironment (TME), which leads to the elimination of FRET. Its application as a glucose sensor is indicated with a linear relationship in the range of 0.01-1.0 mM of glucose and limit of detection is calculated by 6 µM. This nanoplatform also has a TME-responsive antitumor effect and fluorescence imaging functionality, which provide a new idea for cancer therapy together with glucose monitoring in diabetes.

One-step G-quadruplex-based fluorescence resonance energy transfer sensing method for ratiometric detection of uracil-DNA glycosylase activity.

Uracil-DNA glycosylase (UDG) is a crucial enzyme in base excision repair (BER) pathway. It can repair the uracil-induced DNA lesions and maintain the integrity of genome. In this paper, we developed a facile and ratiometric strategy for UDG activity detection using fluorescence resonance energy transfer (FRET). One double-stranded DNA (dsDNA) substrate consisting of strand 1 (dual-fluorescent dye-modified G-quadruplex sequence single-stranded DNA (ssDNA)), carboxyfluorescein (FAM) acted as donor and tetramethylrhodamine (TAMRA) as acceptor) and strand 2 (the complementary sequence of strand 1 containing three mismatched bases and three uracil bases) was introduced. When the UDG-catalyzed uracil is removed from dsDNA, the thermo-stability of dsDNA is decreased and the dual-fluorescent dye-modified G-quadruplex sequence ssDNA is released. Then, the ssDNA transforms into a G-quadruplex comformation, which brings the labeled FAM and TAMRA into close proximity, resulting in a strong FRET signal. In the absence of UDG, the relatively stable dsDNA separates the labeled FAM and TAMRA, giving a weak FRET signal. Thus, by measuring the system fluorescence intensity and exploiting FRET signal difference, UDG activity can be detected in a simple process. The detection limit is 0.087 U/mL without requiring additional signal amplification process. Besides, our developed strategy can also be used for screening the UDG inhibitors in a ratiometric fluorescence detection way.

Enhanced in Vivo Blood-Brain Barrier Penetration by Circular Tau-Transferrin Receptor Bifunctional Aptamer for Tauopathy Therapy.

The lack of blood-brain barrier (BBB) penetrating ability has hindered the delivery of many therapeutic agents for tauopathy treatment. In this study, we report the synthesis of a circular bifunctional aptamer to enhance the in vivo BBB penetration for better tauopathy therapy. The circular aptamer consists of one reported transferrin receptor (TfR) aptamer to facilitate TfR-aptamer recognition-induced transcytosis across BBB endothelial cells, and one Tau protein aptamer that we recently selected to inhibit Tau phosphorylation and other tauopathy-related pathological events in the brain. This novel circular Tau-TfR bifunctional aptamer displays significantly improved plasma stability and brain exposure, as well as the ability to disrupt tauopathy and improve traumatic brain injury (TBI)-induced cognitive/memory deficits in vivo, providing important proof-of-principle evidence that circular Tau-TfR aptamer can be further developed into diagnostic and therapeutic candidates for tauopathies.

Label-free fluorescent assay of T4 polynucleotide kinase phosphatase activity based on G-quadruplexe-thioflavin T complex.

T4 polynucleotide kinase phosphatase (T4 PNKP) is a bifunctional tool enzyme with 5'-kinase and 3'-phosphatase activities. Considering its important roles in the repair of strand breaks, assay of T4 PNKP activity is of great importance. In this work, we proposed a novel label-free sensing strategy for T4 PNKP activity based on G-quadruplexe-thioflavin T fluorescent indicator. In the assay, we used a single stranded oligonucleotide with 3'-phosphoryl and 5'-phosphoryl ends as enzyme substrate. Upon the addition of T4 PNKP, the 3'-PO3 of the substrate was changed to 3'-OH which initiated the polymerization in the presence of terminal deoxynucleotidyl transferase and G-rich dNTP substrates. The resultant elongated DNA can form G-quadruplex in the inducement of K+, resulting in strong fluorescence signal when using thioflavin T as a G-quadruplex-specific light-up fluorescent probe. The detection limit of this method is as low as 0.2U/mL. Additionally, the inhibition of T4 PNKP activity by the inhibitor heparin is demonstrated. This method is easy and convenient to operate in homogeneous solution, and the whole assay process can be completed in a single tube.

Rapid and sensitive detection of potassium ion based on K(+)-induced G-quadruplex and guanine chemiluminescence.

A simple and rapid method for detection of potassium ion (K(+)) based on a guanine chemiluminescence (CL) system is presented. In this system, one guanine-rich DNA molecule is used as the recognition element. K(+) can cause the guanine-rich DNA to form a G-quadruplex conformation, resulting in remarkable quenching of the guanine CL intensity of guanine-rich DNA. The CL intensity of this CL system decreased with increasing K(+) concentration, revealing a linear relationship in K(+) concentration range from 3 × 10(-5) to 1 × 10(-3) M. A complete detection process can be accomplished in about 5 min. Other common cations (such as Na(+), NH4 (+), Mg(2+), Ca(2+), Zn(2+), and Pb(2+)) did not notably interfere with K(+) detection. The mechanism of this strategy is also discussed. The sensing strategy is low cost and simple without the requirement of complex labeling of probe DNA. The scheme is applicable to the detection of other guanine-rich aptamer-binding chemicals or biomolecules.

[Comparison of bacterial diversity of polluted and unpolluted sediment by brominated flame retardant].

OBJECTIVE: By using brominated flame retardant we compared the bacterial diversity of highly polluted river sediment with that of nearby unpolluted lake. METHOD: Total DNA was extracted from unpolluted and highly polluted sediment sample by brominated flame retardant in Guiyu of China. The 16S rRNA gene was amplified by PCR using bacterial primer 27F and 1500R. The plasmid libraries of the amplicons were constructed. The positive clones with insert were screened on plates with IPTG/X-gal/Ap. Amplified ribosmal DNA restriction analysis (ARDRA) was carried out with restriction enzymes Hha I and Hinf I. Representative clones of each operational taxonomic unit based on the ARDRA patterns were selected to be sequenced. After proof reading and careful comparison to remove the chimeric sequences, the partial sequence of 16S rRNA gene were used for construction of the phylogenetic tree. RESULT: The result of blast searching showed that clones from unpolluted sediment sample belonged to alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, delta-Proteobacteria, Planctomycetes, Acidobacteria, Actinobacteria, Chloroflexi, Verrucomicrobia, Firmicutes, the predominant bacteria (30.2% of total clones) is Acidobacteria; most clones from polluted sediment belonged to alpha-Proteobacteria, beta-Proteobacteria, epsilon-Proteobacteria, delta-Proteobacteria, Planctomycetes, Acidobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, Firmicutes, candidate division 0P01, candidate division OP08, the predominant bacteria (44.9% of total clones) are epsilon-Proteobacteria and Chloroflexi. CONCLUSION: Bacterial community structure of polluted sediment has distinguished feature and obviously different from the unpolluted sediment sample, which is mainly reflected in the dominant position of epsilon-Proteobacteria and Chloroflexi in the bacterial flora.