In Vivo Incorporation of Azide Groups into DNA by Using Membrane‐Permeable Nucleotide Triesters

Metabolic incorporation of bioorthogonal functional groups into cellular nucleic acids can be impeded by insufficient phosphorylation of nucleosides. Previous studies found that 5azidomethyl‐2′‐deoxyuridine (AmdU) was incorporated into the DNA of HeLa cells expressing a low‐fidelity thymidine kinase, but not by wild‐type HeLa cells. Here we report that membrane‐permeable phosphotriester derivatives of AmdU can exhibit enhanced incorporation into the DNA of wild‐type cells and animals. AmdU monophosphate derivatives bearing either 5′‐bispivaloyloxymethyl (POM), 5′‐bis‐(4‐acetoxybenzyl) (AB), or “Protide” protective groups were used to mask the phosphate group of AmdU prior to its entry into cells. The POM derivative “POM‐AmdU” exhibited better chemical stability, greater metabolic incorporation efficiency, and lower toxicity than “AB‐AmdU”. Remarkably, the addition of POM‐AmdU to the water of zebrafish larvae enabled the biosynthesis of azide‐modified DNA throughout the body.     

DNA Primer Extension with Cyclopropenylated 7‐Deaza‐2′‐deoxyadenosine and Efficient Bioorthogonal Labeling in Vitro and in Living Cells

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1‐methylcyclopropene (1MCP) group at the 7‐position of 7‐deaza‐2′‐deoxyadenosine and applied for primer extension experiments. The real‐time kinetic data reveals that this 1MCP‐modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP‐modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site‐specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.     

Synthesis,Characterization, and Initial Biological Evaluation of [99mTc]Tc‐Tricarbonyl‐labeled DPA‐α‐MSH Peptide Derivatives for Potential Melanoma Imaging

α‐Melanocyte stimulating hormone (α‐MSH) derivatives target the melanocortin‐1 receptor (MC1R) specifically and selectively. In this study, the α‐MSH‐derived peptide NAP‐NS1 (Nle‐Asp‐His‐d ‐Phe‐Arg‐Trp‐Gly‐NH₂) with and without linkers was conjugated with 5‐(bis(pyridin‐2‐ylmethyl)amino)pentanoic acid (DPA‐COOH) and labeled with [^99mTc]Tc‐tricarbonyl by two methods. With the one‐pot method the labeling was faster than with the two‐pot method, while obtaining similarly high yields. Negligible trans‐chelation and high stability in physiological solutions was determined for the [^99mTc]Tc‐tricarbonyl–peptide conjugates. Coupling an ethylene glycol (EG)‐based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [^99mTc]Tc‐tricarbonyl–peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the ^99mTc‐labeled peptides for in vivo imaging.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.     

DNA编码和混沌神经网络的两相流型辨识

利用DNA编码和混沌神经网络技术，构造了基于DNA编码的混沌神经网络，通过对航空发动机轴承腔气液两相流型的辨识研究，建立了气液两相流型预测模型，从而为航空发动机轴承腔内润滑油气液两相流型辨识提供了技术支持。     

Stimuli‐Responsive Nucleic Acid‐Based Polyacrylamide Hydrogel‐Coated Metal–Organic Framework Nanoparticles for Controlled Drug Release

The synthesis of doxorubicin‐loaded metal–organic framework nanoparticles (NMOFs) coated with a stimuli‐responsive nucleic acid‐based polyacrylamide hydrogel is described. The formation of the hydrogel is stimulated by the crosslinking of two polyacrylamide chains, P_A and P_B, that are functionalized with two nucleic acid hairpins ( 4 ) and ( 5 ) using the strand‐induced hybridization chain reaction. The resulting duplex‐bridged polyacrylamide hydrogel includes the anti‐ATP (adenosine triphosphate) aptamer sequence in a caged configuration. The drug encapsulated in the NMOFs is locked by the hydrogel coating. In the presence of ATP that is overexpressed in cancer cells, the hydrogel coating is degraded via the formation of the ATP–aptamer complex, resulting in the release of doxorubicin drug. In addition to the introduction of a general means to synthesize drug‐loaded stimuli‐responsive nucleic acid‐based polyacrylamide hydrogel‐coated NMOFs hybrids, the functionalized NMOFs resolve significant limitations associated with the recently reported nucleic acid‐gated drug‐loaded NMOFs. The study reveals substantially higher loading of the drug in the hydrogel‐coated NMOFs as compared to the nucleic acid‐gated NMOFs and overcomes the nonspecific leakage of the drug observed with the nucleic‐acid‐protected NMOFs. The doxorubicin‐loaded, ATP‐responsive, hydrogel‐coated NMOFs reveal selective and effective cytotoxicity toward MDA‐MB‐231 breast cancer cells, as compared to normal MCF‐10A epithelial breast cells.     

DNA‐Templated Magnetic Nanoparticle‐Quantum Dot Polymers for Ultrasensitive Capture and Detection of Circulating Tumor Cells

Circulating tumor cell (CTC) enumeration and analysis has emerged as an important platform for cancer diagnosis and prognosis. A great challenge, however, is to efficiently capture low abundant CTCs with high purity from blood samples in a rapid and high‐throughput manner for accurate and sensitive CTC detection. Herein, a new class of DNA‐templated magnetic nanoparticle‐quantum dot (QD)‐aptamer copolymers (MQAPs) is developed for rapid magnetic isolation of CTCs from human blood with high capture efficiency and purity approaching 80%. The phenotype of CTCs is simultaneously profiled with QD photoluminescence (PL) at single cell level. These MQAPs are constructed through hybridization chain reaction to achieve amplified magnetic response, extraordinary binding selectivity for target cells over background cells, and ultra bright ensemble QD PL for single cell detection. MQAPs are free from nonspecific binding that would otherwise compromise the capture purity of target cells. As a result, facile isolation and enumeration of rare CTCs in blood samples could be achieved in 20 min with high sensitivity and accuracy.     

DNA Computing Boosted by a Cationic Copolymer

The huge information storage capability of DNA and its ability to self‐assemble can be harnessed to enable massively parallel computing in a small space. DNA‐based logic gates are designed that rely on DNA strand displacement reactions; however, computation is slow due to time‐consuming DNA reassembly processes and prone to failure as DNA is susceptible to degradation by nucleases and under certain solution conditions. Here, it is shown that the presence of a cationic copolymer boosts the speed of DNA logic gate operations that involve multiple and parallel strand displacement reactions. Two kinds of DNA molecular operations, one based on a translator gate and one on a seesaw gate, are successfully enhanced by the copolymer without tuning of computing conditions or DNA sequences. The copolymer markedly reduces operation times from hours to minutes. Moreover, the copolymer enhances nuclease resistance.