A TriPPPro-Nucleotide Reporter with Optimized Cell-Permeable Dyes for Metabolic Labeling of Cellular and Viral DNA in Living Cells

The metabolic labeling of nucleic acids in living cells is highly desirable to track the dynamics of nucleic acid metabolism in real-time and has the potential to provide novel insights into cellular biology as well as pathogen-host interactions. Catalyst-free inverse electron demand Diels–Alder reactions (iEDDA) with nucleosides carrying highly reactive moieties such as axial 2-trans-cyclooctene (2TCOa) would be an ideal tool to allow intracellular labeling of DNA. However, cellular kinase phosphorylation of the modified nucleosides is needed after cellular uptake as triphosphates are not membrane permeable. Unfortunately, the narrow substrate window of most endogenous kinases limits the use of highly reactive moieties. Here, we apply our TriPPPro (triphosphate pronucleotide) approach to directly deliver a highly reactive 2TCOa-modified 2′-deoxycytidine triphosphate reporter into living cells. We show that this nucleoside triphosphate is metabolically incorporated into de novo synthesized cellular and viral DNA and can be labeled with highly reactive and cell-permeable fluorescent dye-tetrazine conjugates via iEDDA to visualize DNA in living cells directly. Thus, we present the first comprehensive method for live-cell imaging of cellular and viral nucleic acids using a two-step labeling approach.     

Construction of Organelle-Like Architecture by Dynamic DNA Assembly in Living Cells

The design of controllable dynamic systems is vital for the construction of organelle-like architectures in living cells, but has proven difficult due to the lack of control over defined topological transformation of self-assembled structures. Herein, we report a DNA based dynamic assembly system that achieves lysosomal acidic microenvironment specifically inducing topological transformation from nanoparticles to organelle-like hydrogel architecture in living cells. Designer DNA nanoparticles are constructed from double-stranded DNA with cytosine-rich stick ends (C-monomer) and are internalized into cells through lysosomal pathway. The lysosomal acidic microenvironment can activate the assembly of DNA monomers, inducing transformation from nanoparticles to micro-sized organelle-like hydrogel which could further escape into cytoplasm. We show how the hydrogel regulates cellular behaviors: cytoskeleton is deformed, cell tentacles are significantly shortened, and cell migration is promoted.     

用于检测细胞内谷胱甘肽的比率型荧光探针

设计合成了1种用于检测生物巯基的比率型荧光探针(4),并考察了其对谷胱甘肽的识别作用.在4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中,探针4可与谷胱甘肽快速反应,溶液颜色由淡黄色变为粉红色,从而实现"裸眼"检测,且在608 nm处的荧光信号增强.在1.6×10-5~2×10-4mol/L范围内,探针4能够定量检测谷胱甘肽,检出限为8.9×10-7mol/L.此外,探针4还可用于MCF-7细胞中谷胱甘肽的成像.     

Chromophore-Assisted Proximity Labeling of DNA Reveals Chromosomal Organization in Living Cells

The spatial arrangement of chromosome within the nucleus is linked to genome function and gene expression regulation. Existing genome-wide mapping methods often rely on chemically crosslinking DNA with protein baits, which raises concerns of artifacts being introduced during cell fixation. By genetically targeting a photosensitizer protein to specific subnuclear locations, we achieved blue-light-activated labeling of local DNA with a bioorthogonal functional handle for affinity purification and sequence identification through next-generation sequencing. When applied to the nuclear lamina in human embryonic kidney 293T cells, it revealed lamina-associated domains (LADs) that cover 37.6 % of the genome. These LADs overlap with heterochromatin hallmarks and are depleted with CpG islands. This simple labeling method avoids the harsh treatment of chemical crosslinking and is generally applicable to the genome-wide high-resolution mapping of the spatial chromosome organization in living cells.     

Targeting Non‐B‐Form DNA in Living Cells

Under certain conditions, repetitive DNA motifs have the potential to adopt non‐B‐form DNA structures, such as hairpins, triplexes, Z‐DNA, quadruplexes, and i‐motifs. Some non‐B‐form DNAs have been proposed to cause mutations and, consequently, participate in several biologically important processes, including regulation, evolution, and human disease. Advancement in the knowledge of specific interactions between molecules and non‐B‐form DNAs at the molecular level in living cells is important for understanding their biological functions. In this review, we describe the latest studies on molecules that target non‐B‐form DNAs in vivo, with a focus on Z‐DNA, G‐quadruplexes, triplexes, i‐motifs, and hairpins.     

A Smart Deoxyribozyme-Programmable Catalytic DNA Circuit for High-Contrast MicroRNA Imaging

Synthetic catalytic DNA circuits have been recognized as a promising signal amplification toolbox for sensitive intracellular imaging, yet their selectivity and efficiency are always constrained by uncontrolled off-site signal leakage and inefficient on-site circuitry activation. Thus, the endogenously controllable on-site exposure/activation of DNA circuits is highly desirable for achieving the selective imaging of live cells. Herein, an endogenously activated DNAzyme strategy was facilely integrated with a catalytic DNA circuit for guiding the selective and efficient microRNA imaging in vivo. To prevent the off-site activation, the circuitry constitute was initially caged without sensing functions, which could be selectively liberated by DNAzyme amplifier to guarantee the high-contrast microRNA imaging in target cells. This intelligent on-site modulation strategy can tremendously expand these molecularly engineered circuits in biological systems.     

A Universal and Programmable Platform based on Fluorescent Peptide-Conjugated Probes for Detection of Proteins in Organelles of Living Cells

Realizing protein analysis in organelles of living cells is of great significance for developing diagnostic and therapeutic methods of diseases. Fluorescent-labeled antibodies with well imaging performance and high affinity are classical biochemical tools for protein analysis, while due to the inability to effectively enter into cells, not to mention organelles and the uncontrollable reaction sites that might cause antibodies inactivation when chemically modification, they are hard to apply to living cells. Inspired by the structure of fluorescent-labeled antibodies, we designed as a universal detection platform that was based on the peptide-conjugated probes (PCPs) and consisted of three parts: a) a rotor type fluorescent molecular scaffold for conjugation and signal output; b) the cell penetration protein recognition unit; c) the subcellular organelle targeting unit. In living cells, PCPs could firstly localize at organelles and then proceed protein specific recognition, thus jointly leading to the restriction of twisted intramolecular charge transfer and activation of fluorescence signal. As a proof-of-concept, six different proteins in three typical intracellular organelles could be detected by our platform through simply replacing the recognition sequence of proteins and matching organelle targeting units. The position and intensity of fluorescence signals demonstrated specificity of PCPs and universality of the platform.     

A Strategy for Specific Fluorescence Imaging of Monoamine Oxidase A in Living Cells

Monoamine oxidase (MAO) has two isoforms, MAO‐A and MAO‐B, which show different functions, and thus selective fluorescence imaging is important for biological studies. Currently, however, specific detection of MAO‐A remains a great challenge. Herein, we report a new strategy for specific imaging of MAO‐A through the design of fluorogenic probes combining the characteristic structure of an inhibitor of the target enzyme along with propylamine as a recognition moiety. The high specificity of our representative probe is demonstrated by imaging MAO‐A in different live cells such as SH‐SY5Y (high levels of MAO‐A) and HepG2 (high levels of MAO‐B), and further validated by western blot analyses. The superior specificity of the probe may enable the accurate detection of MAO‐A in complex biosystems. Importantly, the use of the characteristic structure of an inhibitor, as demonstrated in this work, may serve as a general strategy to design specific recognition moieties for fluorogenic probes for enzymes.     

钌配合物与G-四链体DNA的相互作用研究进展

人体端粒由富含鸟嘌呤(G)的DNA重复序列组成,该序列在一定条件下可以形成G-四链体DNA结构。小分子化合物诱导该结构的形成并使之稳定,可以抑制端粒酶活性而达到抗肿瘤的目的。因此,G-四链体DNA稳定剂的设计和筛选是近年来生物无机化学的重要前沿研究领域之一。在金属配合物中,钌配合物由于具有丰富的光化学、光物理特性以及生物活性,其作为G-四链体DNA稳定剂引起人们的高度关注。本文以近年一些代表性的研究工作为例,对钌配合物与G-四链体DNA相互作用方面的研究进展进行了综述。     

再生碳纤维电极的新方法

本文根据火焰熔融法自制碳纤维电极的特点，提出了一种简便、易行的火焰灼烧再生电极的新方法。由此法再生电极的各种性能与原电极相当，结果令人满意。     

A NIR Programmable In Vivo miRNA Magnifier for NIR-II Imaging of Early Stage Cancer

Aberrant expressions of biomolecules occur much earlier than tumor visualized size and morphology change, but their common measurement strategies such as biopsy suffer from invasive sampling process. In vivo imaging of slight biomolecule expression difference is urgently needed for early cancer detection. Fluorescence of rare earth nanoparticles (RENPs) in second near-infrared (NIR-II) region makes them appropriate tool for in vivo imaging. However, the incapacity to couple with signal amplification strategies, especially programmable signal amplification strategies, limited their application in lowly expressed biomarkers imaging. Here we develop a 980/808 nm NIR programmed in vivo microRNAs (miRNAs) magnifier by conjugating activatable DNAzyme walker set to RENPs, which achieves more effective NIR-II imaging of early stage tumor than size monitoring imaging technique. Dye FD1080 (FD1080) modified substrate DNA quenches NIR-II downconversion emission of RENPs under 808 nm excitation. The miRNA recognition region in DNAzyme walker is sealed by a photo-cleavable strand to avoid “false positive” signal in systemic circulation. Upconversion emission of RENPs under 980 nm irradiation activates DNAzyme walker for miRNA recognition and amplifies NIR-II fluorescence recovery of RENPs via DNAzyme catalytic reaction to achieve in vivo miRNA imaging. This strategy demonstrates good application potential in the field of early cancer detection.     

A Solution-Processable Porphyrin-Based Hydrogen-Bonded Organic Framework for Photoelectrochemical Sensing of Carbon Dioxide

Detecting CO₂ in complex gas mixtures is challenging due to the presence of competitive gases in the ambient atmosphere. Photoelectrochemical (PEC) techniques offer a solution, but material selection and specificity remain limiting. Here, we constructed a hydrogen-bonded organic framework material based on a porphyrin tecton decorated with diaminotriazine (DAT) moieties. The DAT moieties on the porphyrin molecules not only facilitate the formation of complementary hydrogen bonds between the tectons but also function as recognition sites in the resulting porous HOF materials for the selective adsorption of CO₂. In addition, the in-plane growth of FDU-HOF-2 into anisotropic molecular sheets with large areas of up to 23000 μm² and controllable thickness between 0.298 and 2.407 μm were realized in yields of over 89 % by a simple solution-processing method. The FDU-HOF-2 can be directly grown and deposited onto different substrates including silica, carbon, and metal oxides by self-assembly in situ in formic acid. As a proof of concept, a screen-printing electrode deposited with FDU-HOF-2 was fabricate as a label-free photoelectrochemical (PEC) sensor for CO₂ detection. Such a signal-off PEC sensor exhibits low detection limit for CO₂ (2.3 ppm), reusability (at least 30 cycles), and long-term working stability (at least 30 days).     

Real-time Imaging of Nascent DNA in Live Cells by Monitoring the Fluorescence Lifetime of DNA-Incorporated Thiazole Orange-Modified Nucleotides

A modified 2′-deoxycytidine triphosphate derivative ( dC^TOTP ) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dC^TOTP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dC^TOTP . When dC^TOTP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics.     

Optimized Fluorescent Probe for Specific Imaging of Glutathione S‐Transferases in Living Cells and Mice

GSTP1 has been considered to be a marker for malignancy in many tissues. However, the existing GST fluorescent probes are unfavorable for in vivo imaging because of the limited emission wavelength or insufficient fluorescence enhancement (six‐fold). The limited fluorescence enhancement of GST fluorescent probes is mainly ascribed to the high background signals resulting from the spontaneous reaction between GSH and the probes. In this work, a highly specific GST probe with NIR emission has been successfully developed through optimization of the essential unit of the probe to repress the spontaneous reaction. The novel GST probe exhibits over 100‐fold fluorescence enhancement upon incubation with GSTP1/GSH and high selectivity over other potential interference. In addition, the probe has been proved to be capable of tracking endogenous GST in A549 cells. Finally, the in vivo imaging results demonstrate that the probe can be used for effective imaging of endogenous GST activity in subcutaneous tumor mouse with high contrast.     

A regenerative ratiometric electrochemical biosensor for selective detecting Hg based on Y-shaped/hairpin DNA transformation

Inspired by dual-signaling ratiometric mechanism which could reduce the influence of the environmental change, a novel, convenient, and reliable method for the detection of mercury ions (Hg²⁺) based on Y-shaped DNA (Y-DNA) was developed. Firstly, the Y-DNA was formed via the simple annealing way of using two different redox probes simultaneously, omitting the multiple operation steps on the electrode. The Y-DNA was immobilized on the gold electrode surface and then an obvious ferrocene (Fc) signal and a weak methylene blue (MB) signal were observed. Upon addition of Hg²⁺, the Y-DNA structure was transformed to hairpin structure based on the formation of T-Hg²⁺-T complex. During the transformation, the redox MB gets close to and the redox Fc gets far away from the electrode surface, respectively. This special design allows a reliable Hg²⁺ detection with a detection range from 1 nM to 5 μM and a low detection limit down to 0.094 nM. Furthermore, this biosensor exhibits good selectivity and repeatability, and can be easily regenerated by using l-cysteine. This study offers a simple and effective method for designing ratiometric biosensors for detecting other ions and biomolecules.     

Improved Stabilities of Labeling Probes for the Selective Modification of Endogenous Proteins in Living Cells and In Vivo

To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging.     

Physicochemical and Cytochemical Investigations on the Binding of Ethidium and Acridine Dyes to DNA and to Organelles in Living Cells

Ethidium and acridine dyes are classical model substances for studying the binding of small, pharmacologically active molecules to DNA. Intercalation between the DNA base pairs is nearly always proposed as the most important type of binding. According to our investigations, however, there is a second type of binding, which also occurs when the concentration of the bound molecules is low and will be referred to here as external or preintercalative binding. The experimental binding isotherms show that the binding constant for intercalation K_S1 is considerably smaller than that for external binding K_S2 (K_S1 > K_S2). This surprising result is not due to the binding enthalpy (ΔH ≈ ΔH) but to the binding entropy (ΔS > ΔS). Electrostatic interactions between the dye and the DNA represent the most important contribution to both types of binding; they are supplemented by hydrogen bonds and hydrophobic interactions. The behavior of a substance in living cells, however, cannot be reliably predicted from its in vitro binding to DNA. Very few substances are bound to the DNA of the nuclear chromatin in cell culture; for example, dyes often accumulate instead in the lysosomes. In some cases the dye binds specifically and very efficiently to the mitochondria of the living cell, especially to the mitochondrial membranes, the sites of oxidative phosphorylation.     

光致清洁电极可见光在线更新与细胞实时监测

电化学传感器在用于细胞实时监测过程中,电极界面污染严重影响其检测性能.通过将纳米光催化剂与电化学传感材料复合,构建光致清洁电化学传感器,为电极界面的高效及无损更新提供了新思路.然而光催化产生的活性氧自由基导致细胞损伤,限制了细胞培养及检测过程中电极界面的实时更新.为此,我们在PEDOT@CdS/TiO₂/ITO可见光致更新电极表面旋涂明胶薄层,在保持电极良好的光致清洁和电化学传感性能同时,利用明胶薄层阻碍光催化产生的活性氧自由基扩散至细胞表面,显著降低了细胞损伤.此外,明胶优良的生物相容性有利于细胞的黏附及增殖.利用该电极,我们实现了人脐静脉内皮细胞（HUVECs）培养过程中,电极的在线更新以及细胞释放一氧化氮的实时监测.     

Activating Bi p-orbitals in Dispersed Clusters of Amorphous BiOx for Electrocatalytic Nitrogen Reduction

Catalytic strategies based on main group metals are significantly less advanced than those of transition metal catalysis, leaving untapped areas of potentially fruitful research. We here demonstrate an effective approach for the modulation of Bi 6p energy levels during the construction of atomically dispersed clusters of amorphous BiO_x. Bi oxidation state is proposed to strongly affects the nitrogen fixation activity, with the half-occupied p_z orbitals of the Bi²⁺ ions being highly efficient toward electron injection into the inert N₂ molecule. With sufficient catalytic sites to adsorb and activate N₂, the bonding between N₂ and catalyst is able to be in situ identified. The catalyst shows an outstanding Faraday efficiency (≈30 %) and high yield (≈113 μg h⁻¹ mg⁻¹_cat) in NH₃ production, outperforming most of the existing catalysts in aqueous solution. These results lay the basis for developing the potential of p-block elements for catalysis of multi-electron reactions.