In Situ Self-Assembly of Fluorogenic RNA Nanozipper Enables Real-Time Imaging of Single Viral mRNA Translation

Real-time visualization of individual viral mRNA translation activities in live cells is essential to obtain critical details of viral mRNA dynamics and to detect its transient responses to environmental stress. Fluorogenic RNA aptamers are powerful tools for real-time imaging of mRNA in live cells, but monitoring the translation activity of individual mRNAs remains a challenge due to their intrinsic photophysical properties. Here, we develop a genetically encoded turn-on 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)-binding RNA nanozipper with superior brightness and high photostability by in situ self-assembly of multiple nanozippers along single mRNAs. The nanozipper enables real-time imaging of the mobility and dynamic translation of individual viral mRNAs in live cells, providing information on the spatial dynamics and translational elongation rate of viral mRNAs.     

Fluorogenic and Cell-Permeable Rhodamine Dyes for High-Contrast Live-Cell Protein Labeling in Bioimaging and Biosensing

The advancement of fluorescence microscopy techniques has opened up new opportunities for visualizing proteins and unraveling their functions in living biological systems. Small-molecule organic dyes, which possess exceptional photophysical properties, small size, and high photostability, serve as powerful fluorescent reporters in protein imaging. However, achieving high-contrast live-cell labeling of target proteins with conventional organic dyes remains a considerable challenge in bioimaging and biosensing due to their inadequate cell permeability and high background signal. Over the past decade, a novel generation of fluorogenic and cell-permeable dyes has been developed, which have substantially improved live-cell protein labeling by fine-tuning the reversible equilibrium between a cell-permeable, nonfluorescent spirocyclic state (unbound) and a fluorescent zwitterion (protein-bound) of rhodamines. In this review, we present the mechanism and design strategies of these fluorogenic and cell-permeable rhodamines, as well as their applications in bioimaging and biosensing.     

Fluorophore‐Promoted RNA Folding and Photostability Enables Imaging of Single Broccoli‐Tagged mRNAs in Live Mammalian Cells

Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli‐DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli‐BI complexes in cells. Broccoli‐BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis‐BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.     

Ultrafast Detection of Monoamine Oxidase A in Live Cells and Clinical Glioma Tissues Using an Affinity Binding-Based Two-Photon Fluorogenic Probe

Abnormal expression of monoamine oxidase A (MAO−A) has been implicated in the development of human glioma, making MAO−A a promising target for therapy. Therefore, a rapid determination of MAO−A is critical for diagnosis. Through in silico screening of two-photon fluorophores, we discovered that a derivative of N,N-dimethyl-naphthalenamine ( pre-mito ) can effectively fit into the entrance of the MAO−A cavity. Substitutions on the N-pyridine not only further explore the MAO−A cavity, but also enable mitochondrial targeting ability. The aminopropyl substituted molecule, CD1 , showed the fastest MAO−A detection (within 20 s), high MAO−A affinity and selectivity. It was also used for in situ imaging of MAO−A in living cells, enabling a comparison of the MAO−A content in human glioma and paracancerous tissues. Our results demonstrate that optimizing the affinity binding-based fluorogenic probes significantly improves their detection rate, providing a general approach for rapid detection probe design and optimization.     

Highly Bright Near-Infrared Chemiluminescent Probes for Cancer Imaging and Laparotomy

Near-infrared (NIR) chemiluminescence imaging holds potential for sensitive imaging of cancer due to its low background; however, few NIR chemiluminophores are available, which share the drawback of low chemiluminescence quantum yields (Φ_CL). Herein, we report the synthesis of NIR chemiluminophores for cancer imaging and laparotomy. Molecular engineering of the electron-withdrawing group at the para-position of the phenol-dioxetane leads to a highly bright NIR chemiluminophore (DPT), showing the Φ_CL (4.6×10⁻² Einstein mol⁻¹) that is 3 to 5-fold higher than existing NIR chemiluminophores. By caging the phenol group of DPT with a cathepsin B (CatB) responsive moiety, an activatable chemiluminescence probe (DPT_CB) is developed for real-time turn-on detection of deeply buried tumor tissues in living mice. Due to its high brightness, DPT_CB permits accurate chemiluminescence-guided laparotomy.     

Development of a Highly Selective and Sensitive Fluorescent Probe for Imaging RNA Dynamics in Live Cells

RNA imaging is of great importance for understanding its complex spatiotemporal dynamics and cellular functions. Considerable effort has been devoted to the development of small-molecule fluorescent probes for RNA imaging. However, most of the reported studies have mainly focused on improving the photostability, permeability, long emission wavelength, and compatibility with live-cell imaging of RNA probes. Less attention has been paid to the selectivity and detection limit of this class of probes. Highly selective and sensitive RNA probes are still rarely available. In this study, a new set of styryl probes were designed and synthesized, with the aim of upgrading the detection limit and maintaining the selectivity of a lead probe QUID−1 for RNA. Among these newly synthesized compounds, QUID−2 was the most promising candidate. The limit of detection (LOD) value of QUID−2 for the RNA was up to 1.8 ng/mL in solution. This property was significantly improved in comparison with that of QUID−1. Further spectroscopy and cell imaging studies demonstrated the advantages of QUID−2 over a commercially available RNA staining probe, SYTO RNASelect, for highly selective and sensitive RNA imaging. In addition, QUID−2 exhibited excellent photostability and low cytotoxicity. Using QUID−2, the global dynamics of RNA were revealed in live cells. More importantly, QUID−2 was found to be potentially applicable for detecting RNA granules in live cells. Collectively, our work provides an ideal probe for RNA imaging. We anticipate that this powerful tool may create new opportunities to investigate the underlying roles of RNA and RNA granules in live cells.     

Low Bandgap 2D Perovskite Single Crystal with Anomalous-large Charges/ions Collection Ratio for Ultra-sensitive and Stable X-ray Detectors

The low-dimensional halide perovskites have attracted increasing attention due to their improved moisture stability, reduced defects, and suppressed ions migration in many optoelectronic devices such as solar cells, light-emitting diodes, X-ray detectors, and so on. However, they are still limited by their large band gap and short charge carriers’ diffusion length. Here, we demonstrate that the introduction of metal ions into organic interlayers of two-dimensional (2D) perovskite by cross-linking the copper paddle-wheel cluster-based lead bromide ([Cu(O₂C−(CH₂)₃−NH₃)₂]PbBr₄) perovskite single crystals with coordination bonds can not only significantly reduce the perovskite band gap to 0.96 eV to boost the X-ray induced charge carriers, but can also selectively improve the charge carriers’ transport along the out-of-plane direction and blocking the ions motion paths. The [Cu(O₂C−(CH₂)₃−NH₃)₂]PbBr₄ single-crystal device can reach a record charges/ions collection ratio of 1.69×10¹⁸±4.7 % μGy_air⁻¹ s, and exhibit a large sensitivity of 1.14×10⁵±7% μC Gy_air⁻¹ cm⁻² with the lowest detectable dose rate of 56 nGy_air s⁻¹ under 120 keV X-rays irradiation. In addition, [Cu(O₂C−(CH₂)₃−NH₃)₂]PbBr₄ single-crystal detector exposed to the air without any encapsulation shows excellent X-ray imaging capability with long-term operational stability without any attenuation of 120 days.     

Activatable Near-Infrared Versatile Fluorescent and Chemiluminescent Dyes Based on the Dicyanomethylene-4H-pyran Scaffold: From Design to Imaging and Theranostics

Activatable fluorescent and chemiluminescent dyes with near-infrared emission have indispensable roles in the fields of bioimaging, molecular prodrugs, and phototheranostic agents. As one of the most popular fluorophore scaffolds, the dicyanomethylene-4H-pyran scaffold has been applied to fabricate a large number of versatile activatable optical dyes for analytes detection and diseases diagnosis and treatment by virtue of its high photostability, large Stokes shift, considerable two-photon absorption cross-section, and structural modifiability. This review discusses the molecular design strategies, recognition mechanisms, and both in vitro and in vivo bio-applications (especially for diagnosis and therapy of tumors) of activatable dicyanomethylene-4H-pyran dyes. The final section describes the current shortcomings and future development prospects of this topic.     

Identification and relative quantification of adenosine to inosine editing in serotonin 2c receptor mRNA by CE

A new method has been developed allowing the identification and relative quantification of different forms of mRNA after RNA editing. This method was applied to the serotonin 2c receptor mRNA that potentially exhibits 32 different forms after adenosine to inosine editing at five different sites located in a row of 13 nucleotides. CE was used to characterize fluorescently labeled ssDNA molecules on the basis of their conformational polymorphism. The relative amount of these 32 mRNA forms has been estimated by measuring the fluorescence intensity of each individual DNA strand. Accuracy of quantification was established by diluting one form into another or into a mixture of cDNA, showing linear and precise proportion of each form (0.06 相似文献   

Donor and Ring-Fusing Engineering for Far-Red to Near-Infrared Triphenylpyrylium Fluorophores with Enhanced Fluorescence Performance for Sensing and Imaging

Fluorescent probes have become an indispensable tool in the detection and imaging of biological and disease-related analytes due to their sensitivity and technical simplicity. In particular, fluorescent probes with far-red to near-infrared (FR-NIR) emissions are very attractive for biomedical applications. However, many available FR-NIR fluorophores suffer from small Stokes shifts and sometimes low quantum yields, resulting in self-quenching and low contrast. In this work, we describe the rational design and engineering of FR-NIR 2,4,6-triphenylpyrylium-based fluorophores ( TPP-Fluors ) with the help of theoretical calculations. Our strategy is based on the appending of electron-donating substituents and fusing groups onto 2,4,6-triphenylpyrylium. In contrast to the parent TPP with short emission wavelength, weak quantum yields, and low chemical stability, the obtained novel TPP-Fluors display some favorable properties, such as long-wavelength emission, large Stokes shifts, moderate to high quantum yields, and chemical stability. TPP-Fluors demonstrate their biological value as mitochondria-specific labeling reagents due to their inherently positive nature. In addition, TPP-Fluors can also be applied to develop ratiometric fluorescent probes, as the electron-donating ability of the 2,6-phenyl substituents is closely correlated with their emission wavelength. A proof-of-concept ratiometric probe has been developed by derivatizing the amino groups of TPP-Fluor for the detection and imaging of nitroreductase in vitro and in hypoxic cells.     

银纳米粒子簇的设计、 制备和表面增强拉曼光谱

通过匹配激光光斑直径与胶体微球的尺寸, 设计制备了银纳米粒子的表面增强拉曼散射(SERS)基底, 并将其用于研究单个银纳米粒子簇的表面增强拉曼光谱. 在制备纳米粒子的过程中, 考察了等离子体刻蚀时间与银沉积厚度对“单”银纳米粒子结构与形貌的影响. 将吡啶、 巯基苯和罗丹明R6G作为SERS探针分子, 研究了其SERS效应, 通过荧光共振能量转移(FRET)机理, 实现了染料分子在单银纳米粒子簇上的SERS效应. SERS光谱测试与相关计算结果表明, 单个银纳米粒子簇的拉曼增强因子能够达到约10⁶.     

Synthesis,spectral, thermal and BVS investigations on ZnS4N^N/N coordination environment: Single crystal X-ray structures of bis(dibenzyldithiocarbamato)(N^N)Zinc(II) complexes (N^N = 1,10-phenanthroline,tetramethylethylenediamine and 4,4′-bipyridine)

The current paper describes the synthesis and spectral investigations on the adducts of [Zn(dbzdtc)₂] (1) with 1,10-phen (2), tmed (3), 2,2′-bipy (4) and 4,4′-bipy (5) (where, dbzdtc = dibenzyldithiocarbamate anion, 1,10-phen = 1,10-phenanthroline, tmed = tetramethylethylenediamine, 2,2′-bipy = 2,2′-bipyridine, 4,4′-bipy = 4,4′-bipyridne) and single crystal X-ray structures of [Zn(dbzdtc)₂(1,10-phen)] (2) and [Zn(dbzdtc)₂(tmed)] (3) and [Zn(dbzdtc)₂(4,4′-bipy)] (5). ¹H and ¹³C NMR spectra of 1,10-phen, tmed, 2,2′-bipy and 4,4′-bipy adducts were recorded. ¹H NMR spectra of the complexes show the drift of electrons from the nitrogen of the substituents forcing a high electron density towards sulfur via the thioureide π-system. In the ¹³C NMR spectra, the most important thioureide (N¹³CS₂) carbon signals are observed in the region: 206–210 ppm. Fluorescence spectra of complexes (2) and (4) show intense fluorescence due to the presence of rigid conjugate systems such as 1,10-phenanthroline and 2,2′-bipyridine. The observed fluorescence maxima for complexes with an MS₄N₂ chromophore in the visible region are assigned to the metal-to-ligand charge transfer (MLCT) processes. Single crystal X-ray structural analysis of (2) and (3) showed that the zinc atom is in a distorted octahedral environment. Bond Valence Sum was found to be equivalent to 1.865 for (2), 1.681 for (3) supporting the correctness of the determined structure. BVS of (3) deviates from the formal oxidation number of zinc due to the non-aromatic, sterically hindering tetramethyl bonding end of tmed. Thermal studies on the compounds show the formation of Zn(NCS)₂ as an intermediate during the decay.     

Correlated Secondary Ion Mass Spectrometry-Laser Scanning Confocal Microscopy Imaging for Single Cell-Principles and Applications

Secondary ion mass spectrometry (SIMS) as a powerful surface analysis technique has been widely applied in semiconductor industry and geology research. Recently, with the development of instrumental technology, SIMS is attracting more and more attention in life sciences. SIMS can provide surface MS spectra, 2D/3D chemical images and depth profiling of substances simultaneously. The minimal lateral resolution of 2D SIMS imaging is 80–100 nm, and the longitudinal resolution of 3D SIMS imaging is about 1–5 nm. However, owing to lack of specific ions to render the structures of organelles, SIMS imaging for single cells still have great challenges. Optical microscopy, in particular laser scanning confocal microscopy (LSCM), has been emerged to be an indispensable technique for single cell imaging and can obtain high spatial 2D/3D imaging to visualize the structures of organelles. Thus, the combinational use of SIMS and LSCM, which takes advantages of SIMS for molecular imaging and LSCM for morphological imaging, has greatly extended the application of SIMS imaging and ensured its accuracy at single cells level, providing novel insights into better understanding of the biological events inside cells. In this review, we focus on the development and application of SIMS imaging and the correlated SIMS and LSCM imaging in the research of cell biology and drug discovery. We anticipate that the combinational use of SIMS and LSCM imaging has promising future in biomedicine and life sciences.     

Electrophilic RNA for Peptidyl‐RNA Synthesis and Site‐Specific Cross‐Linking with tRNA‐Binding Enzymes

RNA functionalization is challenging due to the instability of RNA and the limited range of available enzymatic reactions. We developed a strategy based on solid phase synthesis and post‐functionalization to introduce an electrophilic site at the 3′ end of tRNA analogues. The squarate diester used as an electrophile enabled sequential amidation and provided asymmetric squaramides with high selectivity. The squaramate‐RNAs specifically reacted with the lysine of UDP‐MurNAc‐pentapeptide, a peptidoglycan precursor used by the aminoacyl‐transferase FemX_Wv for synthesis of the bacterial cell wall. The peptidyl‐RNA obtained with squaramate‐RNA and unprotected UDP‐MurNAc‐pentapeptide efficiently inhibited FemX_Wv. The squaramate unit also promoted specific cross‐linking of RNA to the catalytic Lys of FemX_Wv but not to related transferases recognizing different aminoacyl‐tRNAs. Thus, squaramate‐RNAs provide specificity for cross‐linking with defined groups in complex biomolecules due to its unique reactivity.