Reconfigurable Bioinspired Framework Nucleic Acid Nanoplatform Dynamically Manipulated in Living Cells for Subcellular Imaging

In nature, the formation of spider silk fibers begins with dimerizing the pH‐sensitive N‐terminal domains of silk proteins (spidroins) upon lowering pH, and provides a natural masterpiece for programmable assembly. Inspired by the similarity of pH‐dependent dimerization behaviors, introduced here is an i‐motif‐guided model to mimic the initial step of spidroin assembly at the subcellular level. A framework nucleic acid (FNA) nanoplatform is designed using two tetrahedral DNA nanostructures (TDNs) with different branched vertexes carrying a bimolecular i‐motif and a split ATP aptamer. Once TDNs enter acidic lysosomes within living cells, they assemble into a heterodimeric architecture, thereby enabling the formation of a larger‐size framework and meanwhile subcellular imaging in response to endogenous ATP, which can be dynamically manipulated by adjusting intracellular pH and ATP levels with external drug stimuli.     

Selenium‐Doped Carbon Quantum Dots for Free‐Radical Scavenging

Heteroatom doping is an effective way to adjust the fluorescent properties of carbon quantum dots. However, selenium‐doped carbon dots have rarely been reported, even though selenium has unique chemical properties such as redox‐responsive properties owing to its special electronegativity. Herein, a facile and high‐output strategy to fabricate selenium‐doped carbon quantum dots (Se‐CQDs) with green fluorescence (quantum yield 7.6 %) is developed through the hydrothermal treatment of selenocystine under mild conditions. Selenium heteroatoms endow the Se‐CQDs with redox‐dependent reversible fluorescence. Furthermore, free radicals such as ^.OH can be effectively scavenged by the Se‐CQDs. Once Se‐CQDs are internalized into cells, harmful high levels of reactive oxygen species (ROS) in the cells are decreased. This property makes the Se‐CQDs capable of protecting biosystems from oxidative stress.     

Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

Single‐molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real‐time single‐molecule measurement of the structure‐changing dynamics of nucleic acids.     

Chemiluminescent Probe for the In Vitro and In Vivo Imaging of Cancers Over‐Expressing NQO1

Activatable (turn‐on) probes that permit the rapid, sensitive, selective, and accurate identification of cancer‐associated biomarkers can help drive advances in cancer research. Herein, a NAD(P)H:quinone oxidoreductase‐1 (NQO1)‐specific chemiluminescent probe 1 is reported that allows the differentiation between cancer subtypes. Probe 1 incorporates an NQO1‐specific trimethyl‐locked quinone trigger moiety covalently tethered to a phenoxy‐dioxetane moiety through a para‐aminobenzyl alcohol linker. Bio‐reduction of the quinone to the corresponding hydroquinone results in a chemiluminescent signal. As inferred from a combination of in vitro cell culture analyses and in vivo mice studies, the probe is safe, cell permeable, and capable of producing a “turn‐on” luminescence response in an NQO1‐positive A549 lung cancer model. On this basis, probe 1 can be used to identify cancerous cells and tissues characterized by elevated NQO1 levels.     

Imaging Endogenous Metal Ions in Living Cells Using a DNAzyme–Catalytic Hairpin Assembly Probe

DNAzymes are a promising platform for metal ion detection, and a few DNAzyme‐based sensors have been reported to detect metal ions inside cells. However, these methods required an influx of metal ions to increase their concentrations for detection. To address this major issue, the design of a catalytic hairpin assembly (CHA) reaction to amplify the signal from photocaged Na⁺‐specific DNAzyme to detect endogenous Na⁺ inside cells is reported. Upon light activation and in the presence of Na⁺, the NaA43 DNAzyme cleaves its substrate strand and releases a product strand, which becomes an initiator that trigger the subsequent CHA amplification reaction. This strategy allows detection of endogenous Na⁺ inside cells, which has been demonstrated by both fluorescent imaging of individual cells and flow cytometry of the whole cell population. This method can be generally applied to detect other endogenous metal ions and thus contribute to deeper understanding of the role of metal ions in biological systems.     

Induction of a Four‐Way Junction Structure in the DNA Palindromic Hexanucleotide 5′‐d(CGTACG)‐3′ by a Mononuclear Platinum Complex

Four‐way junctions (4WJs) are supramolecular DNA assemblies comprising four interacting DNA strands that in biology are involved in DNA‐damage repair. In this study, a new mononuclear platinum(II) complex 1 was prepared that is capable of driving the crystallization of the DNA oligomer 5′‐d(CGTACG)‐3′ specifically into a 4WJ‐like motif. In the crystal structure of the 1 –CGTACG adduct, the distorted‐square‐planar platinum complex binds to the core of the 4WJ‐like motif through π–π stacking and hydrogen bonding, without forming any platinum–nitrogen coordination bonds. Our observations suggest that the specific molecular properties of the metal complex are crucially responsible for triggering the selective assembly of this peculiar DNA superstructure.     

Vancomycin‐Dependent Response in Live Drug‐Resistant Bacteria by Metabolic Labeling

The surge in drug‐resistant bacterial infections threatens to overburden healthcare systems worldwide. Bacterial cell walls are essential to bacteria, thus making them unique targets for the development of antibiotics. We describe a cellular reporter to directly monitor the phenotypic switch in drug‐resistant bacteria with temporal resolution. Vancomycin‐resistant enterococci (VRE) escape the bactericidal action of vancomycin by chemically modifying their cell‐wall precursors. A synthetic cell‐wall analogue was developed to hijack the biosynthetic rewiring of drug‐resistant cells in response to antibiotics. Our study provides the first in vivo VanX reporter agent that responds to cell‐wall alteration in drug‐resistant bacteria. Cellular reporters that reveal mechanisms related to antibiotic resistance can potentially have a significant impact on the fundamental understanding of cellular adaption to antibiotics.     

Control Mechanism for Carbon‐Chain Length in Polyunsaturated Fatty‐Acid Synthases

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are essential fatty acids. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts. However, detailed biosynthetic mechanisms for controlling final product profiles have been obscure. Here, the bacterial DHA and EPA synthases were carefully dissected by in vivo and in vitro experiments. In vitro analysis with two KS domains (KS_A and KS_C) and acyl‐acyl carrier protein (ACP) substrates showed that KS_A accepted short‐ to medium‐chain substrates while KS_C accepted medium‐ to long‐chain substrates. Unexpectedly, condensation from C₁₈ to C₂₀, the last elongation step in EPA biosynthesis, was catalyzed by KS_A domains in both EPA and DHA synthases. Conversely, condensation from C₂₀ to C₂₂, the last elongation step for DHA biosynthesis, was catalyzed by the KS_C domain in DHA synthase. KS_C domains therefore determine the chain lengths.