AND Logic Gate-Regulated DNAzyme Nanoflower for Monitoring the Activity of Multiple DNA Repair Enzymes.

Despite the progress that has been made in diverse DNA-based nanodevices to in situ monitor the activity of the DNA repair enzymes in living cells, the significance of improving both the sensitivity and specificity has remained largely neglected and understudied. Herein, we propose a regulatable DNA nanodevice to specifically monitor the activity of DNA repair enzymes for early evaluation of cancer mediated by genomic instability. Concretely, an AND logic gate-regulated DNAzyme nanoflower was rationally designed by the self-assembly of the DNA duplex modified with both apurinic/apyrimidinic (AP) site and methyl lesion site. The DNAzyme nanoflower could be reconfigured under the repair of AP sites and O6-methylguanine sites by apurinic/apyrimidinic endonuclease 1 (APE1) and O6-methylguanine methyltransferase (MGMT) to produce a fluorescent signal, realizing the sensitive monitoring of the activity of APE1 and MGMT. Compared to the free DNAzyme duplex, the fluorescent response of the DNAzyme nanoflower increased by 60%, due to the effective enrichment of the DNA probes by the nanoflower structure. More importantly, we have demonstrated that the dual-enzyme activated strategy allows imaging of specific cancer cells in the AND logic gate manner using MCF-7 as a cancer cell model, improving the specificity of cancer cell imaging. This AND logic gate-regulated multifunctional DNAzyme nanoflower provides a simple tool for simultaneously visualizing multiple DNA repair enzymes, holding great potential in early clinical diagnosis and drug discovery.

Multienzymatic Orthogonal Activation of DNA Codec Enables Tumor-Specific Imaging of Base Excision Repair Activity.

Accurate monitoring of base excision repair (BER) activity in cancer cells is critical for advancing the comprehension of DNA repair processes, gaining insights into cancer development, and guiding treatment strategies. However, current assay techniques for assessing BER activity in cancer cells face challenges due to the heterogeneous origins and diversity of BER enzymes. In this work, we present a highly reliable triple loop-interlocked DNA codec (GATED) that enables precise assessment of BER activity in cancer cells through signal amplification mediated by multienzyme orthogonal activation. The GATED device features a dumbbell-shaped DNA probe to encode two BER enzymes for BER-related signal conversion as well as two bound circular DNA to decode the apurinic/apyrimidinic sites for apurinic/apyrimidinic endonuclease 1 (APE1)-mediated signal amplification. Importantly, GATED is orthogonally activated by multiple target BER enzymes (i.e., uracil DNA glycosylase, thymine DNA glycosylase, and APE1), resulting in a unified fluorescent signal that significantly improves the detection specificity and sensitivity to BER enzymes. Additionally, we demonstrate that the GATED has exceptional biostability within complex biological systems, where it was successfully employed to monitor BER activity in cancer cells with high specificity and enabled cell-based high-throughput screening for BER inhibitors. The GATED provides a much-needed tool for the real-time monitoring of BER activity and the screening of BER inhibitors in cancer cells, potentially advancing both the investigation and clinical application of BER biology.

Engineering of a Multi-Modular DNA Nanodevice for Spatioselective Imaging and Evaluation of NK Cell-Mediated Cancer Immunotherapy.

Granzyme A (GzmA) secreted by natural killer (NK) cells has garnered considerable interest as a biomarker to evaluate the efficacy of cancer immunotherapy. However, current methodologies to selectively monitor the spatial distribution of GzmA in cancer cells during NK cell-targeted therapy are extremely challenging, primarily due to the existence of diverse cell populations, the low levels of GzmA expression, and the limited availability of GzmA probes. Herein we develop a multi-modular, structurally-ordered DNA nanodevice for evaluating NK cell-mediated cancer immunotherapy (MODERN), that permits spatioselective imaging of GzmA in cancer cells through GzmA-induced apurinic/apyrimidinic endonuclease 1 (APE1) inactivation. The MODERN incorporates multiple functional modules, including an APE1-gated recognition module, a photo-activated amplification module, an aptamer-mediated tumor-target module, and a polycatenane DNA module, enabling improved sensitivity and specificity towards intracellular GzmA. The MODERN was activated (on) in cancer cells due to the overexpression of APE1, whereas it remained silent (off) in the NK-treated cancer cells owing to the GzmA-induced APE1 inactivation. Furthermore, we demonstrated that GzmA-induced APE1 inactivation blocks the cellular repair of target cells, resulting in efficient cell death. This MODERN that relies on the specific inactivation of APE1 by GzmA should be beneficial for evaluating the efficacy of cancer immunotherapy.

Engineering Molecular Emission Centers of Carbon Dots to Boost the Electrochemiluminescence for the Detection of Cancer Cells.

Despite the fact that electrochemiluminescent (ECL) performance of carbon dots (CDs) could be improved by modulating their surface defects, they are still restricted by inferior controllability and poor reproducibility. In this work, we disclosed a new approach for synthesizing luminescent groups rich in CDs (Lu-CDs) by engineering the luminol as molecular emission centers into the CDs, which exhibited an 80-fold stronger ECL intensity at an ECL onset potential of 0.6 V than the CDs without pre-implanted luminol. Different from the significant deviation between the ECL and fluorescence emission of other surface state-dominated CDs, the ECL emission of Lu-CDs was nearly consistent with its fluorescence emission at 465 nm, which was defined as the molecular emission dominated-ECL CDs herein. To prove this principle, the Lu-CDs were employed to construct an ECL biosensor for MCF-7 cell analysis based on the cell direct recognition and amplification strategy, which made the MCF-7 cells as nanomachines via specific binding with aptamer signal probes on the DNA triangular scaffold. The proposed biosensor displayed a wide detection range from 101 to 104 cell mL-1 and a low detection limit of 8.91 cells mL-1. Overall, this work not only presents a new strategy for preparing CDs with high controllability and excellent reproducibility but also provides a platform for tumor cell sensing.

An Affinity-Enhanced DNA Intercalator with Intense ECL Embedded in DNA Hydrogel for Biosensing Applications.

Herein, an amphiphilic perylene derivative (denoted as PTC-DEDA) was explored as DNA intercalators endowed with an enhanced affinity and intense electrochemiluminescence (ECL) to construct a target-induced DNA hydrogel biosensing platform for the sensitive detection of microRNA let-7a (miRNA let-7a). Specifically, the DNA hydrogel with numerous dendritic DNA structures was in situ generated via a target-induced nonlinear hybrid chain reaction in the presence of miRNA let-7a, which possessed a large loading capacity to entrap massive DNA intercalators. Then, the PTC-DEDA with positive charges could easily intercalate into the DNA grooves due to the inherent amphipathic structure, achieving a strong ECL signal. Using the proposed PTC-DEDA as both DNA intercalators and ECL emitters, the DNA hydrogel biosensing platform exhibited a high stability and an excellent sensitivity for miRNA let-7a, with a desirable linear range (10 fM to 10 nM) and a low detection limit (1.49 fM). Significantly, the work provides a potential alternative to develop simple and high-efficiency ECL platforms for biochemical analysis applications.

[Structure and biological functions of mammalian LEM-domain proteins].

During the process of open mitosis in higher eukaryotic cells, the nuclear envelope (NE) is disassembled and reassembled with highly organized and periodical dynamic morphological changes. Recent studies demonstrated that LEM-domain protein family mediates interactions among inner nuclear membrane, nuclear lamina protein and chromatin by interacting with barrier-to-autointegration-factor (BAF). The structure and function of the ternary complex formed by LEM-domain protein, nuclear lamina protein and BAF are dependent on each other. Moreover, the network formation based on this structure and function is critical for the development of basic biological processes of nuclear, and it plays important roles in chromatin separation in late metaphase and anaphase, NE reassembly after mitosis, morphological maintenance of nuclear and NE in interphase, regulation of DNA replication and DNA damage repair, regulation of gene expression and signaling pathway, and infection of retrovirus. Mutations in genes encoding LEM family proteins have important impacts on development and progression of laminopathic diseases and tumorigenesis. This review provides a detailed summary of structural and functional studies of the LEM family proteins.

Dynamic surface reconstruction of individual gold nanoclusters by using a co-reactant enables color-tunable electrochemiluminescence.

Here we report for the first time the phenomenon of continuously color-tunable electrochemiluminescence (ECL) from individual gold nanoclusters (Au NCs) confined in a porous hydrogel matrix by adjusting the concentration of the co-reactant. Specifically, the hydrogel-confined Au NCs exhibit strong dual-color ECL in an aqueous solution with triethylamine (TEA) as a co-reactant, with a record-breaking quantum yield of 95%. Unlike previously reported Au NCs, the ECL origin of the hydrogel-confined Au NCs is related to both the Au(0) kernel and the Au(i)-S surface. Surprisingly, the surface-related ECL of Au NCs exhibits a wide color-tunable range of 625-829 nm, but the core-related ECL remains constant at 489 nm. Theoretical and experimental studies demonstrate that the color-tunable ECL is caused by the dynamic surface reconstruction of Au NCs and TEA radicals. This work opens up new avenues for dynamically manipulating the ECL spectra of core-shell emitters in biosensing and imaging research.

Covalent organic frameworks as micro-reactors: confinement-enhanced electrochemiluminescence.

Electrochemiluminescence (ECL) micro-reactors with enhanced intensity and extreme stability were first established by the assembly of tris(2,2'-bipyridyl) ruthenium(ii) (Ru(bpy)3 2+) onto covalent organic frameworks (COFs), in which a type of imine-linked COF (denoted as COF-LZU1) was employed as a model for ECL micro-reactors. Compared with the dominant ECL system of Ru(bpy)3 2+/tri-n-propylamine (TPrA) (TPrA as a co-reactant), the intensity of the COF-LZU1 micro-reactor-based electrode was significantly increased nearly 5-fold under the same experimental conditions, which is unprecedented in other Ru(bpy)3 2+-based ECL systems. This enhancement can be attributed to the large surface area, delimited space, and stable and hydrophobic porous structure of COF-LZU1, which not only enabled a huge amount of Ru(bpy)3 2+ to be loaded in/on COF-LZU1, but also enriched a large amount of TPrA from the aqueous solution into its inner hydrophobic cavity due to the lipophilicity of TPrA. More importantly, with its hydrophobic porous nanochannels, COF-LZU1 could act as micro-reactors to provide a delimited reaction micro-environment for the electrochemical oxidation of TPrA and the survival of TPrA˙, achieving significant confinement-enhanced ECL. To prove this principle, these Ru@COF-LZU1 micro-reactors were developed to prepare an ECL aptasensor for aflatoxin M1 (AFM1) detection with a wide detection range and a low detection limit. Overall, this work is the first report in which ECL micro-reactors are constructed with COFs to enhance the intensity and stability of the Ru(bpy)3 2+-based ECL system, and opens a new route to the design of other ECL micro-reactors for bioanalysis applications.

Hemin as electrochemically regenerable co-reaction accelerator for construction of an ultrasensitive PTCA-based electrochemiluminescent aptasensor.

In this work, hemin was firstly used as electrochemically regenerable co-reaction accelerator for signal amplification to develop an ultrasensitive aptasensor for Aflatoxin M1 (AFM1) detection. Initially, the perylenetetracarboxylic acid (PTCA) was directly employed as luminophore to construct the ECL sensing nano-platform by combining Au nanoparticles (Au NPs) for immobilizing thiol-terminated hairpin probe (H1). Then with the help of hairpin H2, H3, the AFM1-catalyzed hairpin assembly (CHA) was executed to produce the H1-H3 duplex, which could further initiate the hybridization chain reaction (HCR) to generate dendritic DNA polymers consisting of G-rich sequence for capturing large quantities of hemin on the electrode surface. Herein, hemin as electrochemically regenerable co-reaction accelerator could interact with the co-reactant (S2O82-) to obviously improve the luminous efficiency of the PTCA. Therefore, a strong and stable ECL signal was achieved by the employment of hemin as electrochemically regenerable co-reaction accelerator. The proposed aptasensor determined AFM1 down to 0.09pgmL-1 within a linear range of 0.4pgmL-1 to 400ngmL-1. With excellent sensitivity and stability, the strategy provided an efficient and simple method for the trace detection of biomolecules in clinical analysis.