Chemically Inducible DNAzyme Sensor for Controllable Imaging of Metal Ions.

RNA-cleaving DNAzymes have emerged as a promising tool for metal ion detection. Achieving spatiotemporal control over their catalytic activity is essential for understanding the role of metal ions in various biological processes. While photochemical and endogenous stimuli-responsive approaches have shown potential for controlled metal ion imaging using DNAzymes, limitations such as photocytotoxicity, poor tissue penetration, or off-target activation have hindered their application for safe and precise detection of metal ions in vivo. We herein report a chemically inducible DNAzyme in which the catalytic core is modified to contain chemical caging groups at the selected backbone sites through systematic screening. This inducible DNAzyme exhibits minimal leakage of catalytic activity and can be reactivated by small molecule selenocysteines, which effectively remove the caging groups and restore the activity of DNAzyme. Benefiting from these findings, we designed a fluorogenic chemically inducible DNAzyme sensor for controlled imaging of metal ions with tunable activity and high selectivity in live cells and in vivo. This chemically inducible DNAzyme design expands the toolbox for controlling DNAzyme activity and can be easily adapted to detect other metal ions in vivo by changing the DNAzyme module, offering opportunities for precise biomedical diagnosis.

Orthogonal Control of Nucleic Acid Function via Chemical Caging-Decaging Strategies.

The ability to precisely control the function of nucleic acids plays an important role in biosensing and biomedicine. In recent years, novel strategies employing biological, physical, and chemical triggers have been developed to modulate the function of nucleic acids spatiotemporally. These approaches commonly involve the incorporation of stimuli-responsive groups onto nucleic acids to block their functions until triggers-induced decaging restore activity. These inventive strategies deepen our comprehension of nucleic acid molecules' dynamic behavior and provide new techniques for precise disease diagnosis and treatment. Focusing on the spatiotemporal regulation of nucleic acid molecules through the chemical caging-decaging strategy, we here present an overview of the innovative triggered control mechanisms and accentuate their implications across the fields of chemical biology, biomedicine, and biosensing.

Dual Spatially Localized DNA Walker for Fast and Efficient RNA Detection.

DNA walkers, which are synthetic nanodevices that drive the processive movement of nucleic acids along a well-designed track, have emerged as a powerful tool in biosynthesis, biocomputing, and biosensing due to their exquisite programmability, good biocompatibility, and efficient signal amplification capacity. However, many existing approaches are still hindered by limited reaction kinetics. Herein, we designed a dual spatially localized DNA walker that utilized bipedal catalysts to drive high-speed stochastic movement along three-dimensional tracks via a proximity-driven catalytic hairpin assembly. We demonstrated that the dual colocalization of autocatalytic circuits significantly increased their local concentrations and accelerated reaction kinetics through proximity. We also showed that the use of bipedal catalysts further improved reaction rates compared with unipedal catalysts. Taking advantage of these unique features, we constructed an RNA-responsive PCHA walker for mRNA imaging in live cells, providing a novel and efficient tool for biomolecule detection and biological functions regulation.

Site-Specific Bioorthogonal Activation of DNAzymes for On-Demand Gene Therapy.

RNA-cleaving DNAzymes hold great promise as gene silencers, and spatiotemporal control of their activity through site-specific reactions is crucial but challenging for on-demand therapy. We herein report a novel design of a bioorthogonally inducible DNAzyme that is deactivated by site-specific installation of bioorthogonal caging groups on the designated backbone sites but restores the activity via a phosphine-triggered Staudinger reduction. We perform a systematical screening for installing the caging groups on each backbone site in the catalytic core of 10-23 DNAzyme and identify an inducible DNAzyme with very low leakage activity. This design is demonstrated to achieve bioorthogonally controlled cleavage of exogenous and endogenous mRNA in live cells. It is further extended to photoactivation and endogenous stimuli activation for spatiotemporal or targeted control of gene silencing. The bioorthogonally inducible DNAzyme is applied to a triple-negative breast cancer mouse model using a lipid nanoparticle delivery system, demonstrating high efficiency in knockdown of Lcn2 oncogenes and substantial suppression of tumor growth, thus highlighting the potential of precisely controlling the DNAzyme functions for on-demand gene therapy.

Perioperative Chemotherapy Could Not Improve the Prognosis of Gastric Cancer Patients With Mismatch Repair Deficiency: A Multicenter, Real-World Study.

INTRODUCTION: To date, the role of deficient mismatch repair (dMMR) remains to be proven in gastric cancer, and it is difficult to judge its value in clinical application. Our study aimed to investigate how MMR status affected the prognosis in patients with gastrectomy, as well as the efficacy of neoadjuvant chemotherapy and adjuvant chemotherapy in patients with dMMR with gastric cancer. MATERIALS AND METHODS: Patients with gastric cancer with certain pathologic diagnosis of dMMR or proficient MMR (pMMR) using immunohistochemistry from 4 high-volume hospitals in China were included. Propensity score matching was used to match patients with dMMR or pMMR in 1:2 ratios. Overall survival (OS) and progression-free survival (PFS) curves were plotted using the Kaplan-Meier method and compared statistically using the log-rank test. Univariate and multivariate Cox proportional hazards models based on hazard ratios (HRs) and 95% confidence intervals (CIs) were used to determine the risk factors for survival. RESULTS: In total, data from 6176 patients with gastric cancer were ultimately analyzed, and loss of expression of one or more MMR proteins was observed in 293 patients (293/6176, 4.74%). Compared to patients with pMMR, patients with dMMR are more likely to be older (≥66, 45.70% vs. 27.94%, P < .001), distal location (83.51% vs. 64.19%, P < .001), intestinal type (42.21% vs. 34.46%, P < .001), and in the earlier pTNM stage (pTNM I, 32.79% vs. 29.09%, P = .009). Patients with gastric cancer with dMMR showed better OS than those with pMMR before PSM (P = .002); however, this survival advantage was not observed for patients with dMMR after PSM (P = .467). As for perioperative chemotherapy, results of multivariable Cox regression analysis showed that perioperative chemotherapy was not an independent prognostic factor for PFS and OS in patients with dMMR with gastric cancer (HR = 0.558, 95% CI, 0.270-1.152, P = .186 and HR = 0.912, 95% CI, 0.464-1.793, P = .822, respectively). CONCLUSION: In conclusion, perioperative chemotherapy could not prolong the OS and PFS of patients with dMMR with gastric cancer.

Reversible Acylation of RNA Enables Activatable Biosensing.

There is a high demand to develop chemical tools to control the property and function of RNA. Current methods mainly rely on ultraviolet light-based caging strategies, which may cause phototoxicity in live cell-based experiments. We herein report an endogenous stimulus-responsive RNA acylation approach by introducing boronate ester (BE) groups to 2'-hydroxyls through postsynthetic modification. Treatment with hydrogen peroxide (H2O2) yields a phenol derivative which undergoes a 1,6-eliminaton for the traceless release of 2'-hydroxyl. We demonstrated that the acylation of crRNA enabled conditional regulation of CRISPR/Cas13a activity for activatable detection of target RNA. We also showed that the highly specific acylation of the single RNA in 8-17 DNAzyme allowed reversible control of the catalytic activity of DNAzyme, which was further applied to the cell-selective imaging of metal ions in cancer cells. Thus, our strategy provides a simple, general, and cell-selective method to control RNA activity, affording great potential in the construction of activatable RNA sensors and pre-RNA medicines.

DNA-Templated Bioorthogonal Reactions via Catalytic Hairpin Assembly for Precise RNA Imaging in Live Cells.

There has been a significant interest in developing proximity-induced bioorthogonal reactions for nucleic acid detection and imaging, owing to their high specificity and tunable reaction kinetics. Herein, we reported the first design of a fluorogenic sensor by coupling a bioorthogonal reaction with a DNA cascade circuit for precise RNA imaging in live cells. Two DNA hairpin probes bearing tetrazines or vinyl ether caged fluorophores were designed and synthesized. Upon target mRNA triggering catalytic hairpin assembly, the chemical reaction partners were brought in a spatial proximity to yield high effective concentrations, which dramatically facilitated the bioorthogonal reaction efficiency to unmask the vinyl ether group to activate fluorescence. The proposed fluorogenic sensor was demonstrated to have a high signal-to-noise ratio up to ∼30 fold and enabled the sensitive detection of target mRNA with a detection limit of 4.6 pM. Importantly, the fluorogenic sensor presented low background signals in biological environments due to the unique "click to release" feature, avoiding false positive results caused by unspecific degradation. We also showed that the fluorogenic sensor could accurately image mRNA in live cells and distinguish the relative mRNA expression levels in both tumor and normal cells. Benefiting from these significant advantages, our method provides a useful tool for basic studies of bioorthogonal chemistry and early clinical diagnosis.

Exceeding 30 ELNs is strongly recommended for pT3-4N0 patients with gastric cancer: A multicenter study of survival, recurrence, and prediction model.

The argument concerning the exact minimum number of examined lymph nodes (ELNs) has continued for a long time among various regions, and no consensus has been reached for stratified pathological T stages for data to date. Data from 4607 pN0 patients with gastric cancer were analyzed. Kaplan-Meier analysis showed the similar overall survival (OS) outcomes among the 3 groups (ELNs ≤ 15, 16 ≤ ELNs ≤ 29 and ELNs ≥ 30, P = .171). However, the ELNs ≥ 30 group had a better disease-free survival (DFS) outcome compared with the others (all P < .05). An increased ELN group (ELNs ≥ 30) showed an improved OS only for pT3 patients (hazard ratio [HR] = 0.397, 95% confidence interval (CI): 0.182-0.866, P = .020), while an improved DFS for pT3 patients (HR = 0.362, 95%CI: 0.152-0.860, P = .021) and pT4 patients (HR = 0.484, 95%CI: 0.277-0.844, P = .011) in the multivariate analysis. A well discriminated and calibrated nomogram was constructed to predict the probability of the OS and DFS, with the C-index for OS and DFS prediction of 0.782 (95%CI: 0.735 to 0.829) and 0.738 (95%CI: 0.685 to 0.791), respectively. This study provides new and useful insights into the impact of ELN count on reducing stage migration and postoperative recurrence of pN0 patients with gastric cancer in 2000-2017. In conclusion, a larger number of ELNs is suggested for surgeons to prolong the prognosis of pN0 gastric cancer, especially for pT3 patients.

Digital Loop-Mediated Isothermal Amplification-Based Absolute Methylation Quantification Revealed Hypermethylated DAPK1 in Cervical Cancer Patients.

The aberrant methylation of many genes has been reported to be associated with various carcinomas. Accurate detection of the methylation level could provide critical insights into the diagnostic analysis of diseases. Here, a sensitive HpaII-edited absolute droplet loop-mediated isothermal amplification (HEADLAMP) method based on methylation-sensitive restriction enzyme (MSRE) HpaII was developed for the digital quantification of DNA methylation. Methylation levels of the death-associated protein kinase 1 (DAPK1) gene that is associated with many cancers were studied using ß-actin as an internal reference. DAPK1 (2.5 pM) with 0.01% methylation (250 aM) can be detected with the conventional HpaII-edited LAMP assay. Using HEADLAMP, as low as 1% methylation level can be distinguished with an estimated limit of detection of 5 aM (ca. 3 copies/µL). Moreover, HEADLAMP can detect low levels of methylated DAPK1 in normal L-02 cells, while the conventional assay cannot. Finally, HEADLAMP was applied to the detection of DAPK1 methylation in 20 clinical tissue samples, which revealed hypermethylated DAPK1 in cervical cancer patients. We envisage potential applications of this robust, specific, and sensitive HEADLAMP assay in epigenetic studies and early clinical diagnosis.

"Repaired and Activated" DNAzyme Enables the Monitoring of DNA Alkylation Repair in Live Cells.

Direct measurement of DNA repair is critical for the annotation of their clinical relevance and the discovery of drugs for cancer therapy. Here we reported a "repaired and activated" DNAzyme (RADzyme) by incorporating a single methyl lesion (O6 MeG, 3MeC, or 1MeA) at designated positions through systematic screening. We found that the catalytic activity of the RADzyme was remarkably suppressed and could be restored via enzyme-mediated DNA repair. Benefit from these findings, a fluorogenic RADzyme sensor was developed for the monitoring of MGMT-mediated repair of O6 MeG lesion. Importantly, the sensor allowed the evaluation of MGMT repair activity in different cells and under drugs treatment. Furthermore, another RADzyme sensor was engineered for the monitoring of ALKBH2-mediated repair of 3MeC lesion. This strategy provides a simple and versatile tool for the study of the basic biology of DNA repair, clinical diagnosis and therapeutic assessment.

Cascade Circuits on Self-Assembled DNA Polymers for Targeted RNA Imaging In Vivo.

DNA molecular probes have emerged as a powerful tool for RNA imaging. Hurdles in cell-specific delivery and other issues such as insufficient stability, limited sensitivity, or slow reaction kinetics, however, hinder the further application of DNA molecular probes in vivo. Herein, we report an aptamer-tethered DNA polymer for cell-specific transportation and amplified imaging of RNA in vivo via a DNA cascade reaction. DNA polymers are constructed through an initiator-triggered hybridization chain reaction using two functional DNA monomers. The prepared DNA polymers show low cytotoxicity and good stability against nuclease degradation and enable cell-specific transportation of DNA circuits via aptamer-receptor binding. Moreover, assembling the reactants of hairpins C1 and C2 on the DNA polymers accelerates the response kinetics and improves the sensitivity of the cascade reaction. We also show that the DNA polymers enable efficient imaging of microRNA-21 in live cells and in vivo via intravenous injection. The DNA polymers provide a valuable platform for targeted and amplified RNA imaging in vivo, which holds great implications for early clinical diagnosis and therapy.

Recombinant Fusion Streptavidin as a Scaffold for DNA Nanotetrads for Nucleic Acid Delivery and Telomerase Activity Imaging in Living Cells.

Efficient platforms for intracellular delivery of nucleic acids are essential for biomedical imaging and gene regulation. We develop a recombinant fusion streptavidin as a novel protein scaffold for DNA nanotetrads for highly efficient nucleic acid delivery and telomerase activity imaging in living cells via cross-linking hybridization chain reaction (cHCR). The recombinant streptavidin protein is designed to fuse with multiple SV40 NLS (nuclear localization signal) and NES (nuclear export signal) domains and prepared through Escherichia coli expression. The recombinant NLS-SA protein allows facile assembly with four biotinylated DNA probes via high-affinity noncovalent interactions, forming a well-defined DNA tetrad nanostructure. The DNA nanotetrads are demonstrated to confer efficient cytosolic delivery of nucleic acid via a caveolar mediated endocytosis pathway, allowing efficient escape from lysosomal degradation. Moreover, the nanotetrads enable efficient cHCR assembly in response to telomerase in vitro and in cellulo, affording ultrasensitive detection and spatially resolved imaging for telomerase with a detection limit as low as 90 HeLa cells/mL. The fluorescence brightness obtained in live cell imaging is found to be dynamically correlated to telomerase activity and the inhibitor concentrations. Therefore, the proposed strategy may provide a highly efficient platform for nucleic acid delivery and imaging of biomarkers in living cells.

Self-accelerating Airy-Laguerre-Gaussian light bullets in a two-dimensional strongly nonlocal nonlinear medium.

We report a self-accelerating wave packets eigenmode solution of a two-dimensional (2D) nonlocal nonlinear Schrödinger equation (NNLSE) with an Airy-beam time-dependence, and present their spatiotemporal profiles. The behaviours of such Airy-Laguerre-Gaussian light bullets, as propagated in a strongly nonlocal nonlinear medium (SNNM), are investigated both analytically and numerically. We found that the generation, control, and manipulation of the NL spatiotemporal light bullets are affected by the radial mode number and the azimuthal mode number, as well as the modulation depth. Our scheme is quite different from the linear light bullets, in which the wave propagates in a NL medium and is an eigenmode of NLSE.

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.

Near-Infrared Photothermally Activated DNAzyme-Gold Nanoshells for Imaging Metal Ions in Living Cells.

DNAzymes have enjoyed success as metal ion sensors outside cells. Their susceptibility to metal-dependent cleavage during delivery into cells has limited their intracellular applications. To overcome this limitation, a near-infrared (NIR) photothermal activation method is presented for controlling DNAzyme activity in living cells. The system consists of a three-stranded DNAzyme precursor (TSDP), the hybridization of which prevents the DNAzyme from being active. After conjugating the TSDP onto gold nanoshells and upon NIR illumination, the increased temperature dehybridizes the TSDP to release the active DNAzyme, which then carries out metal-ion-dependent cleavage, resulting in releasing the cleaved product containing a fluorophore. Using this construct, detecting Zn2+ in living HeLa cells is demonstrated. This method has expanded the DNAzyme versatility for detecting metal ions in biological systems under NIR light that exhibits lower phototoxicity and higher tissue penetration ability.

Solitons of four-wave mixing in competing cubic-quintic nonlinearity.

We experimentally investigate the soliton formation and dynamics in the nonlinear propagation of the generated signal and probe beams in four-wave mixing (FWM) process with atomic coherence in a three-level atomic system, under the competition between focusing and defocusing nonlinearities, as well as between gain and dissipation, due to the third- and fifth-order nonlinear susceptibilities with opposite signs. With multi-parameter controllability and nonlinear competition in the system, fundamental, dipole, and azimuthally-modulated vortex FWM solitons can transform mutually from one to the other. Such investigations have potential applications in optical pattern formation and control, and all-optical communication.

Highly sensitive and selective strategy for microRNA detection based on WS2 nanosheet mediated fluorescence quenching and duplex-specific nuclease signal amplification.

MicroRNAs (miRNAs) play vital roles in physiologic and pathologic processes and are significant biomarkers for disease diagnostics and therapeutics. However, rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their short length, sequence homology, and low abundance. Herein, we report for the first time that WS2 nanosheet can exhibit differential affinity toward short oligonucleotide fragment versus ssDNA probe and act as an efficient quencher for adsorbed fluorescent probes. This finding is utilized to develop a new strategy for simple, sensitive, and selective detection of miRNA by combining WS2 nanosheet based fluorescence quenching with duplex-specific nuclease signal amplification (DSNSA). This assay exhibits highly sensitive and selective with a detection limit of 300 fM and even discriminate single-base difference between the miRNA family members. The result indicates that this simple and cost-effective strategy holds great potential application in biomedical research and clinical diagnostics.

Rydberg dressing evolution via Rabi frequency control in thermal atomic vapors.

We report for the first time the theoretical and experimental research on Rydberg electromagnetically induced transparency and second-order fluorescence dressing evolution by Rabi frequency control in thermal atomic vapors, in which the controlled results are well explained by the dressing effect and the Rydberg excitation blockade. Based on the certification of the Rydberg excitation blockade fraction through the dependence on principle quantum number n, we obtain dressing evolution curves, consisting of single-dressing and double-dressing in local and nonlocal blockade samples by scanning the probe and dressing fields. In addition, the competition between the Rydberg dressing second-order fluorescence and fourth-order fluorescence is first investigated. A corresponding theory is presented, which is consistent with the experimental results. Such blockade evolution regularity has potential applications in quantum control, and the Rydberg dressing may be useful for investigating multiple-body interactions, as well as for inducing short range interactions in Bose-Einstein condensates.

Synthesis of Zr2ON2 via a urea-glass route to modulate the bifunctional catalytic activity of NiFe layered double hydroxide in a rechargeable zinc-air battery.

The development of a highly efficient, stable, and low-cost bifunctional catalyst is imperative for facilitating the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). However, significant challenges are involved in extending its applications to rechargeable zinc-air batteries. This study presents a bifunctional catalyst, Zr2ON2@NiFe layered double hydroxide (LDH), that was developed by utilizing a urea-glass route for synthesizing the Zr2ON2 precursor, followed by riveting NiFe LDH nanosheets using a hydrothermal method. Specifically, the vertical distribution of NiFe LDH on the Zr2ON2 surface ensures the maximization of the number of accessible active sites and interfacial catalysis of NiFe LDH. Notably, Zr2ON2@NiFe LDH demonstrates ORR and OER bifunctional electrocatalytic behavior and high stability owing to its heterostructure and composition. Furthermore, a rechargeable zinc-air battery using a Zr2ON2@NiFe LDH electrocatalyst as the air cathode demonstrated a high peak power density (172 mW cm-2) and galvanostatic charge-discharge cycle stability (5 mA cm-2 over 443 h). Thus, this study presents an efficient and cost-effective strategy for the design of bifunctional electrocatalysts.