Chemical biology investigation of a triple-action,smart-decomposition antimicrobial booster based-combination therapy against“ESKAPE”pathogens

The global antibiotic resistance crisis necessitates urgent solutions.One innovative approach involves potentiating antibiotics and non-antibiotic drugs with adjuvants or boosters.A major drawback of these membrane-active boosters is their limited biocompatibility,as they struggle to differentiate between prokaryotic and eukaryotic membranes.This study reports the chemical biology investigation of a dual-action oligoamidine(OA1)booster with a glutathione-triggered decomposition mechanism.OA1,when combined with other antimicrobial molecules,exhibits a triple-targeting mechanism including cell membrane disruption,DNA targeting,and intracellular enzyme inhibition.This multi-targeting mechanism not only enhances the in vitro and in vivo eradication of antibiotic-resistant“ESKAPE”pathogens,but also suppresses the development of bacterial resistance.Furthermore,OA1 maintains its activity in bacterial cells by creating an oxidative environment,while it quickly decomposes in mammalian cells due to high glutathione levels.These mechanistic insights and design principles may provide a feasible approach to develop novel antimicrobial agents and effective anti-resistance combination therapies.

STRAP binds to and promotes the repair of N1-methyldeoxyadenosine in DNA

N1-methyladenosine(m1A)is an important RNA modification that functions in various biological processes by interacting with cellular proteins.However,the binding proteins of N1-methyldeoxyadenosine(1mdA)in DNA remain largely unknown.Herein,we employed a quantitative proteomics strategy to identify the potential binding proteins of 1mdA in human cells.Our results revealed that serine–threonine kinase receptor-associated protein(STRAP)can bind to 1mdA-carrying DNA.We further demonstrated that STRAP participates in alkylating agent-induced DNA damage response and can promote the repair of 1mdA embedded in DNA.Moreover,we investigated the effects of STRAP on 1mdA-induced perturbation in transcription using a shuttle vector-and next-generation sequencing-based assay,and found that STRAP is involved in the transcriptional bypass of 1mdA in human cells.Together,our study revealed STRAP as a novel 1mdA-binding protein in human cells and provided new insight into the biological implications of STRAP and 1mdA modification in human diseases.

Monitoring of Cell Membrane Microenvironment Based on DNA Nanodevices

The cell membrane is a critical barrier for cellular homeostasis,integral to signaling and intercellular communication,and vital for understanding cellular functions and disease mechanisms.Investigating its microenvironment is crucial for uncovering the molecular basis of physiological and pathological processes associated with the cell membrane,driving the development of bioanalytical toolkits capable of dynamically monitoring the cell surface microenvironment.With the continuous advancement of functional nucleic acids and dynamic DNA nanotechnology,DNA nanodevices with controllable nanosized geometry,specific molecular recognition,and selective membrane-localization properties offer a versatile platform for probing the cell membrane microenvironment.In this review,we summarize the current biosensing and membrane-anchoring mechanisms of DNA nanodevices and highlight their use in studying key cell membrane events,including membrane lipid dynamics,transmembrane transport,receptor dimerization,and signal transduction.Furthermore,we discuss the challenges and potential future applications of DNA nanodevices in advancing cell membrane biology research and biomedical applications.

Hot hole multiplication from silver plasmon-resonated gold interband transitions for enhanced photoelectrochemical sensing

Compared with the widespread exploitation of hot electrons in plasmonic nanoparticles(NPs),hot holes generated from plasmonic metal interband transitions,are often overlooked in photoelectrochemistry,including photoelectrochemical sensing.Motivated by the subtle spectral overlap between the characteristic plasmonic bands of Ag NPs and interband transitions of Au,herein,we construct unusual core-shell Ag@Au NPs via an anti-galvanic reaction to promote the generation of hot holes.Benefiting from the unique plasmon resonances of Ag cores in specific wavelength regimes,Ag@Au can excite multiplied hot holes while Au cannot under the same conditions.With satisfactory accuracy and good practicability,the photoelectrochemical sensing platform based on Ag@Au NPs possesses a detection limit of 77 nmol/L for glucose,exhibiting significantly higher sensitivity compared to that using Au NPs.This work exemplifies the applications of interband hot-hole accumulation initiated by plasmons and may inspire more strategies to explore the utilization of hot holes in photoelectrochemistry.

Development and Application of a Chemical Labeling-Based Biosensing Assay for Rapid Detection of 8-Oxoguanine and Its Repair in vitro and in Human Cells

Living cells are constantly threatened by endogenous and environmental agents that can induce various DNA lesions including 8-oxoguanine(8-oxoG).Increasing evidence has suggested that 8-oxoG is not only a biomarker of oxidative stress,but also a novel epigenetic-like modification involved in transcriptional regulation in mammalian cells.Measurement of DNA damage and repair is useful for both basic research and clinical applications,but current methods for 8-oxoG detection still suffer from some problems such as poor selectivity,time consuming and being expensive.Here,we developed a fast and simple biosensing approach for quantitative analysis of 8-oxoG in DNA,which was based on the selective chemical biotinylation of 8-oxoG in conjunction with biotin-streptavidin enzyme-linked immunosorbent assay.We have also successfully applied this method to achieve efficient detection of the repair activities of DNA glycosylases Fpg and hOGG1 toward 8-oxoG in vitro and in human cells.This newly developed biosensing assay should be generally applicable for rapid detection of 8-oxoG and its repair in other organisms.

Coupling of proteolysis-triggered transcription and CRISPR-Cas12a for ultrasensitive protease detection

The efficient signal amplification capacity of several class 2 CRISPR-Cas systems with trans-cleavage activity has exhibited great value in molecular diagnostics,but its potential application for non-nucleic-acid targets is yet underdeveloped.Here,we deploy CRISPR-Cas system for the ultrasensitive detection of protease biomarkers by the coupling of proteolysis-triggered transcription.In this strategy,a protease-activatable RNA polymerase is adopted for the conversion of each protease-catalyzed proteolysis event into the output of multiple programable RNA sequences by in vitro transcription,and the transcribed RNA subsequently serves as the guide RNA of Cas12a proteins with trans-cleavage activity.The rational design of the transcribed RNA efficiently couples the signal conversion and amplification of proteolysis-triggered transcription and the self-signal amplification of CRISPR-Cas12a,resulting in a two-stage amplified detection of target protease.The versatility of this strategy has been demonstrated in the detection of protease biomarkers including MMP-2 and thrombin with femtomolar sensitivity,which is 5–6 orders of magnitude lower than that of the standard peptide-based methods.Moreover,the proposed method has been further applied in the analysis of MMP-2 secreted by different cancer cell lines as well the assessment of MMP-2 activity in clinical serum samples,providing a generic method for the ultrasensitive detection of protease biomarkers in biochemical research and clinical diagnosis.

Comprehensive profiling of CTP-binding proteins using a biotinylated CTP affinity probe

Recent studies have shown that CTP may act as a ligand to regulate the activity of its target proteins in many biological processes.However,proteome-wide identification of CTP-binding proteins remains challenging.Here,we employed a biotinylated CTP affinity probe coupled with stable isotope labeling by amino acids in cell culture(SILAC)-based quantitative proteomics approach to capture,identify and quantify CTP-binding proteins in human cells.By performing two types of competitive SILAC experiments with high vs.low concentrations of CTP probe(100 vs.10µmol/L)or with CTP probe in the presence of free CTP,we identified 90 potential CTP-binding proteins which are involved in a variety of biological processes,including protein folding,nucleotide binding and cell-cell adhesion.Together,we developed a chemical proteomic method for uncovering the CTP-binding proteins in human cells,which could be widely applicable for profiling CTP-binding proteins in other biological samples.

Next-generation sequencing-based analysis of the effect of N^(6)-methyldeoxyadenosine modification on DNA replication in human cells

N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replication in human cells.Herein,we combined next-generation sequencing with shuttle vector technology to explore how 6 mdA affects the efficiency and accuracy of DNA replication in human cells.Our results showed that 6 mdA neither blocked DNA replication nor induced mutations in human cells.Moreover,we found that the depletion of translesion synthesis DNA polymerase(Pol) κ,Pol η,Pol ι or Pol ζ did not significantly change the biological consequences of 6 mdA during replication in human cells.The negligible impact of 6 mdA on DNA replication is consistent with its potential role in epigenetic gene expression.

Dynamics and biological relevance of epigenetic N^(6)-methyladenine DNA modification in eukaryotic cells

DNA methylation represents a major type of DNA modifications that play key roles in diverse biological processes.With the recent development of highly selective and sensitive bioanalytical techniques,N^(6)-methyladenine(6mA)has been characterized as an important internal DNA modification dynamically occurring in multiple eukaryotes including humans.Increasing evidence has indicated that 6mA may act as a novel epigenetic modification involved in regulation of development,stress response and diseases such as cancer and neurodegenerative disorders.We review herein the recent advances in the detection and functional studies of 6mA modification,with special emphasis on its biological consequences and human health relevance as well as its dynamic regulation by various types of methyltransferases,demethylases and 6mA-binding proteins.It can be envisaged that further chemical and biological studies of 6mA modification will lead to a better understanding about its potentially important roles in normal and pathological biological processes.

Advances in the Integration of Nucleic Acid Nanotechnology into CRISPR-Cas System

The microbial adaptive immune systems composed of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated protein(Cas),have been repurposed as revolutionary tool kits in many fields,including gene editing,transcriptional regulation,bioimaging and biosensing.Owing to the unprecedented programmability of base paring in nucleic acids,the progress in nucleic acid nanotechnology has brought new inspiration to CRISPR-Cas system.In this mini review,we summarized the research progress of the integration of nucleic acid nanotechnology into CRISPR-Cas system,including delivery of Cas proteins by DNA nanovehicles,conditional CRISPR-Cas system based on dynamic RNA nanotechnology,coupling of CRISPR-Cas and DNA origami.We also discussed the development prospects of the potential combinations of CRISPR-Cas system and nucleic acid nanotechnology.

Hydrogen-Bonding-Induced H-Aggregation of Charge-Transfer Complexes for Ultra-Efficient Second Near-Infrared Region Photothermal Conversion

Aggregation plays a critical role in modulating the photophysical process of organicmolecules.However,the rational control of the construction of a functionoriented stacking mode for efficient photothermal(PT)conversion in the second near-infrared region(NIR-II;1000-1700 nm)remains a challenge.Herein,an H-aggregation of 3,3′,5,5′-Tetramethylbenzidine(TMB)-TMB dication(TMB++)complexes in linear agarose(H-TTC/LAG)with narrowed band gap(0.96 eV)was fabricated through intermolecular hydrogenbonding interactions between the amino groups of TTC and the peripheral hydroxyl groups of LAG.Charge-transfer mechanism and H-aggregation ensured NIR-Ⅱ absorption of the complex at>1400 nm.The H-aggregation also promoted a non-radiation relaxation pathway and improved the thermal stability of TTC,which together favored the constructed H-TTC/LAG with ultra-efficient PT conversion that increased rapidly to 140℃ in 15 s under the NIR-Ⅱ laser(1064 nm,1.0 W cm^(−2))irradiation.Such a unique H-TTC/LAG with good biocompatibility was used to demonstrate a superior PT therapy via high-efficie ncy tumor growth inhibition in mouse mammary carcinoma(4T1)the BALB/c mice tumor-bearing xenografts.This is the first established H-aggregation of charge-transfer complexes in a noncovalent system,which not only provides a new strategy to develop ultra-efficient NIR-Ⅱ PT materials but also paves the way for constructing functional materials with aggregates of charge-transfer complexes.

Multivalent Display of Lipophilic DNA Binders for Dual-Selective Anti-Mycobacterium Peptidomimetics with Binary Mechanism of Action

We made oligoamidine-based peptidomimetics highly specific for mycobacteria eradication by introducing and arraying lipophilic DNA binding motifs on macromolecular backbones.The short poly(amidino-phenylindole)(PAPI)structures feature an alternating amphiphilic structure with cationic,lipophilic DNA-binding moieties,enabling fast and selective eradication of mycobacteria through binary,membrane-and DNA-selective mechanisms of action.More importantly,PAPIs address the primary treatment challenge by combating mycobacteria in eukaryotic cells and working as a sensitizer for conventional antibiotics,in bothways promoting more thorough removal of pathogens and reducing the mycobacteria’s resistance generation rate during treatment.Structural optimizationwas achieved to counter specific pathogens,including Mycobacterium tuberculosis,in the Mycobacterium genus.One of the hit peptidomimetics was evaluated in a zebrafish-based aquatic infection model using Mycobacterium fortuitum and a mice tail infection model using Mycobacterium marinum,both revealing excellent in vivo performance.

Zinc-substituted hemoglobin with specific drug binding sites and fatty acid resistance ability for enhanced photodynamic therapy

Precisely designed protein-based nanodrugs, as a kind of colloidal drug system, have attracted significant attention in tumor therapy because of their refined drug loading ratio, controlled delivery efficacy and natural biocompatibility. However, most drugs are conjugated to the protein carriers randomly without specific binding sites. Moreover, such sites could easily be replaced by lipophilic molecules in the physiological environment and result in low delivery efficiency. With strong and specific binding locations especially comparatively narrow spatial binding sites and nonflexible structure, hemin (FePPIX)-free hemoglobin or apohemoglobin (apoHb), as a natural metalloporphyrin protein carrier, represents great potential in bioapplication. Therefore, we herein introduce a folate acid (FA) modified, zinc-substituted hemoglobin (ZnPHb-FA) as a naturally occurring protein matrix-based photosensitizer for cancer photodynamic therapy (PDT). Noncovalent inserted ZnPPIX molecules in apoHb possess an extremely stable property and significant recovered photoproperties with superior biocompatibility and phototoxicity, both in vitro and in vivo. This stability was verified by molecular docking analysis and calculation of binding constant, representing a total of five drug binding sites of apoHb for ZnPPIX molecules, four of which are energetically favorable (△G value of -11.9 kcal/mol), and one which is energetically acceptable (△G value of -9 kcal/mol). Folate acid modification has been shown to efficiently enhance the internalization and retention time of ZnPHb nanodrug. ZnPHb-FA is also an efficient depressor of hemin oxygenase-1 (HO-1), which could, in turn, lower the antioxidant ability of cancer cells by decreasing the production of biiirublin. Results in vitro and in vivo both indicated that the firmly combination of apoHb and ZnPPIX described here represents a novel and efficient protein nanodrug systems for cancer therapy.

Versatile metal graphitic nanocapsules for SERS bioanalysis

The novel graphitic nanomaterial of metal graphitic nanocapsules(MGNs) with superior stability, unique optical properties and biocompatibility possess great potential in biomedical and bioanalytical applications. The graphitic shell can quench the background fluorescence interference from external environments via a fluorescence resonance energy transfer(FRET) process and even avoid unnecessary reactions catalyzed by inner metal core. The graphitic shell with several characteristic Raman bands itself can act as Raman signal probe or internal standard(IS), especially the 2D-band within the cellular Raman-silent region helps to reduce the interference signals from external conditions. The present context attempts to give a comprehensive overview about the preparation and unique properties of MGNs as well as their applications in SERS biodetection and bioimaging.

A photonanozyme with light-empowered specific peroxidasemimicking activity

Although nanozymes have been widely developed,directly utilizing light to drive catalytic reactions like natural photoenzymes still remains challenging.Herein,we propose that photonanozymes(PNZs),as a novel kind of nanozyme,exclusively possess enzyme-mimicking activity under illumination.Only in the presence of visible light,the as-synthesized TiO_(2) proposed in this contribution shows excellent specificity of peroxidase-like without any oxidase-or catalase-like activity.The driving force of the light-empowered peroxidase-like photonanozymatic activity is explicated in terms of the photogenerated hot charge carriers in TiO_(2) PNZs and the accompanied reactive oxygen species.The co-substrates for photonanozymatic reaction over TiO_(2) PNZs facilitate the formation of the precarious and reactive peroxo-oxygen bridge between TiO_(2) and H_(2)O_(2),enabling the catalytic specificity.With the TiO_(2) PNZ-based biosensing platform for visual glucose detection exemplifying the concept of the application of PNZs,this work may evoke more inspirations to explore strategies for enlarging the scope of photoenzyme mimics.

Responsive MXene nanovehicles deliver CRISPR/Cas12a for boolean logic-controlled gene editing

Programmable and precise regulation of genetic information is crucial in bioengineering and biomedicine;however, it remains challenging to implement this objective. Here we deployed DNA-functionalized MXenes as a smart delivery system for spatiotemporally controllable genome editing. The MXene nanovehicles rationally integrated photothermal effect with nucleic acid strand displacement reaction, thereby allowing for the binary logic gate-controlled release of Cas ribonucleoprotein complexes in response to different input patterns of NIR light and nucleic acids. This system was highly programmable and could be harnessed to construct 2-input(AND, OR, and N-IMPLY) and 3-input(AND/OR and N-IMPLY/OR) logic gates for precise gene editing in mammalian cells. Moreover, an AND logic gate-controlled delivery system achieved selective induction of tumor cell death in a xenograft mice model using tissue-penetrating NIR light and cancer-relevant microRNA as the inputting cues.Therefore, the MXene nanovehicles adopted both the external and endogenous signals as the stimuli to precisely control gene editing under logic computation, presenting a helpful strategy for therapeutic genome editing.

A Reactive Oxygen Species-Tyrosinase Cascade-Activated Prodrug for Selectively Suppressing Melanoma

“Off-target effect”is one of the obstacles for targeted prodrugs in chemotherapy.To circumvent this issue,herein we propose a dual biomarker cascadeactivated prodrug strategy based on high levels of reactive oxygen species(ROS)and tyrosinase(TYR)in melanoma cells.A representative prodrug,Coumarin-Quinazolinone-phenylBoronic acid pinacol ester(CQB),was prepared.The prodrug contained Coumarin-Quinazolinone(CQ)as mitochondria targeting and monitoring moiety and phenylboronic acid pinacol ester as a cascade-activated prowarhead.After activation by the endogenous ROS and subsequent TYR in melanoma cells,CQB can be converted to the final active reagent Coumarin-Quinazolinone-o-Quinone(CQQ)that contains o-quinone group as the reactive species.CQQ may interact with cellular nucleophiles to exert its genotoxic effect and disrupt the cellular redox balance.This enables CQB to selectively suppress melanoma.CQB accumulates in the mitochondria and causes mitochondrial dysfunction via affecting mitochondrial DNA integrity.This cascade-activated prodrug may provide a precise strategy for melanoma theranostics.

Molecularly Engineered Aptamers Targeting Tumor Tissue and Cancer Cells for Efficient in Vivo Recognition and Enrichment

Molecular engineering of aptamers can confer exogenous biomedical properties that may be beneficial for various applications.In this study,a tumor-homing peptide modification strategy was developed to considerably enhance the accumulation and penetration abilities of the Sgc8c aptamer.Notably,the S2PM conjugate induced a much higher level of morphological variation in three-dimensional tumor microspheres(HCT116 cells)than in control groups,highlighting the importance of the homing and penetrating abilities derived from peptide.