RSC Chemical Biology最新文献

Nanchangmycin is a natural product with broad-spectrum activity against various organisms, exhibiting antibiotic, antiviral, anticancer, and antifibrotic effects. Nanchangmycin belongs to the family of polyether ionophores and is proposed to exert its therapeutic effects by altering ion gradients across biological membranes. Although this therapeutic mechanism has been well characterised in cancer models, it does not fully explain how nanchangmycin inhibits Zika virus infection, as recently reported. The specific molecular targets responsible for mediating nanchangmycin's antiviral activity remain unknown. Here, we designed a photoreactive clickable nanchangmycin probe and employed chemical proteomics to identify protein targets of nanchangmycin related to Zika virus infection in human cells. Among the most prominent targets was the protein SEC11A, a key component of the signal peptidase complex, which is essential for cleaving and processing Zika virus proteins. We showed that nanchangmycin blocks the cleavage of a Zika virus polyprotein, suggesting a novel mechanism for nanchangmycin-mediated inhibition of Zika virus infection.

Riboswitches are widespread regulatory RNA modules in bacteria, with many different classes already identified and even more yet to be discovered. Traditionally, the identification of riboswitches has relied on bioinformatic analyses and genetic screens. In this work, we explored the possibility of identifying and characterizing predicted and novel riboswitches using an affinity purification-based approach with a functionalized preQ₁ ligand. We successfully enriched a predicted preQ₁ riboswitch from L. monocytogenes total RNA. Biophysical characterization revealed that this riboswitch can simultaneously bind two ligand molecules and functions as a regulator of translation in vivo. Furthermore, a transcriptome-wide pull-down experiment resulted in strong preQ₁-dependent enrichment of several candidate sequences. Characterization of the lmo2684 candidate mRNA revealed a preQ₁ riboswitch-like sequence in its 5′ untranslated region. Notably, preQ₁ allowed translation of an upstream open reading frame in this region by promoting stop codon readthrough. Our findings highlight the utility of ligand-based pull-down strategies for enriching mRNAs with aptamers that elude computational detection and may possess undiscovered functions.

DNA methylation is a key epigenetic modification involved in genomic imprinting, X-chromosome inactivation, and repression of repetitive element transcription and transposition. Despite its biological significance, the impact of epigenetic modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) on the structural and UV-induced dynamics of DNA remains poorly understood. Here, we employed the fluorescent nucleobase analogue 2-aminopurine (2Ap) in combination with steady-state and time-resolved spectroscopy, molecular dynamics, and quantum mechanical calculations to investigate these effects. Our findings reveal distinct differences in base stacking and helical stability between mC and hmC-modified DNA. mC-modified DNA predominantly adopts a stacked conformation, promoting efficient fluorescence quenching of 2Ap. In contrast, hmC-modified DNA displays both stacked and non-stacked conformations, leading to reduced base stacking and a more hydrophobic local environment, as indicated by blue-shifted emission spectra. Furthermore, although charge-transfer quenching occurs in all systems, hmC shows weaker charge-transfer character, resulting in higher fluorescence quantum yields and longer lifetimes. These results highlight the subtle but crucial role of hmC in modulating local DNA conformation and stability. Moreover, they demonstrate the effectiveness of 2Ap as a sensitive probe for detecting epigenetic modifications, offering deeper insights into the molecular mechanisms of DNA methylation and demethylation pathways.

The glycoprotein α-dystroglycan is essential in establishing cell-matrix interactions and is implicated in the pathology of muscular dystrophies. Novel tools are needed to study its rare and intriguing O-mannosyl glycans. This report describes the synthesis and evaluation of alkyne-tagged ribitol-5-phosphate derivatives for the metabolic labelling of α-dystroglycan in mammalian cells.

Cell-specific control of the function of antisense oligonucleotides (ASOs) is highly desirable for precise gene therapy while minimizing adverse effects in normal cells. Herein, we report a novel class of chemically inducible ASOs (iASOs) that achieve tumor-cell-selective gene silencing through hydrogen peroxide (H₂O₂)-triggered activation. Through post-synthetic incorporation of phenylboronic acid (BO) caging groups at the backbone positions, we developed iASOs that remain functionally inactive until the H₂O₂-triggered removal of the BO groups caused activation. Using EGFP as a reporter system, we demonstrated that the optimal BO-modified iASO exhibited slight gene silencing activity in normal cells but achieved >80% knockdown of the target mRNA in tumor cells. The BO-modified iASO was further applied to target the endogenous Bcl₂ gene, demonstrating its ability for controlling gene silencing and inducing cell death. This study establishes a simple and effective platform for conditional gene regulation and the development of cell-specific ASO therapeutics.

The synthesis of ester bonds using lipases is one of the most frequently performed reactions in biocatalysis, yet examples of the enzymatic synthesis of phenyl benzoate esters are comparatively rare. In this report we show that the ligase ClxA, from Clostridium cavendishii, initially reported to have roles in amide bond formation in the biosynthesis of benzoxazole antibiotics, is an effective catalyst for the formation of phenyl benzoate esters from acid and phenol substrates using ATP in an aqueous medium. The structure of ClxA in a complex with both AMP and 3,4-aminohydroxybenzoic acid was determined by X-ray crystallography to 2.15 Å resolution and used as a platform to engineer the enzyme to create variants N226L and K140A possessing broader substrate specificity for ester formation, and also the ability to enable the synthesis of native amide product oligomers.

Proteins are biomolecules essential for cellular functions, including cell signaling and regulation. Protein misfolding or mislocalisation can result in various diseases. Peroxidase-mediated proximity labelling has emerged as a powerful tool for studying subcellular proteome and protein–protein interactions. However, the traditional probe, biotin-phenol, suffers from limitations including low protein enrichment efficiency, and the formation of oxidised and polymerised products, complicating the downstream analysis. To address these challenges, a novel probe, N-(4-amino-3,5-dimethylbenzyl)desthiobiotinamide (DBA-Me), for protein labelling in living cells was developed. Western blot analysis demonstrated efficient labelling of bovine serum albumin in vitro. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data confirmed the formation of one-to-one adducts from the in vitro labelling reaction. Notably, this novel probe (DBA-Me) also exhibited labelling activity towards nucleic acids. Moreover, DBA-Me also permits APEX2-mediated labelling within the mitochondrial matrix of HEK293FT cells, and demonstrated improved recovery of labelled proteins after streptavidin enrichment compared to the conventional biotin-phenol (BP) probe, highlighting its superior potential application in cellulo. This facilitates peroxidase-mediated proximity labelling applications in subcellular localisation of proteins, and protein structures, with broader implications for understanding cellular processes and disease mechanisms.

Current tissue engineering strategies primarily focus on replicating mechanical properties of the extracellular matrix (ECM); however, several studies have underscored the critical role of the ECM biochemical cues in developing functional tissue substitutes. Among these, glycans are known to play a key role in regulating cell fate. In this study we developed ECM mimics glyco-conjugated with two different glycans, α-D-glucopyranose (αGlc) and β-D-galactopyranose (βGal), to investigate their influence on morphology and cell behaviour. The ECM mimics were generated crosslinking glycosylated gelatin samples, functionalised with the glycans by reductive amination, and hyaluronic acid. The crosslinking was performed by previous functionalization of gelatin and hyaluronic acid with tyramine, to enable enzymatic phenol-phenol coupling via horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The glyco-conjugated hydrogels exhibited markedly different morphologies, characterized by increased fibrous content, smaller pore sizes, and more wrinkled surfaces. Bone marrow-derived mesenchymal stem cells (BM-MSCs) seeded in hydrogels functionalized αGlc and βGal exhibited a more elongated morphology and differential glycosignature compared to controls.

Identifying cell type-specific molecular markers on the cancer cell surface is essential for understanding cancer progression and for discovering critical neoantigens relevant to immunotherapy. Nucleic acid aptamers serve as powerful tools for probing complex and dynamic cell surface characteristics. Here, we introduce a streamlined comparative aptamer profiling methodology that enables side-by-side analysis of cell surface remodeling. Using cell-SELEX (systematic evolution of ligands by exponential enrichment), we generated a modified base-incorporated aptamer library directly from cells, which was then employed to explore the surface states of normal and mutant protein-expressing cells. Differential analysis of aptamer enrichment using next-generation sequencing revealed distinct aptamer signatures that correlated with cell types. Our analysis demonstrated that mutant K-Ras expression dynamically altered cell surface composition. Individual aptamers showed specific binding to mutant K-Ras-expressing cells without requiring sequence optimization. Moreover, target identification of one aptamer revealed abnormal translocation of a mitochondrial matrix protein to the cell surface without detectable changes in mRNA or protein levels upon altered cellular signaling. These findings highlight the dynamic modulation of cell surface states by aberrant cellular signaling. Overall, we present a useful comparative strategy to investigate cell surface alterations. This approach may help uncover previously unrecognized cell surface markers associated with oncogenic signaling.

Heterobifunctional molecules that induce targeted degradation have emerged as powerful tools in chemical biology, target validation, and drug discovery. Despite their promise, the field is constrained by the relative paucity of ligands available for E3 ligases. Expanding the ligand repertoire for E3 ligases and other components of ubiquitin-proteasome system could significantly broaden the scope of the targeted degradation field. In this study, we report the identification of ligands for non-essential E3 ligases that are preferentially expressed in cancer tissues relative to normal tissues. Using a protein-observed NMR-based fragment screen, an ideal technique for this purpose, we identified fragment ligands and characterized their binding modes by X-ray crystallography. These ligands represent promising starting points for further optimization toward the discovery of tumor-selective degraders that may enhance the therapeutic window targeting proteins for which inhibition or degradation is associated with systemic toxicity.