RSC Chemical Biology最新文献

Labeling the plasma membrane for advanced imaging remains a significant challenge. For time-lapse live cell imaging, probe internalization and photobleaching are major limitations affecting most membrane-specific dyes. In fixed or permeabilized cells, many membrane probes either lose signal after fixation or fail to remain localized to the plasma membrane. Thus, improved probes are critically needed for applications in spatial biology. In this study, we systematically compared a range of custom-synthesized and commercially available lipid-based probes for their efficiency in labeling the plasma membrane in live, fixed, and permeabilized cells. We identified a superior probe, which outperformed others due to its lipid structure. This comparison provides insights into ideal lipid probes for visualizing the plasma membrane using advanced imaging techniques.

The bacterial DNA damage (SOS) response promotes DNA repair, DNA damage tolerance, and survival in the setting of genotoxic stress, including stress induced by antibiotics. In E. coli, translesion DNA synthesis can be fulfilled by Y-family DNA polymerases, including DNA polymerase IV (DinB). DinB features a more open active site and lacks proofreading ability, promoting error-prone replication. While DinB is known to tolerate damaged nucleobases like 8-oxo-guanine (8-oxoG), its ability to accommodate sugar-modified nucleotides has been underexplored, a question of importance given that such analogs are commonly used to inhibit viral and other polymerases. To explore DinB's selectivity, we screened a variety of sugar-modified noncanonical nucleotide triphosphates (nNTPs) and determined that DinB is intolerant of most 3′-modifications but can incorporate a subset of 2′-modifications. In particular, arabinosyl nucleotide triphosphates (araNTPs) showed efficient incorporation and limited extension. Furthermore, araNTPs can effectively compete with natural nucleotide triphosphates leading to stalled replication by DinB. We show that this tolerance extends to combined nucleobase and sugar modifications, with preferred misincorporation of 2′-fluoroarabinosyl-8-oxo-GTP opposite A more than C. Overall, our work highlights the potential for exploiting substrate promiscuity to target DinB and, thereby, slow bacterial adaptation to antibiotics.

Sirtuins are an evolutionarily conserved family of NAD⁺-dependent deacylases whose catalytic mechanism remains under active investigation. While previous studies have captured sirtuin reaction intermediates using thioacetyl-lysine analogs, here we report six crystal structures of human SIRT2 in complex with native myristoylated peptides and NAD⁺, revealing the sequence of changes from initial NAD⁺ binding to the formation of intermediate I. Our structures provide direct structural evidence for: (1) zinc-binding domain shift during NAD⁺ entry, (2) water-mediated hydrogen-bond formation that disrupts nicotinamide aromaticity preceding cleavage, and (3) the formation of intermediate I. Additionally, we determined the structures of two functionally critical mutants (SIRT2^F96A and SIRT2^H187A), demonstrating their roles in stabilizing NAD⁺ in a productive conformation. These findings complete the comprehensive structural framework for the sirtuin deacylation mechanism and highlight key residues governing catalytic efficiency.

The use of β-lactam antibiotics is threatened by antibiotic resistant bacteria that produce β-lactamases. These enzymes not only protect the bacteria that produce them but also shelter other bacteria in the same environment that would otherwise be susceptible. While this phenomenon is of clinical significance, many of the factors that contribute to β-lactamase-mediated antibiotic sheltering have not been well-studied. We report the development of a luminescence assay to directly monitor the survival of β-lactam-susceptible bacteria in the presence of β-lactamase-producing bacteria and β-lactam antibiotics. This method provides a rapid and scalable means of quantifying antibiotic sheltering in mixed microbial populations. We applied this assay to investigate the contributions of several factors to sheltering, including the class of β-lactam, the substrate specificity of the β-lactamase, and the cell wall permeability of the β-lactamase-producing bacterium. Our results show that the extent of sheltering that occurs not only depends on the particular combination of β-lactam and β-lactamase, but is also greatly impacted by the ability of a β-lactamase to access its β-lactam substrates.

We developed a nanoparticle based on a cell-penetrating peptide-PROTAC conjugate with a disulfide linker, MZ1-R9, and dextran sulfate, enhancing cellular uptake and BRD4 degradation. This delivery platform significantly improves PROTAC bioavailability and offers a promising strategy to overcome membrane permeability challenges for targeted protein degradation.

Nucleic acids (NA), namely DNA and RNA, dynamically fold and unfold to perform their functions in cells. Functional NAs include NA enzymes, such as ribozymes and DNAzymes. Their folding and target binding are governed by interactions between nucleobases, including base pairings, which follow thermodynamic principles. To elucidate biological mechanisms and enable diverse technical applications, it is essential to clarify the relationship between the primary sequence and the catalytic activity of NA enzymes. Unlike methods for predicting the stability of NA duplexes, which have been widely used for over half a century, predictive approaches for the catalytic activity of NA enzymes remain limited due to the low throughput of activity assays. However, recent advances in genome analysis and computational data science have significantly improved our understanding of the sequence–function relationship in NA enzymes. This article reviews the contributions of data-driven chemistry to understanding the reaction mechanisms of NA enzymes at the nucleotide level and predicting novel NA enzymes with catalytic activity from sequence information. Furthermore, we discuss potential databases for predicting NA enzyme activity under various solution conditions and their integration with artificial intelligence for future applications.

Peptide nucleic acid (PNA) is a unique class of synthetic nucleic acids with a pseudo-peptide backbone, known for its high nucleic acid recognition capability and its ability to directly recognize double-stranded DNA (dsDNA) via the formation of a unique invasion complex. While most natural and artificial nucleic acids form duplexes in an antiparallel configuration due to the general instability of parallel configurations, PNA distinctively forms both antiparallel and parallel duplexes. In this study, we focused on this previously underexplored property of PNA to adopt a parallel duplex configuration and developed a novel double-duplex invasion strategy by leveraging the differences in thermal stability between the antiparallel and parallel orientations of PNA duplexes. Furthermore, we report the first crystal structure of a parallel PNA duplex, which was found to exhibit different structural features compared to the previously characterized antiparallel PNA duplex. This study highlights the potential of artificial nucleic acids in dsDNA recognition and demonstrates that the parallel architecture may serve as a conceptual foundation for advancing broader methodological innovations in nucleic acid research.

Profiling the full spectrum of protein glycoforms is critical to understanding their functional roles. We developed the differential melting voltage approach using tandem-ion mobility/tandem-mass spectrometry and applied it to study Ribonuclease B glycoforms. Our results indicate that, in addition to glycan mass and intact protein size, the glycan structure plays a role in regulating the stability of Ribonuclease B.

As both chemical and biological engineering approaches continue to expand, the landscape of biomolecular technologies is rapidly evolving, affording new opportunities from basic science to real-world applications. This themed collection brings together engineered biomolecule-based technologies spanning small molecules, nucleic acids, and proteins, with applications in biocatalysis, biosensing, and synthetic biology. Each study showcases the modular and tunable nature of biomolecular design to tailor properties for function in both aqueous solutions and biological environments, as summarized below.

The poly(A) tail plays a crucial role in mRNA stability and translation efficiency. Chemical modification of the poly(A) tail is a promising approach for stabilizing mRNA against deadenylation. In this study, we investigated the effect of poly(A) chemical modifications using phosphorothioate (PS), 2′-fluoro (2′-F), 2′-O-methyl (2′-OMe), and 2′-O-methoxyethyl (2′-MOE) modifications. Notably, PS, 2′-OMe, and 2′-MOE modifications conferred resistance to CAF1, an enzyme responsible for deadenylation. Interestingly, only the PS modification retained the poly(A)-binding protein (PABP) binding activity, which is critical for translation, whereas 2′-F, 2′-OMe, and 2′-MOE modifications abolished this activity. Beyond the PS modification, the combination of 2′-F, 2′-OMe, and 2′-MOE modifications resulted in enhanced resistance to both CAF1 and other nucleases. Based on these results, a 12-nucleotide unmodified poly(A) sequence was inserted upstream of the modified poly(A) to confer both nuclease resistance and PABP-binding activity. Notably, the resulting poly(A) formulation significantly prolonged protein expression in cultured cells and mouse skin when applied to epidermal growth factor-encoding therapeutic mRNA. Collectively, this study presents a design concept for poly(A) chemical modifications to achieve durable protein expression from mRNA, offering a promising strategy for enhancing the function of mRNA-based therapeutics.