RSC Chemical Biology最新文献

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.

Phage display is a powerful platform for ligand evolution, but conventional phage display libraries are confined to the twenty canonical amino acids, greatly limiting the chemical space that these libraries can be used to explore. Here we present an approach to expand the molecular diversity of phage-displayed peptides that combines unnatural amino acid mutagenesis with chemical post-translational modification. By incorporating azide-functionalized unnatural amino acids into phage-displayed peptides and applying optimized conditions for copper-catalysed azide–alkyne cycloaddition, we achieve quantitative and selective peptide modification with a series of alkyne-functionalized small molecules. This approach provides a general platform for constructing chemically augmented phage-displayed libraries with broad utility in ligand discovery.

Analysis of ligand-induced structural changes in proteins is challenging due to the lack of experimental methods suited for detection and characterisation of both ligand binding and induced structural changes. We have explored biosensors with different detection principles to study interactions between ligands and acetylcholine binding proteins (AChBPs), soluble homologues of Cys-loop ligand gated ion channels (LGICs) that undergo similar structural changes as LGICs upon ligand binding. X-ray crystallography was used to identify binding sites and establish if the detected conformational changes involved small changes in loop C or major structural changes in the pentamer associated with ion channel opening. Experiments were initially focused on ligands exhibiting complex surface plasmon resonance (SPR) biosensor sensorgrams or detected by second harmonic generation (SHG) biosensor analysis. Surface acoustic wave (SAW) and SHG biosensors confirmed that complexities in SPR data were indeed due to ligand-induced conformational changes. Grating coupled interferometry (GCI) biosensor sensorgrams were less complex, despite similar detection principles. switchSENSE biosensor analysis revealed that ligands resulted in either a compaction or expansion of the protein structure. X-ray crystallography of the protein–ligand complexes was only successful for 7 out of 12 ligands, despite nM–μM affinities. Crystals were not obtained for the two compounds shown by SHG analysis to induce large structural changes, while electron densities were not seen in the structures for some ligands. The work presented herein shows that several biosensor technologies have a unique capability to detect and discriminate binding and ligand induced conformational changes in proteins, also when interactions are rapid, weak and structural changes are small. However, they are complementary and provide different information.

Near-infrared photoimmunotherapy (NIR-PIT) employing an antibody labeled with a silicon phthalocyanine dye, IR700, was approved as a minimally invasive treatment for unresectable recurrent head and neck cancer in Japan in 2020. However, further derivatization of IR700 is needed to increase the efficiency of cancer treatment. Here, we developed SiPc-1 as an IR700 analog, in which the linker was constructed using click chemistry to simplify the synthetic scheme and its position was switched from α to β on the benzene ring of phthalocyanine to eliminate intramolecular steric repulsion. We evaluated the cleavage rate of the water-soluble axial moieties of SiPc-1 upon photoirradiation, the cytotoxicity, and the morphological change (blebbing) of treated cells upon photoirradiation. We performed gene expression and protein expression analyses to find a target antigen selectively expressed on cells infected with human T-cell lymphotropic virus type 1 (HTLV-1), the causative virus of adult T-cell leukemia/lymphoma (ATL), and identified CD25 as a suitable target antigen. An anti-CD25 antibody, basiliximab, labeled with SiPc-1 (bas-SiPc-1) showed selective toxicity towards HTLV-1-infected cultured cells and ATL patients’ peripheral blood mononuclear cells upon photoirradiation.