{"title":"Peptide nucleic acids in parallel orientation form invasion complexes with double-stranded DNA","authors":"Masanari Shibata, Hiroshi Sugimoto, Masaki Hibino, Osami Shoji and Yuichiro Aiba","doi":"10.1039/D5CB00172B","DOIUrl":"10.1039/D5CB00172B","url":null,"abstract":"<p >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) <em>via</em> 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.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 10","pages":" 1566-1575"},"PeriodicalIF":3.1,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12400189/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144994075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Differential melting voltage by tandem-trapped ion mobility spectrometry: glycan structure influences glycoprotein stability.","authors":"Mengqi Chai, Christian Bleiholder, Fanny C Liu","doi":"10.1039/d5cb00127g","DOIUrl":"10.1039/d5cb00127g","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12394896/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144973040","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Introduction to “Biomolecular Technologies”","authors":"Sheel C. Dodani and Ariel Furst","doi":"10.1039/D5CB90031J","DOIUrl":"https://doi.org/10.1039/D5CB90031J","url":null,"abstract":"<p >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.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 9","pages":" 1364-1365"},"PeriodicalIF":3.1,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2025/cb/d5cb90031j?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144909464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Characterization of nuclease stability and poly(A)-binding protein binding activity of chemically modified poly(A) tail for in vivo applications","authors":"Atsushi Hashimoto, Yuma Kunitomo, Ittoku Kikuchi, Hiroki Yamada, Keiko Kobayashi, Kazuhiro Soshiroda, Hiromi Aman, Yasuaki Kimura, Junichiro Yamamoto, Yasuhisa Shiraishi, Satoshi Uchida, Hiroshi Abe and Hiroto Iwai","doi":"10.1039/D5CB00137D","DOIUrl":"10.1039/D5CB00137D","url":null,"abstract":"<p >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′-<em>O</em>-methyl (2′-OMe), and 2′-<em>O</em>-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.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 10","pages":" 1616-1624"},"PeriodicalIF":3.1,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12381655/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144973007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Olabode Dawodu, Cody A. White, Caitlin Specht, Alejandro Tapia and Jeffery M. Tharp
{"title":"Copper-catalysed azide–alkyne cycloaddition on live M13 bacteriophage for expanding the molecular diversity of phage-displayed peptide libraries","authors":"Olabode Dawodu, Cody A. White, Caitlin Specht, Alejandro Tapia and Jeffery M. Tharp","doi":"10.1039/D5CB00140D","DOIUrl":"10.1039/D5CB00140D","url":null,"abstract":"<p >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.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 10","pages":" 1555-1565"},"PeriodicalIF":3.1,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12363981/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144973045","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Edward A. FitzGerald, Daniela Cederfelt, Daria Kovryzhenko, Pierre Boronat, Bjarte Aarmo Lund, Doreen Dobritzsch, Sven Hennig, Pablo Porragas Paseiro, Iwan J. P. de Esch and U. Helena Danielson
{"title":"Detection and characterisation of ligand-induced conformational changes in acetylcholine binding proteins using biosensors and X-ray crystallography","authors":"Edward A. FitzGerald, Daniela Cederfelt, Daria Kovryzhenko, Pierre Boronat, Bjarte Aarmo Lund, Doreen Dobritzsch, Sven Hennig, Pablo Porragas Paseiro, Iwan J. P. de Esch and U. Helena Danielson","doi":"10.1039/D5CB00041F","DOIUrl":"10.1039/D5CB00041F","url":null,"abstract":"<p >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.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 10","pages":" 1625-1639"},"PeriodicalIF":3.1,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12394895/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144973019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Development of a silicon phthalocyanine analogue for near-infrared photoimmunotherapy and its application to HTLV-1-infected leukemic cells","authors":"Yoshikazu Fuse, Eita Sasaki, Masaharu Tamaki, Shunto Kawamura, Hisashi Ohno, Sota Yamada, Masahiro Yasunaga, Hideo Takakura, Hirofumi Hanaoka, Hisataka Kobayashi, Hideki Nakasone and Kenjiro Hanaoka","doi":"10.1039/D5CB00150A","DOIUrl":"10.1039/D5CB00150A","url":null,"abstract":"<p >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 <strong>SiPc-1</strong> 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 <strong>SiPc-1</strong> 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 <strong>SiPc-1</strong> (bas-<strong>SiPc-1</strong>) showed selective toxicity towards HTLV-1-infected cultured cells and ATL patients’ peripheral blood mononuclear cells upon photoirradiation.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 10","pages":" 1576-1584"},"PeriodicalIF":3.1,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12360215/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144972993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Exploring the significance of palmitoylation using an artificial protein lipidation system","authors":"Kazuki Uchida, Naofumi Shimokawa, Rie Wakabayashi, Shohei Shiomoto, Kiyohiro Toyofuku, Nozomu Ogushi, Masahiro Goto, Masaru Tanaka, Masahiro Takagi and Noriho Kamiya","doi":"10.1039/D5CB00143A","DOIUrl":"10.1039/D5CB00143A","url":null,"abstract":"<p >Protein lipidation, particularly palmitoylation (attachment of a 16-carbon fatty acid), regulates cellular behaviors by controlling protein function at lipid membranes. In this study, we prepared a series of lipidated green fluorescent proteins (“EGFP–lipids”) with various alkyl chain lengths (C8 to C22). Using model lipid membranes and Jurkat (human T lymphocyte) cells, we evaluated how lipidation affects the membrane interactions and vesicular transport from the membrane of these protein–lipid constructs. Our findings demonstrate that elongation of the alkyl chain profoundly affects both lateral membrane diffusion and vesicular transport of the EGFP–lipids. Only artificially lipidated proteins that mimic <em>in vivo</em> lipidation exhibited cellular dynamics in response to external signals, which highlights the significance of the natural selection of palmitic acid to maximize the function of proteins on lipid membranes. This insight can also be useful in membrane engineering using artificial protein lipidation techniques, potentially accelerating medical and industrial developments.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 9","pages":" 1483-1496"},"PeriodicalIF":3.1,"publicationDate":"2025-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12351397/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144875801","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Translocation of penetratin-like peptides involving calcium-dependent interactions between glycosaminoglycans and phosphocholine headgroups of the membrane lipid bilayer","authors":"Bingwei He, Sonia Khemaissa, Sébastien Cardon, Rodrigue Marquant, Françoise Illien, Delphine Ravault, Fabienne Burlina, Emmanuelle Sachon, Astrid Walrant and Sandrine Sagan","doi":"10.1039/D5CB00099H","DOIUrl":"10.1039/D5CB00099H","url":null,"abstract":"<p >Cell-penetrating peptides (CPPs) can internalize ubiquitously in cells. To explore the specific targeting issue of CPPs, we used glycosaminoglycan (GAG)-binding peptides previously identified in Otx2 and En2 homeoproteins (HPs). The Otx2 sequence preferentially recognizes highly sulfated chondroitin (CS) and the En2 one, heparan sulfates (HS) GAGs. The two HPs internalize in specific cells thanks to their GAG-targeting sequence. We studied the capacity of chimeric peptides containing a GAG-targeting and a penetratin-like sequences to enter into various cell lines known to express different levels and types of GAGs. Since GAGs are found at the vicinity the membrane lipid bilayer, we also analyzed the putative binary and ternary interactions between heparin (HI), (4S,6S)-CS (CS-E), zwitterionic phosphocholine (PC) model membranes and those chimeric peptides. Altogether, our results demonstrate the existence of Ca<small><sup>2+</sup></small>-dependent interactions between GAGs and PC lipid bilayers, the major phospholipid headgroup found in animal cell plasma membrane. In addition, the interaction of CS-E (but not HI), with PC favors the binding of the chimeric CS-E-recognition motif-penetratin-like peptide and its subsequent crossing of the lipid membrane to access directly to the cytosol of cells. Altogether, this study brings further understanding of translocation mechanism of CPPs, which requires specific GAGs at the cell-surface. It also shed light on the role of GAGs in the cell transfer specificity and paracrine activity of HPs.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 9","pages":" 1391-1402"},"PeriodicalIF":3.1,"publicationDate":"2025-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12332767/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144817851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yuchen Su, Ruoyu Cheng, Bowei Du, Mai O. Soliman, Hongbo Zhang and Shiqi Wang
{"title":"The impact of conjugation strategies and linker density on the performance of the Spermine-AcDex nanoparticle–splenocyte conjugate","authors":"Yuchen Su, Ruoyu Cheng, Bowei Du, Mai O. Soliman, Hongbo Zhang and Shiqi Wang","doi":"10.1039/D5CB00104H","DOIUrl":"10.1039/D5CB00104H","url":null,"abstract":"<p >A common approach in living medicine engineering is modifying cell surfaces with nanomedicines to form nanoparticle–cell conjugates. Despite various available strategies, limited research has examined how conjugation strategies affect the efficiency and stability of the delivery systems. Herein, we prepared polymeric nanoparticles (NPs) with protein payloads and modified them with different linkers. These NPs were conjugated to primary splenocytes using either covalent or electrostatic interactions, followed by flow cytometry analysis to evaluate the conjugating efficiency and stability. The results demonstrated that electrostatic interactions were more effective in achieving conjugation, whereas covalent interactions provided greater stability. Furthermore, the linker density on the nanoparticle surface also affected the stability. After three days of <em>in vitro</em> culture, NPs with fewer linkers were predominantly internalized by the splenocytes, whereas those with more linkers partially remained on the cell surface. Overall, this study provides fundamental insights into nanoparticle–cell conjugation, thereby contributing to living medicine design and engineering for therapeutic applications.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" 10","pages":" 1546-1554"},"PeriodicalIF":3.1,"publicationDate":"2025-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12352615/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144875802","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}