RSC Chemical Biology最新文献

{"title":"Development of PCR primers enabling the design of flexible sticky ends for efficient concatenation of long DNA fragments†","authors":"Kohei Nomura, Kaoru Onda, Hirotaka Murase, Fumitaka Hashiya, Yukiteru Ono, Goro Terai, Natsuhisa Oka, Kiyoshi Asai, Daisuke Suzuki, Naho Takahashi, Haruka Hiraoka, Masahito Inagaki, Yasuaki Kimura, Yoshihiro Shimizu, Naoko Abe and Hiroshi Abe","doi":"10.1039/D3CB00212H","DOIUrl":"10.1039/D3CB00212H","url":null,"abstract":"<p >We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing <em>o</em>-nitrobenzyl photo-cleavable groups with a <em>tert</em>-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00212h?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140010884","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":"Tracking the cellular uptake and phototoxicity of Ru(ii)-polypyridyl-1,8-naphthalimide Tröger's base conjugates†","authors":"Sandra A. Bright, MariaLuisa Erby, Fergus E. Poynton, Daniel Monteyne, David Pérez-Morga, Thorfinnur Gunnlaugsson, D. Clive Williams and Robert B. P. Elmes","doi":"10.1039/D3CB00206C","DOIUrl":"10.1039/D3CB00206C","url":null,"abstract":"<p >Ruthenium(<small>II</small>) complexes are attracting significant research attention as a promising class of photosensitizers (PSs) in photodynamic therapy (PDT). Having previously reported the synthesis of two novel Ru(<small>II</small>)-polypyridyl-1,8-naphthalimide Tröger's base compounds <strong>1</strong> and <strong>2</strong> with interesting photophysical properties, where the emission from either the Ru(<small>II</small>) polypyridyl centres or the naphthalimide moieties could be used to monitor binding to nucleic acids, we sought to use these compounds to investigate further and in more detail their biological profiling, which included unravelling their mechanism of cellular uptake, cellular trafficking and cellular responses to photoexcitation. Here we demonstrate that these compounds undergo rapid time dependent uptake in HeLa cells that involved energy dependent, caveolae and lipid raft-dependent mediated endocytosis, as demonstrated by confocal imaging, and transmission and scanning electron microscopy. Following endocytosis, both compounds were shown to localise to mostly lysosomal and Golgi apparatus compartments with some accumulation in mitochondria but no localisation was found to the nucleus. Upon photoactivation, the compounds increased ROS production and induced ROS-dependent apoptotic cell death. The photo-activated compounds subsequently induced DNA damage and altered tubulin, but not actin structures, which was likely to be an indirect effect of ROS production and induced apoptosis. Furthermore, by changing the concentration of the compounds or the laser used to illuminate the cells, the mechanism of cell death could be changed from apoptosis to necrosis. This is the first detailed biological study of Ru(<small>II</small>)-polypyridyl Tröger's bases and clearly suggests caveolae-dependent endocytosis is responsible for cell uptake – this may also explain the lack of nuclear uptake for these compounds and similar results observed for other Ru(<small>II</small>)-polypyridyl complexes. These conjugates are potential candidates for further development as PDT agents and may also be useful in mechanistic studies on cell uptake and trafficking.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00206c?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139923941","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":"A covalent compound selectively inhibits RNA demethylase ALKBH5 rather than FTO†","authors":"Gan-Qiang Lai, Yali Li, Heping Zhu, Tao Zhang, Jing Gao, Hu Zhou and Cai-Guang Yang","doi":"10.1039/D3CB00230F","DOIUrl":"10.1039/D3CB00230F","url":null,"abstract":"<p > <em>N</em> <small>⁶</small>-Methyladenosine (m<small>⁶</small>A) is the most prevalent mRNA modification and is required for gene regulation in eukaryotes. ALKBH5, an m<small>⁶</small>A demethylase, is a promising target, particularly for anticancer drug discovery. However, the development of selective and potent inhibitors of ALKBH5 rather than FTO remains challenging. Herein, we used a targeted covalent inhibition strategy and identified a covalent inhibitor, <strong>TD19</strong>, which selectively inhibits ALKBH5 compared with FTO demethylase in protein-based and tumor cell-based assays. <strong>TD19</strong> irreversibly modifies the residues C100 and C267, preventing ALKBH5 from binding to m<small>⁶</small>A-containing RNA. Moreover, <strong>TD19</strong> displays good anticancer efficacy in acute myeloid leukemia and glioblastoma multiforme cell lines. Thus, the ALKBH5 inhibitor developed in this study, which selectively targets ALKBH5 compared with FTO, can potentially be used as a probe for investigating the biological functions of RNA demethylase and as a lead compound in anticancer research.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00230f?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139901963","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":"Strategies for converting turn-motif and cyclic peptides to small molecules for targeting protein–protein interactions","authors":"Deanne Hayward and Andrew M. Beekman","doi":"10.1039/D3CB00222E","DOIUrl":"10.1039/D3CB00222E","url":null,"abstract":"<p >The development of small molecules that interact with protein–protein interactions is an ongoing challenge. Peptides offer a starting point in the drug discovery process for targeting protein-interactions due to their larger, more flexible structure and the structurally diverse properties that allow for a greater interaction with the protein. The techniques for rapidly identifying potent cyclic peptides and turn-motif peptides are highly effective, but this potential has not yet transferred to approved drug candidates. By applying the properties of the peptide–protein interaction the development of small molecules for drug discovery has the potential to be more efficient. In this review, we discuss the methods that allow for the unique binding properties of peptides to proteins, and the methods deployed to transfer these qualities to potent small molecules.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00222e?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139755860","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":"Biomedical applications, perspectives and tag design concepts in the cell – silent Raman window†","authors":"Martha Z. Vardaki, Vasilis G. Gregoriou and Christos L. Chochos","doi":"10.1039/D3CB00217A","DOIUrl":"10.1039/D3CB00217A","url":null,"abstract":"<p >Spectroscopic studies increasingly employ Raman tags exhibiting a signal in the cell – silent region of the Raman spectrum (1800–2800 cm<small>⁻¹</small>), where bands arising from biological molecules are inherently absent. Raman tags bearing functional groups which contain a triple bond, such as alkyne and nitrile or a carbon–deuterium bond, have a distinct vibrational frequency in this region. Due to the lack of spectral background and cell-associated bands in the specific area, the implementation of those tags can help overcome the inherently poor signal-to-noise ratio and presence of overlapping Raman bands in measurements of biological samples. The cell – silent Raman tags allow for bioorthogonal imaging of biomolecules with improved chemical contrast and they have found application in analyte detection and monitoring, biomarker profiling and live cell imaging. This review focuses on the potential of the cell – silent Raman region, reporting on the tags employed for biomedical applications using variants of Raman spectroscopy.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00217a?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139755950","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":"Unusual cysteine modifications in natural product biosynthesis","authors":"Yaojie Gao, Yuhao Zhu, Takayoshi Awakawa and Ikuro Abe","doi":"10.1039/D4CB00020J","DOIUrl":"10.1039/D4CB00020J","url":null,"abstract":"<p > <small>L</small>-Cysteine is a highly reactive amino acid that is modified into a variety of chemical structures, including cysteine sulfinic acid in human metabolic pathways, and sulfur-containing scaffolds of amino acids, alkaloids, and peptides in natural product biosynthesis. Among the modification enzymes responsible for these cysteine-derived compounds, metalloenzymes constitute an important family of enzymes that catalyze a wide variety of reactions. Therefore, understanding their reaction mechanisms is important for the biosynthetic production of cysteine-derived natural products. This review mainly summarizes recent mechanistic investigations of metalloenzymes, with a particular focus on recently discovered mononuclear non-heme iron (NHI) enzymes, dinuclear NHI enzymes, and radical-SAM enzymes involved in unusual cysteine modifications in natural product biosynthesis.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00020j?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139755851","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":"Genetically encodable biosensors for Ras activity","authors":"Ryan Weeks, Sohum Mehta and Jin Zhang","doi":"10.1039/D3CB00185G","DOIUrl":"10.1039/D3CB00185G","url":null,"abstract":"<p >Genetically encoded Ras biosensors have been instrumental in illuminating the spatiotemporal dynamics of Ras activity since the beginning of the imaging revolution of the early 21st century. In general, these sensors employ Ras sensing units coupled with fluorescent proteins. These biosensors have not only helped elucidate Ras signalling dynamics at the plasma membrane but also revealed novel roles for Ras signalling within subcellular compartments such as the Golgi apparatus. In this review, we discuss the different classes of biosensors used to measure Ras activity and discuss their importance in uncovering new roles for Ras activity in cellular signalling and behavior.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00185g?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139755947","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":"Sortase mediated protein ubiquitination with defined chain length and topology†","authors":"Nicole R. Raniszewski, Jenna N. Beyer, Myles I. Noel and George M. Burslem","doi":"10.1039/D3CB00229B","DOIUrl":"10.1039/D3CB00229B","url":null,"abstract":"<p >Ubiquitination is a key post-translational modification on protein lysine sidechains known to impact protein stability, signal transduction cascades, protein–protein interactions, and beyond. Great strides have been made towards developing new methods to generate discrete chains of polyubiquitin and conjugate them onto proteins site-specifically, with methods ranging from chemical synthetic approaches, to enzymatic approaches and many in between. Previous work has demonstrated the utility of engineered variants of the bacterial transpeptidase enzyme sortase (SrtA) for conjugation of ubiquitin site-specifically onto target proteins. In this manuscript, we’ve combined the classical E1/E2-mediated polyubiquitin chain extension approach with sortase-mediated ligation and click chemistry to enable the generation of mono, di, and triubiquitinated proteins sfGFP and PCNA. We demonstrate the utility of this strategy to generate both K48-linked and K63-linked polyubiquitins and attach them both N-terminally and site-specifically to the proteins of interest. Further, we highlight differential activity between two commonly employed sortase variants, SrtA 5M and 7M, and demonstrate that while SrtA 7M can be used to conjugate these ubiquitins to substrates, SrtA 5M can be employed to release the ubiquitin from the substrates as well as to cleave C-terminal tags from the ubiquitin variants used. Overall, we envision that this approach is broadly applicable to readily generate discrete polyubiquitin chains of any linkage type that is accessible <em>via</em> E1/E2 systems and conjugate site-specifically onto proteins of interest, thus granting access to bespoke ubiquitinated proteins that are not currently possible.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00229b?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139755948","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":"Reversible oxidative dimerization of 4-thiouridines in tRNA isolates†","authors":"Larissa Bessler, Jonathan Groß, Christopher J. Kampf, Till Opatz and Mark Helm","doi":"10.1039/D3CB00221G","DOIUrl":"10.1039/D3CB00221G","url":null,"abstract":"<p >The occurrence of non-canonical nucleoside structures in RNA of biological or synthetic origin has encountered several recent boosts in attention, namely in the context of RNA modifications, and with an eye to RNA vaccines. New nucleoside structures introduce added functionality and function into biopolymers that are otherwise rather homogenous in their chemical structure. Here, we report the discovery of a presumed RNA modification that was identified by combination of liquid chromatography–tandem mass spectrometry (LC–MS/MS) with stable isotope labelling as a dimer of the known RNA modification 4-thiouridine (s<small>⁴</small>U). The disulfide-linked structure, which had previously been synthetically introduced into RNA, was here formed spontaneously in isolates of <em>E. coli</em> tRNA. Judicious application of stable isotope labelling suggested that this presumed new RNA modification was rather generated <em>ex vivo</em> by oxidation with ambient oxygen. These findings do not only underscore the need for caution in the discovery of new RNA modifications with respect to artifacts, but also raise awareness of an RNA vulnerability, especially to oxidative damage, during its transport or storage.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00221g?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139657032","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":"Cell penetration of oxadiazole-containing macrocycles†","authors":"Sungjoon Huh, Nefeli Batistatou, Jing Wang, George J. Saunders, Joshua A. Kritzer and Andrei K. Yudin","doi":"10.1039/D3CB00201B","DOIUrl":"10.1039/D3CB00201B","url":null,"abstract":"<p >Passive membrane permeability is an important property in drug discovery and biological probe design. To elucidate the cell-penetrating ability of oxadiazole-containing (Odz) peptides, we employed the Chloroalkane Penetration Assay. The present study demonstrates that Odz cyclic peptides can be highly cell-penetrant depending on the position of specific side chains and the chloroalkane tag. Solution NMR shows that Odz cyclic peptides adopt a β-turn conformation. However, despite observing high cell penetration, we observed low passive permeability in experiments with artificial membranes. These findings highlight the complexity of controlling cell penetration for conformationally sensitive macrocycles and suggest that Odz cyclic peptides may provide a framework for designing cell-penetrant cyclic peptides.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d3cb00201b?page=search","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139469819","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}