{"title":"用光可溶解荧光蛋白对纳米抗体介导的蛋白质活性调节进行光学控制。","authors":"Mizuki Endo, Saki Tomizawa, Qiaoyue Kuang, Takeaki Ozawa","doi":"10.1039/d4an00433g","DOIUrl":null,"url":null,"abstract":"<p><p>Antibodies are crucial in various biological applications due to their specific binding to target molecules, altering protein function and structure. The advent of single-chain antibodies such as nanobodies has paved the way for broader applicability in both research and therapies due to their small size and efficient tissue penetration. Recently, several approaches have been reported to optically control the antigen-binding affinity of nanobodies. Here, we show an alternative strategy for creating photo-activatable nanobodies. By fusing the photocleavable protein PhoCl with the N-terminus of the nanobody (named optoNb60), we successfully demonstrated light-dependent restoration of the antigen-binding ability and the following modulation of the activity of a target protein, the beta-2 adrenergic receptor. Moreover, the activation of optoNb60 was monitored by the fluorescence changes upon photoconversion. The compatibility of the uncaging design with the previously reported optogenetic molecules using nanobodies will contribute to the further optimization of the response capabilities of existing optogenetic tools, thereby expanding their applicability.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":null,"pages":null},"PeriodicalIF":3.6000,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Optical control of nanobody-mediated protein activity modulation with a photocleavable fluorescent protein.\",\"authors\":\"Mizuki Endo, Saki Tomizawa, Qiaoyue Kuang, Takeaki Ozawa\",\"doi\":\"10.1039/d4an00433g\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Antibodies are crucial in various biological applications due to their specific binding to target molecules, altering protein function and structure. The advent of single-chain antibodies such as nanobodies has paved the way for broader applicability in both research and therapies due to their small size and efficient tissue penetration. Recently, several approaches have been reported to optically control the antigen-binding affinity of nanobodies. Here, we show an alternative strategy for creating photo-activatable nanobodies. By fusing the photocleavable protein PhoCl with the N-terminus of the nanobody (named optoNb60), we successfully demonstrated light-dependent restoration of the antigen-binding ability and the following modulation of the activity of a target protein, the beta-2 adrenergic receptor. Moreover, the activation of optoNb60 was monitored by the fluorescence changes upon photoconversion. The compatibility of the uncaging design with the previously reported optogenetic molecules using nanobodies will contribute to the further optimization of the response capabilities of existing optogenetic tools, thereby expanding their applicability.</p>\",\"PeriodicalId\":63,\"journal\":{\"name\":\"Analyst\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.6000,\"publicationDate\":\"2024-06-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analyst\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1039/d4an00433g\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analyst","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1039/d4an00433g","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0
摘要
抗体能与目标分子特异性结合,改变蛋白质的功能和结构,因此在各种生物应用中至关重要。单链抗体(如纳米抗体)因其体积小、组织穿透力强,为更广泛地应用于研究和治疗铺平了道路。最近,有几种方法被报道用于光学控制纳米抗体的抗原结合亲和力。在这里,我们展示了另一种制造光活化纳米抗体的策略。通过将可光裂解蛋白 PhoCl 与纳米抗体(命名为 optoNb60)的 N 端融合,我们成功地证明了光依赖性抗原结合能力的恢复,以及随后对靶蛋白--β-2 肾上腺素能受体--活性的调节。此外,我们还通过光电转换时的荧光变化监测了 optoNb60 的活化情况。开笼设计与之前报道的使用纳米抗体的光遗传分子的兼容性将有助于进一步优化现有光遗传工具的反应能力,从而扩大其适用范围。
Optical control of nanobody-mediated protein activity modulation with a photocleavable fluorescent protein.
Antibodies are crucial in various biological applications due to their specific binding to target molecules, altering protein function and structure. The advent of single-chain antibodies such as nanobodies has paved the way for broader applicability in both research and therapies due to their small size and efficient tissue penetration. Recently, several approaches have been reported to optically control the antigen-binding affinity of nanobodies. Here, we show an alternative strategy for creating photo-activatable nanobodies. By fusing the photocleavable protein PhoCl with the N-terminus of the nanobody (named optoNb60), we successfully demonstrated light-dependent restoration of the antigen-binding ability and the following modulation of the activity of a target protein, the beta-2 adrenergic receptor. Moreover, the activation of optoNb60 was monitored by the fluorescence changes upon photoconversion. The compatibility of the uncaging design with the previously reported optogenetic molecules using nanobodies will contribute to the further optimization of the response capabilities of existing optogenetic tools, thereby expanding their applicability.