Laboratory of Bio-Organic Chemistry, Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands.
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
Bioconjug Chem. 2023 Nov 15;34(11):2089-2095. doi: 10.1021/acs.bioconjchem.3c00390. Epub 2023 Oct 19.
Oligonucleotide conjugation has emerged as a versatile molecular tool for regulating protein activity. A state-of-the-art labeling strategy includes the site-specific conjugation of DNA, by employing bioorthogonal groups genetically incorporated in proteins through unnatural amino acids (UAAs). The incorporation of UAAs in chemokines has to date, however, remained underexplored, probably due to their sometimes poor stability following recombinant expression. In this work, we designed a fluorescent stromal-derived factor-1β (SDF-1β) chemokine fusion protein with a bioorthogonal functionality amenable for click reactions. Using amber stop codon suppression, p-azido--phenylalanine was site-specifically incorporated in the fluorescent N-terminal fusion partner, superfolder green fluorescent protein (sfGFP). Conjugation to single-stranded DNAs (ssDNA), modified with a photocleavable spacer and a reactive bicyclononyne moiety, was performed to create a DNA-caged species that blocked the receptor binding ability. This inhibition was completely reversible by means of photocleavage of the ssDNA strands. The results described herein provide a versatile new direction for spatiotemporally regulating chemokine-receptor interactions, which is promising for tissue engineering purposes.
寡核苷酸缀合已成为调节蛋白质活性的多功能分子工具。一种最先进的标记策略包括通过使用通过非天然氨基酸 (UAA) 在蛋白质中遗传掺入的生物正交基团，对 DNA 进行特异性缀合。然而，迄今为止，趋化因子的 UAA 掺入仍未得到充分探索，这可能是由于它们在重组表达后有时稳定性较差。在这项工作中，我们设计了一种具有生物正交功能的荧光基质衍生因子-1β (SDF-1β) 趋化因子融合蛋白，适用于点击反应。使用琥珀终止密码子抑制，将 p-叠氮基-苯丙氨酸特异性掺入荧光 N 端融合伴侣超折叠绿色荧光蛋白 (sfGFP) 中。通过与光可裂解间隔物和反应性双环壬炔部分修饰的单链 DNA (ssDNA) 进行缀合，创建了一种 DNA 笼状物质，该物质阻断了受体结合能力。通过光裂解 ssDNA 链可以完全逆转这种抑制作用。本文所述的结果为时空调节趋化因子-受体相互作用提供了一个新的多功能方向，有望用于组织工程目的。
