Anderson David M, Logan Matthew G, Patty Sara S, Kendall Alexander J, Borland Christina Z, Pfeifer Carmem S, Kreth Jens, Merritt Justin L
Division of Biomaterial and Biomedical Sciences, Oregon Health & Science University, Portland, OR, USA.
Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, USA.
bioRxiv. 2023 Oct 2:2023.10.02.560575. doi: 10.1101/2023.10.02.560575.
The human microbiome is predominantly composed of facultative and obligate anaerobic bacteria that live in hypoxic/anoxic polymicrobial biofilm communities. Given the oxidative sensitivity of large fractions of the human microbiota, green fluorescent protein (GFP) and related genetically-encoded fluorophores only offer limited utility for live cell imaging due the oxygen requirement for chromophore maturation. Consequently, new fluorescent imaging modalities are needed to study polymicrobial interactions and microbiome-host interactions within anaerobic environments. The fluorescence-activating and absorption shifting tag (FAST) is a rapidly developing genetically-encoded fluorescent imaging technology that exhibits tremendous potential to address this need. In the FAST system, fluorescence only occurs when the FAST protein is complexed with one of a suite of cognate small molecule fluorogens. To expand the utility of FAST imaging, we sought to develop a modular platform (Click-FAST) to democratize fluorogen engineering for personalized use cases. Using Click-FAST, investigators can quickly and affordably sample a vast chemical space of compounds, potentially imparting a broad range of desired functionalities to the parental fluorogen. In this work, we demonstrate the utility of the Click-FAST platform using a novel fluorogen, Blaze-alkyne, which incorporates the widely available small molecule ethylvanillin as the hydroxybenzylidine head group. Different azido reagents were clicked onto Blaze-alkyne and shown to impart useful characteristics to the fluorogen, such as selective bacterial labeling in mixed populations as well as fluorescent signal enhancement. Conjugation of an 80 Å PEG molecule to Blaze-alkyne illustrates the broad size range of functional fluorogen chimeras that can be employed. This PEGylated fluorogen also functions as an exquisitely selective membrane permeability marker capable of outperforming propidium iodide as a fluorescent marker of cell viability.
人类微生物群主要由兼性厌氧菌和专性厌氧菌组成，它们生活在缺氧/无氧的多微生物生物膜群落中。鉴于人类微生物群的很大一部分对氧化敏感，绿色荧光蛋白（GFP）和相关的基因编码荧光团由于发色团成熟需要氧气，因此在活细胞成像中仅具有有限的用途。因此，需要新的荧光成像方法来研究厌氧环境中的多微生物相互作用以及微生物群与宿主的相互作用。荧光激活和吸收转移标签（FAST）是一种快速发展的基因编码荧光成像技术，在满足这一需求方面具有巨大潜力。在FAST系统中，只有当FAST蛋白与一组同源小分子荧光原之一复合时才会产生荧光。为了扩大FAST成像的用途，我们试图开发一个模块化平台（Click-FAST），以使荧光原工程能够用于个性化应用案例。使用Click-FAST，研究人员可以快速且经济地对大量化合物的化学空间进行采样，从而可能为亲本荧光原赋予广泛的所需功能。在这项工作中，我们使用一种新型荧光原Blaze-炔烃展示了Click-FAST平台的实用性，该荧光原将广泛可用的小分子乙基香草醛作为羟基亚苄基头基。将不同的叠氮基试剂与Blaze-炔烃进行点击反应，结果表明这些试剂赋予了荧光原一些有用的特性，例如在混合群体中进行选择性细菌标记以及增强荧光信号。将一个80 Å的聚乙二醇（PEG）分子与Blaze-炔烃偶联，说明了可以使用的功能性荧光原嵌合体的广泛尺寸范围。这种聚乙二醇化的荧光原还可作为一种极其选择性的膜通透性标记物，其作为细胞活力荧光标记物的性能优于碘化丙啶。
