Engineering photostable fluorescent proteins and biosensors using transcriptomic mining and massive-throughput single-cell screening

使用转录组挖掘和大通量单细胞筛选来工程光稳定荧光蛋白和生物传感器

• 批准号: 10610472
• 负责人: Francois St-Pierre
• 金额: $ 60.14万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10610472
• 关键词: Address; Adopted; Agar; Aging; Attention; Benchmarking; Biological; Biological Assay; Biomedical Research; Biophysics; Biosensor; Bulla; Calcium; Cell Cycle; Cell Death; Cell Shape; Cell physiology; Cells; Color; Coupling; Cytometry; Dedications; Detection; Development; Disease; Electrophysiology (science); Embryo; Engineering; Equipment; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Fluorescent Probes; Functional disorder; Gene Expression; Gene Proteins; Genetic Engineering; Health; Image; Imaging Device; Individual; Jellyfish; Label; Laboratories; Lasers; Learning; Light; Lighting; Mammalian Cell; Marine Invertebrates; Medicine; Membrane; Metagenomics; Methods; Microscopy; Mining; Modeling; Morphology; Mutagenesis; Neurons; Nuclear; Optics; Pattern; Photobleaching; Photons; Procedures; Productivity; Property; Protein Engineering; Proteins; Protocols documentation; Rapid screening; Reagent; Recovery; Reporter; Reporter Genes; Reporting; Research Proposals; Resolution; Resource Sharing; Sampling; Science; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structure; Syncope; System; Techniques; Technology; Tissue imaging; Variant; cell type; cellular imaging; chromophore; cofactor; college; design; experimental study; fluorophore; high throughput screening; imaging modality; imaging probe; improved; interest; invention; metagenome; millisecond; monomer; neuroimaging; new technology; novel; nucleic acid localization; red fluorescent protein; screening; single molecule; temporal measurement; transcriptome; transcriptomics; two-photon; voltage

PROJECT SUMMARY/ABSTRACT Fluorescent proteins are ubiquitous reagents in the biomedical sciences for reporting gene expression, protein and nucleic acid localization, cell shape, and cellular activity. However, fluorescent proteins (FPs) become progressively dimmer — they photobleach — with repeated or prolonged illumination. Photobleaching limits multiple types of biological experiments where photostability is essential, such as single-molecule biophysics and timelapse imaging of cellular activity during development, learning, and aging. Photobleaching often cannot simply be addressed by increasing the excitation light, as high illumination power can induce membrane blebbing, nuclear fragmentation, alterations in the cell cycle, changes to the concentration of intracellular calcium, and, ultimately, cell death. While over two decades of FP engineering has led to a toolbox of bright FPs, less attention has been devoted to improving photostability because of the greater difficulty and lower throughput endured when screening for photostable FPs. Moreover, few studies have attempted to improve photophysical properties under two-photon illumination — a method of choice for deep-tissue imaging — because of technical challenges associated with screening under this imaging modality. The overall objective of this research proposal is, therefore, to develop and apply a color palette of bright and photostable FPs for one- and two-photon imaging in mammalian cells. Our proposal leverages two specialized and synergistic approaches to FP discovery and engineering: (1) SPOTlight, a new all- optical screening approach developed in Dr. St-Pierre's lab that circumvents technical hurdles and enables rapid screening of both brightness and photostability at the single-cell level under one- and two-photon illumination; and (2) transcriptomic and metagenomic mining for novel FPs from marine invertebrates, a technique pioneered by Dr. Shaner’s lab. SPOTlight relies on light patterning technology to selectively illuminate individual cells labeled with fluorophores that can be photoactivated from a dim to a bright state. The cells are therefore tagged with a unique fluorescence signature that can then be distinguished and retrieved using Fluorescence Activated Cell Sorting (FACS). SPOTlight thus enables screening in dense mixed cultures with single-cell resolution, thereby eclipsing the throughput of traditional well-based approaches. Mining for novel FPs in marine invertebrate transcriptomes and metagenomes will allow us to rapidly identify and characterize hundreds of novel FPs. From this pool of new FPs, we will select the most photostable for engineering with the SPOTlight pipeline. We will also model their structures to guide site-directed mutagenesis. We propose to leverage these new technologies and assays to develop FPs of different colors that are bright, monomeric, and sufficiently photostable for long-term imaging experiments. We also propose to apply these new FPs to increase the photostability of genetically encoded voltage indicators (GEVIs), which are fluorescent biosensors whose brightness reports changes in voltage. While GEVIs are proposing tools for imaging neural electrical activity with exquisite temporal resolution, they require high illumination power for detection and typically bleach in seconds or minutes. Overall, we anticipate that this project will produce bright and photostable fluorophores and biosensors of broad utility for illuminating cellular dynamics and that our procedures will inspire further multi-parameter engineering of imaging probes for long-term imaging.

项目摘要/摘要 荧光蛋白是生物医学科学中无处不在的试剂，用于报告基因表达，蛋白质和核酸 酸定位，细胞形状和细胞活性。但是，荧光蛋白（FPS）变得逐渐变暗 - 他们光漂白 - 反复或长时间照明。光漂白限制了多种类型的生物学实验 其中具有光稳定性是必不可少的，例如单分子生物物理学和在细胞活性的时间解体成像 发展，学习和衰老。光漂白通常不能简单地通过增加兴奋的光来解决，因为 高照明能力会诱导膜出现，核破碎，细胞周期的改变，变为 细胞内钙的浓度，最终是细胞死亡。虽然超过二十年的FP工程导致了 明亮FPS的工具箱，由于更大的难度和较低 筛选光稳定FPS时忍受的吞吐量。此外，很少有研究试图改善光物理 由于技术挑战，在两光量照明下的属性 - 一种深层组织成像的选择方法 与此成像方式下的筛选有关。因此，该研究建议的总体目标是 在哺乳动物细胞中开发并应用明亮和光稳定FPS的调色板，以进行单光子成像。我们的 建议利用两种专业和协同的方法来发现和工程：（1）聚光灯，新的全能 在圣皮埃尔博士实验室中开发的光学筛查方法，该实验室绕过技术障碍并实现快速筛选 在单细胞和两光子照明下单细胞水平的亮度和光稳定性； （2）转录组 以及来自海洋无脊椎动物的新型FPS的元基因组采矿，该技术由Shaner博士实验室率先进行。聚光灯 依靠光图案技术来有选择地照亮用荧光团标记的单个细胞 从昏暗状态到光活化。因此，将细胞用独特的荧光特征标记 使用荧光激活的细胞分选（FACS）进行区分和检索。因此，聚光灯可以筛选 具有单细胞分辨率的密集的混合培养物，从而黯然失色地基于传统的良好方法。 海洋无脊椎动物转录组和宏基因组中新型FP的开采将使我们能够快速识别和 表征数百个新颖的FPS。从这个新的FPS池中，我们将选择用于工程技术的最光稳定 聚光灯管道。我们还将对其结构进行建模，以指导定向的诱变。我们建议利用 这些新技术和测定法以开发出明亮，单体和足够光稳定的不同颜色的FPS 用于长期成像实验。我们还建议应用这些新的FP，以提高一般的光稳定性 编码的电压指示器（GEVI）是荧光生物传感器的亮度报告的电压变化。尽管 Gevis提出了用于通过独家临时分辨率成像神经电活动的工具，它们需要高 用于检测的照明功率，通常在几秒钟或几分钟内进行漂白。总的来说，我们预计这个项目将 产生明亮的光子荧光团和广泛实用性的生物传感器，以照亮细胞动力学，并说我们 程序将激发成像问题的进一步多参数工程，以实现长期成像。