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.

项目概要/摘要 荧光蛋白是生物医学中普遍存在的试剂，用于报告基因表达、蛋白质和核酸 然而，荧光蛋白（FP）逐渐变暗。 它们进行光漂白——重复或长时间照射限制了多种类型的生物实验。 光稳定性至关重要的领域，例如单分子生物物理学和细胞活动的延时成像 发育、学习和衰老通常不能简单地通过增加激发光来解决。 高照明功率会引起膜起泡、核碎片、细胞周期的改变、 20 多年来，FP 工程已导致细胞内钙浓度的增加，并最终导致细胞死亡。 明亮 FP 的工具箱中，由于难度较大且较低，因此对提高光稳定性的关注较少 此外，很少有研究尝试提高光物理性能。 由于技术挑战，双光子照明下的特性（深部组织成像的一种选择方法） 因此，本研究提案的总体目标是与这种成像方式下的筛查相关。 开发并应用明亮且耐光的 FP 调色板，用于哺乳动物细胞中的单光子和双光子成像。 该提案利用两种专业且协同的方法来进行 FP 发现和工程：(1) SPOTlight，一种新的全 St-Pierre 博士实验室开发的光学筛查方法可克服技术障碍并实现快速筛查 一光子和双光子照明下单细胞水平的亮度和光稳定性；以及（2）转录组学 以及从海洋无脊椎动物中寻找新型 FP 的宏基因组挖掘，这是 Shaner 博士实验室首创的技术。 依靠光图案技术选择性地照亮标有荧光团的单个细胞，这些细胞可以 因此，这些细胞被光激活，从昏暗状态变为明亮状态，然后可以使用独特的荧光特征进行标记。 使用荧光激活细胞分选 (FACS) 进行区分和检索，从而能够进行筛选。 具有单细胞分辨率的密集混合培养物，从而使传统基于孔的方法的通量黯然失色。 在海洋无脊椎动物转录组和宏基因组中挖掘新型 FP 将使我们能够快速识别和 从这个新的 FP 库中，我们将选择最耐光的用于工程设计。 我们还将对其结构进行建模，以指导定点突变。 这些新技术和检测方法可开发不同颜色的 FP，这些 FP 明亮、单体且具有足够的光稳定性 我们还建议应用这些新的 FP 来提高遗传的光稳定性。 编码电压指示器（GEVI），这是一种荧光生物传感器，其亮度报告电压变化。 GEVI 正在提出以精细的时间分辨率对神经电活动进行成像的工具，它们需要高 总体而言，我们预计该项目将用于检测并通常在几秒钟或几分钟内漂白。 产生明亮且光稳定的荧光团和具有广泛用途的生物传感器，用于阐明细胞动力学，并且我们的 程序将进一步激发用于长期成像的成像探针的多参数工程。