Fluorescent gammaPNA Miniprobes for Imaging Telomeric RNA

用于端粒 RNA 成像的荧光 gammaPNA 微型探针

• 批准号: 10595069
• 负责人: Bruce A. ARMITAGE
• 金额: $ 17.93万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-21 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595069
• 关键词: Affinity; Binding; Biological Markers; Cancerous; Cell physiology; Cells; Characteristics; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Cytosol; DNA; DNA Probes; Data; Detection; Development; Diagnosis; Endosomes; Engineering; Exhibits; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent in Situ Hybridization; Functional disorder; Genetic Transcription; Glutamates; Growth; Image; Investigation; Label; Length; Libraries; Link; Malignant Neoplasms; Methods; Molecular; Monitor; Nature; Nucleotides; Pathway interactions; Polymers; Process; RNA; RNA Sequences; RNA-Binding Proteins; Role; Signal Transduction; Source; Specificity; Tail; Telomerase; Telomere Maintenance; Telomere Pathway; Time; Tissues; Transcript; Untranslated RNA; Visualization; Work; cancer cell; cancer diagnosis; cancer therapy; cell fixing; cellular imaging; design; experimental study; improved; insight; live cell imaging; novel; novel therapeutic intervention; overexpression; success; telomere; tissue fixing; tool; vector

Project Summary Altered expression of non-coding RNAs are hallmarks of many cancers. One unique characteristic of telomerase- free cancer cells that use an alternative lengthening of telomere (ALT) pathway for telomere maintenance is the overexpression of telomere repeat-containing RNA (TERRA). TERRA is therefore an attractive target for ALT cancer diagnosis and therapy. TERRA is transcribed from a subset of telomeres but forms foci on other telomeres and non-telomere loci. Manipulation of TERRA binding proteins leads to mis-regulation in TERRA localization and telomere dysfunction, suggesting TERRA function is highly linked to its localization. Visualizing TERRA in fixed tissue and live cells would therefore provide insights needed for the development of TERRA as a biomarker for ALT diagnosis or a target for ALT cancer therapy. Current methods for labeling TERRA suffer from either high background, low efficiency (only label bright TERRA foci), or low selectivity (cannot differentiate TERRA from telomere DNA). Our preliminary data suggest that fluorescent gPNA miniprobes can detect TERRA in fixed cells with high efficiency. This is because the high affinity of gPNA allows us to use shorter probes, leading to hybridization of more probes per TERRA and therefore improving the brightness of TERRA foci. This improved brightness will also allow detection of shorter TERRA transcripts than is possible with conventional DNA TERRA FISH probes. In this work, we aim to further improve this method for TERRA visualization in fixed cells and extend the capacity into live cell imaging. In Aim 1, we will systematically vary gPNA probe length to achieve high selectivity in labeling TERRA vs telomeric DNA, following up on promising preliminary data indicating that a 9mer gPNA can accomplish this whereas a 12mer cannot. In Aim 2, we will achieve low background in TERRA labeling by designing fluorescent gPNA pairs that will emit signal through Förster Resonance Energy Transfer (FRET) only when they bind to TERRA close to each other. The unbound gPNA probes will not FRET and thus exhibit low background. In Aim 3, we will use a synthetic cationic polymer vector to deliver gPNA to live cells to track TERRA dynamics in real time. The optimized gPNA probes that detect TERRA with higher efficiency, high selectivity and low background can be used to detect TERRA in fixed tissue to aid ALT cancer therapy. The ability to monitor TERRA dynamics in live cells can be used to understand how TERRA contributes to telomere elongation and other cellular functions in ALT cancer cells and inspire novel therapeutic strategies targeting TERRA. Beyond TERRA, the design principles can be used to visualize other non-coding RNAs linked to various cancers, particularly those implicated in dynamic processes such as chromatin organization that are challenging to study with current methods. Simultaneously, by designing a library of probes for different non-coding RNAs, our methods can be used to dissect how different non-coding RNAs work with each other to promote the growth of cancerous cells.

项目摘要 非编码RNA的表达改变是许多癌症的标志。端粒酶的一个独特特征 - 使用替代端粒途径（ALT）维持途径的替代癌细胞是 端粒重复RNA（Terra）的过表达。因此，Terra是Alt的吸引力目标 癌症诊断和治疗。 Terra是从端粒的一部分转录的，但将重点放在其他端粒上 和非telomere本地化。操纵Terra结合蛋白会导致Terra定位的错误调节 和端粒功能障碍，表明Terra功能与其本地化高度相关。可视化Terra 因此，固定的组织和活细胞将提供作为生物标志物的陶土发展所需的见解 用于ALT诊断或ALT癌症治疗的靶标。当前标记Terra患者的方法 高背景，低效率（仅标签明亮的Terra Foci）或低选择性（无法区分Terra 来自端粒DNA）。我们的初步数据表明，荧光GPNA微型型可以检测到固定的Terra 效率高的细胞。这是因为GPNA的高亲和力使我们能够使用较短的问题，导致 杂交每个Terra的更多问题，因此改善了Terra Foci的亮度。这有所改善 亮度还将允许检测到较短的Terra转录本，而不是常规DNA Terra的可能性 鱼问题。在这项工作中，我们旨在进一步改善固定细胞和 将容量扩展到活细胞成像中。在AIM 1中，我们将系统地改变GPNA探针长度以达到高 标记Terra与远程DNA的选择性，跟进有希望的初步数据，表明9mer GPNA可以完成此操作，而12mer不能。在AIM 2中，我们将在Terra标签中获得低背景 通过设计将通过Förster共振能量转移（FRET）发射信号的荧光GPNA对 只有当它们与Terra彼此接近时。未结合的GPNA探针不会烦恼，因此表现出 低背景。在AIM 3中，我们将使用合成阳离子聚合物载体向活细胞传递GPNA以跟踪 实时台。优化的GPNA问题，这些问题检测到效率较高的Terra，高 选择性和低背景可用于检测固定组织中的Terra以帮助ALT癌症治疗。 可以使用监测活细胞中的Terra动力学的能力来了解Terra如何对端粒贡献 ALT癌细胞中的伸长和其他细胞功能，并激发针对性的新型治疗策略 Terra。除了Terra之外，设计原理可用于可视化与各种相关的其他非编码RNA 癌症，尤其是在动态过程中暗示的癌症，例如染色质组织，受到挑战 使用当前方法研究。同时，通过为不同的非编码RNA设计问题库， 我们的方法可用于剖析不同的非编码RNA如何相互作用以促进增长 取消细胞。