Fluorescent gammaPNA Miniprobes for Imaging Telomeric RNA

用于端粒 RNA 成像的荧光 gammaPNA 微型探针

基本信息

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
项目类别：
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

项目摘要

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.

项目概要非编码 RNA 表达的改变是许多癌症的标志之一。使用另一种端粒延长（ALT）途径来维持端粒的游离癌细胞是因此，含端粒重复序列的 RNA (TERRA) 的过度表达是 ALT 的一个有吸引力的目标。 TERRA 是从端粒子集转录而来，但在其他端粒上形成病灶。 TERRA 结合蛋白的操作导致 TERRA 定位的错误调节。和端粒功能障碍，表明 TERRA 功能与其定位高度相关。因此，固定组织和活细胞将为 TERRA 开发作为生物标志物提供所需的见解用于 ALT 诊断或 ALT 癌症治疗目标的当前标记 TERRA 的方法存在以下问题：高背景、低效率（仅标记明亮的 TERRA 焦点）或低选择性（无法区分 TERRA 我们的初步数据表明荧光 gPNA 微型探针可以检测固定的 TERRA。这是因为 gPNA 的高亲和力允许我们使用更短的探针，从而导致每个 TERRA 杂交更多探针，从而提高 TERRA 焦点的亮度。亮度还可以检测比传统 DNA TERRA 更短的 TERRA 转录本在这项工作中，我们的目标是进一步改进固定细胞和 TERRA 可视化的这种方法。将能力扩展到活细胞成像在目标 1 中，我们将系统地改变 gPNA 探针长度以实现高水平。标记 TERRA 与端粒 DNA 的选择性，后续有希望的初步数据表明 9mer gPNA 可以实现这一点，而 12mer 则不能。在目标 2 中，我们将在 TERRA 标记中实现低背景。通过设计荧光 gPNA 对，通过福斯特共振能量转移 (FRET) 发射信号仅当它们与 TERRA 彼此靠近结合时，未结合的 gPNA 探针才不会 FRET，从而表现出。在目标 3 中，我们将使用合成的阳离子聚合物载体将 gPNA 传递至活细胞以进行追踪。实时 TERRA 动力学优化的 gPNA 探针，可更高效、高水平地检测 TERRA。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。监测活细胞中 TERRA 动态的能力可用于了解 TERRA 如何促进端粒 ALT 癌细胞的延长和其他细胞功能，并激发针对靶向的新治疗策略 TERRA。除了 TERRA 之外，该设计原理还可用于可视化与各种相关的其他非编码 RNA。癌症，特别是那些与动态过程有关的癌症，例如具有挑战性的染色质组织同时，通过设计不同非编码 RNA 的探针库，我们的方法可用于剖析不同的非编码 RNA 如何相互作用来促进生长癌细胞。