Fluorescent gammaPNA Miniprobes for Imaging Telomeric RNA

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- 使用端粒延长（ALT）途径维持端粒的游离癌细胞是 含端粒重复序列的RNA（TERRA）的过度表达。因此，TERRA是ALT的一个有吸引力的目标 癌症诊断和治疗。TERRA转录自端粒的一个子集，但在其他端粒上形成病灶 和非端粒基因座。TERRA结合蛋白的操纵导致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。可视化TERRA 因此，固定的组织和活细胞将为TERRA作为生物标志物的发展提供所需的见解 用于ALT诊断或ALT癌症治疗的靶点。目前用于标记TERRA的方法受到以下两个方面的影响： 高背景、低效率（仅标记亮TERRA焦点）或低选择性（无法区分TERRA 端粒DNA）。我们的初步数据表明，荧光gPNA微探针可以检测TERRA在固定 电池效率高。这是因为gPNA的高亲和力允许我们使用更短的探针，导致 每个TERRA可以杂交更多的探针，从而提高TERRA焦点的亮度。这种改进 亮度也将允许检测比常规DNA TERRA可能的更短的TERRA转录本 FISH探针。在这项工作中，我们的目标是进一步改善这种方法的TERRA可视化在固定的细胞， 扩展到活细胞成像的能力。在目标1中，我们将系统地改变gPNA探针长度以实现高的 选择性标记TERRA与端粒DNA，后续有希望的初步数据表明，9聚体 gPNA可以实现这一点，而12聚体不能。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Förster共振能量转移（FRET）发射信号， 只有当它们与TERRA紧密结合时才能如此。未结合的gPNA探针将不进行FRET，因此表现出 低背景在目标3中，我们将使用合成的阳离子聚合物载体将gPNA递送到活细胞中以跟踪 真实的TERRA动态。优化的gPNA探针检测TERRA的效率更高， 选择性和低背景可用于检测固定组织中的TERRA以辅助ALT癌症治疗。的 在活细胞中监测TERRA动力学的能力可用于了解TERRA如何影响端粒 延伸和其他细胞功能，并启发新的治疗策略， 泰拉除了TERRA之外，设计原则还可以用于可视化其他非编码RNA，这些非编码RNA连接到各种 癌症，特别是那些涉及动态过程的癌症，如具有挑战性的染色质组织 用现有的方法研究。同时，通过设计针对不同非编码RNA的探针文库， 我们的方法可以用来剖析不同的非编码RNA如何相互作用，以促进生长， 癌细胞。

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.