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特征。端粒酶的一个独特特征-- 使用另一种延长端粒(ALT)途径来维持端粒的游离癌细胞是 端粒重复序列RNA(Terra)的过表达。因此，Terra是Alt的一个有吸引力的目标 癌症诊断和治疗。Terra是从端粒的一个子集转录而来，但在其他端粒上形成焦点 和非端粒基因座。操纵Terra结合蛋白导致Terra定位中的错误调节 和端粒功能障碍，表明Terra功能与其定位高度相关。可视化中的Terra 因此，固定的组织和活细胞将为Terra作为生物标记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。当前标记Terra的方法受到以下两种情况之一的影响 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 来自端粒DNA)。我们的初步数据表明，荧光gPNA微型探针可以检测到固定的 高效的细胞。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杂交更多的探针，从而提高Terra焦点的亮度。这一点改进了 亮度还将允许检测到比传统DNA Terra更短的Terra转录 鱼探头。在这项工作中，我们的目标是进一步改进这种方法，在固定单元和 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，以实现更高的 标记Terra与端粒DNA的选择性，跟踪有希望的初步数据表明9聚体 GPNA可以做到这一点，而12聚体则不能。在目标2中，我们将在Terra标注中实现低背景 通过设计可通过Förster共振能量转移(FRET)发出信号的荧光gPNA对 只有当它们彼此紧密地结合在一起的时候。未绑定的gPNA探针将不会烦恼，从而展示 背景低。在目标3中，我们将使用一种合成的阳离子聚合物载体将gPNA运送到活细胞进行追踪 实时的Terra动态。优化的gPNA探针对Terra的检测效率更高， 选择性和低本底可用于检测固定组织中的Terra，以辅助ALT癌症治疗。这个 监测活细胞中Terra动态的能力可以用来理解Terra对端粒的贡献 ALT癌细胞的延长和其他细胞功能，并启发新的靶向治疗策略 泰拉。除了Terra，设计原则还可以用来可视化链接到各种 癌症，尤其是那些涉及染色质组织等动态过程的癌症，具有挑战性 用当前的方法进行研究。同时，通过为不同的非编码RNA设计探针库， 我们的方法可以用来剖析不同的非编码RNA如何相互作用来促进生长 癌细胞的数量。

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.