Fluorescent gammaPNA Miniprobes for Imaging Telomeric RNA

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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.