Poster abstracts

Poster number 36 submitted by Ruwani Madushika Dalath

Imaging lncRNA TERRA with fluorescent γPNA probes

Meng Xu (Department of Biological Sciences, Carnegie Mellon University ), Nisha Hasija (Department of Chemistry, Carnegie Mellon University ), Ruwani Madushika Dalath (Department of Biological Sciences, Carnegie Mellon University ), Mackenzie Riley (Department of Chemistry, Carnegie Mellon University ), Bruce Armitage (Department of Chemistry, Carnegie Mellon University ), Huaiying Zhang (Department of Biological Sciences, Carnegie Mellon University )

Abstract:
Telomere repeat-containing RNA (TERRA) is a long noncoding RNA (lncRNA) transcribed from chromosome ends(Azzalin et al. 2007). TERRA plays important roles in telomere function in both normal cells and in cancer cells that use either telomerase or alternative lengthening of telomere (ALT) pathway for telomere maintenance(Luke and Lingner 2009). However, mechanistic understanding of TERRA function is still lacking, partially due to the lack of tools to track endogenous TERRA in live cells. Previously we used Cas13 based fluorescent proteins to image TERRA, which achieved high specificity, but the efficiency was low(Xu et al. 2022). Here we report our effort to image TERRA with fluorescent gamma peptide nucleic acid (γPNA) probes. γPNA is a second-generation analogue of PNA which features higher affinity and better water solubility(Canady et al. 2020). Fluorescent γPNA would be appropriate for imaging the repetitive TERRA since a single probe can bind multiple times to each TERRA, leading to bright labeling. Indeed, we found that fluorescent γPNA of 8-10 nucleobases in length successfully stain TERRA in fixed cells, and both the brightness and the selectivity for TERRA versus the isosequential telomeric DNA depends on the length of the probe. In addition, we tested two different γPNA structures, distinguished by the nature of the substituent on the γPNA backbone, with one having a hydroxymethyl and the other having a methoxymethyl group at this position. We observed similar results for TERRA labeling for these two structures, suggesting the nature of the substituent on the γPNA backbone does not affect selectivity, potentially allowing us to introduce other modifications such as cell-penetrating motifs for live imaging. Taken together, our work demonstrates that fluorescent γPNA probes are effective at detecting TERRA in fixed cells, paving the way for future effort to expand this technology to image TERRA in live cells.

References:
Azzalin, Claus M. et al. 2007. “Telomeric Repeat–Containing RNA and RNA Surveillance Factors at Mammalian Chromosome Ends.” Science 318(5851): 798–801.

Canady, Taylor D. et al. 2020. “Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.” Molecules 25(4): 970.

Luke, Brian, and Joachim Lingner. 2009. “TERRA: Telomeric Repeat-Containing RNA.” The EMBO Journal 28(17): 2503–10.

Xu, Meng et al. 2022. “CRISPR Cas13-Based Tools to Track and Manipulate Endogenous Telomeric Repeat-Containing RNAs in Live Cells.” Frontiers in Molecular Biosciences 8: 785160

Keywords: PNA, TERRA, Alternative lengthening of telomeres