Poster abstracts
Poster number 38 submitted by Ruwani Madushika Dalath
Imaging lncRNA TERRA using γPNA probes
Ruwani Madushika Dalath (Department of Biological Sciences, Carnegie Mellon University ), Mackenzie Riley (Department of Chemistry, Carnegie Mellon University ), Meng Xu ( Department of Biological Sciences, Carnegie Mellon University ), Nisha Hasija (Department of Chemistry, Carnegie Mellon University), Bruce Armitage (Department of Chemistry, Carnegie Mellon University ), Huaiying Zhang (Department of Biological Sciences, Carnegie Mellon University )
Abstract:
Telomeric repeat-containing RNA (TERRA) is a long, noncoding RNA transcribed from subtelomeric and telomeric regions of chromosomes (Azzalin et al., 2007). Decades of research conducted on TERRA has established its functional roles in both cancerous and noncancerous cells. However, the mechanistic understanding of TERRA functions remains elusive due to the unavailability of a proper and reliable imaging tool. This work introduces a novel Gamma Peptide Nucleic Acid (γPNA) based fluorescent imaging tool to track endogenous TERRA in cells. Considering γPNA's superior binding affinity, enhanced water solubility, and extended cellular half-life due to its resistance to enzymatic degradation, we identified it as the most promising candidate for TERRA imaging (Andrasi et al., 2006). So far, we have designed and evaluated five different lengths of γPNA (ranging from 8-12 bases), two different backbone structures (γSerine and γMethoxy), and a modified version of γPNA with an attached miniPEG sequence between the backbone and the fluorescent tag to enhance signal intensity. Our findings revealed a length-dependent increase in TERRA puncta detected per nucleus and a rise in total signal intensity, though selectivity toward RNA diminished beyond ten bases. The specificity of the designed probes was further validated by knocking down TERRA using LNA Gapmers. In our efforts to optimize a compatible RNA Fluorescent in situ hybridization (FISH) protocol for the designed gamma PNA, we realized that our TERRA probes could provide a maximum detection at a Formamide concentration of 20% in the hybridization buffer within just a 2-hour incubation time, significantly improving the efficiency compared to the prevailing protocols. After successfully implementing the modified tool for fixed cell imaging, we demonstrated its sensitivity by showing reduced TERRA levels in Promyelocytic leukemia (PML) knockout cells, underscoring its robustness and potential as a transformative tool for TERRA research.
References:
Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E., & Lingner, J. (2007). Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science (New York, N.Y.), 318(5851), 798–801.
Dragulescu-Andrasi, A., Rapireddy, S., Frezza, B. M., Gayathri, C., Gil, R. R., & Ly, D. H. (2006). A simple gamma-backbone modification preorganizes peptide nucleic acid into a helical structure. Journal of the American Chemical Society, 128(31), 10258–10267
Keywords: TERRA, PNA, PML