Poster abstracts

Poster number 34 submitted by Yi-Meng Lo

bPNA probes for intracellular RNA tracking

Yi-Meng Lo (Chemistry & Biochemistry)

Abstract:
New methods for labeling and tracking intracellular RNAs are needed. Herein, we describe a novel RNA-centric method called URL-tagging that can label RNPs using bifacial peptide nucleic acids (bPNAs) to triplex hybridize with U-rich internal loops (URILs) in structured RNA. Prior work has demonstrated that bPNA outfitted with fluorogenic dye (thiazole orange) can be used for intracellular tracking of mammalian RNPs in a manner that is both comparable to and compatible with MS2 labeling, the gold standard in the field. In the present study, we demonstrate URIL-tagging of each of the 4 stem-loop structures in U1 spliceosomal RNA (U1snRNA), which plays a crucial role in several neurodegenerative diseases. Five distinct URIL-tagged U1snRNA encoding constructs were created and packaged within lentiviral vectors that place a URIL motif in each of the 4 stem-loops (URIL-SL1, URIL-SL2, URIL-SL3, URIL-SL4) as well at the 5’ end of U1 snRNA (URIL-5’). These lentiviral vectors were used to transduce HEK 293 cells to generate five distinct cell lines that constitutively express URIL-tagged U1snRNAs. Each cell line with the designed mutant was treated with 10 μM of fluorogenic bPNA, which has been shown to selectively label the intracellular URIL-tagged RNA. Compared to wild-type HEK293 cells (non-transduced with URIL-U1snRNA lentivirus) URIL-tagged cell lines showed a much stronger fluorescence signal, showing successful intracellular U1snRNA tracking.

References:
(1) Liang, Y.; Willey, S.; Chung, Y.-C.; Lo, Y.-M.; Miao, S.; Rundell, S.; Tu, L.-C.; Bong, D. Nat. Commun. 2023, 14 (1), 2987.
(2) Bai, B. Aging Neurosci. 2018, 10, 75.
(3) Chen, P.-C.; Han, X.; Shaw, T. I.; Fu, Y.; Sun, H.; Niu, M.; Wang, Z.; Jiao, Y.; Teubner, B. J. W.; Eddins, D.; Beloate, L. N.; Bai, B.; Mertz, J.; Li, Y.; Cho, J.-H.; Wang, X.; Wu, Z.; Liu, D.; Poudel, S.; Yuan, Z.-F.; Mancieri, A.; Low, J.; Lee, H.-M.; Patton, M. H.; Earls, L. R.; Stewart, E.; Vogel, P.; Hui, Y.; Wan, S.; Bennett, D. A.; Serrano, G. E.; Beach, T. G.; Dyer, M. A.; Smeyne, R. J.; Moldoveanu, T.; Chen, T.; Wu, G.; Zakharenko, S. S.; Yu, G.; Peng, J. Nat Aging 2022, 2 (10), 923–940.

Keywords: U1 snRNA, RNA tracking, bPNA