Talk abstracts
Talk on Saturday 11:00-11:15am submitted by Yi-Meng Lo
Efficient Zero-footprint Intracellular RNA Labeling via URIL-Stem Replacement and Triplex-guided Covalent Crosslinking
Yi-Meng Lo (Chemistry and Biochemistry, The Ohio State University), Dennis Bong (Chemistry and Biochemistry, The Ohio State University)
Abstract:
Deciphering RNAprotein and RNARNA interactions are crucial for regulating cellular functions and understanding disease mechanisms, such as neurodegeneration. Here, we present a highly optimized RNA labeling platform using cell-permeable, bifacial peptide nucleic acids (bPNAs) that selectively target U-rich internal loops (URILs) engineered into RNAs of interest (ROI). Building on prior work where fluorogenic (thiazole orange) bPNAs successfully tracked mammalian U1 spliceosomal RNAs with a 5-to-10-fold signal over background, we have optimized both URIL sequence constraints and bPNA probe chemistry to achieve an unprecedented intracellular signal of >100-fold brightness. These dramatic gains derive from increased probe retention driven by light-activated [2+2] photocycloaddition, covalently crosslinking the bPNA to the URIL pyrimidine bases. Furthermore, we achieved this labeling with a "zero-footprint" targeting motifreplacing existing 4-base-pair duplex stems in the ROI with 818 nucleotide URILsresulting in the minor impact of just 0 to 10 additional nucleotides. This enhanced probe retention expands compatibility beyond fluorogenic labels to highly accessible conventional dyes like Cy3 and Cy5. Finally, we discovered that UV irradiation triggers quality-control RNA degradation via the Zak-kinase-driven ribotoxic stress response (RSR). By co-treating cells with a Zak inhibitor, we successfully mitigated this stress response, significantly enhancing labeling brightness and mitigating the effect of RSR. Combining RSR inhibition with covalent photo-crosslinking yields a >100-fold signal enhancement with a near-zero molecular footprint, greatly broadening the utility of this method for advanced intracellular RNA imaging and synthetic modification.
References:
(1) Bai, B. U1 SnRNP Alteration and Neuronal Cell Cycle Reentry in Alzheimer Disease. Front. Aging Neurosci. 2018, 10, 75.
(2) Chen, P.-C.; et al. Alzheimers Disease-Associated U1 SnRNP Splicing Dysfunction Causes Neuronal Hyperexcitability and Cognitive Impairment. Nat Aging 2022, 2 (10), 923940.
(3) Liang, Y.; Willey, S.; Chung, Y.-C.; Lo, Y.-M.; Miao, S.; Rundell, S.; Tu, L.-C.; Bong, D. Intracellular RNA and DNA Tracking by Uridine-Rich Internal Loop Tagging with Fluorogenic BPNA. Nat. Commun. 2023, 14 (1), 2987.
Keywords: Intracellular RNA labeling, bifacial peptide nucleic acid (bPNA)), Intracellular photo-crosslinking