Poster abstracts
Poster number 119 submitted by Dijana Beganovic
Fluorogenic strand-displacement probes for live-cell imaging of timeless RNA processing and trafficking
Dijana Beganovic (Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA; PRIME Postbaccalaureate Research Education Program, Michigan Medicine, University of Michigan, Ann Arbor, Michigan, USA), Ziming Wang (Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA), AlexanderJohnson-Buck (Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA), Nils G. Walter (Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA)
Abstract:
Circadian clocks generate approximately 24-hour rhythms in gene expression, physiology, and behavior. In Drosophila melanogaster, the core clock gene timeless (tim) encodes TIMELESS (TIM), an essential regulator of circadian rhythms. Humans also possess a TIMELESS homolog with established roles in DNA replication and genome stability, and altered TIMELESS expression has been associated with cancer, underscoring the biomedical relevance of its regulation. Recent work from the Yadlapalli laboratory demonstrates that tim transcription is rhythmic and spatially organized near the nuclear periphery in clock neurons. Moreover, delayed removal of a rate-limiting tim intron supports a robust 24-hour rhythm, whereas removal of this intron from the gene shortens circadian period. Despite the importance of this processing step, fluorescence in situ hybridization requires fixed tissue and cannot resolve tim RNA processing or transport in real time. To overcome this limitation, we are developing fluorogenic RNA probes to visualize tim pre-mRNA splicing and, ultimately, messenger RNA export from the nucleus to the cytoplasm in living cells. Our design combines strand displacement with a fluorophorequencher pair: target binding generates fluorescence, whereas unbound probes remain quenched to minimize background. Strand displacement promotes rapid probe exchange, potentially sustaining target-associated signal and reducing the impact of photobleaching during extended imaging. Current probe designs produce up to a 47-fold fluorescence enhancement upon target binding in vitro, demonstrating strong target-dependent activation. Ongoing studies are optimizing probe kinetics, specificity, and photostability for eventual use in living cells. This platform could directly reveal how tim RNA processing contributes to circadian timing while establishing a broadly applicable approach for visualizing RNA maturation and trafficking in living cells.
References:
Yuan Y, Linskens A, Gouvea R, Liu H, Xiao Y, Suresh S, Hu I, Yadlapalli S. An intron-based timer for circadian rhythms. bioRxiv [Preprint]. 2025 Nov 2:2025.10.27.684876. doi: 10.1101/2025.10.27.684876. PMID: 41279210; PMCID: PMC12636426.
David Yu Zhang, Erik Winfree; Control of DNA Strand Displacement Kinetics Using Toehold Exchange. Journal American Chemical Society 2 December 2009; 131 (47): 1730317314.
Keywords: RNA splicing, Fluorogenic RNA probes, Live-cell RNA imaging