Poster abstracts
Poster number 24 submitted by Ziyao Zhu
Engineering a Fluorogenic RNA Biosensor toward Selective Imaging of Flavin Adenine Dinucleotide in Living Cells
Ziyao Zhu (Department of Chemistry and Biochemistry, University of Notre Dame), Kaiyu Fu (Department of Chemistry and Biochemistry, University of Notre Dame), Saurja DasGupta (Department of Chemistry and Biochemistry, University of Notre Dame)
Abstract:
Flavin adenine dinucleotide (FAD) is an essential redox cofactor and metabolic regulator whose cellular availability influences flavoprotein function. FAD has also been identified as a noncanonical RNA 5′ cap, linking metabolism with RNA regulation. However, selective tools for monitoring free FAD in living cells remain limited.
We developed a genetically encoded fluorogenic RNA biosensor that couples a FAD-binding aptamer to the Red Broccoli reporting aptamer through a transducer that converts ligand-induced RNA folding into fluorescence activation. We first optimized a previously reported FAD aptamer through stem truncation. ITC shows that a 7-bp truncation is the minimal construct that retains weak residual interaction. We also confirmed that intrinsic FAD fluorescence is spectrally separated from the Red Broccoli–OBI signal, with no overlapping fluorescence emission.
We next engineered 19 transducer architectures and identified 5 lead constructs with ligand-dependent fluorescence responses. Selectivity profiling showed that these sensors respond to FAD, FMN, and riboflavin, while remaining unresponsive to NAD, NMN, and ATP. Mg²⁺ concentration and temperature were further optimized to improve in vitro fluorescence output. Ongoing work focuses on transducer refinement and FAD-selective aptamer evolution using rational design and Capture-SELEX.
This platform establishes a foundation for FAD-selective fluorogenic RNA biosensors. Ultimately, an FAD-specific fluorogenic RNA biosensor could enable real-time imaging of FAD dynamics in living cells and provide a tool for investigating how free FAD and FAD-capped RNAs respond to changes in cellular metabolic state.
References:
1. Samuelian, J. S., Gremminger, T. J., Song, Z., et al. (2022). An RNA aptamer that shifts the reduction potential of metabolic cofactors. Nature chemical biology , 18(11), 1263-1269.
2. Song, W., Filonov, G. S., Kim, H., et al. (2017). Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex. Nature chemical biology , 13(11), 1187-1194.
3. Li, X., Mo, L., Litke, J. L., et al. (2020). Imaging Intracellular SAM dynamics in Live Mammalian Cells with a Genetically Encoded Red Fluorescent RNA-based Sensor. Journal of the American Chemical Society , 142(33), 14117.
4. Boussebayle, A., Groher, F., & Suess, B. (2019). RNA-based Capture-SELEX for the selection of small molecule-binding aptamers. Methods , 161, 10-15.
Keywords: Fluorogenic RNA biosensor, Aptamer engineering, Metabolic sensing