Poster abstracts
Poster number 74 submitted by Yichen He
Short RNA duplex formation kinetics in artificial cytoplasms
Yichen He (Dept of Chemistry, Penn State Univ, Univ Pk, PA 16802, Center for RNA Molecular Biology, Penn State Univ, Univ Pk, PA 16802), Elizabeth A. Jolley (Dept of Chemistry, Penn State Univ, Univ Pk, PA 16802, Center for RNA Molecular Biology, Penn State Univ, Univ Pk, PA 16802), Philip C. Bevilacqua (Dept of Chemistry, Penn State Univ, Univ Pk, PA 16802, Center for RNA Molecular Biology, Penn State Univ, Univ Pk, PA 16802, Dept of Biochemistry and Molecular Biology, Penn State Univ, Univ Pk, PA)
Abstract:
The dynamics of RNA folding in cells has not been extensively studied. Previous research from our lab led to the development of a minimal Escherichia coli artificial cytoplasm called Eco80, which has 80% of the metabolites from E. coli [1]. The binding thermodynamics of a diverse set of RNA sequences has been assessed in Eco80 and generally show weaker stability [2], but their binding kinetics have not been studied. By using stopped-flow rapid mixing on several short RNA duplexes with fluorophore and quencher labels, we were able to collect kinetic data under different solution conditions. By performing quenching and chasing experiment using labeled and unlabeled RNA, we were able to collect kinetic constant data of the duplex formation and diassociation. Comparing the ΔΔG data of the RNA duplex formation obtained from stopped-flow experiments in 1 M NaCl buffer and in Eco80 to our previous thermodynamic data, we found that they are in good agreement. By comparing the binding kinetics of the RNA in Eco80 and physiological background ions, we found that metabolites in Eco80 can change both association rate and dissociation rate of the duplex formation. Our future experiments will focus on identifying which components in Eco80 lead to these kinetic differences.
References:
1. Sieg, J. P., McKinley, L. N., Huot, M. J., Yennawar, N. H. & Bevilacqua, P. C. (2022). The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability. Biochemistry 61, 2579-2591.
2. Sieg, J. P., Jolley, E. A., Huot, M. J., Babitzke, P. & Bevilacqua, P. C. (2023). In vivo-like nearest neighbor parameters improve prediction of fractional RNA base-pairing in cells. Nucleic Acids Res 51, 11298-11317.
Keywords: RNA kinetics, Artificial cytoplasm