Poster abstracts
Poster number 112 submitted by Zachary Parada
Impact of Molecular Crowding Agents on RNA Aptamer Biosensors
Zachary L. Parada (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota), Katarzyna P. Adamala (Department of Genetics, Cell Biology, and Development), Aaron E. Engelhart (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota)
Abstract:
Molecular crowding agents replicate cellular environments in cell-free systems with poly-ethylene glycol (PEG) and Ficoll being two of the most commonly used crowding agents. PEG, a flexible, hydrophilic polymer, modulates solution viscosity, while Ficoll, a highly branched polymer, mimics osmotic conditions with minimal biomolecular interaction. Traditionally considered inert, recent studies suggest that PEG and Ficoll may influence biomolecules beyond spatial restriction.
This study investigates how varying concentrations and molecular weights of PEG (7.5k, 20k) and Ficoll (Ficoll-70, Ficoll-400) affect the binding affinity, stability, and fluorescence of SAM RNA aptamer-based biosensors (Corn and Pepper aptamers). We assessed the effects of these agents through fluorescence endpoint assays and UV-visible spectroscopy to measure dissociation constants (KD) and the optical properties of the cognate dyes (DFHO and HBC-620).
Our findings indicate that PEG treatments significantly increased KD values, reducing binding affinity at higher RNA aptamer concentrations. Ficoll-70 had a less pronounced effect, while Ficoll-400 at lower concentrations enhanced ligand interaction. Changes in the dyes' absorption spectra suggest potential interactions with crowding agents, influencing biosensor fluorescence. Importantly, in vitro transcription was unaffected, confirming that the crowding agents influenced binding properties without altering RNA production.
These results highlight the substantial impact of crowding agents on RNA aptamer biosensors, emphasizing the need for careful agent selection in biosensor design. By simulating crowded environments, the performance of RNA biosensors can be optimized, contributing to their potential use in biological and clinical applications.
Keywords: crowding agent, RNA aptamer, biosensor optimization