Thermodynamic Parameters of Non-Watson-Crick Base Paired Structures in In-vitro and In-Vivo-Like Conditions
Restricted (Penn State Only)
Author:
Winemiller, Atlas
Area of Honors:
Chemistry
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
Philip C Bevilacqua, Thesis Supervisor Stewart Anthony Mallory, Thesis Honors Advisor Elizabeth A Jolley, Faculty Reader
Keywords:
RNA thermodynamics non-Watson-Crick in vivo-like
Abstract:
Thermodynamic stability of an RNA duplex is impacted by its environment and the specific structures within the RNA duplex. To explore the effects of RNA structure, non-Watson-Crick base paired structures such as bulges and internal loops were incorporated into the duplex. Two environments were studied: in vitro experiments in 1M NaCl and in vivo-like experiments using our artificial cytoplasm of Eco80 (80% Escherichia coli metabolites) which is an approximation of cellular conditions that makes it possible to draw conclusions about how RNA behaves differently within the cell. Two methods were utilized: UV melts and fluorescence determined binding isotherms (FDBI). Eco80 interferes with absorbance measurements, so comparisons between in vitro and in vivo-like conditions could only be made using FDBI. For these experiments, fluorescein amidite was attached to the 5’ end of one strand and black hole quencher attached to the 3’ end of the other. The fluorescence of the fluorophore is quenched when the duplex is folded together. Data were analyzed in MeltR9, where free energy can be calculated to determine the impacts of structure and environment on the stability of the duplex. In general, the artificial cytoplasm used for the in vivo-like conditions has a destabilizing effect, tending to decrease the stability of the duplex by between 1 and 2 kcal/mol. Results indicate that bulge loops and internal loops further destabilize RNA duplexes in both 1M NaCl and Eco80 environments, although the effect is reduced in artificial cytoplasm. A major conclusion is that structural imperfections are more readily tolerated (i.e. are less penalizing) under in vivo-like conditions.
