ELUCIDATING THERMODYNAMIC RELATIONSHIPS IN THE RNA STRUCTURAL HIERARCHY: MOLECULAR ORIGINS OF STABILITY

Open Access
Author:
Blose, Joshua Matthew
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 10, 2009
Committee Members:
  • Philip C. Bevilacqua, Dissertation Advisor
  • Philip C. Bevilacqua, Committee Chair
  • Sharon Hammes Schiffer, Committee Member
  • Tae Hee Lee, Committee Member
  • Kenneth Charles Keiler, Committee Member
Keywords:
  • Cooperative Folding
  • Thermophilic Adaptation
  • Tetraloop
  • RNA
  • Nonlinear Poisson-Boltzmann
  • NLPB
  • Electrostatics
Abstract:
RNA participates in a wide range of cellular functions, which are often predicated on the folding of diverse tertiary structures from preformed secondary structures. Hairpins are the most common RNA secondary structures, and tetraloops are the most common hairpin loops. UNCG and GNRA loop families are exceptionally stable with CG closing base pairs (cbp). We propose that, despite significant structural differences, these loops present the same functionalities to the CG cbp. Thermodynamic contributions of this molecular mimicry were investigated using nucleobase and functional group substitutions. Effects of substitutions as well as the salt dependence of loop stability were consistent with molecular mimicry. Nonlinear Poisson-Boltzmann (NLPB) calculations revealed that both loop families have increased surface charge density with a GC cbp, which leads to stronger interactions with solvent and salt, explaining the correlation between experimental and calculated salt dependencies. The r(GNRA) tetraloops were also compared to d(GCA) triloops. Effects of substitutions and salt dependence of loop stability were similar for the RNA and DNA loops and consistent with electrostatic interactions identified through NLPB calculations. The similarity of loop-cbp interactions shows portability of the loop-cbp motif between DNA and RNA, and suggests convergence on similar molecular solutions for stability. The molecular mechanism of how secondary structure stability affects stability of functional nucleic acids was investigated using an intramolecularly folding DNA triplex model system. A tunable region adjacent to the triplex-forming portion of the helix allowed secondary structure strength to be modulated, and formation of C+•GC base triples allowed control of folding cooperativity via pH. We found a linear relationship between the free energy of folding of the functional structure and the stability of the tunable region, but only when folding of secondary and tertiary structure is cooperative. The ability to increase the melting temperature of tertiary structure by strengthening base-pairing interactions separate from tertiary interactions provides a simple means for evolving thermostability in functional RNAs. Preliminary experiments on the folding of tRNAs suggest that the model for thermophilic adaptation will be supported by tRNA melting, especially in the presence of molecular crowding agents that mimic cellular conditions.