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The PreQ1 riboswitch is a subset of riboswitches found mainly in eubacteria. It plays an essential role in the biosynthesis of preQ1, a precursor to queuosine (Q). Q is a hypermodified guanine nucleotide that is universally found at the wobble position of certain tRNAs. PreQ1 forms a pseudoknot structure upon binding to preQ1. We use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry (DSC) to determine the unfolding thermodynamics of a DNA analog (PREQ1) and its control hairpin at two different salt concentrations, 16 mM and 116 mM Na+. Furthermore, we use DSC and fluorescence spectroscopy techniques to determine binding affinities, Kb, for the interaction of preq1 ligand with these DNA analogs. Both oligonucleotides unfold with TMs independent of strand concentration, indicating the formation of hairpin structures. The unfavorable unfolding free energy terms resulted from the typical compensation of an unfavorable enthalpy contributions (disruption of base-pair stacks) and favorable entropy contributions (release of ions and water molecules). The ligand preQ1 yielded a Kb of 5.7 x 105. Supported by NSF Grant (MCB-1912587).
Riboswitch, ligand, binding affinity, thermodynamic profile
Sullivan, Grace; Khutsishvili, Irine G.; Reiling, Calliste; and Marky, Luis, "Unfolding and Binding Thermodynamics of the PREQ1 DNA Analog" (2021). Posters: 2021 Summer Undergraduate Research Program. 56.