Crystal Structure-Driven Investigations of a Fluorogenic RNA Aptamer with Divalent Cation Dependence

Pepper is a fluorogenic aptamer tag that is able to bind a variety of benzylidene-cyanophenyl (HBC) derivatives with tight affinity and activate their fluorescence. To investigate how Pepper RNA folds to create a binding site for HBC, I used antibody-assisted crystallography to determine the structures of Pepper bound to HBC530 and HBC599 to 2.3 and 2.7 Å resolution, respectively. The structural data show that Pepper folds into an elongated structure and organizes nucleotides within an internal bulge to create the ligand-binding site, assisted by tertiary interactions from an adjacent bulge. Pepper does not use a G-quadruplex to form a binding pocket for HBC and instead uses a unique base-quadruple stack and a G•U wobble pair to sandwich the ligand. In addition, Pepper exhibits a structural Mg2+ dependence, with one Mg2+ shown binding to a specific location in the Pepper structure to allow folding. This thesis provides deep insight into the structural features that allow the Pepper aptamer to bind HBC and shows how Pepper's function may expand to allow the in-vivo detection of other small molecules and metals.