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The article focuses on the changes in the conformation of tRNAPhe by using an integrated biochemistry laboratory course. Students use three experimental techniques including gel electrophoresis, UV-vis spectrophotometry, and fluorescence spectroscopy. The class examines the changes in the structure of a ligand that affect its ability to bind and change the tRNAPhe structure. Discussions concerning the projects probing tRNAPhe structure using various assay methods are also presented.

The ruthenium-based anticancer agent NAMI-A (ImH[ trans -RuCl 4 (dmso)(Im)], where Im = imidazole) has been shown to interact with RNA in vivo and in vitro. We hypothesized that the similarly structured drug KP1019 (IndH[ trans -RuCl 4 (Ind) 2 ], where Ind = indazole) binds to RNA as well. Fluorescence spectroscopy was employed to assay the interactions between either NAMI-A or KP1019 and tRNA Phe through an intrinsic fluorophore wybutosine (Y) base and by extrinsic displacement of the intercalating agent ethidium bromide. In both the intrinsic Y-base and extrinsic ethidium bromide studies, KP1019 exhibited tighter binding to phenylalanine-specific tRNA (tRNA Phe ) than NAMI-A. In the ethidium bromide study, reducing both drugs from Ru III to Ru II resulted in a significant decrease in binding. Our findings suggest that the relatively large heteroaromatic indazole ligands of KP1019 intercalate in the π-stacks of tRNA Phe within structurally complex binding pockets. In addition, NAMI-A appears to be sensitive to destabilizing electrostatic interactions with the negative phosphate backbone of tRNA Phe . Interactions with additional tRNA molecules and other types of RNA require further evaluation to determine the role of RNA in the mechanisms of action for KP1019 and to better understand how Ru drugs fundamentally interact with biomolecules that are more structurally sophisticated than short DNA oligonucleotides. To the best of our knowledge, this is the first study to report KP1019 binding interactions with RNA. [ABSTRACT FROM AUTHOR]

This article presents a study regarding the fluorescence spectroscopy of tRNAPhe Y base in the presence of magnesium2+ and small molecule ligands. It discusses that tRNAPhe contains a unique Y base with three planar aromatic rings and serves as a fluorophore. It serves as an intrinsic probe to study the conformational changes of tRNAPhe in the presence of magnesium ions and small molecule ligands because Y base fluorescence is exquisitely sensitive to the local environment.

This article presents a study regarding the metal-catalyzed cleavage of tRNAPhe. It discusses that Lead(II)-catalyzed hydrolysis is an effective assay of tRNAPhe tertiary structure because it catalyzes the cleavage of native, folded tRNAPhe at a single site in the D loop resulting in a large fragment and a small fragment. In addition, ligand binding alters the conformation of tRNAPhe inhibiting lead-catalyzed cleavage and metals other than lead may also cleave tRNA.

This article presents a study regarding the UV thermal melting curves of tRNAPhe in the presence of ligands. It discusses that native secondary and tertiary structures of tRNAPhe are only stable below about 50-70 degrees celcius because of the positive entropy of unfolding. A laboratory project was designed to investigate the influence of equilibrium ligand binding on the conformational stability of tRNAPhe via UV melting curves. The melting temperature is derived by calculating the derivatives of the absorbance versus temperature curves using the Cary program.