Speaker: Dan Fabris, University of Maryland-Baltimore County, Baltimore, MD

Topic: Applications of Electrospray Mass Spectrometry to the Investigation of Covalent Modifications and Non-Covalent Interactions Involving Nucleic Acids

Place: Building 426, Conference Room, NCI-Frederick, Frederick, MD

Time: Tuesday, May 8, 2001, at 2:00 PM

Abstract: The interactions of nucleic acids with cellular components and xenobiotics can be of both covalent and non-covalent nature. Electrospray ionization (ESI) mass spectrometry is capable of providing valuable information on covalent modifications of nucleic acids as well as on non-covalent interactions of nucleic acids with proteins and metals.

For example, ESI-MS has been employed in our lab to characterize the covalent products of the reaction between the anti-tumor drug 2-crotonyloxymethyl-2-cyclohexenone (COMC) and nucleic acids, both in presence and absence of glutathione (GSH) and glutathione-S-transferase (GST). These data can be used to elucidate the mechanism of alkylation and to investigate possible nucleotide specificities.

In another application, ESI-MS has been applied to monitor the enzymatic hydrolysis of adenine from susceptible RNA hairpins performed by the plant toxin ricin A chain. The kinetics of depurination of the mammalian sarcin-ricin loop and that of the HIV-1 SL4 have been determined, suggesting the possibility of using ESI for the investigation of sequence effects on enzyme-substrate recognition and catalysis.

The ability of ESI to preserve weak non-covalent associations has been tested in the investigation of the complexes of HIV-1 stem-loop hairpins SL2, SL3, and SL4 with the nucleocapid protein p7 (NC). The observation of the chelation of Cd2+ ions by the ribosomal GTPase Associated Region (GAR) RNA has provided proof of the ability of ESI-MS to determine the binding stoichiometry in metal-nucleic acids complexes.