Synthesis of Fluorescently‐Labeled Tobacco Etch Virus (TEV) RNA and Its Interactions with Pokeweed Antiviral Protein (PAP)

Abstract

Fluorescence spectroscopy has been extensively used to investigate protein‐protein and protein‐nucleic acid interactions. Changes in fluorescence intensity provide vital information about these macromolecular interactions. Availability of extrinsic fluorophores became invaluable for researchers monitoring these interactions. Here, we successfully synthesize a fluorescent anthranioyl‐m7GpppG‐capped tobacco etch virus (TEV) RNA, and examine its properties pertaining to interaction with pokeweed antiviral protein (PAP). PAP is a ribosome inactivating protein (RIP) and is an RNA N‐glycosidase that removes purines from the sarcin/ricin loop of large rRNA. This leads to the translational termination. PAP possesses antiviral properties, and it is thought to bind to the 5'‐cap of viral RNA and depurinate it down the stream of the cap. RIPs are thought to play an important role in plant defense mechanisms. We employ fluorescence spectroscopy and HPLC techniques to investigate and characterize Ant‐m7GpppG‐capped TEV RNA with PAP.