title: Using an emissive uridine analogue for assembling fluorescent HIV-1 TAR constructs.
abstract: Emissive nucleoside analogues that are sensitive to their microenvironment can serve as probes for exploring RNA folding and recognition. We have previously described the synthesis of an environmentally sensitive furan-containing uridine and its triphosphate, and have demonstrated that T7 RNA polymerase recognizes this modified ribonucleoside triphosphate as a substrate in in vitro transcription reactions. Here we report the enzymatic preparation of fluorescently tagged HIV-1 TAR constructs and study their interactions with a Tat peptide. Two extreme labeling protocols are examined, where either all native uridine residues are replaced with the corresponding modified fluorescent analogue, or only key residues are site-specifically modified. For the HIV-1 Tat-TAR system, labeling all native uridine residues resulted in relatively small changes in emission upon increasing concentrations of the Tat peptide. In contrast, when the two bulge U residues were site-specifically labeled, a reasonable fluorescence response was observed upon Tat titration. The scope and limitations of such fluorescently tagged RNA systems are discussed.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0358.
authors.forename: Seergazhi G
authors.initials: SG
authors.lastname: Srivatsan
authors.forename: Yitzhak
authors.initials: Y
authors.lastname: Tor