Journal
TETRAHEDRON
Volume 63, Issue 17, Pages 3601-3607Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.tet.2007.01.074
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Funding
- NIAID NIH HHS [R01 AI047673-07, R01 AI047673] Funding Source: Medline
- NIGMS NIH HHS [R01 GM069773-04, R01 GM069773] Funding Source: Medline
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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. (c) 2007 Elsevier Ltd. All rights reserved.
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