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Single-Molecule FRET Studies of Important Intermediates in the Nucleocapsid-Protein-Chaperoned Minus-Strand Transfer Step in HIV-1 Reverse Transcription

Hsiao-Wei Liu ^*, Gonzalo Cosa ^*, Christy F. Landes ^*, Yining Zeng ^*, Brandie J. Kovaleski , Daniel G. Mullen , George Barany , Karin Musier-Forsyth  and Paul F. Barbara ^*

^* Department of Chemistry and Biochemistry, Center for Nano and Molecular Science and Technology, University of Texas, Austin, Texas 78712; and Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455

Correspondence: Address reprint requests to Paul F. Barbara, E-mail: p.barbara{at}mail.utexas.edu.

The minus-strand transfer step of HIV-1 reverse transcription is chaperoned by the nucleocapsid protein (NC), which has been shown to facilitate the annealing between the transactivation response element (TAR) RNA and complementary TAR DNA stem-loop structures. In this work, potential intermediates in the mechanism of NC-chaperoned TAR DNA/TAR RNA annealing have been examined using single-molecule fluorescence resonance energy transfer. The interaction between TAR DNA and various DNA oligonucleotides designed to mimic the initial annealing step was monitored to capture potential intermediates along the reaction pathway. Two possible mechanisms of annealing were examined, namely nucleation through the 3'/5' termini, termed the "zipper" complex, or nucleation through the hairpin loops in a "kissing" complex. Intermediates associated with both mechanisms were observed in the presence of NC, and the kinetics of formation of these intermediates were also measured. Thus, the single-molecule experiments support the notion that NC-assisted annealing of TAR DNA:TAR RNA may occur through multiple pathways.

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