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J. Biol. Chem., Vol. 276, Issue 3, 1968-1973, January 19, 2001

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Specific RNA Binding by a Single C₂H₂ Zinc Finger^*

Westley J. Friesen and Martyn K. Darby^§

From the Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Zinc finger proteins with high affinity for human immunodeficiency virus Rev responsive element stem loop IIB (RRE-IIB) were previously isolated from a phage display zinc finger library. Zinc fingers from one of these proteins, RR1, were expressed individually and assayed for RRE-IIB affinity. The C-terminal zinc finger retained much of the binding affinity of the two-finger parent and was disrupted by mutations predicted to narrow the RRE-IIB major groove and which disrupt Rev binding. In contrast, the N-terminal zinc finger has a calculated affinity at least 1000-fold lower. Despite the high affinity and specificity of RR1 for RRE-IIB, binding affinity for a 234-nucleotide human immunodeficiency virus Rev responsive element (RRE₂₃₄) was significantly lower. Therefore, zinc finger proteins that bind specifically to RRE₂₃₄ were constructed using an in vitro selection and recombination approach. These zinc fingers bound RRE₂₃₄ with subnanomolar dissociation constants and bound the isolated RRE-IIB stem loop with an affinity 2 orders of magnitude lower but similar to the affinity of an arginine-rich peptide derived from Rev. These data show that single C₂H₂ zinc fingers can bind RNA specifically and suggest that their binding to stem loop IIB is similar to that of Rev peptide. However, binding to RRE₂₃₄ is either different from stem loop IIB binding or the tertiary structure of stem loop IIB is changed within the Rev responsive element.

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