Abstract
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.
Highlights
C2H2 zinc finger proteins comprise a diverse family of DNAand RNA-binding proteins
To explore the potential for designed RNA-binding proteins, we previously reported the isolation of zinc finger proteins with high affinity for HIV Rev responsive element stem loop IIB (RRE-IIB) and 5 S rRNA through a combination of phage display and gene shuffling [16]
We report that RR1, a zinc finger protein previously selected for binding to RRE-IIB, and a single zinc finger derived from it bind RRE-IIB with similar RNA sequence and structural requirements as the arginine-rich Rev peptide
Summary
C2H2 zinc finger proteins comprise a diverse family of DNAand RNA-binding proteins. NMR and crystallographic data show clearly that zinc fingers bind DNA through ␣-helical contacts in a slightly enlarged DNA major groove [1, 2]. To explore the potential for designed RNA-binding proteins, we previously reported the isolation of zinc finger proteins with high affinity for HIV RRE-IIB and 5 S rRNA through a combination of phage display and gene shuffling [16]. These zinc fingers most likely make critical contacts through amino acids in ␣-helices because these amino acids were randomized in the displayed zinc finger library. In light of these results, we constructed high affinity zinc fingers that bind RRE234 by phage display and in vitro recombination of ␣-helix randomized zinc fingers
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