Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published September 24, 1996 | public
Journal Article

Effects of the A·T/T·A Degeneracy of Pyrrole−Imidazole Polyamide Recognition in the Minor Groove of DNA

Abstract

Pairing rules have been developed to predict the sequence specificity of minor groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids. An Im/Py pair distinguishes G·C from C·G and both of these from A·T/ T·A base pairs. A Py/Py pair appears not to distinguish A·T from T·A base pairs. To test the extent of this degeneracy, the affinity and binding orientation of the hairpin polyamide ImPyPy-γ-PyPyPy-β-Dp were measured for eight possible five base pair 5'-TG(A,T)_3-3' match sites. Affinity cleavage experiments using a polyamide with an EDTA·Fe(II) moiety at the carboxy terminus, ImPyPy-γ-PyPyPy-β-Dp-EDTA·Fe(II), are consistent with formation of an oriented 1:1 hairpin polyamide complex at all eight 5'-TG(A,T)_3-3' binding sites [20 mM HEPES, 200 mM NaCl, 50 mg/ml glycogen, pH 7.0, 22 °C, 5 mM DTT, 1 mM Fe(II)]. Quantitative DNase I footprint titration experiments reveal that ImPyPy-γ-PyPyPy-β-Dp binds all eight 5'-TG(A,T)_3-3' target sites with only a 12-fold difference in the equilibrium association constants between the strongest site, 5'-TGTTT-3' (K_a = 2.1 × 10^8 M^(-1)), and the weakest site, 5'-TGAAT-3' (K_a = 1.8 × 10^7 M^(-1)) (10 mM Tris·HCl, 10 mM KCl, 10 mM MgCl_2, 5 mM CaCl_2, pH 7.0, 22 °C). This relatively small range indicates that the Py/Py pair is approximately degenerate for recognition of A,T base pairs, affording generality with regard to targeting sequences of mixed A·T/T·A composition.

Additional Information

© 1996 American Chemical Society. Received March 27, 1996; Revised Manuscript Received July 30, 1996. Publication Date (Web): September 24, 1996. This research was supported by National Institutes of Health Grant GM-27681, a National Science Foundation predoctoral fellowship to S.W., and a Howard Hughes Medical Institute predoctoral fellowship to E.E.B.

Additional details

Created:
August 19, 2023
Modified:
October 18, 2023