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Design of a Hairpin Polyamide, ZT65B, for Targeting the Inverted CCAAT Box (ICB) Site in the Multidrug Resistant ( MDR1 ) Gene
Author(s) -
Buchmueller Karen L.,
Taherbhai Zarmeen,
Howard Cameron M.,
Bailey Suzanna L.,
Nguyen Binh,
O’Hare Caroline,
Hochhauser Daniel,
Hartley John A.,
Wilson W. David,
Lee Moses
Publication year - 2005
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500179
Subject(s) - caat box , microbiology and biotechnology , inverted repeat , biology , binding site , dna , promoter , gene , moiety , chemistry , genetics , gene expression , stereochemistry , genome
Abstract A novel hairpin polyamide, ZT65B, containing a 3‐methylpicolinate moiety was designed to target the inverted CCAAT box (ICB) of the human multidrug resistance 1 gene ( MDR1 ) promoter. Binding of nuclear factor‐Y (NF‐Y) to the ICB site upregulates MDR1 gene expression and is, therefore, a good target for anticancer therapeutic agents. However, it is important to distinguish amongst different promoter ICB sites so that only specific genes will be affected. All ICB sites have the same sequence but they differ in the sequence of the flanking base pairs, which can be exploited in the design of sequence‐specific polyamides. To test this hypothesis, ten ICB‐containing DNA hairpins were designed with different flanking base pairs; the sequences ICBa and ICBb were similar to the 3′‐ICB site of MDR1 (TGGCT). Thermal‐denaturation studies showed that ZT65B effectively targeted ICBa and ICBb (Δ T M =6.5 and 7.0 °C) in preference to the other DNA hairpins (<3.5 °C), with the exception of ICBc (5.0 °C). DNase I‐footprinting assays were carried out with the topoisomerase IIα‐promoter sequence, which contains five ICB sites; of these, ICB1 and ICB5 are similar to the ICB site of MDR1. ZT65B was found to selectively bind ICB1 and ICB5; footprints were not observed with ICB2, ICB3, or ICB4. A strong, positive induced ligand band at 325 nm in CD studies confirmed that ZT65B binds in the DNA minor groove. The selectivity of ZT65B binding to hairpins that contained the MDR1 ICB site compared to one that did not (ICBd) was confirmed by surface‐plasmon studies, and equilibrium constants of 5×10 6 –1×10 7 and 4.6×10 5 M −1 were obtained with ICB1, ICB5,and ICB2 respectively. ZT65B and the previously published JH37 (J. A. Henry, et al. Biochemistry 2004 , 43 , 12 249–12 257) serve as prototypes for the design of novel polyamides. These can be used to specifically target the subset of ubiquitous gene elements known as ICBs, and thereby affect the expression of one or a few proteins.