Design of a hairpin polyamide, ZT65B, for targeting the inverted CCAAT box (ICB) site in the multidrug resistant (MDR1) gene.
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 (DeltaTM=6.5 and 7.0 degrees C) in preference to the other DNA hairpins (<3.5 degrees C), with the exception of ICBc (5.0 degrees C). DNase I-footprinting assays were carried out with the topoisomerase IIalpha-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 5x10(6)-1x10(7) and 4.6x10(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.
Buchmueller
Karen L
KL
Department of Chemistry, Furman University, Greenville, SC 29613, USA.
Taherbhai
Zarmeen
Z
Howard
Cameron M
CM
Bailey
Suzanna L
SL
Nguyen
Binh
B
O'Hare
Caroline
C
Hochhauser
Daniel
D
Hartley
John A
JA
Wilson
W David
WD
Lee
Moses
M
D000951:Antigens, Neoplasm / Q000235:genetics; D000970:Antineoplastic Agents / Q000138:chemical synthesis; D001483:Base Sequence; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D015195:Drug Design; D018432:Drug Resistance, Multiple / Q000187:drug effects; D019450:Genes, MDR / Q000187:drug effects*; D006801:Humans; D009690:Nucleic Acid Conformation; D009757:Nylons / Q000138:chemical synthesis* / Q000494:pharmacology; D009841:Oligonucleotides / Q000138:chemical synthesis; D010847:Picolines / Q000138:chemical synthesis* / Q000494:pharmacology; D010848:Picolinic Acids; D011401:Promoter Regions, Genetic / Q000187:drug effects*
D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D009757:Nylons; D009841:Oligonucleotides; D010847:Picolines; D010848:Picolinic Acids; C514965:ZT65B; D004250:DNA Topoisomerases, Type II; C030614:picolinic acid