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Sequence‐Selective DNA Recognition with Peptide–Bisbenzamidine Conjugates
Author(s) -
Sánchez Mateo I.,
Vázquez Olalla,
Vázquez M. Eugenio,
Mascareñas José L.
Publication year - 2013
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201300519
Subject(s) - zinc finger , dna , electrophoretic mobility shift assay , dna binding protein , peptide , binding site , dna binding domain , dna binding site , bzip domain , chemistry , transcription factor , protein–dna interaction , biochemistry , plasma protein binding , biology , promoter , gene expression , gene
Transcription factors (TFs) are specialized proteins that play a key role in the regulation of genetic expression. Their mechanism of action involves the interaction with specific DNA sequences, which usually takes place through specialized domains of the protein. However, achieving an efficient binding usually requires the presence of the full protein. This is the case for bZIP and zinc finger TF families, which cannot interact with their target sites when the DNA binding fragments are presented as isolated monomers. Herein it is demonstrated that the DNA binding of these monomeric peptides can be restored when conjugated to aza‐bisbenzamidines, which are readily accessible molecules that interact with A/T‐rich sites by insertion into their minor groove. Importantly, the fluorogenic properties of the aza‐benzamidine unit provide details of the DNA interaction that are eluded in electrophoresis mobility shift assays (EMSA). The hybrids based on the GCN4 bZIP protein preferentially bind to composite sequences containing tandem bisbenzamidine–GCN4 binding sites (TCAT ⋅ AAATT). Fluorescence reverse titrations show an interesting multiphasic profile consistent with the formation of competitive nonspecific complexes at low DNA/peptide ratios. On the other hand, the conjugate with the DNA binding domain of the zinc finger protein GAGA binds with high affinity ( K D ≈12 n M ) and specificity to a composite AATTT ⋅ GAGA sequence containing both the bisbenzamidine and the TF consensus binding sites.