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Background  It is well known that the  C- terminal zinc-finger-3 in transcription factor Sp1 contributes more than the  N- terminal zinc-finger-1 in determining Sp1’s DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the  C- terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of  p16  promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced  p16 . These 7ZFPs contain one  N- terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and  C- terminal regions.    Results  In the present study, sets of variants for several representative 7ZFPs with the  p16- binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the  N -terminal fingers,  C -terminal fingers, and linker peptide, respectively. Their  p16- binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the  C- terminal 2ZF domain completely abolished their  p16- binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between  finger-7  and the 6 × Histidine-tag in the  C- terminal also dramatically abolished their binding affinity. In contrast, the replacement of the  finger-3  in the  N -terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.    Conclusions  Altogether, the present study show that the  C- terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity