Evaluating the binding affinities of NF-kappaB protein to the single-nucleotide mismatch DNA binding sites by using double-stranded DNA microarray.
Protein-DNA sequence-specific interaction plays an essential role in many biological processes. Here we immobilized a series of double-stranded DNA probes on an agarose coated slide to investigate the binding affinity of NF-kappaB p50 homodimer to the single-nucleotide mismatches (G<-->A or T<-->C) of the 10 base pair (bp) protein binding sites. The results demonstrated that the nucleotides at different positions contribute differently to the p50p50/DNA binding interaction. Within the 10 bp binding sites, the 5tG or 6cA mismatch has less effect on the protein-DNA binding affinity. Even the 5tG mismatch may have the ability to enhance the protein-DNA interaction (5t/w = 1.07). On the other hand, the 7cA or 10tG mismatch blocked the protein-DNA interaction more significantly than other six single-nucleotide mismatches. (7c/W = 0.37, 10t/W = 0.35). It also indicated that the duplex DNA probes immobilized on the agarose-coated surface were apt to be recognized by DNA-binding proteins, and this method would provide a reliable method for exploring the binding affinities of DNA-binding proteins with a larger number of DNA targets.
Bai
Yunfei
Y
State Key Laboratory for Bioelectronics, Southeast University, Nanjing, China.
Ge
Qinyu
Q
Liu
Quanjun
Q
Li
Tongxiang
T
Wang
Jinke
J
Lu
Zuhong
Z
D020137:Base Pair Mismatch; D001665:Binding Sites; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry*; D016328:NF-kappa B / Q000032:analysis / Q000737:chemistry*; D009711:Nucleotides / Q000032:analysis / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D009711:Nucleotides; D004247:DNA