Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RR_Tres is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1–69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain R_TRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of R_TRES at the −1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic,in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RR_Tres The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1 × 10⁻¹² mol/L-1 × 10¹¹ mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1 × 10⁻⁹ mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor R_TRESR_TRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recognition properties previously determined for the R_TREs domain. These operators containing the consensus sequenceGTAAGAAARNTTACN orGGAAGAAARNTTCCN (R is A or G) were recognized by R_TRESR_TRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA interactions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.