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

Single-chain repressor RRTRES is a derivative of bacteriophage 434 represso r, which contains covalently dimerized DNA-binding domains (amino acids 1-6 9) 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 RTRES at t he -1, 1, 2, and 5 positions of the alpha3 helix are T, R, E, S respectivel y. By using a randomized DNA pool containing the central sequence -CATACAAG AAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed . The selected population was cloned and the individual members were charac terized by determining their binding affinities to RRTRES The results showe d that the optimal operators contained the TTAC or TTCC sequences in the un derlined positions as above, and that the Kd values were in the 1x10(-12) m ol/L - 1x10(-11)mol/L concentration range. Since the affinity of the natura l 434 repressor to its natural operator sites is in the 1x10(-9) mol/L rang e, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the op timal tetramer binding sites, especially by the base at the 5 ' position. W e constructed a new homodimeric single-chain repressor RTRESRTRES and its D NA-binding specificity was tested by using a series of new operators design ed according to the recognition properties previously determined for the RT RES domain. These operators containing the consensus sequence GTAAGAAARNTTA CN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifica lly, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high a ffinity protein-DNA interactions with new specificity. This method can pote ntially be used to obtain new binding specificity for other DNA-binding pro teins.