Design And Selection Of High Affinity DNA Ligands For Mutant Single-chain Derivatives Of The Bacteriophage 434 Repressor

I. Design and selection of thc high affinity DNA ligands for singIe-chain reprcssorSingle-chain repressor RRn., is a derivative of bacteriophage 434 repressor,which contains covalent1y dimerized DNA-binding domains (axnino acids 1-69) of thephage 434 repressor. In this single-chain molecule, the wi1d type domain R isconnected to the mutant domain Rr., by a recombinant linker in a head-to-tai1arrangement. The DNA~contacting amino acids of Rr.., at the -1, 1, 2, and 5 positionsof the a3 helix are T, R, E, S respective1y By using a randomized DNA poo1cofltaining the cetTtTal sequence --C ATA C AA GAAA -- T TT -, a cy cI ic, invitrO DNA-binding site se1ection was perfOrmed. The selected popu1ation was clonedand the individua1 members were characterized by determining their binding affinitiesto RR.,S' The results showed that the optima1 operators colltained the TTAC orTTCC sequences in the underlined positions as above, and that the Kd va1ues were inthe 1-10pM concentTation range. The operator, which has selected random regionTMG, had the highest binding affinity to RR,.,, as 1pM in Kd value, the averageKd value of TTAC group was around 3pM, and the Kd value of TTCC group was in5-l0pM concentration range. Since the affinity of the natural 434 repressor to itsnatural operator sites is in the nM range, the observed binding affinity increase isremarkable. It was also fOund that binding affinity was strongly affected by theflanking bases of the optima1 tetramer binding sites, especially by the base at the 5'position.3We constructed a new homodimeric sing1e-chain repressor RTas,Rm, and itsDNA-binding specificity was tested by using a series of new operators designedaccording tO the recognition properties previously determined fOr the RT.,, domain.These operators containing the consensus sequence caGAAAANm orWGNANM (R is A or G) were re c o gnized b y Rr.,,Rr.. S sp ec ific al l y,and With high binding affinity the Kd value was in 5-40pM concentration range. Andthe operator sequence unGAAA GTixG bind in g affinity t o R., S R.., was5pM in Kd value. A new heterodimeric single-chain repressor R*Rr.,, wasconstructed also, but the binding affinity to its designed operators was not very high,around 100pM in Kd value. Thus, by using a combination of random selection andrational design principles, we have discovered novel, high affinity protein-DNAinteractions with new specificities. This methOd can potentia1ly be used to obtain newbinding specificities for other DNA-binding proteins.II. Exploring non-radioactivc method to select the optimal DNA-bindingsequencc fOr singIe-chain reprcssorCloning and expression of single-chain repressors with cysteine tai1 wereperformed and immobilized these protein in the solid surface by using the standardmicrowell plates Which have been activated to contain maleimide grouPs, al1owing tobind sulthydryl-containing protein on its surface. Se1ection of the optimal DNA-binding sites of single-chain repressor R..,,Rr., was performed in viho, andobtained some selected sequences with not very high binding affinity, the Kd valuewas in nM range, This method needs to be optimized trihermore.