Length-encoded multiplex binding site determination: application to zinc finger proteins.
The screening of combinatorial libraries is becoming a powerful method for identifying or refining the structures of ligands for binding proteins, enzymes, and other receptors. We describe an oligonucleotide library search procedure in which the identity of each member is encoded in the length of oligonucleotides. This encoding scheme allows binding-site preferences to be evaluated via DNA length determination by denaturing gel electrophoresis. We have applied this method to determine the binding-site preferences for 18 Cys2His2 zinc finger domains as the central domain within a fixed context of flanking zinc fingers. An advantage of the method is that the relative affinities of all members of the library can be estimated in addition to simply determining the sequence of the optimal or consensus ligand. The zinc finger domain specificities determined will be useful for modular zinc finger protein design.
Desjarlais
J R
JR
Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218.
Berg
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides