Defining the sequence-recognition profile of DNA-binding molecules.
Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis.
Warren
Christopher L
CL
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Kratochvil
Natasha C S
NC
Hauschild
Karl E
KE
Foister
Shane
S
Brezinski
Mary L
ML
Dervan
Peter B
PB
Phillips
George N
GN
Ansari
Aseem Z
AZ
D000818:Animals; D004396:Coloring Agents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004331:Drosophila melanogaster; D005821:Genetic Techniques; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D009757:Nylons / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000737:chemistry; D014982:Xenopus laevis
D004396:Coloring Agents; D004268:DNA-Binding Proteins; D008024:Ligands; D009757:Nylons; D014157:Transcription Factors; D004247:DNA