Quadruple 9-mer-based protein binding microarray with DsRed fusion protein.
BACKGROUND
The interaction between a transcription factor and DNA motif (cis-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Recently, the universal protein binding microarray (PBM) was introduced to determine if a DNA motif interacts with proteins in a genome-wide manner.


RESULTS
We facilitated the PBM technology using a DsRed fluorescent protein and a concatenated sequence of oligonucleotides. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, permitting unequivocal interpretation of the cis-acting elements. The complimentary DNA strands of the features were synthesized with a primer and DNA polymerase on microarray slides. Proteins were labeled via N-terminal fusion with DsRed fluorescent protein, which circumvents the need for a multi-step incubation. The PBM presented herein confirmed the well-known DNA binding sequences of Cbf1 and CBF1/DREB1B, and it was also applied to elucidate the unidentified cis-acting element of the OsNAC6 rice transcription factor.


CONCLUSIONS
Our method demonstrated PBM can be conveniently performed by adopting: (1) quadruple 9-mers may increase protein-DNA binding interactions in the microarray, and (2) a one-step incubation shortens the wash and hybridization steps. This technology will facilitate greater understanding of genome-wide interactions between proteins and DNA.
Kim
Min-Jeong
MJ
GreenGene Biotech Inc, Myongji University, Yongin 449-728, Korea. kim750a11@gmail.com
Lee
Tae-Ho
TH
Pahk
Yoon-Mok
YM
Kim
Yul-Ho
YH
Park
Hyang-Mi
HM
Choi
Yang Do
YD
Nahm
Baek Hie
BH
Kim
Yeon-Ki
YK
D020816:Amino Acid Motifs; D001665:Binding Sites; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D012275:Oryza / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding
D008164:Luminescent Proteins; D009841:Oligonucleotides; D010940:Plant Proteins; C413663:fluorescent protein 583