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Published online on February 20, 2007, 10.1073/pnas.0611075104
PNAS | February 27, 2007 | vol. 104 | no. 9 | 3061-3066


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BIOLOGICAL SCIENCES / BIOCHEMISTRY
Quantifying DNA–protein binding specificities by using oligonucleotide mass tags and mass spectroscopy

Lingang Zhang*,{dagger}, Simon Kasif{dagger},{ddagger},§, and and Charles R. Cantor*,{dagger},¶,||

*Center for Advanced Biotechnology, {dagger}Department of Biomedical Engineering, {ddagger}Bioinformatics Program, and §Center for Advanced Genomic Technology, Boston University, Boston, MA 02215; and SEQUENOM, Inc., 3595 John Hopkins Court, San Diego, CA 92121

Contributed by Charles R. Cantor, January 1, 2007 (received for review September 28, 2006)

The ability to determine the relative binding affinity of different transcription-factors (TF) to their DNA binding sites is fundamentally important for a comprehensive understanding of gene regulation. Here we present a general approach for multiplex quantification of DNA-TF binding specificities in vitro using oligonucleotide mass tag (OMT) labeling and mass spectroscopic quantification. An OMT is a short nucleic acid sequence with a distinct mass that can be resolved by a mass spectrometer. Each putative binding sequence is labeled with a unique OMT, and PCR amplification of OMTs is performed after removing nonbound DNA. Subsequently, a primer extension reaction is carried out, and the extension products are quantified by MALDI-TOF mass spectroscopy. Using the TF NF-{kappa}B P50, we have quantified the binding specificities of up to 15 binding sequences in a single assay. The results from the multiplex assay are consistent with data from the traditional gel shift assay. The approach allows the competitive binding of multiple DNA sequences to the given protein in a homogeneous reaction. By using the commercially available homogeneous MassEXTEND platform (SEQUENOM), it is scalable for high-throughput DNA-TF binding applications, including genome-wide TF binding site mapping and analyses of SNPs in promoter regions.

polymerase chain reaction | transcription factor

Author contributions: L.Z., S.K. and C.R.C designed research; L.Z. performed research; and L.Z., S.K., and C.R.C. wrote the paper.

Conflict of interest statement: C.R.C. is a SEQUENOM employee.

This article contains supporting information online at www.pnas.org/cgi/content/full/0611075104/DC1.

Note.

||To whom correspondence should be addressed. E-mail: ccantor{at}sequenom.com

© 2007 by The National Academy of Sciences of the USA


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[Abstract] [Full Text] [PDF]