Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Sep 25;33(17):e141.
doi: 10.1093/nar/gni139.

Combining SELEX with quantitative assays to rapidly obtain accurate models of protein-DNA interactions

Jiajian Liu  1 , Gary D Stormo
Affiliations

Combining SELEX with quantitative assays to rapidly obtain accurate models of protein-DNA interactions

Jiajian Liu et al. Nucleic Acids Res. .

Abstract

Models for the specificity of DNA-binding transcription factors are often based on small amounts of qualitative data and therefore have limited accuracy. In this study we demonstrate a simple and efficient method of affinity chromatography-SELEX followed by a quantitative binding (QuMFRA) assay to rapidly collect the data necessary for more accurate models. Using the zinc finger protein EGR as an e.g. we show that many bindings sites can be obtained efficiently with affinity chromatography-SELEX, but those sequences alone provide a weight matrix model with limited accuracy. Using a QuMFRA assay to determine the quantitative relative affinity for only a subset of the sequences obtained by SELEX leads to a much more accurate model. Application of this method to variants of a transcription factor would allow us to generate a large collection of quantitative data for modeling protein-DNA interactions that could facilitate the determination of recognition codes for different transcription factor families.

PubMed Disclaimer

Figures

Figure 1
Figure 1
In vitro selection for Zif268 finger 1 with the affinity chromatography-SELEX procedure. (A) The DNA template used for in vitro selection in this study. The fixed flanking sequences bind to the PCR primers and contain restriction sites for cloning. The capitalized sequence in the center is the consensus for interacting with fingers 2 and 3 of Zif268. The ‘xxxx’ are the randomized positions. (B) The sequences for 14 selected DNA sites obtained from the first-round of selection; (C) The sequences for 22 selected DNA sites obtained from the second-round of selection.
Figure 2
Figure 2
SELEX based model for representing DNA specificity for Zif268 finger 1. (A) The frequency matrix for Zif268 finger 1 that was obtained from the alignment of all 36 sites shown in Figure 1. (B) The weight matrix for Zif268 finger 1 that was obtained from the frequency matrix. (C) The sequence logo for Zif268 finger 1 from the weight matrix. For the logo logarithms were converted to base 2 to display the results in bits.
Figure 3
Figure 3
Binding model from quantitative data. (A) The weight matrix with the optimum parameters obtained by multiple regression on the binding affinity data. (B) The sequence logo for Zif268 finger 1 from that weight matrix.
Figure 4
Figure 4
Strategy for obtaining quantitative data for a family of TFs. For any specific TF the SELEX data followed by the QuMFRA assay will provide the necessary information for a quantitative modeling of its specificity. Then creating variants in the TF sequence and performing the same experimental steps will allow for the development of models in which both the protein sequence and the binding site sequences are variable. Such a model constitutes a ‘probabilitistic recognition code’ that can be used to predict binding affinities for any combination of binding site and TF sequence from that family (25,26).
See this image and copyright information in PMC

References

    1. Stormo G.D. DNA binding sites: representation and discovery. Bioinformatics. 2000;16:16–23. - PubMed
    1. Matys V., Fricke E., Geffers R., Gossling E., Haubrock M., Hehl R., Hornischer K., Karas D., Kel A.E., Kel-Margoulis O.V., et al. TRANSFAC: transcriptional regulation, from patterns to profiles. Nucleic Acids Res. 2003;31:374–378. - PMC - PubMed
    1. Sandelin A., Alkema W., Engstrom P., Wasserman W.W., Lenhard B. JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 2004;32:D91–D94. - PMC - PubMed
    1. Alexander M.K., Bourns B.D., Zakian V.A. One-hybrid systems for detecting protein–DNA interactions. Methods Mol. Biol. 2001;177:241–259. - PubMed
    1. Ren B., Robert F., Wyrick J.J., Aparicio O., Jennings E.G., Simon I., Zeitlinger J., Schreiber J., Hannett N., Kanin E., et al. Genome-wide location and function of DNA binding proteins. Science. 2000;290:2306–2309. - PubMed

Publication types

MeSH terms