EMSA-SELEX-seq method for analysis of binding site sequences in DNA-protein complexes

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The BOB1 protein (OBF1, OCA-B) is a transcriptional coactivator of two POU domain proteins — OCT1, expressed in all cells, and lymphoid-specific OCT2. The interaction of BOB1 with OCT1/2 plays an important role in the regulation of immune responses in both physiological and pathological contexts. BOB1 is known to form a ternary complex with OCT1/2 bound to DNA in monomeric and certain dimeric configurations, changing the sequence specificity of the binding. To analyze DNA sequences from these complexes, in this work we proposed the EMSA-SELEX-seq method, based on the separation of OCT/BOB1 complexes of various compositions in a non-denaturing polyacrylamide gel (EMSA) followed by the isolation and amplification of the oligonucleotides that they contain (SELEX). Based on several rounds of the enrichment followed by the NGS sequencing and bioinformatics analysis, the DNA sequences were determined and the relevance of this approach was confirmed. Thus, the proposed EMSA-SELEX-seq method allows the analysis of DNA sequences in DNA-protein complexes with varying dimensions of its protein components.

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Sobre autores

I. Nazarov

Institute of Cytology RAS

Autor responsável pela correspondência
Email: i.nazarov@incras.ru
Rússia, St. Petersburg, 194064

M. Gordeev

Institute of Cytology RAS

Email: i.nazarov@incras.ru
Rússia, St. Petersburg, 194064

A. Kuzmin

Institute of Cytology RAS

Email: i.nazarov@incras.ru
Rússia, St. Petersburg, 194064

D. Zilov

Institute of Cytology RAS

Email: i.nazarov@incras.ru
Rússia, St. Petersburg, 194064

E. Potapenko

University of Haifa

Email: i.nazarov@incras.ru

Institute of Evolution, Department of Evolutionary and Environmental Biology

Israel, Haifa, 3498838

A. Tomilin

Institute of Cytology RAS

Email: a.tomilin@incras.ru
Rússia, St. Petersburg, 194064

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