Abstract
The goal of present paper is to develop a reliable DNA-based method for isolation of protein complexes bound to DNA (Isolation of DNA Associated Proteins: IDAP). We describe a robust and versatile procedure to pull-down chromatinized DNA sequences-of-interest by formation of a triple helix between a sequence tag present in the DNA and a complementary triple helix forming oligonucleotide (TFO) coupled to a desthiobiotin residue. Following optimization to insure efficient recovery of native plasmids via TFO probe in vitro, the procedure is shown to work under various experimental situations. For instance, it allows capture proteins associated to plasmids hosted in E. coli, and is also successfully applied to recovering nucleosomes in vitro opening many possibilities to study post translational modifications of histones in a genuine nucleosome context. Incubation in human nuclear extracts of a plasmid carrying a NF-κB model promoter is shown to pull-down a specific transcription factor. Finally, isolation of a specific locus from human genomic chromatin has been successfully achieved (Chromatin-of-Interest Fragment Isolation: CoIFI). In conclusion, the methodology can be implemented for capturing proteins that specifically bind to any sequence-of-interest, DNA adduct or secondary structure provided a short sequence tag for triple helix formation is located nearby.
Highlights
IntroductionCharacterization of the proteins involved in various DNA transactions (e.g., replication, repair, transcription, nucleosome dynamics) is an essential requirement for the proper understanding of how protein networks control gene expression in fundamental as well as in clinical areas
Characterization of the proteins involved in various DNA transactions is an essential requirement for the proper understanding of how protein networks control gene expression in fundamental as well as in clinical areas
Our goal is to develop a robust experimental workflow that makes use of the unique feature of triplex formation to capture, identify and characterize proteins assembled, in vivo or in vitro, on a given DNA sequence-of-interest
Summary
Characterization of the proteins involved in various DNA transactions (e.g., replication, repair, transcription, nucleosome dynamics) is an essential requirement for the proper understanding of how protein networks control gene expression in fundamental as well as in clinical areas For this purpose, numerous in vitro and in vivo experimental approaches have been developed. While biotinylated TFO probes with normal deoxyribonucleotides were shown to capture dsDNA in vitro[17], numerous efforts were deployed to further increase the stability of TFO-mediated triplexes. These improvements involved both chemically altered nucleotides (e.g., peptide nucleic acid (PNA), locked nucleic acid (LNA))[18,19] and the introduction of DNA intercalators (e.g., acridine, psoralen) linked to an extremity of the TFO molecule[20,21,22]. The approach can be applied to any DNA adduct or secondary structure (hairpins, quadruplexes, triplet repeats and so on)
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