Analyzing RNA-DNA Triplex Formation in Chromatin.

Methods in Molecular Biology
Rodrigo Maldonado, Gernot Längst

Abstract

A significant fraction of non-coding RNAs (ncRNAs) is associated with chromatin, shown to regulate gene expression and to organize nuclear architecture. Mechanisms of direct and indirect RNA-chromatin interactions have been described, including the sequence-specific formation of triple helix structures. Triplexes are formed by the sequence-specific binding of RNA to the bases located in the major groove of DNA. We recently showed that triplexes do exist in the context of cellular chromatin and that these structures are stabilized by the histone H3 tail of adjacent nucleosomes. The in vitro characterization of the specificity and binding affinity of triplex sequences next to nucleosomes are essential parameters to identify potential sites of RNA-chromatin interaction in vivo. Here we provide a detailed protocol to determine the influence of nucleosome positioning on triple helix formation. This assay allows the comparative quantification of triplex formation and specificity for triplex targeting sequences relative to the spatial nucleosome position.

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