Direct, Programmable Detection of Epigenetic DNA Cytosine Modifications using TAL Effectors

摘要

The present invention relates to methods for the differentiation of a 5-hydroxymethyl modification of a cytosine residue of interest in a nucleic acid molecule from (i) a 5-methyl modification of said cytosine residue of interest o,r (ii) said unmodified cytosine residue of interest, said methods making use of the capability of transcription-activator-like effector (TALE) proteins to preferentially bind with strong affinity to nucleic acid sequences containing non-modified cytosine residues or 5-methyl modified cytosine residues, and to bind, if at all, with only strongly reduced affinity to nucleic acid sequences containing 5-hydroxymethyl modified cytosine residues. The present invention further relates to respective uses of TALE proteins for the differentiation of a 5-hydroxymethyl modification of a cytosine residue of interest in a nucleic acid molecule from (i) a 5-methyl modification of said cytosine residue of interest or (ii) said unmodified cytosine residue of interest.

主权项

1. A method for the differentiation of a 5-hydroxymethyl modification of a cytosine residue of interest in a nucleic acid molecule from
(i) a 5-methyl modification of said cytosine residue of interest or (ii) said unmodified cytosine residue of interest, said method comprising the steps of: (a) providing a transcription-activator-like effector (TALE) protein, said TALE protein being capable of binding in a sequence- and locus-specific manner to a region of said nucleic acid molecule that includes said cytosine residue of interest; and (b) determining whether said TALE protein binds to said region; wherein (i) a 5-hydroxymethyl modification of said cytosine residue of interest is present when said TALE protein does not bind to said region or does bind to said region with a strongly reduced affinity, and (ii) a 5-methyl modification of said cytosine residue of interest or said unmodified cytosine residue of interest is present when said TALE protein binds to said region.