Direct, programmable detection of epigenetic DNA cytosine modifications using TAL effectors
First Claim
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) an 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, wherein a repeat variable diresidue (RVD) of said TALE protein whose position corresponds to the position of the cytosine residue of interest is a size reduced RVD; and
(b) determining whether said TALE protein binds to said region bydetecting a 5-hydroxymethyl modification of said cytosine residue of interest, wherein(i) said modification 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) said modification is absent when said TALE protein binds to said region,said method allowing for the direct detection of a 5-hydroxymethyl modification of said cytosine residue of interest in said nucleic acid molecule.
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Abstract
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 the cytosine residue of interest or (ii) an unmodified cytosine residue of interest, the 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 the cytosine residue of interest or (ii) the unmodified cytosine residue of interest.
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Citations
8 Claims
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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) an 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, wherein a repeat variable diresidue (RVD) of said TALE protein whose position corresponds to the position of the cytosine residue of interest is a size reduced RVD; and (b) determining whether said TALE protein binds to said region by detecting a 5-hydroxymethyl modification of said cytosine residue of interest, wherein (i) said modification 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) said modification is absent when said TALE protein binds to said region, said method allowing for the direct detection of a 5-hydroxymethyl modification of said cytosine residue of interest in said nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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Specification