Detection of 5-hydroxymethylcytosine by glycosylation
Inventors
Rao, Anjana • Tahiliani, Mamta • Koh, Kian Peng • Agarwal, Suneet • Iyer, Aravind
Assignees
Boston Childrens Hospital • US Department of Health and Human Services
Publication Number
US-9816986-B2
Publication Date
2017-11-14
Expiration Date
2029-09-28
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Abstract
Provided herein are methods and kits for detecting a modified cytosine.
Core Innovation
The invention provides novel methods and kits for detecting a modified cytosine, specifically 5-hydroxymethylcytosine, in nucleic acids. This is based on the identification of a novel catalytic activity of the TET family of enzymes, including TET1, TET2, TET3, and CXXC4, capable of converting 5-methylcytosine to 5-hydroxymethylcytosine by hydroxylation.
The problem addressed arises from the critical biological roles of DNA methylation and demethylation in mammalian development, cell differentiation, aging, and tumorigenesis, where aberrant methylation patterns are linked to diseases such as cancer. Existing methods are insufficient for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine, as 5-hydroxymethylcytosine is not recognized by standard 5-methylcytosine-specific proteins or antibodies, making detection and study challenging.
The invention further provides novel approaches for modulating the methylation status of cells, including improving reprogramming of somatic cells into pluripotent stem cells by enhancing the efficiency and rate of induced pluripotent stem cell generation through delivery or contact with catalytically active TET family enzymes or derivatives. It also offers methods to improve cloning efficiency via nuclear transfer by treating nuclei with TET family enzymes. Additionally, the invention provides methods for generating stable human regulatory Foxp3+ T cells via TET family enzyme treatment. It addresses detection methods using enzymatic conversion and antibody-based techniques that specifically bind 5-hydroxymethylcytosine or its derivatives, as well as assays employing enzymatic glycosylation of 5-hydroxymethylcytosine for improved detection and isolation.
Claims Coverage
The independent claims focus on methods for detecting 5-hydroxymethylcytosine using enzymatic glycosylation with labeled glucose derivatives, and the use of TET enzymes in this process.
Detection of 5-hydroxymethylcytosine by enzymatic glucosylation
A method for detecting 5-hydroxymethylcytosine in a mammalian nucleic acid by contacting it with a beta-glucosyltransferase enzyme that adds a labeled glucose molecule from a uridine diphosphate glucose donor substrate to 5-hydroxymethylcytosine, producing labeled glucosylated 5-hydroxymethylcytosine.
Use of TET enzyme in detection method
The method further comprises treating the contacted sample with a TET enzyme or catalytically active fragment comprising the specific catalytic sequence SEQ ID NO: 1, establishing the presence or formation of 5-hydroxymethylcytosine.
The independent claims disclose a novel enzymatic detection method for 5-hydroxymethylcytosine involving specific glycosyltransferase mediated labeling and the use of catalytically active TET enzymes to improve sensitivity and specificity of detection in mammalian DNA.
Stated Advantages
Provides novel tools and methods for detecting and distinguishing 5-hydroxymethylcytosine from 5-methylcytosine in DNA.
Improves methods for reprogramming somatic cells into induced pluripotent stem cells by increasing efficiency and rate through TET enzyme activity.
Enhances cloning efficiency in nuclear transfer protocols via TET enzyme treatment.
Enables improved production of stable human regulatory Foxp3+ T cells via modulation of DNA methylation status.
Offers improved cancer diagnostic and therapeutic methods by detecting and modulating TET enzyme activity and 5-hydroxymethylcytosine levels.
Documented Applications
Detection of 5-hydroxymethylcytosine in nucleic acids of biological samples for research and diagnostic purposes.
Enhancing reprogramming of somatic cells into induced pluripotent stem cells in vitro and in vivo.
Improving efficiency of cloning mammals by nuclear transfer or nuclear transplantation protocols.
Generation of stable human regulatory Foxp3+ T cells through TET family enzyme treatment.
Cancer diagnosis, prognosis, and treatment by evaluating TET enzyme activity and 5-hydroxymethylcytosine levels in patient samples.
Screening for compounds that modulate TET family enzyme activity for therapeutic use.
Stem cell therapy improvement by contacting stem cells with TET family enzymes or derivatives to modulate pluripotency and differentiation status.
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