Methods for identifying hydroxylated bases
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-10793899-B2
Publication Date
2020-10-06
Expiration Date
2029-09-28
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Abstract
The present invention provides methods for performing an imaging method to detect a moiety associated with labeled hydroxymethylated bases in nucleic acid sequences, such as detecting a moiety associated with glucosylated 5-hydroxmethylated cytosines.
Core Innovation
The invention provides methods for performing an imaging method to detect a moiety associated with labeled hydroxymethylated bases in nucleic acid sequences, particularly the detection of a moiety associated with glucosylated 5-hydroxymethylated cytosines. The invention is based upon identification of a novel and surprising catalytic activity of the TET proteins (TET1, TET2, TET3, and CXXC4) capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.
The invention addresses the problem of lack of prior methods to detect 5-hydroxymethylcytosine in genomes, since 5-hydroxymethylcytosine is not recognized by 5-methylcytosine binding protein MeCP2 or specific antibodies directed against 5-methylcytosine, making its detection and mapping in genomic DNA difficult. The invention also resolves challenges in improving reprogramming of somatic cells into pluripotent stem cells and in diagnosing and treating diseases associated with DNA methylation abnormalities, such as cancers and myeloid disorders, by regulating and detecting DNA methylation status using TET family enzymes.
Claims Coverage
The patent includes one independent claim focusing on a method and device for imaging nucleic acids comprising glucosylated 5-hydroxymethylated cytosines from extracellular fluid samples. Two inventive features are extracted.
Imaging method to detect glucosylated 5-hydroxymethylated cytosine
A method comprising performing an imaging method on nucleic acid from an extracellular fluid sample to detect a moiety associated with glucosylated 5-hydroxymethylated cytosine in the nucleic acid, where the moiety may comprise a fluorophore or biotinyl group.
Imager with nucleic acid containing glucosylated 5-hydroxymethylated cytosine
An imager comprising a nucleic acid or amplicon thereof containing glucosylated 5-hydroxymethylated cytosine from an extracellular fluid sample, wherein the glucosylated 5-hydroxymethylated cytosine is associated with a moiety such as a fluorophore or biotinyl group.
The inventive features relate to specific imaging techniques to detect and isolate glucosylated 5-hydroxymethylated cytosine in nucleic acids from extracellular fluid, including moiety labeling and use of imaging devices with labeled nucleic acids.
Stated Advantages
Improved modulation and regulation of cellular differentiation and pluripotency status.
Enhanced methods for reprogramming somatic cells into pluripotent stem cells with increased efficiency and rate.
Novel sensitive detection methods for 5-hydroxymethylcytosine, resolving limitations of prior 5-methylcytosine detection techniques.
Methods to diagnose and monitor hematologic malignancies and cancers associated with TET family mutations.
Tools for efficient cloning by nuclear transfer with improved efficiency.
Documented Applications
Reprogramming somatic cells to induced pluripotent stem cells (iPS cells) with higher efficiency and rate using catalytically active TET family enzymes.
Generation of stable human regulatory Foxp3+ T cells by delivering catalytically active TET family enzymes to human T cells, optionally with cytokines like TGF-beta.
Improving efficiency of cloning mammals by nuclear transfer or nuclear transplantation by contacting donor nuclei with TET family enzymes.
Detection of glucosylated 5-hydroxymethylated cytosine nucleic acids in extracellular fluid samples for diagnostic imaging, including cancer diagnosis.
Screening for agents that modulate TET enzyme activity for potential therapeutic applications, including cancer treatment.
Kits comprising catalytically active TET family enzymes and associated reagents for use in reprogramming, detection of hydroxymethylcytosine, and cloning enhancement.
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