Methods for the epigenetic analysis of DNA, particularly cell-free DNA
Inventors
Arensdorf, Patrick A. • Spacek, Damek
Assignees
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Publication Number
US-12351859-B2
Publication Date
2025-07-08
Expiration Date
Abstract
Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.
Core Innovation
The invention relates to epigenetic analysis of cell-free DNA (cfDNA) by converting oxidized 5-methylcytosine residues to dihydrouracil (DHU) so they can be read as thymine after amplification and sequencing. In particular, oxidized 5-methylcytosine residues are selected from 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC), and conversion is achieved by contacting cfDNA with pyridine borane.
The invention further addresses conversion of 5-hydroxymethylcytosine (5hmC) to DHU without affecting 5-methylcytosine (5mC) residues. An oxidizing reagent converts 5hmC into oxidized 5hmC residues selected from 5caC and 5fC, without affecting 5mC, and thereafter the nucleic acid is contacted with pyridine borane to provide DHU in place of the at least one 5caC and/or 5fC.
For co-occurrence detection, the invention provides a method for identifying co-occurrence of 5mC and 5hmC in a single DNA fragment in a cell-free DNA sample. The method includes tagging 5hmC residues in fragmented, adapter-ligated cfDNA with an affinity tag enabling removal of the tagged 5hmC-containing fragments, removing the tagged fragments, oxidizing 5mC in the removed fragments to oxidized 5mC residues selected from 5caC and 5fC, treating with pyridine borane to convert the oxidized 5mC to DHU, and sequencing to identify removed fragments containing DHU as template fragments containing both 5mC and 5hmC.
Claims Coverage
The partial content provides three independent claims describing conversion of oxidized 5mC to DHU in cfDNA, conversion of 5hmC to DHU without affecting 5mC, and identification of 5mC/5hmC co-occurrence on a single cfDNA fragment. The inventive features across these claims focus on selective chemical conversion using pyridine borane and, for co-occurrence, selective enrichment of 5hmC-containing fragments via affinity tagging.
Pyridine borane conversion of oxidized 5mC in cfDNA to DHU
A method for converting an oxidized 5-methylcytosine (5mC) residue selected from 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC) to a dihydrouracil (DHU) residue in cell-free DNA (cfDNA) by contacting cfDNA containing at least one oxidized 5mC residue with pyridine borane.
Selective 5hmC-to-DHU conversion using oxidizing reagent followed by pyridine borane without affecting 5mC
A method for converting a 5-hydroxymethylcytosine (5hmC) residue in a nucleic acid to a DHU residue without affecting 5-methylcytosine (5mC) residues, comprising contacting the nucleic acid with an oxidizing reagent effective to convert at least one 5hmC residue to at least one oxidized 5hmC residue selected from 5caC, 5fC, and combinations thereof without affecting 5mC residues, and thereafter contacting the nucleic acid with pyridine borane to provide a DHU residue in place of the at least one 5caC and/or 5fC.
Affinity tagging enrichment to identify 5mC/5hmC co-occurrence in a single DNA fragment
A method for identifying co-occurrence of 5mC and 5hmC in a single DNA fragment in a cell-free DNA sample comprising tagging 5hmC residues with an affinity tag enabling removal of tagged 5hmC-containing DNA fragments, removing the tagged fragments, oxidizing 5mC in the removed tagged fragments to oxidized 5mC residues selected from 5caC and 5fC, treating the removed fragments with pyridine borane to convert the oxidized 5mC to DHU, and sequencing the removed fragments to identify any removed fragments containing DHU as template fragments containing both 5mC and 5hmC.
Across the independent claims, the core coverage is chemical conversion to DHU using pyridine borane: first converting oxidized 5mC residues in cfDNA to DHU, second converting 5hmC to DHU while not affecting 5mC, and third enabling fragment-level 5mC/5hmC co-occurrence identification by affinity tagging and removal of 5hmC-containing fragments followed by oxidation of 5mC and pyridine borane conversion to DHU with subsequent sequencing.
Stated Advantages
Reduces DNA degradation compared with bisulfite.
Avoids complete-conversion false positives arising from incomplete conversion associated with bisulfite.
Allows mild/one-pot processing without intermediate isolation.
Documented Applications
Epigenetic analysis of cell-free DNA (cfDNA) using conversion of oxidized 5-methylcytosine residues to DHU for amplification and sequencing readout.
Detection of 5mC/5hmC patterns in nucleic acid by converting 5hmC to DHU without affecting 5mC.
Identification of co-occurrence of 5mC and 5hmC in a single DNA fragment in a cell-free DNA sample via selective affinity tagging of 5hmC-containing fragments, pyridine borane conversion, and sequencing.
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