Compositions and methods for DNA cytosine carboxymethylation
Inventors
Kohli, Rahul • Wang, Tong • SCHUTSKY, Emily
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12680128-B2
Publication Date
2026-07-14
Expiration Date
Abstract
Compositions and methods for carboxymethylation of cytosine containing DNA and applications thereof for direct sequencing of 5mC are disclosed.
Core Innovation
The invention relates to an isolated genetically modified methyltransferase enzyme having carboxymethyltransferase activity that catalyzes formation of 5-carboxymethylcytosine using carboxy-S-adenosyl-L-methionine (CxSAM) as a substrate. The enzyme includes an active site motif naturally comprising a polar amino acid residue situated adjacent to carbon 5 of a target cytosine present in a nucleic acid of interest, and the polar amino acid is substituted with a positively charged amino acid.
The enzyme has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, with position N374 of SEQ ID NO: 1 able to be either K or R. The disclosure also provides embodiments and workflow concepts for direct methylation sequencing that use the carboxymethyltransferase to generate 5-carboxymethylcytosine in DNA.
This enables bisulfite-free, deaminase-based direct detection that distinguishes 5mC versus 5hmC. In the described approach, conversion products are further processed using deamination with APOBEC3A in related claim coverage and, in some embodiments, glucosylation of 5hmC using a beta-glucosyltransferase and UDP-glucose.
The disclosure further describes identification of 5cxmC, including LC-MS/MS identification of 5cxmC and assays using restriction-digest and in vitro/in vivo evaluations. The described sequencing context includes suitability for low-input and long-read scenarios and discusses third-generation sequencing signature behavior, such as SMRT kinetic signature (IPD) and nanopore sequencing signals, to support direct discrimination of cytosine modifications.
Claims Coverage
The independent claim covers a genetically modified, isolated carboxymethyltransferase enzyme with CxSAM-dependent formation of 5-carboxymethylcytosine, defined by an active-site polar-to-positively charged substitution adjacent to carbon 5, bounded by sequence identity to SEQ ID NO: 1, and a specific N374 (K or R) constraint. The claim set extends into additional residue constraints, sequence tethering, and downstream assay embodiments and kits, including a direct methylation sequencing approach that distinguishes C, 5mC, and 5hmC via methyltransferase labeling plus deamination and optional glucosylation.
CxSAM-dependent carboxymethyltransferase conversion to 5-carboxymethylcytosine
An isolated genetically modified methyltransferase enzyme having carboxymethyltransferase activity that catalyzes formation of 5-carboxymethylcytosine employing carboxy-S-adenosyl-L-methionine (CxSAM) as a substrate.
Active-site polar-to-positively charged substitution adjacent to carbon 5
An active site motif naturally comprising a polar amino acid residue situated adjacent to carbon 5 of a target cytosine in a nucleic acid of interest, wherein the polar amino acid is substituted with a positively charged amino acid.
Sequence identity to SEQ ID NO: 1 with N374 specified as K or R
An amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein position N374 of SEQ ID NO: 1 can be either K or R.
Distinguishing unmodified C, 5mC, and 5hmC by methyltransferase labeling and analysis after deamination and optional glucosylation
A method to resolve and identify unmodified cytosine (C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) in a polynucleotide sample by methyltransferase labeling of unmodified C using CxSAM, optionally glucosylating 5hmC, deaminating 5mC and/or 5hmC with a deaminase, and analyzing the resulting polynucleotide to distinguish each form present.
Use of APOBEC3A deaminase in the C/5mC/5hmC resolution workflow
The method specified to use a methyltransferase containing the amino acid sequence of SEQ ID NO: 1 together with the deaminase enzyme APOBEC3A, acting on a modified cytosine base that is 5pyC.
Kit including the carboxymethyltransferase enzyme and CxSAM
A kit that includes the methyltransferase enzyme together with CxSAM.
Overall, the claim coverage centers on an isolated genetically modified CxSAM-dependent carboxymethyltransferase defined by an active-site polar-to-positively charged substitution adjacent to carbon 5, bounded by sequence identity to SEQ ID NO: 1 and an N374 (K or R) constraint, and extends into a direct methylation sequencing workflow that distinguishes C, 5mC, and 5hmC using methyltransferase labeling, deamination, and optional glucosylation, with embodiments including kits containing the enzyme and CxSAM.
Stated Advantages
Enables bisulfite-free, deaminase-based direct detection that distinguishes 5mC versus 5hmC.
Documented Applications
Epigenetic profiling, including analysis involving cfDNA and cancer tissue-of-origin (reported examples include PDAC/NSCLC with KRAS).
Interested in licensing this patent?