Granted Patent
B2
US 12,680,128 · App. 17/999,260 · Granted Jul 14, 2026
Compositions and methods for DNA cytosine carboxymethylation
Inventors:
Rahul Kohli (Penn Valley, PA); Tong Wang (Philadelphia, PA); Emily Schutsky (Waltham, MA)
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Abstract
Compositions and methods for carboxymethylation of cytosine containing DNA and applications thereof for direct sequencing of 5mC are disclosed.
Claims (30)
1 . An isolated genetically modified methyltransferase enzyme having carboxymethyltransferase activity, which catalyzes formation of 5-carboxymethylcytosine employing carboxy-S-adenosyl-L-methionine (CxSAM) as a substrate, said enzyme having an active site motif naturally comprising a polar amino acid residue that is situated adjacent to carbon 5 of a target cytosine present in a nucleic acid of interest, wherein said polar amino acid is substituted with a positively charged amino acid, wherein said enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and wherein position N374 of SEQ ID NO: 1 can be either K or R.
2 . The methyltransferase enzyme of claim 1 , wherein said enzyme comprises the amino acid sequence as set forth in SEQ ID NO: 1.
3 . The methyltransferase enzyme of claim 1 , wherein said enzyme further comprises one or more amino acid substitutions selected from:
a) substitution of one or both residues at T300 and E305 with S, A, G, Q, D, or N;
b) substitution of one or more residues A323, N306, and Y299 with a positively charged amino acid selected from K, R or H; and
c) substitution of S323 with A, G, K, R or H.
4 . A method for resolving unmethylated cytosine (C), 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in a polynucleotide sample, comprising:
(a) reacting a polynucleotide containing C, 5mC, and/or 5hmC with the methyltransferase enzyme of claim 1 , in the presence of carboxy-S-adenosyl-L-methionine (CxSAM) substrate, thereby labeling any unmodified C in said polynucleotide and rendering it resistant to deaminase action; wherein said 5hmC is also optionally glucosylated;
(b) contacting the polynucleotide of step (a) with a deaminase which deaminates 5mC and/or 5hmC; and
(c) analyzing said polynucleotide sample, to identify each of unmodified C, 5mC, and 5hmC present in said polynucleotide.
5 . The method of claim 4 , wherein said polynucleotide in said sample is fragmented or sheared prior to step (a), and sequence adapters containing modified cytosine bases resistant to deamination, are operably linked to said sheared or fragmented polynucleotide, and wherein said analyzing step of (c) is performed by sequencing.
6 . The method of claim 5 , wherein the sample of step (b) is amplified prior to the sequencing of step (c).
7 . The method of claim 4 , wherein said methyltransferase enzyme comprises the amino acid sequence as set forth in SEQ ID NO: 1, and said deaminase enzyme is APOBEC3A, and modified cytosine base is 5pyC.
8 . The method of claim 4 , wherein said polynucleotide is genomic DNA.
9 . The method of claim 4 , further comprising inclusion of methylated control polynucleotides.
10 . The method of claim 4 , wherein said polynucleotide is present in cell free DNA.
11 . The method of claim 4 , wherein said polynucleotide sample is obtained from cancer cells.
12 . The method of claim 4 , wherein said polynucleotide sample is isolated from the blood of a pregnant woman.
13 . The method of claim 4 , further comprising comparing with results obtained using bisulfite dependent 5mC+5hmC localization and ACE-seq 5hmC localization.
14 . A nucleic acid vector encoding the methyltransferase enzyme of claim 1 .
15 . A host cell with naturally occurring CxSAM and said host cell comprising the nucleic acid vector of claim 14 .
16 . The host cell of claim 15 , wherein said host cell is an E. coli cell.
17 . A kit comprising the methyltransferase enzyme of claim 1 , and CxSAM.
18 . The kit of claim 17 , wherein said kit further comprises a cytosine deaminase enzyme.
19 . The kit of claim 18 , wherein said cytosine deaminase enzyme is APOBEC3A.
20 . The kit of claim 19 , wherein said kit further comprises reagents and enzymes for cleaving or shearing DNA and optionally reagents for amplification of DNA.
21 . A method for identifying S-adenosyl-methionine (SAM) analogs which render cytosine residues present in a polynucleotide resistant to deaminase action, comprising;
a) reacting a polynucleotide containing C, 5mC, and/or 5hmC with the methyltransferase enzyme of claim 1 in the presence of substrates comprising SAM analogs;
b) isolating polynucleotides comprising modified C residues which are resistant to deaminase action, thereby identifying said SAM analog.
22 . The method of claim 21 , wherein said methylase enzyme comprises the amino acid sequence as set forth in SEQ ID NO: 1.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Apr 5, 2023
From: KOHLI, RAHUL; SCHUTSKY, EMILY; WANG, TONG
To: THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA
Reel/Frame 063225/0957 →
Continuity (2)
Provisional Application
63027254
· May 19, 2020
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