# US 11,999,998 B2 · Modification of 5-methylcytosine catalyzed by Cmd1 enzyme and application thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,999,998 B2 - Document ID: US11999998B2 - Status: Granted - Kind: B2 - Application number: 16/327,521 - Filed: 2017-08-24 - Published: 2023-06-15 - Granted: 2024-06-04 - Earliest claimed: 2016-08-24 - Assignees: Center for Excellence In Molecular Cell Science, Chinese Academy of Sciences - Applicants: Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences - Inventors: Guoliang Xu - Web page: https://patentgrep.com/patent/US11999998B2 ## CPC - C12Q 1/6869: Methods for sequencing - C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)} - C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides - C12Q 1/68: involving nucleic acids - C12Q 1/6827: for detection of mutation or polymorphism - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} ## Abstract The present invention relates to a novel modification of a 5-methylcytosine nucleic acid catalyzed by a Cmd1 enzyme and an application thereof. The present inventors have for the first time discovered Cmd1, a 5mC-modifying enzyme, which can link a glyceryl group to a 5mC methyl carbon of a methylated nucleic acid through a carbon-carbon single bond, and this is a new epigenetic modification. ## Claims ### Claim 1 A method for adding a glyceryl group to a methyl group of 5-methylcytosine of a methylated nucleic acid, comprising: treating the methylated nucleic acid with a Cmd1 enzyme, so that the glyceryl group is added to the methyl group of 5-methylcytosine of the methylated nucleic acid; the Cmd1 enzyme comprises: (a) a polypeptide of an amino acid sequence of SEQ ID NO: 1; (b) a polypeptide derived from (a), being formed by subjecting the amino acid sequence of SEQ ID NO: 1 to substitution, deletion or addition of one or more amino acid residues, and having a function of the polypeptide of (a); or (c) a polypeptide derived from (a), having more than 60% homology to the polypeptide sequence defined in (a) and having a function of the polypeptide of (a); and in the sequences defined in (b) and (c), based on the amino acid sequence of SEQ ID NO: 1, the 330th position is A, the 345th position is H, the 347th position is D, and the 350th position is D. ### Claim 2 (depends on 1) The method of claim 1, wherein a glyceryl group-containing compound is used as a glyceryl group donor. ### Claim 3 (depends on 2) The method of claim 2, wherein the glyceryl group-containing compound is vitamin C or an analogue thereof. ### Claim 4 (depends on 2) The method of claim 2, wherein the glyceryl group-containing compound is vitamin C or an analogue thereof; and the glyceryl group is a glyceryl group on vitamin C or an analogue thereof. ### Claim 5 (depends on 1) The method of claim 1, wherein in a reaction system in which the methylated nucleic acid is treated with the Cmd1 enzyme, a ferrous ion is present as a cofactor. ### Claim 6 (depends on 1) The method of claim 1, wherein the methylated nucleic acid is treated with the Cmd1 enzyme, thereby forming a product selected from the following: ### Claim 7 (depends on 1) The method of claim 1, wherein the Cmd1 enzyme is a Cmd1 enzyme derived from algae. ### Claim 8 A method for adding a glyceryl group to a methyl group of 5-methylcytosine of a methylated nucleic acid, comprising: treating the methylated nucleic acid with an up-regulator of a Cmd1 enzyme, so that the glyceryl group is added to the methyl group of 5-methylcytosine of the methylated nucleic acid; the Cmd1 enzyme comprises: (a) a polypeptide of an amino acid sequence of SEQ ID NO: 1; (b) a polypeptide derived from (a), being formed by subjecting the amino acid sequence of SEQ ID NO: 1 to substitution, deletion or addition of one or more amino acid residues, and having a function of the polypeptide of (a); or (c) a polypeptide derived from (a), having more than 60% homology to the polypeptide sequence defined in (a) and having a function of the polypeptide of (a); and in the sequences defined in (b) and (c), based on the amino acid sequence of SEQ ID NO: 1, the 330th position is A, the 345th position is H, the 347th position is D, and the 350th position is D; the up-regulator of the Cmd1 enzyme is an expression vector expressing the Cmd1 enzyme. ### Claim 9 (depends on 8) The method of claim 8, wherein the glyceryl group is attached to a carbon atom of the methyl group of 5-methylcytosine via a carbon-carbon single bond. ### Claim 10 (depends on 3) The method of claim 3, wherein the vitamin C analogue comprises dehydroascorbic acid. ### Claim 11 (depends on 4) The method of claim 4, wherein the vitamin C analogue comprises dehydroascorbic acid.