# US 11,999,768 B2 · Mutant Uracil DNA glycosylase with improved thermal sensitivity

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- Number: US 11,999,768 B2
- Document ID: US11999768B2
- Status: Granted
- Kind: B2
- Application number: 17/264,509
- Filed: 2020-10-22
- Published: 2022-12-15
- Granted: 2024-06-04
- Earliest claimed: 2020-10-22
- Assignees: Enzynomics Co. Ltd.; Daegu Gyeongbuk Institute of Science and Technology
- Applicants: Enzynomics Co. Ltd.; Daegu Gyeongbuk Institute of Science and Technology
- Inventors: Yong Keol Shin; Jeongyeon Yoon; Ki Hoon Nam; Seulki Lim; Wookyung Yu; Seongjun Park; Juhwan Lee; Sangyeol Kim; Moo Seok Kang; Minjae Seo
- Web page: https://patentgrep.com/patent/US11999768B2

## CPC

- C07K 14/245: Escherichia (G)
- C12N 9/2497: {hydrolysing N- glycosyl compounds (3.2.2)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Y 302/02027: Uracil-DNA glycosylase (3.2.2.27)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- C12Y: ENZYMES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Provided is a mutant UDG having improved thermal sensitivity compared to a wild-type UDG. The mutant UDG of the presently claimed subject matter having a high thermal sensitivity has no inhibitory effect on the PCR reaction and thus can be advantageously used for the development of PCR/qPCR Premix and particularly PCR diagnostic kits employing UDG which requires the use of relatively low temperature in melting and amplification steps.

## Claims

### Claim 1

An isolated UDG (Uracil DNA Glycosylase) polypeptide from Escherichia coli consisting of the amino acid sequence set forth in SEQ ID NO: 1 having at least one amino acid substitution selected from the group consisting of E4A, W7A, E13A, Q16A, Y19A, D43X, F48A, F50A, E52A, H67A, K57A, Q71A, H73A, P87A, L96A, E112A, L121A, H134A, E142A, F144A, R156A, F161A, L162A, W164A, H180A, L183A, H202A, G214E, G214W, G214R, W220A, and L224A, in which the number indicates the position of the substituted amino acid and the amino acids are indicated as a single letter code and X indicates any amino acids, in which the codes on the left and right sides of the position indicate a wild type and substituted residues, respectively,
wherein the UDG has an improved thermal sensitivity in comparison to a wild type UDG from E. coli,
wherein the X of D43X is A, C, G, K, H, I, P, R, V or W.

### Claim 2 (depends on 1)

The isolated UDG of claim 1, wherein the UDG polypeptide comprises at least one substitution selected from the group consisting of D43A, D43C, D43H, D43R, D43V, D43W and K57A.

### Claim 3 (depends on 1)

The isolated UDG of claim 1, wherein the UDG polypeptide further comprises E157A or E215A substitution.

### Claim 4 (depends on 1)

The isolated UDG of claim 1, wherein the UDG polypeptide comprises a combination of at least two substations-substitutions and is selected from the group consisting of D43A/K57A, D43A/E157A, D43A/E215A, D43A/K57A/E157A, D43A/E157A/E215A, and D43A/K57A/E157A/E215A.

### Claim 5 (depends on 1)

A kit for removing nucleic acid contaminants in RT (Reverse Transcription), PCR (Polymerase Chain Reaction) or RT-PCR or RT-PCR reaction mixture, comprising the UDG polypeptide according to claim 1.

### Claim 6 (depends on 1)

A premix composition for PCR comprising the UDG polypeptide according to claim 1, a PCR polymerase and a buffer necessary for a PCR reaction.

### Claim 7 (depends on 1)

A premix composition for RT comprising the UDG polypeptide according to claim 1, a reverse transcriptase and a buffer necessary for a RT reaction.

### Claim 8 (depends on 1)

A premix composition for RT-PCR comprising the UDG polypeptide according to claim 1, a PCR polymerase, a reverse transcriptase and a buffer necessary for a RT-PCR reaction.

### Claim 9 (depends on 1)

A nucleic acid encoding the UDG polypeptide according to claim 1.

### Claim 10 (depends on 9)

A vector comprising the nucleic acid of claim 9.

### Claim 11 (depends on 10)

A prokaryotic cell comprising the vector of claim 10.