# US 11,999,980 B2 · Non-specific nucleases from the genus Pseudomonas for use in facilitating flow of cells through a microfluidic channel

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- Number: US 11,999,980 B2
- Document ID: US11999980B2
- Status: Granted
- Kind: B2
- Application number: 17/264,748
- Filed: 2019-07-26
- Published: 2021-09-30
- Granted: 2024-06-04
- Earliest claimed: 2018-07-30
- Assignees: Miltenyi Biotec GmbH
- Applicants: Miltenyi Biotec B.V. & Co. KG
- Inventors: Skander Elleuche; Sarah Schmitz; Stefan Miltenyi; Volker Nolle
- Web page: https://patentgrep.com/patent/US11999980B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention provides a composition comprising an isolated polypeptide having non-specific nuclease (NSN) activity for degrading nucleic acids comprising or consisting of at least 70% amino acid sequence identity to SEQ ID NO:2, wherein said polypeptide has NSN activity in a solution of about 4° C., and a cation complexing agent such as EDTA. Said composition may have preferentially a neutral to basic pH. A kit comprising said composition and a method using said composition are also disclosed.

## Claims

### Claim 1

A method of preparing a cell suspension, comprising:
(a) obtaining a recombinant nuclease,
(b) incubating the cell suspension with the recombinant nuclease until nucleic acids present in the suspension and/or released from cells are removed, and then
(c) flowing the cells through a microfluidic channel;
wherein the recombinant nuclease comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2,
wherein the recombinant nuclease when present in a solution at 4° C. has non-specific nuclease (NSN) activity with a kcat value of at least 10 s−1, and
wherein removing nucleic acids from the suspension in step (b) prevents nucleic acids from clogging the microfluidic channel in step (c).

### Claim 2 (depends on 1)

The method of claim 1, wherein said microfluidic channel is part of a cell sorting apparatus.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amino acid sequence of the nuclease comprises at least one amino acid substitution, addition, or deletion compared with the amino acid sequence set forth in SEQ ID NO:2 wherein the nuclease has increased NSN activity compared with a nuclease having an amino acid sequence consisting of SEQ ID NO:2.

### Claim 4 (depends on 1)

The method of claim 1, wherein the incubating is performed at a temperature between 0° C. and 10° C.

### Claim 5 (depends on 1)

The method of claim 1, wherein the NSN activity has a kcat value of at least 200 s−1.

### Claim 6 (depends on 1)

The method of claim 1, wherein incubating is performed at a pH of 7 to 8.

### Claim 7 (depends on 1)

The method of claim 1, wherein the amino acid sequence of the nuclease comprises the amino acid sequence set forth in SEQ ID NO:3 or in SEQ ID NO:6.

### Claim 8

A method of preparing a cell suspension, comprising:
(a) obtaining a recombinant nuclease, and
(b) incubating the cell suspension with the recombinant nuclease until nucleic acids present in the suspension and/or released from cells are removed, thereby facilitating flow of the cells through a microfluidic channel;
wherein the recombinant nuclease contains an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2, and
wherein the recombinant nuclease when present in a solution at 4° C. has non-specific nuclease (NSN) activity with a kcat value of at least 100 s−1.

### Claim 9

A method of preparing a cell suspension, comprising:
(a) obtaining a recombinant nuclease, and
(b) incubating the cell suspension with the recombinant nuclease until nucleic acids present in the suspension and/or released from cells are removed, thereby facilitating flow of the cells through a microfluidic channel;
wherein the recombinant nuclease contains an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2;
wherein the recombinant nuclease when present in a solution at 4° C. has non-specific nuclease (NSN) activity with a kcat value of at least 10 s−1; and
wherein the method further comprises complexing cations that are present in the cell suspension before or during step (b).

### Claim 10 (depends on 9)

The method of claim 9, wherein cations in the suspension are complexed by adding a cation complexing agent to the cell suspension at a concentration between 0.1 mM and 20 mM.

### Claim 11 (depends on 10)

The method of claim 10, wherein the cation complexing agent is EDTA (ethylenediaminetetraacetic acid).

### Claim 12 (depends on 10)

The method of claim 10, wherein the cation complexing agent is selected from the group consisting of EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), DOTA (1,4,7,10 tetraazacyclododecane-1,4,7,10 tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), MGDA (methylglycine N,N-diacetic acid), GLDA (glutamate diacetate), NTA (nitrilotriacetic acid), IDS (iminodisuccinate), HEEDTA (N-(2-hydroxyethyl)ethylenediamine-N,N′,N′ triacetic acid), STPP (sodium tripolyphosphate), TTHA (triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid), phosphonates, DFOA (desferrioxamine), DMSA (2,3 di-mercaptosuccinic acid), penicillamine, and dimercaprol.

### Claim 13

A kit for facilitating flow of cells in a suspension through a microfluidic channel and/or to prepare cells in a suspension for cell sorting, wherein the kit comprises:
(i) a reagent solution that comprises a recombinant nuclease and a non-naturally occurring cation complexing agent,
wherein the nuclease comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2,
wherein the nuclease has non-specific nuclease (NSN) activity in the reagent solution at 4° C. has a kcat value of at least 100 s−1, and
(ii) instructions for using the solution for removing nucleic acids from the cell suspension to facilitate flow of the cells through the microfluidic channel;
wherein the amount and concentration of the recombinant nuclease in the solution is effective for degrading and removing nucleic acids from the cell suspension when combined and incubated at a temperature between 0° C. and 10° C.

### Claim 14 (depends on 13)

The kit of claim 13, wherein the non-naturally occurring cation complexing agent is present in the reagent solution at a concentration between 0.1 mM and 50 mM.

### Claim 15 (depends on 13)

A method of preparing a cell suspension, the method comprising:
(a) obtaining the kit of claim 13;
(b) incubating the cell suspension with the reagent solution from the kit at a temperature between 0° C. and 10° C. until nucleic acids present in the suspension and/or released from the cells are removed.