# US 12,215,378 B2 · RNA identity method using RNAse H digestion and size fractionating

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- Number: US 12,215,378 B2
- Document ID: US12215378B2
- Status: Granted
- Kind: B2
- Application number: 16/637,935
- Filed: 2018-08-10
- Published: 2020-10-15
- Granted: 2025-02-04
- Earliest claimed: 2017-08-11
- Assignees: Glaxosmithkline Biologicals SA
- Applicants: Glaxosmithkline Biologicals SA; Prakash Koodathingal; Ying Zhang
- Inventors: Prakash Koodathingal; Ying Zhang
- Web page: https://patentgrep.com/patent/US12215378B2

## CPC

- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/55566: {Emulsions, e.g. Freund's adjuvant, MF59}
- 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
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Methods, compositions, kits, and uses are provided herein for detecting a subject nucleic acid in various samples.

## Claims

### Claim 1

A process of manufacturing a vaccine product comprising a subject RNA, the process comprising the steps of:
(a) manufacturing an RNA from a template DNA and obtaining a sample of the RNA;
(b) confirming the identity of the RNA in the sample as that of the subject RNA using a method comprising the steps of:
(A) contacting the sample with a query nucleic acid consisting of a DNA oligonucleotide complementary to a portion of the subject RNA under conditions that the query DNA oligonucleotide specifically hybridizes with the subject RNA vaccine product, when present, to form a hybrid duplex, wherein the formation of the hybrid duplex is carried out at a temperature between 0-10° C.;
(B) adding an enzyme consisting of RNAse H to the sample under conditions that facilitate specific cleavage of the subject RNA vaccine product when the hybrid duplex is present;
(C) size fractionating of the sample by denaturing agarose gel electrophoresis and determining the presence of cleaved subject RNA vaccine product by detecting all cleavage products of the subject RNA vaccine product,
wherein said method can distinguish between a subject RNA vaccine product and a RNA sharing at least 80% sequence homology,
wherein the identity of the RNA is confirmed as that of the subject RNA where (i) all cleavage products of the subject RNA vaccine product are of predicted size and (ii) contaminating template DNA uncleaved products are not detected;
and
(D) utilizing the vaccine product if the identity of the RNA is confirmed.

### Claim 2 (depends on 1)

The method of claim 1, wherein the query nucleic acid has a length of 12, or more, nucleotides.

### Claim 3 (depends on 1)

The method of claim 1, wherein the molar ratio of the query nucleic acid to subject nucleic acid is between 50:1 and 10000:1, inclusive.

### Claim 4 (depends on 3)

The method of claim 3, wherein the molar ratio of the query nucleic acid to subject nucleic acid is about 1000:1.

### Claim 5 (depends on 1)

The method of claim 1, wherein the query nucleic acid comprises between 40-60% G-C content, inclusive.

### Claim 6 (depends on 5)

The method of claim 5, wherein the query nucleic acid comprises about 50% G-C content.

### Claim 7 (depends on 1)

The method of claim 1, wherein the formation of the hybrid duplex is carried out for a period of time between 1-60 minutes.

### Claim 8 (depends on 1)

The method of claim 1, wherein the specific cleavage of the subject nucleic acid is carried out at a temperature of between 0-50° C., inclusive.

### Claim 9 (depends on 8)

The method of claim 8, wherein the specific cleavage of the subject nucleic acid is carried out at a temperature of about 37° C.

### Claim 10 (depends on 1)

The method of claim 1, wherein the products are not subject to a sequencing reaction.

### Claim 11 (depends on 1)

The method of claim 1, wherein the subject RNA encodes a polypeptide of interest.