RNA identity method using RNAse H digestion and size fractionating

Patent metadata

Field	Value
Publication number	US-12215378-B2
Application number	US-201816637935-A
Country	US
Kind code	B2
Filing date	Aug 10, 2018
Priority date	Aug 11, 2017
Publication date	Feb 4, 2025
Grant date	Feb 4, 2025

Abstract

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

First claim

The invention claimed is:

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.

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

3. 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.

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

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

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

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

8. 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.

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

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

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

Assignees

• Glaxosmithkline Biologicals Sa
• Koodathingal Prakash
• Zhang Ying

Inventors

• Koodathingal Prakash
• Zhang Ying

Classifications

• A61K2039/55566

  Emulsions, e.g. Freund's adjuvant, MF59 · CPC title

• A61K2039/53

  DNA (RNA) vaccination · CPC title

• A61K39/00

  Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) · CPC title

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/683 Primary

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title