EP-4739797-A1 - STABILISATION OF PHI29 POLYMERASE

Abstract

The present invention provides a method of stabilising phi29 DNA polymerase by contacting the phi29 DNA polymerase with a stabilisation oligonucleotide protected from 3' exonuclease degradation. The phi29 polymerase exhibits increased temperature stability in the presence of the stabilisation oligonucleotide, in comparison to when it is absent. The method involves preparing a composition comprising phi29 DNA polymerase and a stabilisation oligonucleotide, comprising one or more modified nucleotides. The composition can be utilised in methods of performing a polymerase reaction, replicating a nucleic acid, and detecting a target nucleic acid or analyte in a sample. The composition is of particular use in rolling circle amplification reactions, wherein the target is generated in a proximity ligation assay, particularly a circularised padlock probe.

Inventors

• KLAESSON, Axel
• ELOFSSON, Hampus

Assignees

• Navinci Diagnostics AB

Dates

Publication Date

20260513

Application Date

20240705

Claims (20)

1. 1. A method of stabilising phi29 DNA polymerase, comprising contacting said phi29 DNA polymerase with a stabilisation oligonucleotide, wherein said stabilisation oligonucleotide is protected from 3’ exonuclease degradation.
2. 2. The method of claim 1 , wherein said method comprises preparing a composition comprising phi29 DNA polymerase and the stabilisation oligonucleotide.
3. 3. A method of performing a polymerase reaction, said method comprising contacting a sample comprising a target nucleic acid with a composition comprising phi29 polymerase and a stabilisation oligonucleotide, and allowing a polymerisation reaction to take place, wherein the stabilisation oligonucleotide is protected from 3’ exonuclease degradation.
4. 4. A method of replicating a nucleic acid, said method comprising contacting a target nucleic acid with a composition comprising phi29 polymerase and a stabilisation oligonucleotide, wherein the stabilisation oligonucleotide is protected from 3’ exonuclease degradation.
5. 5. A method of detecting a target nucleic acid in a sample, said method comprising: (i) contacting the sample with a composition comprising phi29 polymerase and a stabilisation oligonucleotide, and allowing the phi29 polymerase to perform a polymerase reaction using the target nucleic acid as template, wherein the stabilisation oligonucleotide is protected from 3’ exonuclease degradation; (ii) detecting the product of the polymerase reaction to detect the target nucleic acid.
6. 6. The method of claim 5, wherein said target nucleic acid is an analyte nucleic acid in the sample, or a copy or amplicon thereof.
7. 7. The method of claim 5, wherein said target nucleic acid is a reporter nucleic acid molecule which is indicative of a target analyte in the sample.
8. 8. The method of any one of claims 3 to 6 or 8, wherein the target nucleic acid is a nucleic acid molecule generated as a detection assay reaction product in a detection assay for detection of a target analyte in a sample.
9. 9. A method of detecting a target analyte in a sample, wherein a detection assay is performed to detect said analyte and said assay generates a target nucleic acid molecule in situ which is detected to detect said analyte, said method comprising: (i) after generation of said target nucleic acid molecule in said sample, contacting said sample with a composition comprising phi29 polymerase and a stabilisation oligonucleotide, and allowing the phi29 polymerase to perform a polymerase reaction using the target nucleic acid molecule as template, wherein the stabilisation oligonucleotide is protected from 3’ exonuclease degradation; (ii) detecting the product of the polymerase reaction to detect the target nucleic acid molecule and thereby the target analyte.
10. 10. The method of any one of claims 2 to 9, wherein the composition is maintained at a temperature above freezing for a period of at least 15 minutes, or more particularly at least 1 hour, prior to use, or prior to said contacting.
11. 11. The method of any one of claims 1 to 10, wherein the stabilisation oligonucleotide has a length of at least 8 or at least 10 nucleotides.
12. 12. The method of any one of claims 1 to 11, wherein the stabilisation oligonucleotide comprises one or more nucleotides with 2’ sugar modifications and/or one or more modified internucleotide linkages.
13. 13. The method of claim 12, wherein the 2’ sugar modification is 2’-O-methyl or 2’ MOE.
14. 14. The method of any one of claims 2 to 13, wherein the composition further comprises dNTPs and a buffer.
15. 15. The method of any one of claims 3 to 14, wherein the target nucleic acid is a circular DNA molecule.
16. 16. The method of any one of claims 3 to 15, wherein the polymerase or replication reaction is an amplification reaction.
17. 17. The method of claim 16, wherein the amplification reaction is rolling circle amplification (RCA).
18. 18. The method of any one of claims 3 to 17, wherein the target nucleic acid is a ligation product, including a circular nucleic acid molecule which has been circularised by ligation.
19. 19. The method of claim 17, wherein the ligation product is generated in a proximity ligation assay (PLA), optionally an in situ PLA (isPLA).
20. 20. The method of any one of claims 18 or 19, wherein the target nucleic acid is a ligated probe.

Description

Stabilisation of Phi29 polymerase Field The present disclosure and invention lie generally in the field of nucleic acid polymerisation, and particularly in the use of the polymerase enzyme phi29, especially its use in the detection of nucleic acids. In particular, the present disclosure and invention relate to the stabilisation of phi29 preparations for such use, and the provision of stabilised preparations of phi29. Background The detection of target nucleic acid sequences has applications in many different fields, including notably clinically, for personalised medicine and in the diagnosis, prognosis and/or treatment of disease, such as cancer, infectious diseases and inherited or genetic disorders, as well as in research and biosecurity. The target nucleic acid sequence may be a target analyte, i.e. it may be contained in a nucleic acid molecule present in a sample, which itself is the target to be detected, for example mRNA, or a copy or amplicon thereof. Alternatively, it may be a nucleic acid molecule generated as a proxy, or signal, or in other words, as a reporter for a target analyte in a sample, which may be a nucleic acid or another molecule. Nucleic acids are typically used or generated as reporter molecules in the detection of proteins, for example in the case of immunoPCR or immunoRCA methods, or in proximity assays using proximity probes comprising antibodies as binding domains, conjugated to nucleic acid domains which interact when the probes are in proximity, for example by ligation and/or hybridisation and extension, to generate a nucleic acid molecule which is detected. Such nucleic acid detection assays frequently involve amplification, and particularly rolling circle amplification (RCA). RCA is an isothermal amplification technique requiring a circular amplification template. Amplification of the circular template provides a concatenated RCA product (RCP), comprising multiple copies of a sequence complementary to that of the amplification template. Such a concatemer typically forms a ball or “blob”, which may readily be visualised and detected, and thus RCA-based assays have been adopted for the detection of nucleic acids, and indeed, more generally, as reporter systems for the detection of any target analyte. Both target nucleic acids, which may themselves be circularised directly, or probes, e.g. padlock probes, or reporter nucleic acids more generally may provide template nucleic acid circles for RCA (for example as used in immunoRCA reactions, or as generated in proximity ligation assays). RCA requires a strand-displacing polymerase which is able to displace the synthesised strand (extended from the primer hybridised to the circular template) and “roll” along the circular template, and the polymerase enzyme almost universally used for this purpose is phi29 DNA polymerase, from Bacillus subtilis phage phi29. The uses of phi29 DNA polymerase (also referred to as phi29 polymerase or phi29 herein) are not, however, limited to RCA and it has applications in other protocols and processes using polymerase extension reactions. In some instances, it is desirable to automate protocols utilising phi29. Automation may require that the phi29 reaction mix is prepared at the start of the automation protocol and sits at ambient temperature before being added to the interrogated sample. This includes, for example, the common tissue slide automation instruments (“autostainers”), which are used for analysis of tissue samples on glass slides, such as e.g. the Leica Bond, Lunaphore Comet, Roche Ventana or Dako Omnis instruments; phi29 is commonly used to amplify detection signals in such tissue samples. Tissue slide instruments typically require that all reagents are loaded before the instrument run, and do not cool the reagents below ambient temperature. Unfortunately, phi29 DNA polymerase demonstrates limited stability, or halflife, at temperatures above freezing, including ambient or elevated temperatures. This limits the use of phi29-based detection protocols in such instruments. Since, as noted above, phi29-based reactions, particularly RCA, have many advantages making them attractive options as detection methodologies, it is desirable to find a solution to this problem of limited stability. Whilst phi29 mutants and variants have been developed which exhibit extended half-life at elevated temperatures, a need still exists for other or improved methods of stabilising phi29, allowing its use in protocols and instruments which operate, or hold reagents, at ambient or higher temperatures for extended periods. Summary We propose the addition of an inert, 3’ exonuclease-protected oligonucleotide to the phi29 enzyme reaction mix in order to stabilise phi29 polymerase, if it needs to be stored or kept at above freezing for an extended period of time. The oligonucleotide is referred to herein as a “stabilisation oligonucleotide” (“stabilisation oligo”), and it creates a “walk-away” solution for reaction