WO2025008523A1

WO2025008523A1 - Stabilisation of phi29 polymerase

Info

Publication number: WO2025008523A1
Application number: PCT/EP2024/069042
Authority: WO
Inventors: Axel KLAESSON; Hampus ELOFSSON
Original assignee: Navinci Diagnostics AB
Current assignee: Navinci Diagnostics AB
Priority date: 2023-07-05
Filing date: 2024-07-05
Publication date: 2025-01-09
Anticipated expiration: 2026-01-05
Also published as: GB202310278D0

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.

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 mixes, or reaction buffers, requiring phi29 enzyme stability. Whilst the stabilisation oligonucleotide is useful for protecting aqueous or liquid preparations of Phi29, it may also find utility in stabilising freeze-dried preparations, notably stabilising the enzyme in the course of preparation of compositions for freeze-drying and/or in reconstituting freeze-dried preparations. In particular it is useful to include the stabilisation oligonucleotide in compositions for freeze-drying.

Accordingly, in a first aspect provided herein is 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.

Such a stabilisation oligonucleotide may be referred to as a 3’ exonuclease- protected oligonucleotide. It is an oligonucleotide comprising one or more modifications which protect it from 3’ exonuclease degradation.

It will be understood that in this context, the contact between the phi29 polymerase and the stabilisation oligonucleotide is maintained. Thus, the phi29 is provided, or kept, in contact with the stabilisation oligonucleotide.

Accordingly, in particular the method comprises providing said phi29 polymerase in a composition, or preparation, comprising the stabilisation oligonucleotide.

In other words, this aspect can be seen to provide a method of stabilising phi29 polymerase by providing it, or formulating it, in combination (or in admixture) with a stabilisation oligonucleotide, wherein said stabilisation oligonucleotide is protected from 3’ exonuclease degradation.

In a second aspect, provided herein is use of an oligonucleotide as a stabilising agent to stabilise phi29 polymerase, wherein the oligonucleotide (referred to herein as a stabilisation oligonucleotide) is protected from 3’ exonuclease degradation.

More particularly, the stabilisation oligonucleotide is used to stabilise the phi29 polymerase in a preparation, or composition, comprising or containing the phi29 polymerase. In other words, the stabilisation oligonucleotide is used to stabilise a composition, or preparation of phi29 polymerase.

In a third aspect, provided herein is a composition comprising phi29 polymerase and a stabilisation oligonucleotide, wherein the stabilisation oligonucleotide is protected from 3’ exonuclease degradation, and acts to stabilise the phi29 polymerase, and wherein in said composition the phi29 is not capable of performing a polymerase extension reaction.

In other words, in all the aspects above, the composition is not, or does not include or comprise, a reaction mix capable of a phi29-catalysed nucleic acid polymerisation reaction.

The composition may be viewed as a reagent mix, or mixture or preparation, comprising phi29 polymerase, but not in a form in which the polymerase reaction can take place. Thus, the composition does not comprise all the components or reagents (or reactants) necessary for a polymerase reaction to take place.

Viewed another way, the composition provided herein may alternatively be defined as comprising phi29 polymerase and a stabilisation oligonucleotide, wherein the stabilisation oligonucleotide is protected from 3’ exonuclease degradation, and acts to stabilise the phi29 polymerase, and wherein said composition does not comprise a sample, or alternatively, does not comprise a substrate, or target, nucleic acid for the polymerase, e.g. a nucleic acid to be amplified or replicated by the phi29 polymerase, or in other words the template nucleic acid.

Thus, this aspect can be seen to provide a reagent composition for use to contact with a sample comprising a nucleic acid to be amplified, replicated, extended and/or detected, i.e. the composition is provided prior to its contact with a sample. It will be understood that the sample is a sample to be subjected to the polymerase, e.g. a sample comprising a target nucleic acid molecule. This may be a nucleic acid molecule to be replicated, or amplified, or extended, or detected.

In an embodiment, the target nucleic acid is a template nucleic acid molecule for a nucleic acid polymerisation reaction.

Further, alternatively, the composition may be defined as not comprising a primer to prime a polymerase reaction by the phi29 polymerase. In particular, in an embodiment the stabilisation oligonucleotide is not itself a primer, or does not itself act as a primer for an extension reaction by the phi29 polymerase (i.e. for a polymerase reaction).

In another embodiment, as noted above, the composition may be defined as not comprising cations necessary for enzymatic activity of phi29 polymerase.

A fourth aspect herein provides 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.

Permissive conditions for the polymerisation reaction will depend on the composition that is brought into contact with the sample, and whether or not further reactants and/or components need to be added to allow enzyme activity.

In particular, the use of the stabilisation oligonucleotide allows the composition to be prepared, and maintained at a temperature above freezing, before use, i.e. before the polymerase reaction is initiated, be that by contacting with the sample, or with a component necessary for the polymerase (e.g. RCA) reaction. This allows the phi29-containing composition (i.e. an RCA reaction mix) to be prepared and to be maintained (i.e. kept or held) for a period of time before the polymerisation (e.g. RCA) reaction takes place, for example in an instrument.

Accordingly, in a particular embodiment the composition comprising phi29 polymerase and stabilisation oligonucleotide is prepared, and is maintained at a temperature above freezing (in particular above 4°C, or at a temperature which is at least ambient or room temperature) for a period of at least 15 minutes, more particularly at least 30 or 45 minutes, or at least 1 hour, before being contacted with the sample and/or before an RCA reaction is initiated. In particular, an aqueous composition is prepared. In other words, a composition comprising phi29 polymerase and stabilisation oligonucleotide in a buffer is prepared.

Thus, it can be seen that such an embodiment provides a method of performing a polymerase reaction, said method comprising preparing an aqueous composition comprising phi29 polymerase and a stabilisation oligonucleotide which is protected from 3’ exonuclease action, and maintaining said composition at a temperature above freezing for a period of at least 15 minutes, before initiating a polymerase reaction.

In another embodiment, the composition comprising phi29 polymerase and stabilisation oligonucleotide is prepared as a freeze-dried composition. In other words, a composition comprising phi29 polymerase and stabilisation oligonucleotide in a buffer is prepared, which is then freeze-dried. In a particular embodiment, the freeze-dried composition comprises phi29 polymerase, stabilisation oligonucleotide and components required to perform the polymerisation reaction (e.g. deoxynucleotides (dNTPs), or a primer for the polymerase) in a buffer or other suitable medium. Other suitable components necessary to perform standard methods of freeze-drying known in the art may also be included in the composition.

Thus, it can be seen that such an embodiment provides a method of freeze- drying the composition, said method comprising preparing an aqueous composition comprising phi29 polymerase and a stabilisation oligonucleotide, optionally including other components or reactants for a polymerisation reaction (e.g. as mentioned above) or to facilitate or aid or improve freeze-drying, and freeze-drying the composition. Freeze-drying may be accomplished according to any suitable technique well known in the art. The composition can then be resuspended by the addition of water, or other suitable liquid (e.g. aqueous) medium to the freeze-dried composition, converting it back to an aqueous composition, known as reconstitution. Due to the presence of the stabilisation oligonucleotide, the phi29 polymerase is stabilised before, during, and after both the freeze-drying and reconstitution processes. Thus, freeze-drying the composition does not impact on the stability of the phi29 polymerase.

The freeze-dried composition is particularly advantageous as it improves the ease of transportation when distributing the composition. In an aqueous composition, the product must be stored and transported at temperatures of -20°C, whereas the freeze-dried composition can be transported at ambient temperatures or 4°C.

In a particular embodiment, the composition is maintained before contacting it with a sample comprising a target nucleic acid (i.e. a nucleic acid to be subjected to said polymerase reaction). In another embodiment the composition comprises the sample, or the target nucleic acid, and another component necessary for the polymerase reaction is omitted from the composition, e.g. a primer, dNTPS, or a component of the reaction buffer/reaction mixture necessary for polymerase activity, e.g. cations. Thus, the composition may be lacking one or more components necessary for polymerase reaction, and said reaction may be initiated by contacting the composition with said one or more components (which may be sample/target nucleic acid and/or other polymerase reaction reagents or polymerase reaction mixture components). The contact with said one or more components, missing from the composition, allows the polymerisation reaction to take place.

Thus, in an embodiment, the sample is contacted with the composition under conditions which allow a polymerisation reaction catalysed by the phi29 polymerase to take place. Such conditions would typically include the presence of deoxynucleotides (dNTPs). A primer for the polymerase may be separately provided, or may be present in the sample, for example in the context of, or as part of, the target nucleic acid. Other conditions, discussed further below, include suitable temperature, and buffer etc.

In an embodiment, the polymerisation reaction is an amplification reaction. More particularly the polymerisation reaction is an RCA reaction.

A fifth aspect herein provides a method of replicating a nucleic acid, said method comprising contacting a target nucleic acid (more particularly a sample comprising said target nucleic acid) with a composition comprising phi29 polymerase and a stabilisation oligonucleotide, wherein the stabilisation oligonucleotide is protected from 3’ exonuclease degradation.

More particularly, the nucleic acid, or sample containing it, is contacted with the composition under conditions which allow the phi29 polymerase to replicate the nucleic acid. Such conditions include at least the presence of dNTPs. As above, a primer may be present in the sample, or pre-hybridised with the target nucleic acid, or may be separately added, for example together with, or as part of, the composition.

As noted above, in an embodiment of this aspect, the composition has been prior-prepared (i.e. as an aqueous composition, including as a reconstituted composition), and maintained at a temperature above freezing for a period of at least 15 minutes, before being brought into contact with the target nucleic acid/sample (i.e. before said contacting step).

The replication reaction may be an amplification reaction, for example RCA. It may be any primer extension reaction, more particularly a primer extension reaction using the target nucleic acid molecule as an extension template.

In an embodiment, the method is a method of amplifying a target nucleic acid and comprises contacting the sample with the composition, as defined or set out above, and amplifying the target nucleic acid to produce an amplification product.

A sixth aspect herein provides 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.

In an embodiment of this aspect, the composition has been maintained at a temperature above freezing for a period of at least 15 minutes, before being brought into contact with the sample (i.e. before said contacting step).

As noted above, the composition may be a reconstituted composition (i.e. a freeze-dried composition which has been reconstituted).

As above, in an embodiment, the polymerisation reaction is an amplification reaction. More particularly the polymerisation reaction is an RCA reaction, and the amplification product is an RCA product (RCP).

In an embodiment the detection method is performed in, or using, an automated tissue slide instrument.

In all the aspects above, the target nucleic acid more particularly may be referred to as a target nucleic acid molecule. The target nucleic acid molecule may comprise a target nucleic acid sequence, e.g. a target sequence to be replicated, amplified and/or detected. The target nucleic acid molecule may be the target analyte of a detection assay (e.g. a target nucleic acid molecule occurring naturally (i.e. present natively) in a sample), or a copy or amplicon thereof, including a complementary copy or amplicon thereof. Alternatively, it may be a nucleic acid molecule used, or detected, in a detection method as a means of detecting a target analyte, or in other words a reporter nucleic acid used or generated in a detection assay for a target analyte. Thus, the target nucleic acid molecule may be a nucleic acid molecule generated as a detection assay reaction product by a detection assay for detection of a target analyte in the sample. Alternatively, it may be nucleic acid molecule provided or used as a tag in the detection method, for example a nucleic acid tag attached to a binding partner for an analyte, which is detected as a means of detecting the binding of the partner (e.g. antibody) to the analyte.

Accordingly, the target nucleic acid molecule may be a reporter nucleic acid molecule which reports on the presence (i.e. is indicative of the presence of) a target analyte in the sample.

In a particular embodiment, the nucleic acid molecule is the product of a detection assay for a target analyte in the sample.

Thus, a seventh aspect provides 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.

As indicated above for other aspects, in an embodiment, the composition is prepared, or reconstituted, and maintained at a temperature above freezing for a period of at least 15 minutes, before said contacting step.

In more particular embodiments of the methods above, where the composition is maintained above freezing for a period of time, the time period is at least 0.5, 0.75 or 1 hour.

In an embodiment of any of the aspects above the target nucleic acid molecule is a probe, or part of a probe, or a product generated from a probe. In an embodiment, the target nucleic acid molecule is a nucleic acid product generated in situ in the sample, for example from a probe. It may be a ligation product (e.g. a ligated probe or ligated probes probe parts), or an extension product (e.g. an extended probe or probe part) or a cleavage product (e.g. a cleaved probe or probe part). In another embodiment, as indicated above the target nucleic acid molecule may be provided in the sample, e.g. as a means of detecting a target analyte in the sample.

In an embodiment the target nucleic acid molecule is a circular nucleic acid molecule. For example, it is a circularised target nucleic acid analyte or a copy or amplicon thereof (including a complementary copy or amplicon). In another embodiment it is a circularised probe, e.g. a circularised padlock probe or molecular inversion probe (MIP).

In an embodiment, the target nucleic acid molecule is a nucleic acid product generated in a proximity assay, for example a proximity ligation assay (PLA). In this embodiment it is a ligation product.

The sample may be any sample comprising a target nucleic acid, whether present natively, added to the sample, or generated in the sample. In an embodiment the sample is a tissue sample, which may be a solid tissue sample, or a blood sample.

In an embodiment, the sample is an immobilised tissue or cell sample, and particular a tissue or cell sample on a slide.

In an embodiment, the target nucleic acid molecule is a nucleic acid molecule generated in situ in a tissue sample, including a blood sample. In a more particular embodiment, the nucleic acid molecule is generated in an in situ PLA (isPLA) reaction.

In the aspects above, the phi29 polymerase exhibits increased stability in the presence of the stabilisation oligonucleotide relative to its stability in the absence in the stabilisation oligonucleotide. Particularly, temperature stability is increased.

Description of drawings

Figure 1 :

Fluorescent microscopy scans of human FFPE colon tissue after performing in situ PLA with the NaveniFlex Tissue kit to detect the interaction between E-cadherin and B-catenin. In situ PLA was ran under four different assay conditions. For each condition, a split, greyscale image of nuclei (DAPI) and the interaction (E-cadherin - B-catenin) is shown. Condition A: In situ PLA ran with freshly prepared RCA reaction mix. Condition B: In situ PLA ran with RCA reaction mix prepared and stored on a lab benchtop for 18 hours at room temperature. Condition C: In situ PLA ran with freshly prepared RCA reaction mix supplemented with 0.05 pm stabilisation oligo. Condition D: In situ PLA ran with RCA reaction mix supplemented with 0.05 pm stabilisation oligo prepared and stored on a lab benchtop for 18 hours at room temperature. RCA reaction mix includes phi29 DNA polymerase, dNTPs in 1X Thermo Scientific Reaction Buffer for phi29 DNA polymerase.

Detailed Description

The methods and uses herein address the problem of stability of phi29 polymerase, and in particular the limited stability which phi29 polymerase exhibits in solutions when kept at ambient or elevated temperatures, or indeed at any temperature greater than freezing, including e.g. greater than 4°C.

It has been found that oligonucleotides which are protected from 3’ exonuclease degradation (that is from digestion by 3’ exonuclease, or from 3’ exonuclease action) may beneficially act as stabilising agents to stabilise phi29 polymerase, and in particular to improve, or increase, its temperature stability.

Thus, according to the methods and uses herein a stabilising agent may be added to, or included in, preparations (i.e. compositions) of phi29 polymerase, wherein the stabilising agent is an oligonucleotide which is protected from 3’ exonuclease degradation.

The term “oligonucleotide” is used herein in accordance with its usual meaning in the art to mean a relatively short nucleic acid molecule. The stabilisation oligonucleotide of concern herein is capable of stabilising phi29 polymerase. Whilst not wishing to be bound by theory, it is believed that this occurs due to binding of the polymerase to the stabilisation oligonucleotide. Accordingly, in particular, the stabilisation oligonucleotide is long enough to bind to phi29 polymerase. In other words, it has a length at least as long as the length corresponding to the minimum phi29 enzyme footprint (i.e. the length of the sequence in the substrate (e.g. template) nucleic acid molecule to which the enzyme binds, or with which it interacts.

In an embodiment the stabilisation oligonucleotide is at least 6, 7, 8, 9 or 10 nucleotides in length.

The maximum length of the stabilisation oligonucleotide is not critical. Typically for ease of synthesis etc, the length does not exceed 200, 150, 100, 90, 80, 70, 60 or 50 nucleotides. Thus, the length of the stabilisation oligonucleotide may be in a range between any of the above-listed integers. For example, the stabilisation oligonucleotide may be from any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 to any one of 25, 28, 30. 35, 40, 45, 50, 55, 60, 65 or 70 nucleotides in length. In particular embodiments, the stabilisation oligonucleotide is any one of 9, 10, or 11 to any one of 20, 25 or 30 nucleotides in length.

The stabilisation oligonucleotide is protected from 3’ exonuclease degradation. This means that it is modified to be resistant to degradation by 3’ exonuclease enzymatic activity, or more particularly to exhibit reduced susceptibility to 3’ exonuclease degradation (whether that exonuclease activity is provided by a stand-alone, or independent, exonuclease enzyme having 3’ exonuclease activity, such as Exonuclease I, III or V for example, or by a polymerase enzyme, or a part or component thereof, having 3’ exonuclease activity). In this regard, the stabilisation oligonucleotide is resistant to 3’ exonuclease degradation by phi29 polymerase.

Modifications to confer 3’ exonuclease resistance to oligonucleotides are well known in the art, and of these may be used. Such modifications are typically incorporated during oligonucleotide synthesis and include modifications to the sugar (deoxyribose or ribose) moiety of the nucleotide, or to the linkage between adjacent nucleotides in the oligonucleotide (“internucleotide linkage”). These latter modifications are also termed “backbone modifications”. Thus, the stabilisation oligonucleotide comprises modified nucleotides, which term is used broadly to refer to a nucleotide which is not found in nature in the context of nucleic acid molecules as they occur in living organisms, i.e. an unnatural or non-native nucleotide. It may be defined as not being a nucleotide as occurs in nature in nucleic acid/DNA. The modification may be in the structure of the nucleotide, including notably in the sugar, or it may be a backbone modification, where the backbone of the nucleotide sequence is modified). Thus, the modified nucleotide may be a nucleotide linked to its adjacent nucleotide by an unnatural bond (that is a bond which is not a phosphodiester bond).

The stabilisation oligonucleotide may comprise one or more modified nucleotides, and this may depend on the nature of the modification and/or length of the oligonucleotide etc. The stabilisation oligonucleotide may comprise a single species or type of modification, or different modifications may be present in combination. The modified nucleotides, particularly where they comprise sugar modifications, are typically present at the 3’ end of the oligonucleotide, but in some embodiments they may be present throughout the length of the oligonucleotide. In an embodiment the 3’-most 1 , 2, 3 or 4 nucleotides are modified, but the stretch of modified nucleotides may be longer. The modified nucleotide may be DNA or RNA, or a synthetic nucleic acid. Modified oligonucleotides comprising modifications to limit 3’ exonuclease degradation may be obtained commercially, for example from Integrated DNA Technologies.

Modifications to the sugar include in particular 2’ substitutions (substitution at position 2 of the ribose), for example 2’ Ribo (2’OH); 2’-O-methyl (2’0Me;

2’methoxy), 2’-O-methoxyethyl (2’MOE) or 2’ fluoro (2’F). Other modifications to the sugar moiety may also or alternatively be made.

Particular examples of nucleotides with sugar modifications include 2’F RNA; 2’0Me nucleotide; LNA (locked nucleic acid); FANA (2’-fluoro-arabinonucleic acid); HNA (hexitol nucleic acid); or 2’-O-methoxy-ethyl (2’ MOE) nucleotides.

In an embodiment, the stabilisation oligonucleotide comprises one or more, e.g. 1 to 3, 4, 5 or 6, 2’-O-modified ribose nucleotides (e.g. 2’0Me or 2’MOE).

The modified nucleotide may comprise any base. In an embodiment the modified nucleotide is 2’-O-methyl uracil.

Backbone modifications, or modified nucleotide linkages, include modifications to the phosphate group in the phosphodiester linkage, including phosphorothioate or phosphorothiolate linkages, or borano-phosphate linkages, or a polyethylene-linker backbone moiety incorporated between nucleotide residues.

As is well known in the art, the phosphorothioate bond modification alters the phosphate linkage between bases by replacing one of the oxygens in the linkage with a sulphur atom. Specifically, in the phosphorothioate modification, a non-bridging oxygen covalently bound to phosphorous is replaced by a sulphur atom. Analogously, in the borano-phosphate modification, a non-bridging oxygen is replaced with BH3. In the phosphorothiolate modification (also known as a 5’-thio nucleoside), a sulphur atom replaces the 5’-bridged oxygen connected to the sugar moiety. In an embodiment, the backbone modification is not, or does not include, a phosphorothiolate modification.

Other known backbone modifications which may be used include phNA (methyl phosphonate, where a non-bridging oxygen in the phosphate linkage is replaced by CH3) or peptide nucleic acid (PNA).

Other backbone (or linkage) modifications include those which involve the sugar moiety of the nucleotide (i.e. so-called sugar/backbone modifications). Such modifications known and described in the art include mirror DNA, ribuloNA, TNA, tPhoNA and dXNA.

Typically, more than 1 modified linkage is needed to confer effective 3’ exonuclease resistance, typically at least 3 linkages, or 3-6 linkages e.g. phosphorothioate linkages. In some embodiments all the linkages in the stabilisation oligonucleotide are modified linkages.

In certain embodiments, the stabilisation oligonucleotide comprises one or more 2’ modified nucleotides and/or phosphorothioate linkages, particularly at least 2, 3, 4, 5, or 6 such modifications.

Other modifications to protect from 3’ exonuclease digestion include inverted dT or ddT. Such modified nucleotides can be incorporated at the 3’ end of the stabilisation oligoncucleotide, leading to a 3’-3’ linkage that inhibits degradation by 3’ exonucleases, as well extension by polymerases.

Still further possible modifications include incorporation of phoshoramidite C3 spacer at the 3’ end of the oligonucleotide, 3’ phosphorylation or 3’ Hexanediol.

In certain embodiments, the stabilisation oligonucleotide comprises only modified nucleotides with a structural modification, particularly to the sugar moiety, and no backbone modifications. In particular embodiments the stabilisation oligonucleotide comprises one or more modified nucleotides comprising a modification at the 2 position of the sugar.

A representative example of a stabilisation oligonucleotide, as used in the Examples below, is:

5’ TGACTGGGAATGTAGGAGCAmUmUmU 3’ (SEQ ID NO: 1)

5’ GGACTACATATCTTACTACGUmUmU 3’ (SEQ ID NO. 2)

The sequence of the stabilisation oligonucleotide is not critical, and the skilled person will know how to design or select an appropriate oligonucleotide sequence, e.g. to minimize unwanted interactions, and to avoid secondary structure etc. The stabilisation oligonucleotide may or may not be able to bind to the generated polymerase product (e.g. RCP). Thus, it may or may not comprise a sequence which is complementary to the polymerase product (or in other words, homologous to the template/target nucleic acid). In an embodiment, the stabilisation oligonucleotide does not hybridise to a polymerase product, particularly an RCP, at a site to which a detection oligonucleotide is intended to bind (i.e. at a so-called detection sequence; as will be explained in more detail further below polymerase products such as RCPs are frequently detected by hybridising to the product a labelled detection probe, referred to herein as a detection oligonucleotide). However, in another embodiment, the stabilisation oligonucleotide may bind at the detection sequence. In another embodiment, the stabilisation oligonucleotide may bind to the polymerase product, e.g. RCP, at a different site to the detection sequence. In a preferred embodiment, the stabilisation oligonucleotide does not comprise a sequence which is complementary to the polymerase product (i.e. the stabilisation oligonucleotide is not homologous to and does not bind to the template/target nucleic acid). As the stabilisation oligonucleotide cannot bind to the template/target nucleic acid, it cannot act as a primer for the phi29 polymerase to amplify the template/target nucleic acid, and a polymerisation product (e.g. an amplification product, for example an RCP) cannot be generated (i.e. the stabilisation oligonucleotide is not or cannot act as a primer). Thus, if the stabilisation oligonucleotide is present in the reaction mix but a phi29 polymerase primer is not, no reaction can take place.

In another embodiment, the stabilisation oligonucleotide does not bind to a nucleotide for use in nucleotide identification and nucleic acid sequencing, or more generally it is not attached to a nucleotide which is incorporated in a polymerase extension reaction. In other words, the stabilisation oligonucleotide is not attached to a nucleotide as a tag. In an embodiment, the stabilisation oligonucleotide does not bind to a nucleotide as a tag. In another embodiment, the stabilisation oligonucleotide does not bind to a nucleotide utilised in an incorporation reaction as a tag. The term “incorporation reaction” is used herein in accordance with its usual meaning in the art to mean a reaction comprising a nucleic acid polymerase catalysing the incorporation of a tagged nucleotide into an oligonucleotide.

Phi29 polymerase from the bacteriophage phi29 is a monomeric enzyme of 66 kDa, a protein-primed DNA-dependent replicase belonging to the eukaryotic-type family of DNA polymerases (family B). Like other DNA polymerases it accomplishes DNA synthesis by adding nucleotides to the 3’ OH group of a growing DNA chain. It contains an exonuclease domain that catalyses 3’^5’ exonucleolysis of mismatched nucleotides (proofreading). Phi29 has distinctive functional features, including strong binding to single-stranded DNA, a very high processivity (it does not require processivity factors to synthesise DNA), and the ability to unwind the parental DNA helix, which allows it to replicate double-stranded genomic DNA without any unwinding factors. Phi29 can be used to amplify any DNA sample - no sequence information is required, and simple random primers can be used. These properties render it useful in a range of applications, including isothermal amplification processes such as RCA, and multiple displacement amplification (MDA). Uses include, as well as RCA- or MDA-based nucleic acid detection methods (e.g. in situ genotyping with padlock probes, or in situ PLA), whole genome amplification (WGA) using a variety of sample types, amplification of DNA from single cells, uncultured microbial cells or viral particles, preparing DNA for SNP or short tandem repeat (STR) detection, protein-primed amplification, cell-free cloning of lethal DNA, and RNA-primed DNA amplification. All such uses and applications are included herein.

Phi29 polymerase (also referred to as phi29 DNA polymerase) is commercially available from various sources, for example New England Biolabs and Thermo Fisher Scientific. As well as the wild-type enzyme, various mutants or derivatives of phi29 have been developed, to increase or improve various properties, to expand the range of possible applications. These include various mutations (amino acid substitutions) in the enzyme. For example, the mutant enzyme EquiPhi29™ DNA polymerase, available from Thermo Scientific, was developed through in vitro protein evolution and exhibits improved thermostability, reaction speed, product yield and amplification bias, while retaining all the benefits of the wild-type enzyme. 4BB™ QualiPhi® is a chimeric form of Phi29 polymerase engineered for enhanced sensitivity and efficiency available from 4basebio SLU, Spain. Phi29-XT RCA, available from New England BioLabs, is another engineered version that generates high product yield with improved thermostability and sensitivity over the wild-type phi29 polymerase.

A number of phi29 mutants and derivatives are reported in the literature. These include, for example, EP2813576 (increased protein stability and increased half-life; EP3854872 (improved thermal stability); and WO 2021/163052 (increased processivity, strand displacement activity, template or primer binding, 3’ exonuclease activity, nucleotide sensitivity or temperature stability, or decreased error rate). Povilaitis et al., 2016, Protein Engineering Design and Selection 29(12), 617-628 describe improved phi29 mutants developed by an isothermal compartmentalized self-replication technique for use in WGA, and Gao et al., 2021 Microbial Biotechnology, 14(4), 1642-1656 describe a chimeric phi29 DNA polymerase with helix-hairpin-helix motifs showing enhanced salt tolerance and replication performance. (All references cited herein are incorporated by reference). Any such mutants or derivatives described in these references, or elsewhere in the literature, or commercially available from any source may be used.

Accordingly, the term “phi29 DNA polymerase” (and the shortened “phi29 polymerase and “phi29”) as used herein includes the wild-type enzyme, and any mutant or derivative thereof, in particular which retains the functions and properties of the wild-type enzyme.

According to the uses and methods herein, the stability of phi29 is improved. Thus, the phi29 polymerase exhibits increased (i.e. enhanced or improved) stability in the presence of the stabilisation oligonucleotide relative to its stability in the absence in the stabilisation oligonucleotide. Thus, stability of phi29 polymerase in a composition may be compared in the presence and absence of a stabilisation oligonucleotide in the composition. “Stability” refers to the ability of the enzyme to retain its activity, namely in this case, polymerase activity. Particularly, temperature stability is increased. The term “temperature stability” is used herein synonymously with “thermal stability” or “thermostability”, and means the ability of the enzyme to maintain its activity at elevated temperature. More particularly, the enzyme is able to maintain its activity at elevated temperature over an extended period of time, e.g. for a period of at least 3 hours, more particularly, at least 4, 5, 6 or 7 hours. In particular embodiments the activity is stabilised for at least 10, 12, 15 or 18, 24 hours. As used herein “elevated temperature” is any temperature above freezing, including particularly ambient or room temperature, e.g. 20 or 21 °C.

Particularly, the references above to stability are in the context of aqueous compositions, e.g. solutions, of the Phi29 enzyme.

In particular, in the presence of the stabilisation oligonucleotide, the phi29 polymerase is able to retain its activity, or better retain its activity, compared to in the absence of the stabilisation oligonucleotide, in a buffered composition (e.g. a reaction buffer) kept at ambient temperature for a period of 3 or more hours. It is well within the routine skill of the skilled person in this field to perform assays of phi29 polymerase activity in order to compare activity levels between the conditions (presence or absence of stabilisation oligonucleotide) at different time periods and at different temperatures. Furthermore, as demonstrated in the examples below, improved stability may also be seen from the improved results obtained in detection assay performance using detection protocols involving a phi29-catalysed RCA reaction, and detecting the RCA product, in the presence of stabilisation oligonucleotide, compared to absence. Improved stability thus includes improved performance of the phi29 polymerase in detection protocols where a target nucleic acid (reporter or analyte) is detected using the phi29 polymerase to generate a detectable product which is detected.

As noted above, the phi29 polymerase is provided, or formulated, in a composition (or in other words a preparation) comprising the phi29 polymerase and the stabilisation oligonucleotide. Typically, the composition comprises the polymerase and oligonucleotide in a buffer. The composition that is provided, or prepared, is thus an aqueous composition. Alternatively, the composition (e.g. aqueous composition) that is provided, or prepared, can be freeze-dried, and then reconstituted into an aqueous composition. Suitable buffers for phi29 polymerase are known in the art, and available commercially, for example Thermo Scientific Reaction Buffer for phi29 DNA polymerase. The buffer may contain components required or optimal for phi29 DNA polymerase activity, such as cations.

A typical storage buffer for phi29 comprises: 50mM Tris-HCI (pH 7.5), 0.1 mM EDTA, 1mM DTT, 100 mM KCI, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 20 and 50% glycerol. Thus, a composition herein may comprise, in addition to the enzyme and stabilisation oligonucleotide, a buffer at around pH 7.5 (e.g. pH 7-8), and optionally, EDTA, a salt, (e.g. KCI), and further optionally a detergent or surfactant and/or glycerol). EDTA is in storage buffers, and since it binds cations, it tends to be inhibitory to activity.

A typical reaction buffer, provided at 10X concentration, comprises: 330 mM Tris-acetate (pH 7.9 at 37 °C, 100 mM Mg acetate, 660 mM K acetate, 1 % Tween 20, 10mM DTT.

It may be convenient according to the methods and uses herein to provide the phi29 and stabilisation in a reaction buffer (i.e. to formulate the composition in a reaction buffer). Thus, the composition may comprise a buffer at around pH 7-8, one or more salts to provide cations, e.g. Mg and K cations, particularly Mg cations. Mg 2⁺ cations are generally required for phi29 activity and are included in reaction buffers. It may further be advantageous to provide a reducing agent, e.g. DTT or similar, as it has been reported that this is beneficial for maximal enzyme activity. Other possible ingredients include detergent or surfactant. The buffer may be a Tris buffer.

The composition may additionally comprise one or more further components, for example other reagents for a polymerase (e.g. amplification) reaction. This may include in particular dNTPs for incorporation. Thus, the composition may be a reaction mix for a polymerase reaction, e.g. an amplification reaction, for example for RCA. In some embodiments, the composition may additionally comprise one or more primers. However, as noted above, in some embodiments a primer is not needed in the composition, as it is separately provided, or is present in the sample to which the composition is added. For example, in some embodiments, the target nucleic acid may itself serve as, or provide, a primer.

However, as noted above, the compositions claimed as such herein do not include a complete reaction mixture for a phi29-catalysed polymerisation/DNA synthesis reaction; the composition is not capable itself of performing a DNA polymerisation reaction. As described above, one or more components essential for a polymerase reaction are omitted. In a particular embodiment, the composition does not include a sample, or a substrate or template nucleic acid. In other words, it does not include a nucleic acid molecule, e.g. a target nucleic acid, which may serve as the subject nucleic acid molecule for the polymerase reaction, for example as a nucleic acid to be replicated (copied) or amplified, or in other words as a template for the polymerase reaction.

In another embodiment, the composition does not comprise cations, or more particularly it does not comprise a source of magnesium cations. In another embodiment, the composition does not comprise a primer.

As noted above, the stabilisation oligonucleotide has utility in preparing and reconstituting freeze-dried (i.e. lyophilised) preparations of phi29. In this regard, the stabilisation oligonucleotide may be included in compositions prepared for freeze- drying. Thus, an aqueous composition may be prepared which is then subjected to freeze-drying. Freeze-dried phi29 preparations prepared according to the methods herein can be stored and/or transported at ambient or refrigerator temperatures (e.g. about 4 °C). Freeze-drying has the advantage that shipping and delivery of the phi29 enzyme product does not require freezing conditions (e.g. -20°C). Inclusion of the stabilisation oligonucleotide stabilises the enzyme during drying and reconstitution.

Thus, the stabilisation oligonucleotide acts to stabilise the aqueous composition during the course of its preparation, and during the freeze-drying process, e.g. whilst it is dispensed into vials or other containers, and whilst awaiting and during the course of freeze-drying. Thus, the stabilisation oligonucleotide protects the enzyme during room temperature production protocols, or protocols which use room temperature reagents. When reconstituted, the stabilisation oligonucleotide stabilises the phi29 enzyme during the course of reconstitution, and in the reconstituted composition.

As noted above, the aqueous composition prepared for freeze-drying may comprise other components, including any of the components mentioned above. These may include a buffer and one or more components or reagents required for a polymerase reaction, or for a detection reaction using the phi20 polymerase. In particular, the composition for freeze-drying may contain all the components necessary for a polymerase reaction.

As well as components or reagents for downstream uses of the phi29 polymerase, the composition prepared for freeze drying may also contain one or more other components, for example excipients or formulation aids, including for example other stabilisers or aids for the freeze-drying process. Cryoprotectants which may be used are well known in the art and reported in the literature and include proteins, carbohydrates and organic polymers. Suitable proteins include inert proteins such as are routinely used as blocking agents, e.g. albumins (e.g. BSA, gelatin or milk proteins. Carbohydrates include particularly sugars and sugar alcohols, such as trehalose, dextrose, sucrose, mannitol, sorbitol, etc. Polymers include polyvinyl pyrrolidone or polyethylene glycol. Other components include for example surfactants, e.g. detergents, including non-ionic detergents such as Tween, Brij or Triton detergents. Any standard cryoprotectants or excipients may be used, including particularly those use for freeze-drying of protein, especially enzyme, preparations.

Similarly, standard procedures for freeze-drying may be used, as are widely known and used in the art, including particularly for proteins, and especially enzymes.

It is within the routine skill of the practitioner skilled in this art to determine an appropriate concentration of stabilisation oligonucleotide to use. However, generally speaking the concentration of stabilisation oligonucleotide in the composition, notably in the phi29 preparation before it is contacted with sample, may be in the range of 0.001 pM - 10 pM, more particularly from any one of 0.002, 0.005, 0.01 or 0.02 to an any one of 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 pM, For example the range may be 0.03 - 0.5 pM, e.g. 0.05 - 0.2 pM or 0.05-0.1 pM.

As noted above, the stabilised phi29 polymerase compositions herein may be used for any application for which phi29 polymerase is useful. They may thus be used in any polymerase reaction, e.g. a replication reaction, an amplification reaction, or indeed any polymerase-catalysed primer extension reaction. This may be in the context of a wider protocol, or process, including any of the applications mentioned above.

In an embodiment, the polymerase reaction is an RCA reaction, and the target nucleic acid is a circular DNA molecule. In another embodiment the polymerase reaction is MDA.

The amplification reaction is in certain embodiments performed in the context of a wider analysis, where an amplification reaction (e.g. RCA) is performed as part of a method (i.e. a method of analysis, or a method of detection etc.)

The various methods above which define stabilising the phi29 polymerase or uses of the stabilised phi29 polymerase generally comprise contacting the phi29 polymerase, or a composition comprising it with another component, e.g. the stabilisation oligonucleotide, or the sample, or a target nucleic acid.

The term “contacting” is used broadly herein to include bringing the reagents in question into contact. Thus, one may be added to the other and vice versa, or they may each be introduced to each other etc. This time or order of addition, or contact with the sample etc., may depend on the precise nature of the method, or method step, which is performed. In the context of a detection or analysis method, for example, where the target nucleic acid is a reporter nucleic acid, the composition may be added or introduced to the sample after the detection assay or analysis method steps have been performed, e.g. after the sample has been contacted with detection reagents for detection of the target analyte of the detection assay method, i.e. after the target nucleic acid has been introduced into the sample (e.g. by means of an assay reagent comprising the target nucleic acid) or generated in the sample (e.g. by a ligation or extension reaction etc). Thus, in an embodiment, the composition may be contacted with a target nucleic acid product after it has been generated. Thus, an assay method may be performed to generate a nucleic acid product as a detection assay reaction product (e.g. a ligation and/or extension product, and after this, the composition comprising the stabilised phi29 may be introduced.

As further noted above, the contacting of the composition with the target nucleic acid may place after a period of delay. In other words, the contacting may be delayed for a period of time, after the composition has been prepared. This period of delay may be a period of storage, e.g. reagents may be prepared in advance of their use.

Thus, the composition may be prepared, or more particularly, an aqueous or freeze-dried composition of the phi29 polymerase and stabilisation oligonucleotide may be prepared and this may be for the purpose purely of storage (i.e in a storage buffer), or the composition may be suitable for use in the intended reaction (i.e. in a reaction buffer). As noted above, suitable buffers are known in the art. The composition may be stored, maintained (e.g. held, or kept) at a temperature above freezing. This includes fridge or “on-ice” temperatures, e.g. at or around 4 °C. Advantageously, the composition may be maintained at ambient or room temperature, or higher.

The period of delay is, as noted above, at least 15 minutes, more particularly at least 30 or 45 minutes. In certain embodiments, it is at least 1 hour, for example, at least 1.5, 2. 2.5 or 3 hours. It may be longer, including for example 4, 5, 6, 7, 8, 9, or more hours. In some embodiments the delayed contact may take place after, 10, 11, 12, 13, 14, 15, 16, 17 or 18 or more hours, e.g. after 20 or more, or 24 hours or more.

This is beneficial in the case of automation. Thus, a composition may be prepared and may loaded into an instrument or automated apparatus, e.g. into an autostainer. It may be kept, or held in the instrument/apparatus, until needed for the polymerase reaction. It may then, in an exemplary embodiment, be automatically dispensed. Such a composition may be a reaction mix ready for use, e.g. for contact with the sample, or it may be a concentrated composition which is diluted before use. As noted above, the stabilised phi29 is advantageously used, according to the methods and uses herein, in the context of detection assays, e.g. for a target analyte. The analyte may be the target nucleic acid itself (i.e. the target of the detection oligonucleotide) or the target nucleic acid may be a reporter (or in other words, a proxy, or tag, or signal) which is detected in order to detect the target analyte.

More particularly, the detection method may be to detect a target nucleic acid sequence in a target nucleic acid molecule. The term “nucleic acid sequence” is used synonymously and interchangeably with “nucleotide sequence”.

Detection assays are frequently performed in multiplex to detect multiple analytes, or targets, in a single sample or mixture. Thus, different nucleic acid molecules present in a sample may be detected in a multiplex assay.

Many detection assays rely on signal amplification to improve the sensitivity and accuracy of the assay, and accordingly comprise an amplification step involving a polymerase. As noted above, RCA is particularly advantageous in this regards it generates a large nucleic acid product, the RCP, which may readily be labelled and detected. RCPs are generated as the ultimate reaction products, or the signals, or markers, of the assay which are detected in many assay methods as a means of detecting the target analyte of the assay.

The nucleic acid detection method may be used to detect variants of target nucleic acid sequences. Target nucleic acid sequences may commonly occur in variant forms, for example allelic variants, or mutant and wild-type sequences, and it may be desirable to detect which variant is present. Thus, the target nucleic acid sequence may be one of a number of different variants of the nucleic acid sequence which may occur in a target nucleic acid molecule. The detection methods may also be used to detect other types of analytes, including for example proteins, including cell surface proteins, and protein-protein interactions (PPIs). In particular embodiments, the target analyte is thus a protein or proteinaceous molecule (i.e. a molecule comprising a protein), or a protein complex or PPI. As noted above, and described further below, such analytes may conveniently be detected by proximity assays.

An RCP represents an attractive detection assay reaction product since it comprises or is made up of monomer units (i.e. the monomer repeats of the concatemeric RCP), this may be at least 100, 200, 300, 400 or 500 monomers. Each monomer may comprise a binding site for a detection oligonucleotide by means of which the RCP may be detected. A detection oligonucleotide is a hybridisation probe which comprises a binding site complementary to the binding site in the RCP monomers, and a detectable moiety, e.g. a label such as a fluorescent, coloured or colorimetric label. An RCP is particularly advantageous since this may be coiled into a ball, or blob, a structure which is a discrete visualisable entity.

Since they are large, RCPs may accommodate multiple copies of the detection oligonucleotides which are used to label them. In other words, they comprise multiple sites for binding of detection oligonucleotide. Such a binding site may be referred to as a detection sequence. The detection sequence can be viewed as a tag sequence.

The term “multiple” or “multiplicity” as used herein means two or more, for example, 3, 4, 5, 6, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 or more. Indeed, in the case of many types of nucleic acid product, this may comprise a thousand or more detection sequences and hence binding sites for labelled detection oligonucleotides. Thus “multiple” in the context of copy number of detection oligonucleotides, or binding/attachment sites for detection oligonucleotides, or detection sequences etc. may include at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000, or multiples of these.

Amplification methods such as MDA may similarly be used to generate branched amplification products which include multiple detection sites for binding of detection oligonucleotides.

The target nucleic acid molecule (e.g. nucleic acid reporter or product, or analyte) is typically a DNA molecule. However, it may also be composed of or may comprise other nucleic acids, natural or synthetic. Thus, it may for example be a chimeric construct comprising both RNA and DNA. The nucleic acid product may be made up of ribonucleotides and/or deoxyribonucleotides as well as synthetic nucleotides that are capable of participating in Watson-Crick type or analogous base pair interactions. Thus, the target nucleic acid product may be or may comprise, e.g. bisulphite-converted DNA, LNA, PNA or any other derivative containing a nonnucleotide backbone.

As indicated above, a detection method herein may include the steps of generating a target nucleic acid molecule and detecting it. The generation steps may be carried out in multiplex. In other words, different target nucleic acid molecules may be generated in the same reaction mixture. However, it is not precluded that separate reactions are performed in parallel to generate each different target nucleic acid molecule, and the molecules are then combined, or pooled.

In a particular embodiment, the target nucleic acid molecules are generated as proxies, or markers, for the detection of target analytes in a detection assay. In particular, they may be generated in the course of a method for detecting target nucleic acid sequences which may occur in one or more target nucleic acid molecules.

The term “detecting” is used broadly herein to include any means of determining the presence of the target nucleic acid. In the present methods the target nucleic acid is detected by detecting the presence or amount of the nucleic acid, and can include detecting simply if it is present or not, or any form of measurement of the target nucleic acid molecule, and this may include detecting the target nucleic acid itself, or a copy or amplicon thereof, including a complementary copy. Accordingly, detecting the target nucleic acid includes determining, measuring, assessing or assaying the presence or absence or amount or location of the target nucleic acid in any way. The presence of a target nucleic acid (i.e. the confirmation of its presence or amount) is indicative or identificatory of the presence of the target nucleic acid.

Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when two or more different target nucleic acid sequences, or target molecules, in a sample are being detected, or absolute. Accordingly, in an embodiment the method may be for quantifying or determining the amount of target nucleic acid which is present. The term "quantifying" when used in the context of quantifying a target nucleic acid in a sample can refer to absolute or to relative quantification. Absolute quantification may be accomplished by inclusion of known concentration(s) of one or more control nucleic acid molecules and/or referencing the detected level of the target nucleic acid with known control nucleic acid molecules or sequences (e.g. through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different target nucleic acids, or different target sequences, to provide a relative quantification of each of the two or more different nucleic acid molecules or sequences, i.e., relative to each other. Thus, as noted above, ratios of target nucleic acids present in sample may be determined. Thus, copy numbers of target nucleic acids may be compared.

As noted above, in an embodiment the target nucleic acid molecule is an analyte in a sample. However, in another embodiment, the target nucleic acid molecule is not itself the target analyte, but rather is detected as part of an assay to detect another target analyte.

The target analyte may thus be any analyte it is desired to detect. The analyte may be a nucleic acid, a protein (which term includes peptides and polypeptides), or any other chemical or biological molecule or moiety, including for example carbohydrates, e.g. such as may occur as glycosyl groups on proteins. The target analyte may thus be a modified protein, for example with a post-translational modification which is detected in an assay for an analyte. The target analyte may also be an interaction, or complex between different molecules, e.g. a PPI as noted above, and the individual members of the interaction may be detected using proximity probes, each specific for a member of the interaction.

In an embodiment, the target analyte may be a protein or component of a proteinaceous molecule which is detected on the surface of a cell, or vesicle, or other cellular or sub-cellular compartment.

As noted above, the target nucleic acid may be any sequence it is desired to detect or identify. It may be DNA or RNA, or a modified variant thereof. Where the target sequence is the analyte it may be any target sequence it is desired to detect, for example a nucleic acid present in a sample, e.g. in a cell or tissue sample or any biological sample etc. Thus, it may be a naturally occurring sequence, or a derivative or copy or amplicon thereof.

Alternatively, as noted above, the target sequence may instead be a reporter for an analyte of an assay. Reporter nucleic acids may be used or generated in the course of an assay for any analyte, for example a protein or other biological molecule, or small molecule, in a sample. Thus, a reporter nucleic acid may be provided as a tag, or label, for a binding probe for an analyte, and may be detected in order to detect the analyte, for example in an immunoassay, e.g. as in an immunoPCR or immunoRCA reaction. A reporter nucleic acid may be generated in the course of an assay, for example by a ligation reaction in a proximity ligation assay (PLA), or an extension reaction in a proximity extension assay (PEA), or by a cleavage reaction, or such like. Such a reporter target nucleic acid may therefore be a synthetic or artificial sequence. It may be a linear or a circular or circularised or circularisable molecule.

In an embodiment, the target nucleic acid is a DNA molecule, natural or synthetic. The target nucleic acid molecule may be coding or non-coding DNA, for example genomic DNA or a sub-fraction thereof, or may be derived from genomic DNA, e.g. a copy or amplicon thereof, or it may be cDNA or a sub-fraction thereof, or an amplicon or copy thereof etc.

In another embodiment, the target nucleic acid molecule is a target RNA molecule. It may be an RNA molecule in a pool of RNA or other nucleic acid molecules for example genomic nucleic acids, whether human or from any source, from a transcriptome, or any other nucleic acid (e.g. organelle nucleic acids, i.e. mitochondrial or plastid nucleic acids), whether naturally occurring or synthetic. The target RNA molecule may thus be or may be derived from coding (i.e. pre-mRNA or mRNA) or non-coding RNA sequences (such as tRNA, rRNA, snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA and long ncRNA). one preferred embodiment, the target nucleic acid molecule is a micro RNA (miRNA). In one embodiment, the target RNA molecule is 16S RNA, for example wherein the 16S RNA is from and identificatory of a microorganism (e.g. a pathogenic microorganism) in a sample. Alternatively, the target RNA molecule may be genomic RNA, e.g. ssRNA or dsRNA of a virus having RNA as its genetic material. Notable such viruses include Ebola, HIV, SARS, SARS-CoV2, influenza, hepatitis C, West Nile fever, polio and measles. Accordingly, the target RNA molecule may be positive sense RNA, negative sense RNA, or double-stranded RNA from a viral genome, or positive-sense RNA from a retroviral RNA genome.

Where the target molecule is an RNA molecule, the method may comprise a preliminary step of generating a cDNA copy of the target RNA molecule.

Methods for generating a target nucleic acid as used herein are well known in the art, and widely described in the literature, as are detection methods employing them.

Thus, RCA is widely known as an amplification technique, and many detection assays have been proposed and described using RCA to generate a detectable product.

The template circle for an RCA reaction may be produced by the circularisation of a probe for a target nucleic acid sequence, notably a padlock probe, according to principles well-known in the art. Padlock probes may take many forms, and may be provided in 1-part form, or multi-part (e.g. 2-part) form. They include gapfill padlock probes (also known as molecular inversion probes (MIPs)). The RCA template may alternatively be a pre-formed circle, which forms part of a targetspecific probe (e.g. is hybridised to a target-specific probe, or to a nucleic acid part or domain thereof), or is used together with a target specific probe, such as in immunoRCA reactions for example. Analogously, it may be a circularisable oligonucleotide which is ligated to form a circle during the course of the assay reactions. Thus, an RCP may be a product of an immunoRCA reaction or any type of detection reaction which comprises a RCA step, for example a proximity probe assay in which a circular nucleic acid molecule is generated - see the Duolink™ PLA of SigmaAldrich for example, and the modified PLA which uses so-called Unfold proximity probes, which comprise hairpins which are opened, or unfolded, by cleavage to release nucleic acid domains which may be circularised to form a RCA template (see Klaesson et al, 2018, Scientific Reports 8, 5400). A typical PLA generates a template circle upon interaction of the nucleic acid domains of proximity probes, when bound in proximity to their target. More specifically, the nucleic acid domains of a pair of proximity probes may, upon binding of the proximity probes in proximity to their respective targets, hybridise to one or more circularisable oligonucleotides (which may be viewed as a padlock probe specific for one or both of the nucleic acid domains of the proximity probe pair), allowing a ligation reaction to be templated to generate a nucleic acid circle which may be subjected to RCA. The proximity probes may be secondary reagents which bind to specific binding partners which are themselves bound to the target analyte. However, the proximity probes may alternatively be primary reagents which bind directly to the target analyte. A single circularisable oligonucleotide (padlock probe) may be used, which hybridises to both nucleic acid domains of the two proximity probes. However, various configurations are possible, including the use of a 2-part padlock probe (2 circularisable oligonucleotides) which are hybridised to the nucleic acid domains, such that the hybridisation brings the respective 5’ and 3’ ends of the oligonucleotides together in juxtaposition for ligation. The ligation may be templated by one or both of the nucleic acid domains. In an embodiment, RCA of the resulting circle may be primed by a nucleic acid domain. In such an embodiment, one nucleic acid domain may template ligation, and the other may prime RCA. In another embodiment both nucleic acid domains may template a ligation, and one nucleic acid domain may also prime RCA. In another embodiment, a separate RCA primer is used.

Accordingly, in more detail, a padlock probe may alternatively be defined as a circularisable probe. The use of padlock or circularisable probes is well known in the art, including in the context of RCA reactions. A circularisable probe comprises one or more linear oligonucleotides which may be ligated together to form a circle. Padlock probes are well known and widely used and are well-reported and described in the literature. Thus, the principles of padlock probing are well understood and the design and use of padlock probes is known and described in the art. A padlock probe is typically a linear circularisable oligonucleotide which hybridizes to its target nucleic acid sequence or molecule in a manner which brings 5’ and 3’ ligatable ends of the probe into juxtaposition for ligation together, either directly or indirectly, with a gap in between. By ligating the hybridized 5' and 3' ends of the probe, the probe is circularized. It is understood that for circularization (ligation) to occur, the ligatable 5’ end of the padlock probe has a free 5' phosphate group.

To allow the juxtaposition of the ends of the padlock probe for ligation, the padlock probe is designed to have the target-binding sites at or near its 5' and 3' ends. That is, the regions of complementarity which allow binding of the padlock probe to its target lie at or near the ends of the padlock probe.

To allow ligation, the 3’ and 5’ ends which are to be ligated (the “ligatable” 3’ and 5’ ends) are hybridized to their target sequence (i.e. to a complementary binding site), which acts as the ligation template. The ligatable ends of a padlock probe may be brought into juxtaposition for ligation in various ways, depending on the probe design. Where the target-binding sites are located at the ends of the padlock probe, the binding of the padlock probe may bring the ends into said juxtaposition. Where the complementary binding sites in the target molecule or sequence lie directly adjacent (or contiguous) to one another, the ends of the padlock probe will hybridise directly adjacent to each other (i.e. with no gap) and may be ligated to each other directly. Thus, in this case the ligatable ends of the probe are provided by the actual ends of the probe. However, in an alternative configuration the padlock probe is a gap-fill padlock probe, and hence the binding sites at the ends of the padlock probe do not hybridise to adjacent binding sites, but rather to non-adjacent (noncontiguous) binding sites in the target sequence. In such an arrangement, the 5’ ligatable end of the probe is provided by the actual 5’ end of the probe. However, the ligatable 3’ end of the probe is generated by extension of the hybridized 3’ end of the probe, using the target sequence as extension template to fill the gap between the hybridized ends of the probe. The extension reaction brings the extended 3’ end of the probe into juxtaposition for ligation. In this case, the ligatable 3’ end of the probe is thus the extended 3’ end of the probe.

Padlock probes may be provided in 2 or more parts that are ligated together. In the context of a proximity probe assay, the nucleic acid domains of a pair of proximity probes may each act as a ligation template. In another embodiment, a 2- part padlock may take the form of a “connector” oligonucleotide with two targetbinding regions at or near the 5’ and 3’ ends respectively, which hybridise to the target with a gap in between them, and a gap oligonucleotide which hybridizes in the gap between the ends. The gap oligonucleotide may partially or fully fill the gap.

Alternatively, the RCA template used to generate the RCP may be a circularised target nucleic acid sequence, or an amplicon, e.g. PCR or other copy thereof. A target nucleic acid molecule, or an amplicon of the target sequence may be circularised using a ligation template which hybridises to the ends of the molecule. Circularisation adaptors, or so-called “Selectors”, for circularisation of target nucleic acid molecules are described in WO 99/049079, WO 2003/012119 and WO 2005/070630. The method may be carried out in heterogenous or homogenous formats. That is, it may be performed on a solid phase (or support), or in solution or suspension (i.e. without a solid phase or support), or indeed both, since a solid phase may be introduced at a later stage.

The format of the method may be selected based on the nature of the sample, or the target nucleic acid molecule, or the desired readout or detection technology used.

Since the target nucleic acid molecule need not itself be the target analyte of the assay, but can be a reporter molecule used or generated in the course of an assay for any desired analyte, the sample need not be a sample which naturally contains nucleic acid, or a source of nucleic acid (e.g. a cell or virus, or biological or clinical material etc.), but can be a synthetic or artificial sample.

The sample may be any sample which contains a nucleic acid it is desired to replicate, amplify, analyse or detect etc. The target molecule may be present in cells in the sample, but this need not necessarily be the case, and the detection of cell- free nucleic acid (e.g. cell-free DNA) is included, for example in a blood sample. The sample may thus be a tissue sample, and this includes solid tissues and blood (blood being classified as a tissue). However, also included are any other samples which may contain cells or nucleic acid, and these may for example be body fluid samples, or other clinical samples which contain cells, for example washings or swabs etc., or cell-containing samples which have been artificially prepared, e.g. cell suspensions, or treated in any way. Fresh, frozen and fixed cell samples are included, e.g. FFPE samples. Accordingly, both natural and synthetic samples included, that is, materials which occur naturally or preparations which have been made. Naturally occurring samples may be treated or processed before being subjected to the methods herein. All cell-containing biological and clinical samples are included, e.g. any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates etc. Environmental samples, e.g. soil and water samples or food samples are also included. The samples may be freshly prepared or they may be prior-treated in any convenient way e.g. for storage.

Representative samples thus include any material which may contain a target nucleic acid molecule, including for example foods and allied products, clinical and environmental samples. The sample may contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Such biological material may thus comprise all types of mammalian and non-mammalian animal cells, plant cells, algae including blue green algae, fungi, bacteria, protozoa etc., or a virus. The cells may be for example human cells, avian cells, reptile cells etc., without limitation.

Representative samples thus include whole blood and blood-derived products which contain cells, blood cells, or any other cell-containing body fluids, tissues, biopsies, cell cultures, cell suspensions, etc. The sample may be pre-treated in any convenient or desired way to prepare for use in the method.

The methods and uses herein are thus particularly suited to detection methods which are performed in situ, that is to detect a target nucleic acid in situ in the native context in which it occurs. Thus, nucleic acids may be detected in situ in cells in which they occur. Thus, advantageously, the sample may be a tissue sample in an in situ detection assay.

The methods and uses herein are further particularly suited to detection methods performed on samples which may contain cancer cells or tumours. The present methods and uses thus find particular utility in detection assays for cancer cells or tumours. The methods may thus be performed in the context of clinical diagnostic assay, i.e. on clinical samples for the detection of cancer cells or tumours.

In an embodiment, the sample is immobilised, or is provided on a solid support. Thus, the method may be performed in a heterogenous, or solid phasebased format. In particular the sample may be provided on a slide or such like However, this is not a requirement, and homogenous, or solution-phase embodiments are included, as indeed are mixed phase methods with both solid phase and in-solution steps.

As indicated above, the sample may be a synthetic or artificial sample. It may accordingly be a sample which has been subjected to a detection assay for an analyte in which a target nucleic has been generated, or to which a target nucleic acid molecule has been added. It may be a reaction mixture, or a reaction product, for example the product resulting from an immunoassay to detect a target analyte, e.g. an immunoPCR, immunoRCA, or proximity assay (e.g. proximity ligation assay (PLA) or proximity extension assay (PEA).

The term "hybridisation" or "hybridises" as used herein refers to the formation of a duplex between nucleotide sequences which are sufficiently complementary to form duplexes via Watson-Crick base pairing, or any analogous base-pair interactions. Two nucleotide sequences are "complementary" to one another when those molecules share base pair organization homology. Hence, a region of complementarity in a molecule or probe or sequence refers to a portion of that molecule or probe or sequence that is capable of forming a duplex. Hybridisation does not require 100% complementarity between the sequences, and hence regions of complementarity to one another do not require the sequences to be fully complementary, although this is not excluded. Thus, the regions of complementarity may contain one or more mismatches. Accordingly, "complementary", as used herein, means "functionally complementary", i.e. a level of complementarity sufficient to mediate a productive hybridisation, which encompasses degrees of complementarity less than 100%. The degree of mismatch tolerated can be controlled by suitable adjustment of the hybridisation conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the respective molecules or probe or detection oligonucleotides etc., ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art. Thus, the design of appropriate probes, or ligation templates or primers etc. for any of the reaction steps describe herein, and binding regions thereof, and the conditions under which they hybridise to their respective targets is well within the routine skill of the person skilled in the art.

A region of complementarity, such as for example to a target sequence in the binding region of a padlock probe, or between a detection sequence and a detection oligonucleotide, or an RCA primer to its template (e.g. circularised padlock probe) etc., may be at least 6 nucleotides long, to ensure specificity of binding, or more particularly at least 7, 8, 9 or 10 nucleotides long. The upper limit of length of the region is not critical, but may for example be up to 50, 40, 35, 30, 25, 20 or 15 nucleotides. A complementary region may thus have a length in a range between any one of the lower length limits and upper length limits set out above. In the case of a padlock probe, the length of an individual target-binding region may be in the lower ranges, so that the total length of the two binding regions when hybridised to their target is within the upper ranges. For example, an individual target binding region may be 5-15, e.g. any one of 6, 7 or 8 to any one of 9,10, 11 or 12 nucleotides, so that the total hybridised length is for example 16-30 nucleotides long, e.g. 20-24. It may be desirable, within the constraints of conformation of the probes, and spacing of the domains, and desired or favoured hybridisations, to minimise the total length of a padlock probe to minimise the size of the circle which is subjected to RCA, and hence to minimise the lengths of the complementary regions where possible.

After the target nucleic acid molecule has been contacted with the phi29 polymerase composition, and the polymerase reaction has been performed, e.g. RCA, the polymerase reaction product, e.g. RCP may be detected using any convenient protocol or detection modality. This may depend on the target sequence to be detected, the purpose of the method, and/or the specific details of the procedures employed in the method

For example, the products may be labelled and detected using any of the well-established methods for analysis of nucleic acid molecules known from the literature which use detection of fluorescence or coloured or colorimetric labels, including for example microscopy, and imaging techniques.

Depending on the level of multiplexing, combinatorial labelling methods may be used, according to techniques well known in the art. For example, ratio labelling may be performed with different fluorescently labelled detection oligonucleotides.

Although various detection modalities may be employed, conveniently the labelled nucleic acid molecules may be detected by microscopy or flow cytometry. In particular, in a microscopy-based method, the labelled molecules (e.g. RCPs) may be detected by imaging.

The use of such detection techniques advantageously allows the nucleic acid molecules to be digitally recorded. Indeed, since the degree of signal amplification afforded by products such as RCPs in the methods herein allows them to be visualised, and they may be detected by a camera or any device including a camera, such as a mobile phone.

To detect products generated in a homogenous format, they may be captured or brought down to a solid support, or surface, to facilitate imaging, or microscopic detection more generally.

As noted above, the improvements to phi29 polymerase-catalysed reactions afforded by the present methods and uses, are particularly useful in the context of performing such reactions in automated instruments where the phi29-containing reagents are held or kept at ambient temperature for periods of time. This allows reagents to be prepared and introduced into the instrument, while minimising loss of phi29 activity. Indeed, it can be seen that this preservation of activity would provide benefits in all types of laboratory or analytical procedures, not just automated ones.

Particular mention may be made of tissue slide automation instruments such as the Leica Bond, Lunaphore Comet, Roche Ventana, or Dako Omnis. Such instruments are now widely used to perform analyte detection reactions on or in tissue or cell samples on slides, including fresh, frozen or fixed samples. Such detection assays may be performed using protocols comprising an amplification step with phi29 polymerase, as described above. For example, as well as the Duolink kits mentioned above, kits for performing PLA detections are commercially available from Navinci Diagnostics AB under the brand name NaveniFlex. These may variously provide proximity probes for use as primary or secondary binding agents for the target analyte and are configured to create a target circle which is amplified by RCA using phi29 polymerase. The RCP is detected using fluorescent detection oligonucleotides or detection oligonucleotides labelled with enzymes reactive with chromogenic substrates, specifically alkaline phosphatase (AP) or horseradish peroxidase (HRP). The high signal to noise ratio enables the detection of separate proximity events, allowing for a resolution down to a single protein or PPI. Example 1 below shows the use of a stabilised phi29 composition in a PLA protocol using such a kit in an autostainer, and the improved results that can be obtained.

Thus, a standard PLA protocol, e.g. isPLA protocol, may be modified according to the methods and principles herein to stabilise the RCA reaction buffer comprising phi29 DNA polymerase.

The method will now be described in more detail with reference to the Figures and non-limiting examples.

Examples

Example 1 - Detection of protein-protein interaction between E-Cadherin and B- Catenin in formalin-fixed, paraffin-embedded (FFPE) human colon tissue using NaveniFlex Tissue

In this experiment, in situ PLA was performed on human FFPE colon tissue using the NaveniFlex Tissue kit. Detection of the interaction between E-Cadherin and B- Catenin in FFPE human colon tissue was tested in various conditions with or without the stabilisation oligonucleotide in the RCA mixture and with or without storing the RCA mixture at room temperature for 18 hours prior to the assay. The in situ PLA was run manually, simulating the storage of reagents in the Leica Bond instrument by keeping the RCA mixture on the lab bench at room temperature. The RCA mixture where either freshly prepared (control) or pre-mixed with and without the stabilization oligo. The composition of the RCA mix was phi29, dNTPs, and 1x Thermo phi29 buffer. After approximately 18 hours on the lab bench at room temperature, the RCA mixture with and without the stabilization oligo was added to the tissue slide at the amplification step of the protocol. Also at this step, a freshly prepared RCA mixture was added as a control, both with and without the stabilization oligo, in order to verify that the stabilization oligo does not interfere with the in situ PLA.

The following steps were performed:

1. Deparafinization and antigen retrieval (60 min) 2. Wash (2 min)

3. Blocking (60 min)

4. Primary antibody incubation (60 min)

5. Wash (15 min)

6. Probe M1 and R2 (antibody proximity probe pair directed against mouse (M) and rabbit (R) (60 min)

7. Wash (15 min)

8. Ligation (30 min)

9. Wash (10 min)

10. RCA (90 min) - in this reaction mixture (1x), stabilization oligo is present at 0.05 pM.

11. Post-block incubation (30 min)

12. Detection (30 min)

13. Wash and stored in TBS-T until slide mounting

In Figure 1 , the results show that when the RCA reaction mixture buffer is freshly prepared and the stabilisation oligo is present, the interaction between E-Cadherin and B-Catenin is clearly visible (Condition C, Figure 1). When the buffer is prepared and stored on a lab benchtop for 18 hours at room temperature and no stabilisation oligo is present, the interaction between the E-Cadherin and B-Catenin is not visible (Condition B, Figure 1). In contrast, when the buffer is prepared and stored on a lab benchtop for 18 hours at room temperature and the 0.05 pM of the stabilisation oligo is present in the reaction mix, the interaction is detectable (Condition D, Figure 1) and the signal detected is comparable to that of Condition A, Figure 1 , where the buffer is freshly prepared but the stabilisation oligo is not present. The observed results were confirmed in different experimental conditions and using a different oligo sequence.

Claims

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. The method of claim 1 , wherein said method comprises preparing a composition comprising phi29 DNA polymerase and the stabilisation oligonucleotide.

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. 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. A method of detecting a target nucleic acid in a sample, said method comprising:

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. 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. 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. 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:

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. 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. 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. The method of claim 12, wherein the 2’ sugar modification is 2’-O-methyl or 2’ MOE.

14. The method of any one of claims 2 to 13, wherein the composition further comprises dNTPs and a buffer.

15. The method of any one of claims 3 to 14, wherein the target nucleic acid is a circular DNA molecule.

16. The method of any one of claims 3 to 15, wherein the polymerase or replication reaction is an amplification reaction.

17. The method of claim 16, wherein the amplification reaction is rolling circle amplification (RCA).

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. The method of claim 17, wherein the ligation product is generated in a proximity ligation assay (PLA), optionally an in situ PLA (isPLA).

20. The method of any one of claims 18 or 19, wherein the target nucleic acid is a ligated probe.

21. The method of claim 20, wherein the target nucleic acid is a circularised padlock probe, optionally wherein the padlock probe comprises one or more parts and is circularised using the nucleic acid domain of one or more proximity probes as a ligation template.

22. The method of any one of claims 1 to 21, wherein the phi29 polymerase exhibits increased temperature stability in the presence of the stabilisation oligonucleotide, compared to the absence of the stabilisation oligonucleotide.

23. A composition comprising phi29 polymerase and a stabilisation oligonucleotide, wherein the stabilisation oligonucleotide is protected from 3’ exonuclease degradation, and acts to stabilise the phi29 polymerase, and wherein in said composition the phi29 is not capable of performing a polymerase extension reaction.

24. The composition of claim 23, wherein the stabilisation oligonucleotide is as defined in any one of claims 11 to 13 and/or the composition is further defined as in claim 14.

25. The composition of claim 23 or claim 24, wherein the composition is freeze- dried.

26. Use of an oligonucleotide as a stabilising agent to stabilise phi29 polymerase, wherein the oligonucleotide is a stabilisation oligonucleotide which is protected from 3’ exonuclease degradation.

27. The use of claim 26, wherein the stabilisation oligonucleotide is as defined in any one of claims 11 to 13, and/or is for use in a method as defined in any one of claims 1 to 22.

28. A method of freeze-drying phi29 polymerase, said method comprising preparing an aqueous composition comprising phi29 polymerase and a stabilisation oligonucleotide, and freeze-drying the composition, wherein said stabilisation oligonucleotide is protected from 3’ exonuclease degradation.

29. The method, composition or use of any one of claims 1 to 28, wherein the phi29 polymerase, or the composition, is used, or is for use, in an automated tissue slide instrument.