Stabilization of Phi-29 polymerase

Abstract

The present invention provides a method for stabilizing phi-29 DNA polymerase by contacting it with a stabilized oligonucleotide protected from 3' exonuclease degradation. The phi-29 polymerase exhibits improved temperature stability in the presence of the stabilized oligonucleotide compared to its absence. The method comprises preparing a composition comprising phi-29 DNA polymerase and a stabilized oligonucleotide containing one or more modified nucleotides. The composition can be used in methods for carrying out polymerase reactions, methods for replicating nucleic acids, and methods for detecting target nucleic acids or analytes in a sample. The composition is particularly useful in proximity ligation assays, especially in rolling circle amplification reactions where the target is generated in a cyclic padlock probe.

Description

Technical Field

[0001] field The present disclosure and invention generally belong to the field of nucleic acid polymerization, and more particularly relate to the use of polymerase enzyme phi29, especially its use in the detection of nucleic acids. Specifically, the present disclosure and invention relate to the stabilization of phi29 preparations for such uses and the provision of stabilized phi29 preparations.

[0002] background The detection of target nucleic acid sequences is applied in many different fields, particularly clinically, for personalized medicine, and in the diagnosis, prognosis and / or treatment of diseases such as cancer, infectious diseases and genetic or inherited diseases, and also in research and biosecurity. The target nucleic acid sequence may be contained in a target analyte, i.e., a nucleic acid molecule present in a sample that is the target to be detected itself, such as mRNA, or a copy or amplicon thereof. Alternatively, it may be a nucleic acid molecule generated as a proxy, or signal, or rather, as a reporter for a target analyte in a sample that may be a nucleic acid or other molecule. Nucleic acids are usually used or generated as reporter molecules in the detection of proteins, for example, in the case of immuno-PCR or immuno-RCA methods, or in proximity assays using proximity probes. The proximity probe contains an antibody as a binding domain, and when the probe is in proximity, it is conjugated to interacting nucleic acid domains, for example, by ligation and / or hybridization and extension, to generate a nucleic acid molecule for detection. Nucleic acid detection assays often involve amplification, particularly rolling circle amplification (RCA). RCA is an isothermal amplification technique that requires a circular amplification template. Amplification of a circular template provides a concatenated RCA product (RCP) containing multiple copies of sequences complementary to the amplification template sequence. Since such concatemers usually form easily visible or detectable balls or "blobs," RCA-based assays are employed for nucleic acid detection, and indeed, more commonly, as reporter systems for the detection of any target analyte. Target nucleic acids, or probes, such as padlock probes, or more commonly, reporter nucleic acids, which can themselves be directly cyclic, provide the circular template nucleic acids for RCA (e.g., those used in immunoRCA reactions or those generated in proximity ligation assays).

[0003] RCA requires a strand-displacing polymerase capable of displacing the synthesized strand (extended from a primer hybridized to a circular template) and "rolling" along the circular template. A polymerase enzyme almost universally used for this purpose is Phi-29 polymerase derived from Bacillus subtilis phage 29. However, the use of Phi-29 DNA polymerase (hereinafter also referred to as Phi-29 polymerase or Phi-29) is not limited to RCA and is also applied in other protocols and processes that utilize polymerase extension reactions. In some cases, it is desirable to automate protocols that utilize Phi-29. Automation requires the Phi-29 reaction mixture to be prepared at the start of the automated protocol and left at room temperature before being added to the sample. Examples include common automated tissue slide instruments ("auto-stainers") used for tissue sample analysis, such as glass slides, e.g., Leica Bond, Lunaphore Comet, Roche Ventana, or Dako Omnis instruments. Phi-29 is commonly used to amplify detection signals in such tissue samples. Tissue slide instruments typically require all reagents to be filled before instrument operation and the reagents should not be cooled below room temperature.

[0004] Unfortunately, phi-29 DNA polymerase exhibits limited stability or half-life at temperatures above freezing, including room temperature and high temperatures. This limits the use of phi-29-based detection protocols in such instruments. As mentioned above, phi-29-based reactions, particularly RCA, have many advantages that make them an attractive detection methodology, so finding a solution to the limited stability problem is desirable. While phi-29 variants and mutants have been developed to exhibit extended half-lives at high temperatures, other or improved methods for stabilizing phi-29 are still needed to enable their use in protocols and instruments that handle or hold reagents for extended periods at room temperature or higher temperatures.

[0005] overview We propose adding an inactive, 3'-exonuclease-protected oligonucleotide to the phi-29 enzyme reaction mixture to stabilize the phi-29 polymerase when it needs to be stored or maintained for extended periods at temperatures below freezing. Hereinafter referred to as “stabilized oligonucleotide” (“stabilized oligo”), it generates a “walk-away” solution for the reaction mixture or reaction buffer requiring phi-29 enzyme stability. While stabilized oligonucleotides are useful for protecting aqueous or liquid preparations of phi-29, they are also useful for stabilizing lyophilized preparations, particularly the enzyme during the preparation of compositions for lyophilization and / or the reconstitution of lyophilized preparations. In particular, it is useful to include stabilized oligonucleotides in compositions for lyophilization.

[0006] Accordingly, a first aspect provided herein is a method for stabilizing phi-29 DNA polymerase, comprising contacting the phi-29 DNA polymerase with a stabilizing oligonucleotide, wherein the stabilizing oligonucleotide is protected from 3' exonuclease degradation. Such stabilized oligonucleotides may be referred to as 3'-exonuclease-protected oligonucleotides. They are oligonucleotides that contain one or more modifications that protect them from 3'-exonuclease degradation. In this context, it will be understood that contact between the phi-29 polymerase and the stabilized oligonucleotide is maintained. Therefore, phi-29 is provided or retained in contact with the stabilized oligonucleotide. Therefore, in particular, this method includes providing the phi-29 polymerase in a composition or preparation containing the stabilized oligonucleotide. In other words, this embodiment can be considered to provide a method for stabilizing phi-29 polymerase by providing (or formulating) a stabilizing oligonucleotide in combination with (or mixed with) the same, where the stabilizing oligonucleotide is protected from 3' exonuclease degradation.

[0007] In a second embodiment, this specification provides the use of an oligonucleotide as a stabilizer for stabilizing phi-29 polymerase, wherein the oligonucleotide (referred to herein as the stabilized oligonucleotide) is protected from 3' exonuclease degradation. More specifically, stabilizing oligonucleotides are used to stabilize phi-29 polymerase in preparations or compositions containing or related to phi-29 polymerase. In other words, stabilizing oligonucleotides are used to stabilize compositions or preparations of phi-29 polymerase.

[0008] In a third embodiment, this specification provides a composition comprising phi-29 polymerase and a stabilizing oligonucleotide, wherein the stabilizing oligonucleotide is protected from 3'-exonuclease degradation and acts to stabilize the phi-29 polymerase, so that in the composition, phi-29 cannot undergo polymerase elongation.

[0009] In other words, in all of the above embodiments, the composition is neither a reaction mixture capable of pha-29-catalyzed nucleic acid polymerization nor does it contain a reaction mixture capable of pha-29-catalyzed nucleic acid polymerization.

[0010] The aforementioned composition may be considered a reagent mixture containing phi-29 polymerase, or a mixture or composition, but not in a form in which a polymerase reaction can be carried out. Therefore, the aforementioned composition does not contain all the components or reagents (or reactants) necessary for the polymerase reaction to be carried out.

[0011] From another perspective, the compositions provided herein can be defined as comprising phi-29 polymerase and a stabilizing oligonucleotide, wherein the stabilizing oligonucleotide is protected from 3' exonuclease degradation and acts to stabilize the phi-29 polymerase, and the compositions do not contain a sample or alternatively a nucleic acid that is a substrate or target for the polymerase, for example, a nucleic acid that is amplified or replicated by the phi-29 polymerase, or in other words, a template nucleic acid.

[0012] Therefore, this embodiment can be considered to provide a reagent composition for use in contact with a sample containing nucleic acids to be amplified, replicated, extended, and / or detected, i.e., the composition is provided before contact with the sample. The sample will be understood to be a sample to be subjected to polymerase, for example, a sample containing a target nucleic acid molecule. This may be a nucleic acid molecule for replication, or amplification, or extension, or detection.

[0013] In one embodiment, the target nucleic acid is a template nucleic acid molecule for the nucleic acid polymerase reaction.

[0014] Furthermore, alternatively, a composition may be defined as one that does not contain a primer that initiates the polymerase reaction by phi-29 polymerase. Specifically, in one embodiment, the stabilized oligonucleotide is neither a primer itself nor acts as a primer for the extension reaction by phi-29 polymerase (i.e., for the polymerase reaction).

[0015] In other embodiments, as described above, the composition may be defined as one that does not contain the cations necessary for the enzymatic activity of phi-29 polymerase.

[0016] A fourth aspect of this specification provides a method for carrying out a polymerase reaction, the method comprising contacting a sample containing a target nucleic acid with a composition containing phi-29 polymerase and a stabilized nucleotide, and causing a polymerization reaction, wherein the stabilized oligonucleotide is protected from 3' exonuclease degradation. The acceptable conditions for polymerization reactions may depend on the composition brought into contact with the sample and whether or not additional reactants and / or compositions need to be added to enable enzyme activity. Specifically, the use of stabilized oligonucleotides makes it possible to prepare a composition and maintain it at a temperature above freezing point before use, i.e., before the polymerase reaction is initiated by contacting it with a sample or a composition required for a polymerase (e.g., RCA) reaction. This makes it possible to prepare a composition containing phi 29 (i.e., an RCA reaction mixture) and maintain it for a predetermined period (i.e., keep or hold) before the polymerization (RCA) reaction takes place.

[0017] Therefore, in certain embodiments, a composition comprising phi-29 polymerase and a stabilized oligonucleotide is prepared and maintained at a temperature above freezing (in particular, above 4°C, or at least ambient temperature or room temperature) for at least 15 minutes, more specifically at least 30 or 45 minutes, or at least 1 hour, before contact with the sample and / or before the RCA reaction is initiated. Specifically, an aqueous composition is prepared. In other words, a composition comprising phi-29 polymerase and a stabilized oligonucleotide in a buffer is prepared. Accordingly, such embodiments provide a method for carrying out a polymerase reaction, the method comprising preparing an aqueous composition containing phi-29 polymerase and a stabilized oligonucleotide protected from 3'-exonuclease action, and maintaining the composition at a temperature above freezing for at least 15 minutes before initiating the polymerase reaction.

[0018] In another embodiment, a composition comprising phi-29 polymerase and a stabilized oligonucleotide is prepared as a lyophilized composition. In other words, a composition comprising phi-29 polymerase and a stabilized oligonucleotide is prepared in a buffer and then lyophilized. In a particular embodiment, the lyophilized composition comprises phi-29 polymerase, a stabilized oligonucleotide, and a composition necessary to carry out the polymerization reaction in the buffer or other suitable medium (e.g., deoxynucleotide (dNTP), or a primer for the polymerase). Other suitable components necessary to carry out lyophilization methods known in the art may also be included in the composition.

[0019] Accordingly, such embodiments can be considered to provide a method for lyophilizing a composition, the method comprising preparing a composition comprising phi-29 polymerase and a stabilized oligonucleotide, and optionally comprising other components or reactants for polymerization reactions (e.g., as described above), or other components or reactants to facilitate, assist, or improve lyophilization, and lyophilizing the composition. Lyophilization can be achieved according to suitable techniques well known in the art. The composition can then be resuspended by adding water or other suitable liquid (e.g., aqueous) media, thereby returning it to an aqueous composition. This is known as reconstitution. Due to the presence of the stabilized oligonucleotide, the phi-29 polymerase is stabilized before, during, and after both the lyophilization and reconstitution processes. Therefore, lyophilizing the composition does not affect the stability of the phi-29 polymerase.

[0020] Lyophilized compositions are particularly advantageous in that they improve the ease of transport during distribution. While aqueous compositions require storage and transport at a temperature of -20°C, lyophilized compositions can be transported at room temperature or 4°C.

[0021] In certain embodiments, the composition is maintained prior to contacting with a sample containing a target nucleic acid (i.e., the nucleic acid to be subjected to the polymerization reaction). In other embodiments, the composition contains the sample or the target nucleic acid and one or more other components necessary for the polymerase reaction, such as primers, dNTPs, or a reaction buffer / reaction mixture necessary for polymerase activity, such as cations, are omitted from the composition. Thus, the composition may lack one or more components necessary for the polymerase reaction, and the reaction may be initiated by contacting the composition with the one or more components (which may be the sample / target nucleic acid and / or other polymerase reaction reagents or polymerase reaction mixture components). The polymerization reaction occurs upon contact with the one or more components lacking in the composition.

[0022] Thus, in one embodiment, the sample is contacted with the composition under conditions that allow a polymerization reaction catalyzed by phi29 polymerase to occur. Such conditions will typically include the presence of deoxynucleotides (dNTPs). Primers for the polymerase may be provided separately or may be present in the sample, for example, in the context of the target nucleic acid or as part of it, in the sample. Other conditions, which are described in more detail below, include appropriate temperature and buffer. <**********> In one embodiment, the polymerization reaction is an amplification reaction. More specifically, the polymerization reaction is an RCA reaction.

[0024] A fifth aspect of the present specification provides a method for replicating a nucleic acid, the method comprising contacting a target nucleic acid (more specifically, a sample containing the target nucleic acid) with a composition comprising phi29 polymerase and a stabilized oligonucleotide, wherein the stabilized oligonucleotide is protected from 3'-exonuclease degradation. More specifically, a nucleic acid or a sample containing the same is contacted with the composition under conditions under which phi29 polymerase can replicate the nucleic acid. Such conditions include, for example, the presence of at least dNTPs. As described above, the primer may be present in the sample or in the target nucleic acid, may be pre-hybridized with the target nucleic acid, or may be added separately, for example, added together with or as part of the composition. As described above, in an embodiment of this aspect, the composition is pre-prepared (i.e., pre-prepared as an aqueous composition such as a reconstituted composition) and maintained at a temperature above freezing for at least 15 minutes before contacting with the target nucleic acid / sample (i.e., before the contacting step).

[0025] The replication reaction may be an amplification reaction, for example, RCA. Any primer extension reaction, more specifically a primer extension reaction using a target nucleic acid molecule as an extension template, may also be used.

[0026] In one embodiment, the method is a method for amplifying a target nucleic acid, and includes contacting a sample with the composition defined or described above, and amplifying the target nucleic acid to generate an amplification product.

[0027] The sixth aspect of the present specification provides a method for detecting a target nucleic acid in a sample, the method including the following. (i) Contacting the sample with a composition containing phi29 polymerase and a stabilized oligonucleotide, and causing a polymerase reaction using the target nucleic acid as a template to be performed by the phi29 polymerase, wherein the stabilized 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 is maintained at a temperature above freezing for at least 15 minutes before contacting with the sample (i.e., before the contacting step). As described above, the composition may be a reconstructed composition (i.e., a reconstructed freeze-dried composition). As described above, in one embodiment, the polymerization reaction is an amplification reaction. More specifically, the polymerization reaction is an RCA reaction, and the amplification product is an RCA product (RCP). In one embodiment, the detection method is performed within or using an automated tissue slide device.

[0028] In all of the above embodiments, the target nucleic acid may more specifically be referred to as a target nucleic acid molecule. The target nucleic acid molecule may include 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 the detection assay (e.g., a target nucleic acid molecule that is naturally present in the sample), or a complementary copy thereof, or a copy or amplicon containing their amplicons. Alternatively, it may be a nucleic acid used or detected in the detection method as a means of detecting the target analyte, in other words, a reporter nucleic acid used or produced in a detection assay for the target analyte. Thus, the target nucleic acid molecule may be a nucleic acid molecule produced as a detection assay reaction product by a detection assay for detecting the target analyte in a sample. Alternatively, it may be a 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 analysis, which is detected as a means of detecting the binding of the partner (e.g., an antibody) to the analyte. Therefore, the target nucleic acid molecule may be a reporter nucleic acid molecule that reports the presence of the target analyte in the sample (i.e., is an indicator of its presence).

[0029] In certain embodiments, the nucleic acid molecule is the product of a detection assay for a target analyte in a sample. Accordingly, the seventh embodiment provides a method for detecting a target analyte in a sample, wherein a detection assay is performed to detect the analyte, the assay detects the analyte by generating the target nucleic acid molecule to be detected in situ, and the method comprises the following: (i) After generating the target nucleic acid molecule in the sample, the sample is brought into contact with a composition comprising phi-29 polymerase and a stabilized oligonucleotide, and the phi-29 polymerase is subjected to a polymerase reaction using the target nucleic acid molecule as a template, wherein the stabilized oligonucleotide is protected from 3'-exonuclease degradation; (ii) Detect the product of the polymerase reaction to detect the target nucleic acid molecule, thereby detecting the target analyte.

[0030] As described above for other embodiments, in one embodiment, the composition is prepared or reconstituted and maintained at a temperature above freezing for at least 15 minutes prior to the contact step. In a more specific embodiment of the above method, if the composition is maintained at a temperature above freezing for a predetermined time, that time is at least 0.5 hours, 0.75 hours, or 1 hour. In any of the above embodiments, the target nucleic acid molecule is a probe, a part of a probe, or a product generated from a probe.

[0031] In one 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 probe parts of a ligated probe), an extension product (e.g., an extended probe or probe part), or a cleavage product (e.g., a cleaved probe or probe part). In other embodiments, as described above, the target nucleic acid molecule may be provided in the sample, for example, as a means for detecting a target analyte in the sample.

[0032] In one embodiment, the target nucleic acid molecule is a cyclic nucleic acid molecule. For example, it is a cyclic target nucleic acid analyte, or a copy or amplicon thereof (including a complementary copy or amplicon). In other embodiments, it is a cyclic probe, for example, a cyclic padlock probe or a molecular inversion probe (MIP).

[0033] In one embodiment, the target nucleic acid molecule is a nucleic acid product generated in a proximity assay, such as a proximity ligation assay (PLA). In this embodiment, the target nucleic acid molecule is a ligation product.

[0034] The sample may be any sample containing the target nucleic acid, regardless of whether it is naturally occurring, added to the sample, or generated within the sample. In one embodiment, the sample may be a tissue sample, and may be a solid tissue sample or a blood sample.

[0035] In one embodiment, the sample is an immobilized tissue sample or cell sample, and in particular a tissue sample or cell sample on a slide.

[0036] In one embodiment, the target nucleic acid molecule is a target nucleic acid molecule generated in situ in a tissue sample, including a blood sample. In a more specific embodiment, the nucleic acid molecule is generated by an in situ PLA (isPLA) reaction.

[0037] In the above embodiment, the stability of phi-29 polymerase increases in the presence of the stabilizing oligonucleotide compared to the absence of the stabilizing oligonucleotide. In particular, its temperature stability increases. [Brief explanation of the drawing]

[0038] [Figure 1]Figure 1: Fluorescence microscopy scan of human FFPE colon tissue after in situ PLA using the NaveniFlex Tissue Kit to detect the interaction between E-cadherin and B-catenin. In situ PLA was performed under four different assay conditions. For each condition, segmented grayscale images of the nucleus (DAPI) and interaction (E-cadherin-B-catenin) are shown. Condition A: In situ PLA was performed using a freshly prepared RCA reaction mixture. Condition B: In situ PLA was performed using an RCA reaction mixture that had been prepared and stored on the bench at room temperature for 18 hours. Condition C: In situ PLA was prepared using a freshly prepared RCA reaction mixture supplemented with 0.05 μm stabilized oligonucleotides. Condition D: In situ PLA was performed using an RCA reaction mixture that had been prepared with 0.05 μm stabilized oligonucleotides and stored on the bench at room temperature for 18 hours. The RCA reaction mixture contains phi-29 DNA polymerase and dNTPs in a 1X Thermo Scientific reaction buffer for phi-29 DNA polymerase.

[0039] Detailed explanation The methods and uses described herein address the stability issues of phi-29 polymerase, specifically the limits of its stability in solution when stored at ambient or elevated temperatures, or when held at any temperature above freezing point (e.g., including temperatures above 4°C). We found that oligonucleotides protected from 3'-exonuclease degradation (i.e., digestion by 3'-exonuclease or degradation by 3'-exonuclease action) can act beneficially as stabilizers for phi-29 polymerase, specifically as stabilizers that improve or increase its temperature stability.

[0040] Accordingly, according to the method and use of the present invention, the stabilizer can be added to or contained in a preparation (i.e., a composition) of phi-29 polymerase, wherein the stabilizer is an oligonucleotide protected from 3'-exonuclease degradation.

[0041] In this specification, the term “oligonucleotide” means a relatively short nucleic acid molecule, in accordance with the common meaning in the art. The stabilized oligonucleotides described herein can stabilize phi-29 polymerase. While not intended to be theoretically bound, this is assumed to occur by the polymerase binding to the stabilized oligonucleotide. Specifically, the stabilized oligonucleotide is long enough to bind to phi-29 polymerase. In other words, its length is at least equivalent to the minimum phi-29 enzyme footprint (i.e., the length of the sequence in the substrate (e.g., template) nucleic acid molecule to which the enzyme binds or interacts).

[0042] In one embodiment, the stabilized oligonucleotide is at least 6, 7, 8, 9, or 10 nucleotides long. The maximum length of a stabilizing oligonucleotide is not critical. From the standpoint of ease of synthesis, the length typically does not exceed 200, 150, 100, 90, 80, 70, 60, or 50 nucleotides. Therefore, the length of a stabilizing oligonucleotide can be within any of the integer ranges listed above. For example, a stabilizing oligonucleotide can be any length from 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 to 25, 28, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides. In certain embodiments, the stabilizing oligonucleotide is any length from 9, 10, or 11 to 20, 25, or 30 nucleotides.

[0043] Stabilized oligonucleotides are protected from 3' exonuclease degradation. This means they are modified to be resistant to degradation by 3' exonuclease enzyme activity, or more specifically, modified to have reduced sensitivity to 3' exonuclease degradation (whether the exonuclease activity is provided by a single or independent exonuclease enzyme having 3' exonuclease activity, such as exonuclease I, III, or V, or by some or a component thereof having 3' exonuclease activity). In this regard, stabilized oligonucleotides are resistant to 3' exonuclease degradation by phi-29 polymerase. Modifications that confer 3'-exonuclease resistance to oligonucleotides are well known in the art and can be used. Such modifications are usually incorporated during oligonucleotide synthesis and include modifications to the sugar (deoxyribose or ribose) portion of the nucleotide, or modifications to the bonds between adjacent nucleotides in the oligonucleotide ("internucleotide bonds"). The latter modification is also called "skeletal modification." Thus, stabilized oligonucleotides include modified nucleotides, and this term is widely used to refer to nucleotides not found in nature, i.e., non-natural or non-inherent nucleotides, in the context of nucleic acid molecules present in living organisms. The modification may be within the structure of the nucleotide, particularly within the structure of a sugar-containing nucleotide, or it may be a skeletal modification (in which the nucleotide skeleton is modified). Thus, a modified nucleotide may be a nucleotide linked to an adjacent nucleotide by a non-natural bond (i.e., a bond other than a phosphated ester bond).

[0044] A stabilized oligonucleotide may contain one or more modified nucleotides, which may depend on the nature and / or length of the modification of the oligonucleotide. The stabilized oligonucleotide may contain a single modified species or type, or a combination of different modifications. The modified nucleotides, especially if they involve sugar modification, are usually located at the 3' end of the oligonucleotide, but in some embodiments, they may be present along the entire length of the oligonucleotide. In one embodiment, 1, 2, 3, or 4 nucleotides are modified from the 3' end, but the stretch of the modified nucleotides may be longer. The modified nucleotides may be DNA, RNA, or synthetic nucleic acids.

[0045] Modified oligonucleotides containing modifications that restrict 3' exonuclease degradation may be commercially available, for example, from Integrated DNA Technologies.

[0046] Specific modifications to sugars include 2' substitutions (substitution at the 2-position of ribose), such as 2'-ribo(2'OH); 2'-O-methyl(2'OMe; 2'methoxy); 2'-O-methoxyethyl(2'MOE); or 2'fluoro(2'F). Other modifications to sugar groups may be carried out similarly or alternatively. Specific examples of nucleotides with sugar modifications include 2'F RNA; 2'OMe nucleotide; LNA (Locke nucleic acid); FANA (2'-fluoroarabino nucleic acid); HNA (hexitol nucleic acid); or 2'-O-methoxyethyl (2'MOE) nucleic acid. In one embodiment, the stabilized oligonucleotide comprises one or more, for example, 1 to 3, 4, 5, or 6 2'-O-modified ribose nucleotides (e.g., 2'OMe, or 2'MOE).

[0047] The modified nucleotide may contain any base. In one embodiment, the modified nucleotide is 2'-O-methyluracil.

[0048] Examples of skeletal modifications or modified nucleotide bonds include modifications to the phosphate group in phosphodiester bonds, and examples of phosphodiester bonds include phosphorothioate or phosphorothiolate bonds, borano-phosphate bonds, or polyethylene linker skeletons incorporated between nucleotide residues. As is well known in the field, phosphorothioate modification alters the phosphate bond between bases by substituting one of the oxygen atoms in the bond with a sulfur atom. Specifically, in phosphorothioate modification, the non-bridged oxygen covalently bonded to phosphorus is substituted with a sulfur atom. Similarly, in borano-phosphate modification, the non-bridged oxygen is substituted with BH3. In phosphorothiolate modification (also known as 5'-thionucleoside), the sulfur atom substitutes the 5'-bridged oxygen bonded to the sugar residue. In one embodiment, the skeletal modification is either not a phosphorothiolate modification or does not include a phosphorothiolate modification.

[0049] Other known skeletal modifications that may be used include phNA (methylphosphonate, in which the non-crosslinked oxygen in the phosphate bond is replaced by CH3) or peptide nucleic acid (PNA). Other skeletal (or binding) modifications include those involving sugar residues in nucleotides (i.e., so-called sugar / skeletal modifications). Such modifications known and described in the art include mirror DNA, libro-NA, TNA, tPhoNA, and dXNA.

[0050] Typically, one or more modified bonds are required to confer effective 3'-exonuclease resistance, usually at least three bonds, or three to six bonds, such as phosphorothioate bonds. In some embodiments, all bonds within the stabilizing nucleotide are modified bonds.

[0051] In certain embodiments, the stabilized oligonucleotide comprises one or more 2'-modified nucleotides and / or phosphorothioate bonds, and in particular comprises at least two, three, four, five, or six such modifications.

[0052] Other modifications to protect against 3' exonuclease degradation include reverse dT or ddT. Such modified nucleotides can be incorporated into the 3' end of a stabilizing oligonucleotide, forming a 3'-3' bond that inhibits both 3' nuclease degradation and polymerase elongation. Further possible modifications include the incorporation of a phosphoramidite C3 spacer, 3' phosphorylation, or 3' hexanediol at the 3' end of the oligonucleotide.

[0053] In certain embodiments, the stabilized oligonucleotide comprises only modified nucleotides having structural modifications, specifically, only modified nucleotides having structural modifications to a sugar group. In certain embodiments, the stabilized oligonucleotide comprises one or more modified nucleotides having a modification at the 2-position of the sugar.

[0054] Typical examples of stabilized oligonucleotides used in the following examples are as follows: 5' TGACTGGGAATGTAGGAGCAmUmUmU 3' (Sequence ID: 1) 5' GGACTACATATCTTACTACGUmUmU 3' (Sequence ID: 2)

[0055] The sequence of the stabilizing oligonucleotide is not important, and those skilled in the art know how to design or select an appropriate oligonucleotide sequence, for example, to minimize undesirable interactions and avoid secondary structures. The stabilizing oligonucleotide may or may not be able to bind to the resulting polymerase product (e.g., RCP). That is, it may or may not contain a sequence that is complementary to (or, in other words, homologous to) the polymerase product. In one embodiment, the stabilizing oligonucleotide does not hybridize to the polymerase product, specifically RCP, at the site where the detection oligonucleotide is intended to bind (i.e., the so-called detection sequence; as will be discussed later, polymerase products such as RCP are often detected by hybridizing the product with a labeled detection probe, which is referred to herein as the detection oligonucleotide). However, in another embodiment, the stabilizing oligonucleotide may bind to the detection sequence. In another embodiment, the stabilizing oligonucleotide may bind to the polymerase product, e.g., RCP, at a site different from the detection sequence.

[0056] In a preferred embodiment, the stabilizing oligonucleotide does not contain a sequence complementary to the polymerase product (i.e., the stabilizing oligonucleotide is not homologous to and does not bind to the template / target nucleic acid). Because the stabilizing oligonucleotide cannot bind to the template / target nucleic acid, the phi-29 polymerase cannot act as a primer for amplifying the template / target nucleic acid, and no polymerization product (e.g., amplification product, e.g., RCP) is produced (i.e., the stabilizing oligonucleotide does not act or cannot act as a primer). Therefore, if the stabilizing oligonucleotide is present in the reaction mixture but the phi-29 polymerase primer is not present, the reaction will not occur.

[0057] In another embodiment, the stabilized oligonucleotide does not bind to the nucleotide used for nucleotide identification and nucleic acid sequencing, and more generally, does not bind to the nucleotide incorporated into the polymerase elongation reaction. In other words, the stabilized oligonucleotide does not bind to the nucleotide as a tag. In one embodiment, the stabilized oligonucleotide does not bind to the nucleotide as a tag. In another embodiment, the stabilized oligonucleotide does not bind to the nucleotide used as a tag in the incorporation reaction. Herein, the term “incorporation reaction” is used herein in accordance with the common sense of the art and means a reaction comprising a nucleic acid polymerase that catalyzes the incorporation of a tagged nucleotide into an oligonucleotide.

[0058] Phi-29 polymerase, derived from bacteriophage phi-29, is a 66 kDa monomeric enzyme and a protein-prime DNA-dependent replicase belonging to the eukaryotic DNA polymerase family (Family B). Like other DNA polymerases, it performs DNA synthesis by adding nucleotides to the 3'OH group of the elongating DNA strand. It contains an exonuclease domain that catalyzes the 3'→5' exonuclease degradation (proofreading) of mismatched nucleotides. Phi-29 possesses distinctive features such as strong binding to single-stranded DNA, extremely high reactivity (requiring no reactive factors for DNA synthesis), and the ability to unwind parental DNA helices, enabling the replication of double-stranded genomic DNA without unwinding factors. Phi-29 can be used to amplify any DNA sample, without the need for sequence information, and can be used with simple random primers. These properties are useful in a variety of applications, including isothermal amplification processes such as RCA and multiple substitution amplification (MDA). Uses include RCA- or MDA-based nucleic acid detection methods (e.g., in situ genotyping analysis using padlock probes, or in situ PLA), whole-genome amplification (WGA) using various sample types, DNA amplification from single cells, uncultured microbial cells, or viral particles, DNA preparation for SNP or short tandem repeat (STR) detection, protein-primed amplification, cell-free cloning of lethal DNA, and RNA-primed DNA amplification. All of these uses and applications are included herein.

[0059] Phi-29 polymerase (also known as Phi-29 DNA) is commercially available from various suppliers, such as New England Biolabs and Thermo Fisher Scientific. In addition to the wild-type enzyme, various variants or derivatives of Phi-29 have been developed to enhance or improve various properties and broaden the range of possible applications. These include various mutations (amino acid substitutions) in enzymes. For example, the mutant enzyme EquiPhi29, available from Thermo Fisher Scientific. TM DNA polymerase was developed through in vitro protein evolution, exhibiting improved thermal stability, reaction rate, product yield, and amplification bias while retaining all the advantages of the wild-type enzyme. 4BB TM QualiPhi (R) This is a chimeric form of Phi29 polymerase designed to improve sensitivity and efficiency, and is available from 4basebio SLU in Spain. Phi29-XT RCA, available from New England Biolabs, is another designed version that has improved thermal stability and sensitivity compared to wild-type Phi29 polymerase and exhibits high product yield.

[0060] Numerous Phi-29 variants and derivatives have been reported in the literature. Examples include EP2813576 (improved protein stability and half-life), EP3854872 (improved thermal stability), and WO2021 / 163052 (improved reaction acceleration, chain displacement activity, template or primer binding, 3'-exonuclease activity, nucleotide sensitivity, or temperature stability, or reduced error rate). Povilaitis et al., 2016, Protein Engineering Design and Selection 29(12), 617-628 describes an improved Phi-29 variant developed by isothermal compartmentalization self-replication technology for use in WGA, and Gao et al., 2021 Microbial Biotechnology, 14(4), 1642-1656 describes a chimeric Phi-29 polymerase with a helix-hairpin-helix motif that exhibits improved salt tolerance and replication performance. (All references cited herein are incorporated by reference). Such variants and derivatives described in these or other documents, or available from any source, may be used.

[0061] Therefore, as used herein, the terms “Phi-29 DNA polymerase” (and the abbreviated “Phi-29 polymerase and Phi-29”) include wild-type enzymes and any variants or derivatives thereof, specifically including variants or derivatives that retain the function and properties of the wild-type enzymes.

[0062] The use and methods described herein improve the stability of Phi 29. Thus, Phi 29 polymerase exhibits increased (i.e., enhanced or improved) stability in the presence of a stabilizing oligonucleotide compared to its stability in the absence of the stabilizing oligonucleotide. Therefore, the stability of Phi 29 polymerase in a composition can be compared with and without the presence of the stabilizing oligonucleotide. “Stability” refers to the ability of an enzyme to retain its activity, i.e., polymerase activity in this case. The term “temperature stability” is used herein as synonymous with “thermal stability” or “heat resistance,” and means the ability of an enzyme to maintain its activity at high temperatures. More specifically, the enzyme can maintain its activity at high temperatures for extended periods, for example, at least 3 hours, and more specifically, at least 4, 5, 6, or 7 hours. In certain embodiments, the activity is stabilized for at least 10, 12, 15, or 18 or 24 hours. “High temperature” as used herein refers to any temperature above freezing point, specifically including ambient temperature or room temperature, e.g., 20°C or 21°C.

[0063] In particular, the above description of stability applies in the context of aqueous compositions of the Phi-29 enzyme, for example, solutions of the Phi-28 enzyme.

[0064] Specifically, in the presence of a stable oligonucleotide, phi-29 polymerase can retain its activity, or retain its activity more effectively, when held in a buffer composition (e.g., a reaction buffer) at room temperature for 3 hours or more, compared to the absence of the stable oligonucleotide. Performing assays of phi-29 polymerase activity to compare activity levels under different time and temperature conditions (with or without the stable oligonucleotide) is within the scope of the ordinary skill of those skilled in the art. Furthermore, as shown in the following examples, the improvement in stability can be confirmed by the improved results obtained from detection assays that detect RCA products using a detection protocol including a phi-29 catalyzed RCA reaction, in the presence of the stable oligonucleotide compared to its absence. Thus, the improvement in stability is attributed to the improved performance of phi-29 polymerase in the detection protocol, in which a target nucleic acid (reporter or analyte) is detected using phi-29 polymerase, and a detectable product is produced.

[0065] As described above, phi29 polymerase is provided or formulated as a composition (in other words, a preparation) comprising phi29 polymerase and a stabilized oligonucleotide. Typically, such composition contains the polymerase and oligonucleotide in a buffer. Therefore, the provided or prepared composition is an aqueous composition. Alternatively, the provided or prepared composition (e.g., an aqueous composition) can be lyophilized and then reconstituted into an aqueous composition. Suitable buffers for phi29 polymerase are known in the art and are commercially available, for example, Thermo Scientific Reaction Buffer for phi29 DNA polymerase.

[0066] The buffer may contain components necessary or optional for phi-29 polymerase activity, such as cations.

[0067] A typical storage buffer for Phi 29 contains 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5% (v / v) nonidet P40, 0.5% (v / v) Tween 20, and 50% glycerol. Therefore, the compositions herein, in addition to the enzyme and stabilizing oligonucleotide, may contain a buffer of approximately pH 7.5 (e.g., pH 7-8), and optionally EDTA, a salt (e.g., KCl), and further optionally a detergent or surfactant and / or glycerol. EDTA, being present in the storage buffer, tends to inhibit activity because it binds to cations.

[0068] The standard storage buffer, supplied at 10x concentration, contains 330 mM Tris-acetic acid (pH 7.9 at 37°C), 100 mM magnesium acetate, 660 mM potassium acetate, 1% Tween 20, and 10 mM DTT.

[0069] According to the methods and uses described herein, it may be convenient to provide phi 29 and stabilization in a buffer (i.e., to formulate the composition in a reaction buffer). Therefore, the composition may contain a buffer at around pH 7-8, one or more salts to provide cations, for example, Mg and K cations, specifically one or more salts to provide Mg cations. 2+ Cations are generally required for Phi-29 activity and are included in the reaction buffer. Furthermore, providing a reducing agent, such as DTT or an analogue, may be advantageous, as it has been reported to be useful for maximizing enzyme activity. Other possible components include detergents or surfactants. The buffer may be a Tris buffer.

[0070] The composition may further contain one or more additional components, such as other reagents for polymerase (e.g., amplification) reactions. Specifically, this may include dNTPs for incorporation. Thus, the composition may contain a reaction mixture for polymerase reactions, such as amplification reactions, such as reaction mixtures for RCA. In some embodiments, the composition may further contain one or more primers. However, as described above, in some embodiments, the primers are not required in the composition and are provided separately or are present in the sample to which the composition is added. For example, in some embodiments, the target nucleic acid may function as a primer itself or provide primers.

[0071] However, as stated above, the compositions claimed herein do not include a complete reaction mixture for a phi 29 catalyst polymerization / DNA synthesis reaction. That is, the compositions themselves do not have the ability to carry out a DNA polymerization reaction. As stated above, one or more components essential for the polymerase reaction are omitted. In certain embodiments, the compositions do not contain a sample, or a substrate or template nucleic acid. In other words, they do not contain a nucleic acid molecule that can function as a target nucleic acid molecule for a polymerase reaction, e.g., a target nucleic acid, e.g., as a nucleic acid to be replicated (copied) or amplified, or in other words, a nucleic acid molecule that can function as a template for a polymerase reaction, e.g., a target nucleic acid.

[0072] In another embodiment, the composition is cation-free, or more specifically, does not contain a magnesium cation source. In another embodiment, the composition does not contain a primer.

[0073] As described above, stabilizing oligonucleotides are useful for the preparation and reconstitution of freeze-dried (i.e., lyophilized) compositions of Phi 29. In this regard, stabilizing oligonucleotides may be included in the composition prepared for freeze-drying. Therefore, an aqueous composition may be prepared and then freeze-dried. Freeze-dried Phi 29 preparations prepared according to the methods described herein can be stored and / or transported at room temperature or refrigerated temperature (e.g., about 4°C). Freeze-drying has the advantage that freezing conditions (e.g., -20°C) are not required for the transport and delivery of Phi 29 enzyme products. The addition of stabilizing oligonucleotides stabilizes the enzyme during drying and reconstitution.

[0074] Therefore, the stabilizing oligonucleotide acts to stabilize the aqueous composition during its preparation and lyophilization processes, for example, while being dispensed into vials or other containers, and during the lyophilization waiting period and the lyophilization process itself. Thus, the stabilizing oligonucleotide protects the enzyme during room temperature preparation protocols or protocols using room temperature reagents. If reconstituted, the stabilizing oligonucleotide stabilizes the Phi 29 enzyme during the reconstitution process and in the reconstituted composition.

[0075] As described above, the aqueous composition prepared for lyophilization may contain any of the above-mentioned components, as well as other components. These may include buffers and one or more components or reagents necessary for the polymerase reaction. Specifically, the lyophilization composition may contain all the components necessary for the polymerase reaction.

[0076] As with components or reagents for downstream use of Phi-29 polymerase, compositions prepared for lyophilization may contain, for example, excipients or formulation aids, such as other stabilizers or aids for the lyophilization process. Available cryopreservatives are well known in the art and described in the literature and include proteins, carbohydrates, and organic polymers. Suitable proteins include inactive proteins routinely used as blocking agents, such as albumin (e.g., BSA, gelatin, or milk protein). Carbohydrates include sugars and sugar alcohols, such as trehalose, dextrose, sucrose, mannitol, and sorbitol. Polymers include polyvinylpyrrolidone or polyethylene glycol. Other components include, for example, surfactants, such as detergents, such as nonionic surfactants like Tween, Brij, or Triton surfactants. All standard cryoprotectants and excipients can be used, particularly those used for lyophilization of proteins, especially those used for lyophilization of enzyme preparations.

[0077] Similarly, standard procedures for freeze-drying can be used as are widely known and used in the field, including procedures used for proteins and, in particular, enzymes.

[0078] Determining the appropriate concentration of the stabilized oligonucleotide to be used is within the ordinary skill of those skilled in the art. However, generally speaking, the concentration of the stabilized oligonucleotide in the composition, particularly in the Phi 29 preparation before contact with the sample, may be in the range of 0.001 μM to 10 μM, more specifically from any of 0.002, 0.005, 0.01 or 0.02 to any of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μM. For example, the range may be 0.03 to 0.5 μM, e.g., 0.05 to 0.2 μM, or 0.05 to 0.1 μM.

[0079] As described above, the stabilized phi-29 polymerases described herein can be used in any application where phi-29 polymerase is useful. Therefore, they can be used in any polymerase reaction, such as replication reactions, amplification reactions, or in fact, any polymerase-catalyzed primer extension reaction. This may be done in the context of a broader protocol or process, including any of the applications described above.

[0080] In one 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 an MDA reaction.

[0081] Amplification reactions are carried out in a broader analytical context in certain embodiments, and amplification reactions (e.g., RCA) are carried out as part of a method (i.e., an analytical method or a detection method, etc.).

[0082] Various methods defining the stabilization of phi-29 polymerase or the use of a stabilized polymerase generally involve contacting phi-29 polymerase or a composition containing it together with other components, for example, with a stabilized oligonucleotide, or a sample, or a target nucleic acid.

[0083] In this specification, the term “contact” broadly includes contacting the reagents in question. Therefore, one may be added to the reagent, or vice versa, or they may be introduced to each other. The timing and order of addition or contact with the sample may depend on the exact nature of the method or steps of the method being performed. In the context of a detection or analytical method, for example, if the target nucleic acid is a reporter nucleic acid, the composition may be added to or introduced to the sample after the detection assay or analytical method steps have been performed, for example, after the sample for detection of the target analyte in a detection assay has been contacted with the detection reagent, i.e., after the target nucleic acid has been introduced into the sample (e.g., by an assay reagent containing the target nucleic acid), or after it has been generated in the sample (e.g., by a ligation reaction or extension reaction, etc.). Therefore, in one embodiment, the composition may be contacted with the target nucleic acid product after the target nucleic acid product has been generated. Therefore, the 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), after which the composition containing stabilized phi 29 may be introduced.

[0084] Furthermore, as mentioned above, contact between the composition and the target nucleic acid may occur after a delay period. In other words, contact may be delayed for a certain period after the composition has been prepared. This delay period may be a storage period, and for example, the reagents may be prepared prior to their use.

[0085] Accordingly, compositions may be prepared, more specifically, aqueous or lyophilized compositions of phi-29 polymerase and stabilized oligonucleotides, which may be purely for preservation purposes (i.e., in a preservation buffer), or the compositions may be suitable for use in the intended reaction (i.e., in a reaction buffer). As mentioned above, suitable buffers are known in the art. The compositions may be stored, maintained (e.g., held, or kept) at temperatures above freezing. This includes refrigerator or "above ice" temperatures, for example, 4°C or about 4°C. Advantageously, the compositions may be maintained at room temperature or room temperature or above.

[0086] The delay period is, as described above, at least 15 minutes, more specifically at least 30 or 45 minutes. In certain embodiments, it is at least 1 hour, for example, at least 1.5 hours, 2 hours, 2.5 hours, or 3 hours. It may be longer, for example, including 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or more. In some embodiments, the delayed contact may occur after 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours or more, for example, after 20 hours or 24 hours or more.

[0087] This is beneficial in automation. Therefore, the composition may be prepared and loaded into an apparatus or automated apparatus, such as an autotainer. It may be held or stored in the apparatus / apparatus until required for the polymerase reaction. Thus, in exemplary embodiments, it may be automatically dispensed. Such a composition may be a reaction mixture prepared for use, for example, prepared for contact with a sample, or a concentrated composition that is diluted before use.

[0088] As described above, stabilized Phi 29 is advantageously used in the context of detection assays according to the methods and uses described herein, for example, in the context of detection assays of target analytes. 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, tag, or signal) detected to detect the target analyte.

[0089] More specifically, the detection method may detect the target nucleic acid sequence in the target nucleic acid molecule. The term "target nucleic acid sequence" is used synonymously and interchangeably with "nucleotide sequence."

[0090] Detection assays are often performed in multiple assays to detect multiple analytes or targets in a single sample or mixture. Therefore, different nucleic acid molecules present in a sample may be detected in multiple assays.

[0091] Many detection assays rely on signal amplification to improve the sensitivity and accuracy of the assay, and accordingly include an amplification step involving polymerase. As mentioned above, RCAs are particularly useful in that they produce large nucleic acid products, RCPs, which can be easily labeled and detected. RCPs are produced as the final reactant, signal, or marker of the assay, and are detected in many assay methods as a means of detecting the target analyte of the assay.

[0092] Nucleic acid detection methods can be used to detect variants of target nucleic acid sequences. Target nucleic acid sequences generally occur in the form of variants, such as allele variants, or in variants and wild-type sequences, and it may be desirable to detect which variants are present. Therefore, the target nucleic acid sequence may be one of several different variants of the nucleic acid sequence that can occur in the target nucleic acid molecule. Detection methods can be used to detect other types of analytes, such as proteins including cell surface proteins, and protein-protein interactions (PPIs). Therefore, in certain embodiments, the target analyte is a protein or protein-like molecule (i.e., a protein-containing molecule), or a protein complex or PPI. As described above or later, such analytes can be conveniently detected by proximity assays.

[0093] Since RCP contains or is composed of monomer units (i.e., monomer repeats of concatemer RCP), it exhibits attractive detection assay reaction products, which may be at least 100, 200, 300, 400, or 500 monomers. Each monomer may have a binding site for a detection oligonucleotide, thereby enabling detection of RCP. The detection oligonucleotide is a hybridization probe that includes a binding site complementary to the binding site in the PCR monomer, and a detectable portion, and includes a label such as fluorescence, color, or colorimetric labeling. RCP is particularly advantageous because it can be coiled into spherical or lumpy forms, which are separate, visible entities.

[0094] Because they are large, RCPs may have multiple copies of the detection oligonucleotide used for labeling. In other words, they contain multiple sites for binding the detection oligonucleotide. Such binding sites can be called detection sequences. Detection sequences can be considered as tag sequences.

[0095] As used herein, the terms “multiple” or “multiple” mean two or more, for example, three, four, five, six, eight, nine, ten, fifteen, twenty, twenty-five, three, four, fifty, sixty, seventy, eighty, ninety, or one hundred or more. In fact, for many types of nucleic acid products, this may include thousands or more detection sequences and binding sites of labeled detection oligonucleotides. Therefore, in the context of the number of copies of detection oligonucleotides, or the binding / attachment sites of detection oligonucleotides, or detection sequences, “multiple” may include at least two hundred, three hundred, four hundred, five hundred, six hundred, seven hundred, eighty, ninety, or one thousand, or multiples thereof.

[0096] Amplification methods such as MDA are similarly used to generate branched amplification products, which include multiple detection sites for the binding of detection oligonucleotides.

[0097] The target nucleic acid molecule (e.g., nucleic acid reporter or product, or analyte) is typically a DNA molecule. However, it may be composed of, or contain, other natural or synthetic nucleic acids. For example, it may be a chimeric construct containing both RNA and DNA. The nucleic acid product may consist of ribonucleotides and / or deoxyribonucleotides, as well as synthetic nucleotides capable of participating in Watson-Crick type or similar base-pair interactions. Therefore, the target nucleic acid product may be, for example, bisulfite-converted DNA, LNA, PNA, or any other derivative containing a non-nucleotide backbone.

[0098] As described above, the detection method described herein may include the steps of generating a target nucleic acid molecule and detecting it. The generation step may be carried out in multiple steps. In other words, different target nucleic acid molecules may be generated in the same reaction mixture. However, this does not prevent separate reactions from being carried out in parallel to generate different target nucleic acid molecules, and then the molecules from being combined or pooled.

[0099] In certain embodiments, target nucleic acid molecules are generated as proxies or markers for the detection of a target analyte in a detection assay. Specifically, they may be generated in the course of a method for detecting a target nucleic acid sequence that may occur in one or more target nucleic acid molecules.

[0100] In this specification, the term “detection” is used in a broad sense, including any means of determining the presence of a target nucleic acid. In this method, the target nucleic acid is detected by detecting the presence or amount of the nucleic acid, which may include simply detecting whether or not it is present, or measuring any form of the target nucleic acid molecule, which may include detecting the target nucleic acid itself, or a copy or amplicon thereof, or a complementary copy. Thus, detecting a target nucleic acid includes determining, measuring, evaluating, or assaying the presence or absence, or the amount or location of the target nucleic acid by any means. The presence of a target nucleic acid (i.e., confirmation of its presence or amount) is an indicator or identification of the presence of the target nucleic acid.

[0101] This includes quantitative and qualitative determinations, measurements, or evaluations, and also includes semi-quantitative ones. Such quantitative and qualitative determinations, measurements, or evaluations may be relative, for example, if two or more different target nucleic acid sequences or target molecules are detected in a sample, they may be relative or absolute. Thus, in one embodiment, the method may be a method for quantifying or determining the amount of target nucleic acid present. In the context of quantifying target nucleic acid in a sample, the term "quantification" can refer to absolute quantification or relative quantification. Absolute quantification can be achieved by including known concentrations of one or more control nucleic acid molecules and / or by referencing the level of the target nucleic acid (e.g., through the generation of a standard curve) with known control nucleic acid molecules or sequences. Alternatively, relative quantification can be achieved by comparing the levels or amounts detected between two or more different target nucleic acids or different target sequences, and can provide relative quantification of two or more different nucleic acid molecules or sequences, i.e., relative quantification of each other. Thus, as described above, the proportion of target nucleic acid present in a sample can be determined. Thus, the copy numbers of target nucleic acids can be compared.

[0102] As described above, in one embodiment, the target nucleic acid molecule is the analyte in the sample. However, in another embodiment, the target nucleic acid molecule is not the target analyte itself, but rather is detected as part of an assay for detecting another target analyte.

[0103] Therefore, the target analyte can be any analyte that is desired to be detected. The analyte may be a nucleic acid, a protein (this term includes peptides and polypeptides), or any other chemical or biological molecule or substructure, such as a carbohydrate, for example, a carbohydrate that may arise as a glycosyl group on a protein. Therefore, the target analyte may be a modified protein, for example, a modified protein that has undergone post-translational modifications to be detected in the assay for the analyte. The target analyte may be an interaction or complex between different molecules, such as a PPI as described above, and the individual components of the interaction may be detected using proximity probes specific to each component of the interaction.

[0104] In one embodiment, the target analyte may be a protein or protein molecule component detected on the surface of a cell, vesicle, or other intracellular or organelle.

[0105] As described above, the target nucleic acid may be any sequence that is desired to be detected or identified. It may be DNA or RNA, or modified variants thereof. If the target sequence is an analyte, it may be any target sequence that is desired to be detected, for example, nucleic acids present in a sample, for example, nucleic acids present in cells or tissues or any biological sample. Therefore, it may be a naturally occurring sequence, or a derivative, copy, or amplicon thereof.

[0106] Alternatively, as described above, the target sequence may instead be a reporter for the assay analyte. The reporter nucleic acid may be used or generated in the course of an assay for any analyte, for example, a protein or other biological molecule in a sample, or for a small molecule. Thus, the reporter nucleic acid may be provided as a tag or label for a binding probe to the analyte and detected to detect the analyte, for example, in an immunoassay, for example, in an immunoPCR or immunoRCA reaction. The 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 or cleavage reaction in a proximity extension assay (PEA), etc. Thus, such a reporter target nucleic acid may be a synthetic sequence or an artificial sequence. It may be a linear, cyclic, cyclic, or cyclizable molecule.

[0107] In one embodiment, the target nucleic acid is a natural or synthetic DNA molecule. The target nucleic acid molecule may be coding DNA or non-coding DNA, such as genomic DNA or a subfraction thereof, or derived from genomic DNA, such as a copy or amplicon thereof, or cDNA or a fraction thereof, or an amplicon or copy thereof, etc.

[0108] In another embodiment, the target nucleic acid molecule is a target RNA molecule. It may be an RNA molecule in the RNA pool or other nucleic acid molecule, for example, a genomic nucleic acid, which may be of human origin or any source, transcriptome origin, or any other nucleic acid (e.g., organelle nucleic acid, i.e., mitochondrial or chloroplast nucleic acid), and may be of natural or synthetic origin. Thus, the target RNA molecule may be a coding RNA sequence (i.e., pre-mRNA or mRNA) or a non-coding RNA sequence (such as tRNA, rRNA, snoRNA, miRNA, siRNA, snRNA, exRNA, piRNA, and long ncRNA), or derived therefrom. In one preferred embodiment, the target nucleic acid molecule is a microRNA (miRNA). In one embodiment, the target RNA molecule is 16S RNA, for example, 16S RNA derived from a microorganism in the sample (e.g., a pathogenic microorganism) that identifies the microorganism. Alternatively, the target RNA molecule may be a genomic RNA, for example, ssRNA or dsRNA of a virus having RNA as its genetic material. Notable viruses include Ebola, HIV, SARS, SARS-CoV-2, influenza, hepatitis C, West Nile fever, polio, and measles. Therefore, the target RNA molecule may be positive-strand RNA, negative-strand RNA, or double-stranded RNA derived from a viral genome, or positive-strand RNA derived from a retroviral RNA genome.

[0109] If the target molecule is an RNA molecule, the method may include a preliminary step of generating a cDNA copy of the target RNA molecule.

[0110] The methods for generating target nucleic acids used herein are well known in the art and, as are the detection methods using them, are widely described in the literature.

[0111] Therefore, RCA is widely known as an amplification technique, and many detection assays using RCA to produce detectable products have been proposed and described.

[0112] The template ring for the RCA reaction may be generated by cyclizing a probe relative to a target nucleic acid sequence, and in particular, by cyclizing a padlock probe according to principles well known in the art. Padlock probes can take various forms and may be provided in a single-part form or a multi-part form (e.g., two parts). These include gap-fill padlock probes (also known as molecular inversion probes (MIPs)). Alternatively, the RCA template may be a pre-formed ring, which constitutes part of a target-specific probe (e.g., hybridized to a target-specific probe, or its nucleic acid portion or domain) or is used in combination with a target-specific probe, for example, in an immunoRCA reaction. Similarly, it may be a cyclic oligonucleotide linked to form a ring during the course of the assay reaction. Thus, the RCP may be the product of an immunoRCA reaction or any type of detection reaction involving an RCA step, for example, a proximity probe assay that generates a cyclic nucleic acid molecule, e.g., SigmaAldrich's Duolink TMSee PLA, and also modified PLA using so-called unfolded proximity probes, which include a hairpin that, upon cleavage, is opened or unfolded to release a nucleic acid domain, which is then cyclized to form an RCA template (see Klaesson et al., 2018, Scientific Reports 8, 5400). Typical PLAs generate a template ring through interaction when the nucleic acid domains of the proximity probe bind in close proximity to their targets. More specifically, when the nucleic acid domains of a pair of proximity probes bind to the proximity probe near their respective targets, they can hybridize with one or more cyclizable oligonucleotides (which can be considered padlock probes specific to one or both of the nucleic acid domains of the proximity probe pair) and, using a ligation reaction as a template, generate a nucleic acid ring to be supplied to the RCA. The proximity probe may be a secondary reagent that binds to a specific partner that binds to the target analyte itself. However, the proximity probe may also be a primary reagent that binds directly to the target analyte. A single cyclizable oligonucleotide (padlock probe) may be used and hybridized to both nucleic acid domains of two adjacent probes. However, various configurations are possible, including the use of a two-part padlock probe (two cyclizable oligonucleotides) hybridized to nucleic acid domains, such hybridization ligating the 5' and 3' ends of each oligonucleotide adjacent to each other. The ligation may be templated by one or both nucleic acid domains. In one embodiment, the RCA of the resulting ring may be primed by a nucleic acid domain. In such an embodiment, one nucleic acid domain may be the template for the ligation and the other may prime the RCA. In other embodiments, both nucleic acid domains may be the templates for the ligation and one nucleic acid domain may be the primer for the RCA. In yet another embodiment, separate RCA primers are used.

[0113] Therefore, more specifically, a padlock probe may be defined alternatively as a cyclizable probe. The use of padlock probes or cyclizable probes is well known in the art, including in the context of RCA reactions. A cyclizable probe comprises one or more linear oligonucleotides that can be ligated together to form a ring. Padlock probes are well known, widely recognized, and well reported and described in the literature. Therefore, the principle of padlock probes is well understood, and the design and use of padlock probes are well known and described in the art. A padlock probe is typically a linear cyclizable oligonucleotide that hybridizes with a target nucleic acid sequence or molecule, with the 5' and 3' ends of the probe positioned adjacent to each other and ligated together directly or indirectly with a gap in between. The probe is cyclized (ligated) by ligating the hybridized 5' and 3' ends of the probe. It is understood that for cyclization (ligation) to occur, the ligable 5' end of the padlock probe must have a free 5' phosphate group.

[0114] To enable ligation by aligning the ends of the padlock probe, the padlock probe is designed to have target binding sites at or near its 5' and 3' ends. In other words, the complementary region that enables the padlock probe to bind to its target is located at or near the ends of the padlock probe.

[0115] To enable ligation, the 3' and 5' ends to be ligated ("ligable" 3' and 5' ends) are hybridized with a target sequence (i.e., a complementary binding site) that serves as a ligation template. The ligable ends of a padlock probe can be adjacent for ligation in various ways, depending on the probe's design. If the target binding site is located at the end of the padlock probe, the binding of the padlock probe may involve adjoining the ends. If the complementary binding sites in the target molecule or sequence are directly adjacent (or continuous) to each other, the ends of the padlock probe can hybridize directly adjacent (i.e., without gaps) to each other and ligate directly. In this case, the ligable 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 therefore the binding site at the end of the padlock probe does not hybridize to an adjacent binding site, but rather to a non-adjacent (discontinuous) binding site in the target sequence. In this configuration, the 5' ligable end of the probe is provided by the actual 5' end of the probe. However, the ligable 3' end of the probe is generated by elongation of the hybridized 3' end of the probe, using the target sequence as an elongation template, and fills the gap between the hybridized ends of the probe. The elongation reaction positions and ligates the elongated 3' end of the probe to an adjacent position. In this case, the ligable 3' end of the probe is the elongated 3' end of the probe.

[0116] A padlock probe may be provided as two or more segments linked together. In the context of proximity probe assays, the nucleic acid domains of a pair of proximity probes may act as a ligation template. In other embodiments, a two-part padlock may take the form of a "connector" oligonucleotide that hybridizes with the target, each having two target-binding regions at or near its 5' and 3' ends with a gap between them, and a gap oligonucleotide that hybridizes into the gap between its ends. The gap oligonucleotide may partially or completely fill the gap.

[0117] Alternatively, the RCA template used to generate RCP may be a circulated target nucleic acid sequence or amplicon, which may be, for example, a PCR or other copy. The target nucleic acid molecule, or amplicon of the target sequence, may be circulated using a ligation template that hybridizes to the ends of the molecule. Circulation adapters, or so-called "selectors," for circulating target nucleic acid molecules are described in WO99 / 049079, WO2003 / 012119 and WO2005 / 070630.

[0118] This method can be carried out in a heterogeneous or homogeneous form. That is, it can be carried out on a solid phase (or carrier), in a solution or suspension (i.e., without a solid phase or carrier), or in practice, both, because the solid phase can be introduced later.

[0119] The form of this method may be selected based on the properties of the sample, the target nucleic acid molecule, or the desired readout or detection technique used.

[0120] The target nucleic acid molecule does not need to be the target analyte of the assay itself, but can be a reporter molecule used or generated in the process of the assay for any desired analyte. Therefore, the sample does not need to be a sample that naturally contains nucleic acids or nucleic acid sources (e.g., cells or viruses, or biological or clinical materials), but may be a synthetic or artificial sample.

[0121] The sample may be any sample containing nucleic acids to be replicated, amplified, analyzed, or detected. The target molecule may, but does not necessarily, be present in cells in the sample, and includes, for example, the detection of cell-free nucleic acids (e.g., cell-free DNA) in a blood sample. Therefore, the sample may also be a tissue sample, which includes solid tissues and blood (blood is classified as a tissue). However, it may also include any other sample containing cells or nucleic acids, which may be, for example, a bodily fluid sample, or other clinical sample containing cells, such as a wash or swab, or an artificially prepared cell-containing sample, such as a cell suspension, or a sample prepared in any way. This includes fresh, frozen, and immobilized cell samples, such as FFPE samples. Therefore, both natural and synthetic samples are included, i.e., naturally occurring substances or prepared preparations. Naturally occurring samples may be processed or modified before being used in the methods described herein. This includes all cell-containing biological and clinical samples, such as any cell or tissue samples of biological origin, or any bodily fluids or preparations derived therefrom, as well as cell cultures, cell preparations, and cell lysates. Environmental samples, such as soil and water samples or food samples, are also included. Samples may be newly prepared or may be pre-treated by any convenient method, for example, for preservation.

[0122] Therefore, typical samples include any substance that may contain the target nucleic acid molecule, such as food and related products, clinical and environmental samples. Samples may also contain any viral or cellular material, such as all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasmas, and organelles. Thus, such biological materials may include all types of mammalian and non-mammalian animal cells, plant cells, algae including cyanobacteria, fungi, bacteria, protozoa, or viruses. Cells may be, for example, human cells, avian cells, reptile cells, etc.

[0123] Therefore, typical samples include blood-derived products containing whole blood and cells, blood cells, or any cell-containing bodily fluids, tissues, biological specimens, cell cultures, cell suspensions, etc. The samples may be pretreated by any simple or desired method and prepared for use in the present invention.

[0124] Therefore, the methods and uses described herein are particularly suitable for detection methods performed in situ, i.e., detection methods that detect target nucleic acids in situ in the environment in which they exist. Thus, nucleic acids can be detected in situ in the cells in which they exist. Conveniently, the sample may be a tissue sample in an in situ detection assay.

[0125] The methods and uses described herein are particularly suitable for detection methods performed on samples that may contain cancer cells or tumors. Therefore, these methods and uses are particularly useful for detection assays for cancer cells or tumors. Accordingly, these methods can be performed in the context of clinical diagnostic assays, i.e., on clinical samples for the detection of cancer cells or tumors.

[0126] In one embodiment, the sample is immobilized or provided on a solid support. Therefore, the method can be carried out in heterogeneous or solid-phase-based forms. Specifically, the sample may be provided on a slide or similar. However, this is not a requirement, and homogeneous or liquid-phase forms are included, and in practice, mixed phases having both solid-phase and liquid-phase processes are included.

[0127] As described above, the sample may be a synthetic or artificial sample. Therefore, it may be a sample that has been subjected to a detection assay of an analyte in which the target nucleic acid has been generated or to which the target nucleic acid molecule has been added. It may also be a reaction mixture or reaction product, for example, a product obtained from an immunoassay for detecting the target analyte, such as immunoPCR, immunoRCA, or proximity assay (e.g., proximity ligation assay (PLA) or proximity extension assay (PEA)).

[0128] As used herein, the terms “hybridization” or “hybridize” refer to the formation of a double helix between nucleotide sequences that are sufficiently complementary to form a double helix via Watson-Crick base pairing, or any similar base pairing interaction. Two nucleotide sequences are “complementary” to each other if their molecules share homology in base pairing configuration; therefore, a complementary region in a molecule, probe, or sequence is the portion of that molecule, probe, or sequence that can form a double helix. Hybridization does not require 100% complementarity between sequences, and therefore does not require, but is not excluded, that the sequences be perfectly complementary in their complementary regions. Thus, as used herein, “complementary” means “functionally complementary,” i.e., a degree of complementarity sufficient to mediate productive hybridization, and includes degrees of complementarity less than 100%. The acceptable degree of mismatch can be controlled by appropriately adjusting the hybridization conditions. Those skilled in the field of nucleic acid technology can empirically determine double-strand stability by considering several variables and following the guidance provided in the art, such as the length and base pair composition of each molecule or probe or detection oligonucleotide, ionic strength, and the rate of mismatch base pair occurrence. Accordingly, in any reaction step described herein, the design of appropriate probes, or ligation templates or primers, their binding regions, and the conditions under which they hybridize to their respective targets are within the scope of the ordinary skill of those skilled in the field.

[0129] The complementary region, such as the target nucleic acid in the binding region of a padlock probe, or the region between the detection sequence and the detection oligonucleotide, or the complementary region of an RCA primer and its template (e.g., a cyclic padlock probe), may be at least 6 nucleotides long, and more specifically at least 7, 8, 9, or 10 nucleotides long, in order to ensure binding specificity. The upper limit of the region length is not critical, but may be, for example, up to 50, 40, 35, 30, 25, 20, or 15 nucleotides. Thus, the complementary region may have a length within the range between one of the lower limits and one of the upper limits set above. In the case of a padlock probe, the length of each target binding region may be within the lower limit range, so the total length of the two binding regions when hybridized to those targets will be within the upper limit range. For example, each target-binding region may be 5 to 15 nucleotides, or for example, from one of 6, 7, or 8 to one of 9, 10, 11, or 12 nucleotides, and therefore the hybridized total length may be, for example, 16 to 30 nucleotides long, or for example, 20 to 24. Within the constraints of the probe conformation, domain spacing, and desirable or preferred hybridization, it is desirable to minimize the size of the ring provided to the RCA, and therefore, where possible, to minimize the length of complementary regions.

[0130] After contacting a target nucleic acid molecule with a Phi-29 polymerase composition, a polymerase reaction, such as RCA, is performed, and the polymerase product, such as RCP, can be detected using any convenient protocol and detection modality. This may depend on the target nucleic acid to be detected, the purpose of the method, and / or the specific details of the procedure used in the method.

[0131] For example, the product can be detected using any established method for the analysis of nucleic acid molecules, such methods are known in the literature that use fluorescence, color, or colorimetric labeling detection, and include, for example, microscopy and imaging techniques.

[0132] Depending on the level of redundancy, combinatorial labeling may be used in accordance with techniques well known in the field. For example, ratio labeling can be carried out using different fluorescently labeled detection oligonucleotides.

[0133] While various detection modalities can be employed, for convenience, labeled nucleic acid molecules can be detected by microscopy or flow cytometry. In particular, in microscopy-based methods, labeled molecules (e.g., RCP) can be detected by imaging.

[0134] The use of such detection techniques has the advantage of being able to digitally record nucleic acid molecules. In fact, the degree of signal amplification given by products such as RCP in this method makes it possible to visualize them and detect them with a camera or any device including a camera such as a mobile phone.

[0135] To detect products produced in a uniform form, they may be captured or settled on a solid support or surface for imaging or, more generally, microscopic detection.

[0136] As described above, the improvements to the phi-29 polymerase catalytic reaction provided by this method and its use are particularly useful in automated equipment where phi-29-containing reagents are kept or stored at room temperature for a certain period. This allows for the preparation and introduction of reagents into the equipment while minimizing the loss of phi-29 activity. In fact, it can be understood that this preservation of activity would be beneficial not only in automated systems but in all types of laboratory or analytical equipment.

[0137] Of particular note are automated tissue slide analyzers such as Leica Bond, Lunaphore Comet, Roche Ventana, or Dako Omnis. Such analyzers are now widely used to perform analyte detection reactions on or within tissue or cell samples on slides, including fresh, frozen, or fixed samples. Such detection assays can be performed using protocols that include an amplification step with phi-29 polymerase, as described above. For example, kits for performing PLA detection, similar to the Duolink kits mentioned above, are commercially available from Navinci Diagnostics AB under the brand name NaveniFlex. These provide proximity probes in various forms for use as primary or secondary binders to the target analyte, configured to form a target ring amplified by RCA using phi-29 polymerase. RCP is detected using a fluorescent detection oligonucleotide, or a detection oligonucleotide labeled with an enzyme reactive with a chromogenic substrate, specifically alkaline phosphatase (AP) or horseradish peroxidase (HRP). The high signal-to-noise ratio enables the detection of individual proximity events and allows for resolution down to a single protein or PPI. Example 1 below demonstrates the use of a stabilized Phi 29 composition in a PLA protocol using such a kit in an autostainer and the improved results obtained.

[0138] Therefore, standard PLA protocols, such as the isPLA protocol, can be modified according to the methods and principles of the present invention to stabilize the RCA buffer containing phi-29 DNA polymerase.

[0139] This method will be described in more detail with reference to the drawings and non-limiting embodiments. [Examples]

[0140] Examples Example 1 - Detection of protein-protein interactions 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. The interaction between E-cadherin and B-catenin in FFPE was tested under various conditions, including the presence or absence of stabilized oligonucleotides in the RCA mixture and whether the RCA mixture was stored at room temperature for 18 hours before assay. In situ PLA was performed manually, simulating reagent storage in a Leica Bond instrument by keeping the RCA mixture on the bench at room temperature. The RCA mixture was either freshly prepared (control) or pre-mixed, with or without stabilized oligonucleotides. The RCA mixture consisted of Phi-29, dNTPs, and 1x Thermo Phi-29 buffer. After approximately 18 hours at room temperature on the bench, the RCA mixture with and without stabilized oligonucleotides were added to tissue slides during the amplification step of the protocol. In this step, a freshly prepared RCA mixture was also added as a control to both the stabilized oligonucleotide-containing and non-stabilized oligonucleotide-containing mixtures to confirm that stabilized oligonucleotides did not interfere with in situ PLA.

[0141] The following steps were taken: 1. Deparaffinization and antigen recovery (60 minutes) 2. Wash (2 minutes) 3. Blocking (60 minutes) 4. Primary antibody incubation (60 minutes) 5. Washing (15 minutes) 6. Probes M1 and R2 (antibody-proximal probe pairs for mouse (M) and rabbit (R)) (60 min) 7. Washing (15 minutes) 8. Ligation (30 minutes) 9. Wash (10 minutes) 10. RCA (9 min) - This reactant (1x) contains a stabilizing oligonucleotide at 0.05 μM. 11. Incubation after blocking (30 minutes) 12. Detection (30 minutes) 13. Clean and store in TBS-T until slide mounted.

[0142] In Figure 1, the results show that when the RCA reaction mixture buffer is freshly prepared and a stabilizing oligonucleotide is present, the interaction between E-cadherin and B-catenin is clearly visualized (Condition C, Figure 1). When the buffer is prepared, stored at room temperature on the bench for 18 hours, and no stabilizing oligonucleotide is present, the interaction between E-cadherin and B-catenin is not visualized (Condition B, Figure 1). In contrast, when the buffer is prepared, stored at room temperature on the bench for 18 hours, and 0.05 μM of the stabilizing oligonucleotide is present in the reaction mixture, the interaction is detectable (Condition D, Figure 1), and the detected signal is equivalent to the signal in Condition A of Figure 1, where the buffer is freshly prepared but no stabilizing oligonucleotide is present. The observed results were confirmed using different experimental conditions and different oligonucleotide sequences.

Claims

1. A method for stabilizing phi-29 DNA polymerase, comprising contacting the phi-29 DNA polymerase with a stabilizing oligonucleotide, wherein the stabilizing oligonucleotide is protected from 3'-exonuclease degradation.

2. The method according to claim 1, comprising preparing a composition containing phi-29 polymerase and the stabilized oligonucleotide.

3. A method for carrying out a polymerase reaction, the method comprising contacting a sample containing a target nucleic acid with a composition containing phi-29 polymerase and a stabilized oligonucleotide, and causing a polymerization reaction, wherein the stabilized oligonucleotide is protected from 3'-exonuclease degradation.

4. A method for replicating nucleic acids, the method comprising contacting a target nucleic acid with a composition comprising phi-29 polymerase and a stabilized oligonucleotide, wherein the stabilized oligonucleotide is protected from 3'-exonuclease degradation.

5. A method for detecting a target nucleic acid in a sample, the method comprising the following: (i) Contacting the sample with a composition comprising phi-29 polymerase and a stabilized oligonucleotide, and allowing the phi-29 polymerase to carry out a polymerase reaction using the target nucleic acid as a template, wherein the stabilized oligonucleotide is protected from 3'-exonuclease degradation; (ii) Detecting the target nucleic acid by detecting the product of the polymerase reaction.

6. The method according to claim 5, wherein the target nucleic acid is the nucleic acid to be analyzed in the sample, or a copy or amplicon thereof.

7. The target nucleic acid is a reporter nucleic acid molecule that serves as an indicator of the target analyte in the sample. The method according to claim 5.

8. The method according to any one of claims 3 to 6 or 8, wherein the target nucleic acid is a nucleic acid molecule produced as a detection assay reaction product in a detection assay for detecting a target analyte in a sample.

9. A method for detecting a target analyte in a sample, wherein a detection assay is performed to detect the analyte, the assay generates a target nucleic acid molecule to be detected in situ, and the method comprises the following: (i) After generating the target nucleic acid molecule in the sample, the sample is brought into contact with a composition comprising phi-29 polymerase and a stabilized oligonucleotide, and the phi-29 polymerase is subjected to a polymerase reaction using the target nucleic acid molecule as a template, wherein the stabilized oligonucleotide is protected from 3'-exonuclease degradation; (ii) Detecting the product of the polymerase reaction to detect the target nucleic acid molecule, thereby detecting the target analyte.

10. The method according to any one of claims 2 to 9, wherein the composition is kept at a temperature above freezing for at least 15 minutes, or more specifically, at least 1 hour, before use or contact.

11. The method according to any one of claims 1 to 10, wherein the stabilized oligonucleotide has a length of at least 8 nucleotides or at least 10 nucleotides.

12. The method according to any one of claims 1 to 11, wherein the stabilizing nucleotide comprises one or more nucleotides having a 2' sugar modification and / or one or more modified internucleotide bonds.

13. The method according to claim 12, wherein the 2' sugar modification is 2'-O-methyl or 2'MOE.

14. The method according to any one of claims 2 to 13, further comprising dNTPs and a buffer.

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

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

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

18. The method according to any one of claims 3 to 17, wherein the target nucleic acid is a ligation product comprising a cyclic nucleic acid molecule cyclized by ligation.

19. The method according to claim 17, wherein the ligation product is generated by proximity ligation assay (PLA) and optionally generated by in situ PLA (isPLA).

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

21. The method according to claim 20, wherein the target nucleic acid is a circularized padlock probe, and optionally, the padlock probe comprises one or more portions and is circularized using the nucleic acid domains of one or more neighboring probes as a ligation template.

22. The method according to any one of claims 1 to 21, wherein the temperature stability of the phi-29 polymerase is increased in the presence of the stabilized oligonucleotide compared to the absence of the stabilized oligonucleotide.

23. A composition comprising phi-29 polymerase and a stabilizing oligonucleotide, wherein the stabilizing oligonucleotide is protected from 3'-exonuclease degradation and acts to stabilize the phi-29 polymerase, and in the composition, phi-29 cannot undergo polymerase elongation.

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

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

26. Use of an oligonucleotide as a stabilizer for stabilizing phi-29 polymerase, wherein the oligonucleotide is a stabilized oligonucleotide protected from 3' exonuclease degradation.

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

28. A method for lyophilizing phi-29 polymerase, comprising preparing an aqueous composition comprising phi-29 polymerase and a stabilized oligonucleotide, and lyophilizing the composition, wherein the stabilized oligonucleotide is protected from 3'-exonuclease degradation.

29. The method, composition, or use according to any one of claims 1 to 28, for use in an automated tissue slide instrument, or for use in an automated tissue slide instrument.

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