PUMA1 polymerases and uses thereof

Abstract

The present disclosure relates, in some aspects, to recombinant polymerases, variant PumA1 polymerases, and compositions thereof. Also provided herein are methods of using the recombinant polymerases and / or variant PumA1 polymerases for nucleic acid amplification (e.g., rolling circle amplification). In some aspects, the compositions and methods disclosed herein provide more robust amplification (e.g., RCA) reactions for improved in vitro and in situ analysis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 397,638, filed on August 12, 2022, entitled “PUMA1 POLYMERASES AND USESTHEREOF,” which is incorporated herein by reference in its entirety for all purposes.

[0003] Citation of sequence listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named 202412016140SeqList.xml created on August 9, 2023, which is 36,613 bytes in size. The information in the electronic form of the sequence listing is incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates in some aspects to compositions and methods using recombinant PumA1 polymerase and variants thereof. Background Art

[0006] Rolling circle amplification (RCA) allows the production of multiple copies of a circular nucleic acid template. Phi29 DNA polymerase is the standard DNA polymerase currently used for rolling circle amplification. However, existing Phi29 DNA polymerases have limited thermal stability and / or show limited sensitivity in assays based on rolling circle amplification. Improved polymerases for rolling circle amplification are needed. Compositions and methods that address such needs and other needs are provided herein. Summary of the invention

[0007] Rolling circle amplification and other methods that benefit from high nucleic acid continuous synthesis capacity (such as multiple displacement amplification) are usually performed with Phi29 polymerase. Phi29 polymerase is a mesophilic DNA polymerase derived from Bacillus subtilis phage Phi29. Phi29 polymerase has 3'-5' exonuclease activity and nucleic acid polymerization activity. RCA can be used for many types of assay reactions, including in situ analysis of biological samples, for endogenous analyte detection at spatially located locations in biological samples. Such exemplary assays are important tools for understanding the molecular basis of the biology of cells and organisms and for evaluating and / or treating diseases. In some cases, Phi29 polymerase has certain limitations, including limited thermal stability, and the optimal reaction temperature is 30°C, which leads to slower reaction kinetics, fewer amplification products, and greater heterogeneity and amplification bias, especially for DNA with high guanine / cytosine (G / C) content.

[0008] In some aspects, novel recombinant polymerase polypeptides and variants of PumA1 polymerase (or PumA1 Pol) derived from PumA1 bacteriophage are provided herein. In some cases, the recombinant polymerase and variant PumA1 polymerase are thermostable, provide high yields of amplified products, and / or can be used in a wider range of nucleic acid amplification methods or samples.

[0009] The application provides a recombinant polymerase comprising an amino acid sequence of a PumA1 polymerase or a variant thereof. A phylogenetic survey shows that the polymerase protein encoded by the PumA1 genome has a sequence identity of 58% with the Phi29 polymerase (Phi29Pol). The recombinant polymerase comprising a PumA1 polymerase or a variant thereof provided herein is soluble and well expressed. In addition, the application provides data proving that a recombinant polymerase comprising a PumA1 polymerase sequence has the characteristics that are conducive to rolling circle amplification. It is noteworthy that the recombinant PumA1 polymerase polypeptide provided herein provides a higher sensitivity than Phi29 in a rolling circle amplification assay (e.g., producing a higher number of detectable rolling circle amplification products under the same conditions). PumA1 polymerase variants are also provided herein. In some aspects, PumA1 polymerase variants exhibit enhanced thermal stability relative to a reference polymerase (e.g., wherein the reference polymerase is a wild-type PumA1 polymerase or Phi29). The present application also provides nucleic acids encoding recombinant polymerases or PumA1 variants (e.g., recombinant nucleic acids and vectors), expression systems and methods for producing the recombinant polymerases or PumA1 variants, and methods and kits using recombinant polymerases comprising PumA1 polymerases or their variants (e.g., for rolling circle amplification).

[0010] In some aspects, provided herein is an isolated recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the isolated recombinant polymerase comprises one or more amino acid substitutions at one or more positions selected from positions 545, 574 and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In any embodiments herein, the isolated recombinant polymerase may comprise one or more amino acid substitutions selected from the group consisting of I545E, A574S and P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO: 1. In any embodiments herein, the isolated recombinant polymerase may comprise the amino acid substitutions I545E, A574S and P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO: 1. In any embodiments herein, the isolated recombinant polymerase may comprise the sequence of amino acid residues 2-30 of SEQ ID NO: 1. In any of the embodiments herein, the isolated recombinant polymerase can comprise an N-terminal deletion of a methionine residue at position 1 corresponding to the position of the sequence shown in SEQ ID NO:1.

[0011] In some aspects, provided herein is an isolated recombinant polymerase comprising the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, provided herein is an isolated recombinant polymerase comprising the amino acid sequence set forth in SEQ ID NO:2.

[0012] In some aspects, provided herein is an isolated recombinant polymerase comprising the amino acid sequence set forth in SEQ ID NO: 10. In some aspects, provided herein is an isolated recombinant polymerase comprising the amino acid sequence set forth in SEQ ID NO:11.

[0013] In any embodiment herein, the recombinant polymerase may further comprise a heterologous sequence. In any embodiment herein, the recombinant polymerase may comprise, from the amino terminus (N terminus) to the carboxyl terminus (C terminus), an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and a heterologous sequence. In any embodiment herein, the recombinant polymerase comprises, from the N terminus to the C terminus, a heterologous sequence and an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0014] In some embodiments of any of the embodiments herein, the recombinant polymerase comprises an affinity tag, a linker and / or a solubility tag. In any of the embodiments herein, the heterologous sequence may comprise a cleavable sequence. In any of the embodiments herein, the heterologous sequence may comprise an affinity tag selected from the group consisting of: a polyhistidine tag (His tag), a glutathione-s-transferase (GST) tag, a maltose binding protein (MBP) tag, a mutated dehalogenase (Halo) tag, a liver fluke 8-kDa antigen (Fh8) tag, a streptavidin binding peptide (SBP) tag, and a Tamavidin tag. In some cases, the affinity tag is a His tag.

[0015] In any embodiment herein, the heterologous sequence may comprise a solubility tag selected from the group consisting of a thioredoxin (Trx) tag, a small ubiquitin modification (SUMO) tag, an IgG domain B1 (GB1) tag of protein G, and a solubility enhancing tag (SET). In any embodiment herein, the solubility tag may be located between the affinity tag and an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In any embodiment herein, the solubility tag may be attached to the N-terminus of an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In any embodiments herein, the affinity tag may be located between the solubility tag and an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In any embodiments herein, the solubility tag may be a SUMO tag. In any embodiments herein, the SUMO tag may comprise the sequence shown in SEQ ID NO: 6.

[0016] In any embodiments herein, the solubility tag may have a net negative charge at pH 7. In any embodiments herein, the solubility tag may have a net negative charge of at least or at least about -6, -10, -12, -14, -15, -16, -17 or -18 at pH 7. In any embodiments herein, the solubility tag may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more acidic amino acid residues. In any embodiments herein, the solubility tag may be between 5 and 20 amino acid residues in length. In any embodiments herein, the solubility tag may be between 5 and 12 amino acid residues in length. In any embodiments herein, the solubility tag may be the sequence S(E / D) n K m (E / D) p G (SEQ ID NO: 12), wherein S is serine, (E / D) is aspartic acid or glutamic acid, K is lysine, G is glycine, n is 5 or 6, m is 1 or 0, and p is an integer between 2 and 6. In any embodiment herein, the solubility tag may be a sequence as shown in any one of SEQ ID NOs: 13-18. In any embodiment herein, the solubility tag may have a sequence as shown in SEQ ID NO: 14.

[0017] In any embodiments herein, the heterologous sequence may comprise a linker.In any embodiments herein, the linker may comprise one or more glycine and / or serine residues.In any embodiments herein, the linker may comprise a sequence selected from the group consisting of SEQ ID NOs: 19-26.

[0018] In any of the embodiments herein, the recombinant polymerase may comprise, from N-terminus to C-terminus, a polyhistidine tag (His tag), a linker, a SUMO tag, and an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In any of the embodiments herein, the recombinant polymerase may comprise, from N-terminus to C-terminus, an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, a linker, a polyhistidine tag (His tag), and a solubility tag.

[0019] In any embodiment herein, the isolated recombinant polymerase can be at least 85%, at least 90% or at least 95% soluble. In any embodiment herein, the apparent molecular weight of the recombinant polymerase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis can be between about 55 kDa and about 80 kDa, or between about 60 kDa and about 70 kDa.

[0020] In some aspects, provided herein is a variant PumA1 polymerase comprising one or more amino acid substitutions at one or more positions selected from positions 545, 574, and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions selected from the group consisting of I545E, A574S, and P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO: 1. In any embodiments herein, the variant PumA1 polymerase may comprise amino acid substitutions I545E, A574S, and P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO: 1. In any embodiments herein, the variant PumA1 polymerase may comprise the sequence of amino acid residues 2-30 of SEQ ID NO: 1. In any embodiments herein, the variant PumA1 polymerase may comprise the sequence of amino acid residues 2-100 of SEQ ID NO: 1. In any of the embodiments herein, the variant PumA1 polymerase may comprise a sequence of amino acid residues 2-150 of SEQ ID NO: 1. In any of the embodiments herein, the variant PumA1 polymerase may comprise a sequence of amino acid residues 2-190 of SEQ ID NO: 1. In any of the embodiments herein, the variant PumA1 polymerase may comprise an N-terminal deletion of a methionine residue at position 1 corresponding to the position of the sequence shown in SEQ ID NO: 1.

[0021] In any of the embodiments herein, the variant PumA1 polymerase may comprise the amino acid sequence of SEQ ID NO: 10. In any of the embodiments herein, the variant PumA1 polymerase may comprise the amino acid sequence of SEQ ID NO:11.

[0022] In any embodiment herein, the variant PumA1 polymerase may exhibit an improved polymerization rate at 42°C compared to a reference PumA1 polymerase. In any embodiment herein, the variant PumA1 polymerase may generate an equivalent density of detected RCA products in a rolling circle amplification (RCA) reaction compared to a reference Phi29 polymerase. In any embodiment herein, the variant PumA1 polymerase may produce a higher signal intensity for the RCA products generated in the RCA reaction at 42°C compared to the RCA products generated by the reference PumA1 polymerase.

[0023] In any embodiment herein, the variant PumA1 polymerase can be at least 85%, at least 90% or at least 95% soluble. In any embodiment herein, the variant PumA1 polymerase can have an apparent molecular weight of between about 55 kDa and about 80 kDa, or between about 60 kDa and about 70 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0024] In some aspects, the present invention provides a composition comprising a recombinant polymerase as described in any one of the claims or a variant PumA1 polymerase as described in any one of the preceding embodiments. In some embodiments, the composition comprises no more than 10 5 No more than 10 4 No more than 10 3 No more than 10 2 or no more than 10 genome equivalents of bacterial or bacteriophage DNA.

[0025] In any embodiment herein, the composition may include about 0.05 μM to about 1 μM of a recombinant polymerase or variant PumA1 polymerase. In any embodiment herein, the composition may include at least 1 μM, 10 μM, 50 μM, or 100 μM of a recombinant polymerase or variant PumA1 polymerase. In any embodiment herein, the recombinant polymerase or variant PumA1 polymerase may be at least 85%, at least 90%, or at least 95% soluble in the composition. In any embodiment herein, the recombinant polymerase or variant PumA1 polymerase may be at least 85%, at least 90%, or at least 95% soluble in the composition at 20°C.

[0026] In any embodiment herein, the pH of the composition may be about 8.5. In any embodiment herein, the composition may comprise about 5 mM to about 10 mM MgCl 2 In any embodiment herein, the composition may comprise about 0.1 mM to about 0.2 mM dNTPs. In any embodiment herein, the composition may comprise about 1 mM to about 4 mM DTT. In any embodiment herein, the composition may comprise about 2 mg / mL BSA. In any embodiment herein, the composition may comprise about 10 mM NH 4 SO 4 .

[0027] In any of the embodiments herein, the variant polymerase or variant PumA1 polymerase has an apparent molecular weight of between about 55 kDa and about 80 kDa, or between about 60 kDa and about 70 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0028] In some aspects, provided herein is a polynucleotide encoding a recombinant polymerase as described in any of the foregoing embodiments or a variant PumA1 polymerase as described in any of the foregoing embodiments. In some embodiments, the polynucleotide may have at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0029] In some aspects, provided herein are recombinant nucleic acid molecules comprising a polynucleotide as described in any of the preceding claims. In any embodiment herein, the recombinant nucleic acid molecule may also include a transcriptional regulatory sequence operably connected to the polynucleotide. In any embodiment herein, the transcriptional regulatory sequence may include a promoter selected from the group consisting of a bacterial promoter, a viral promoter, and a mammalian promoter.

[0030] In some aspects, the present invention provides a vector comprising a polynucleotide or recombinant nucleic acid molecule as described in any one of the foregoing embodiments. In any embodiment herein, the vector can be a plasmid, a phagemid, a viral vector, a cosmid or a transposon.

[0031] In some aspects, a recombinant expression system is provided herein, comprising a polynucleotide, a recombinant nucleic acid molecule or a vector as described in any of the foregoing embodiments. In any embodiment herein, the recombinant expression system can produce a recombinant polymerase or a variant PumA1 polymerase as described in any of the foregoing embodiments. In any embodiment herein, the recombinant expression system can be a cell system or a cell-free system. In any embodiment herein, the recombinant expression system can comprise one or more cell systems selected from the group consisting of bacterial cells, fungal cells, insect cells and mammalian cells.

[0032] In any of the embodiments herein, the polynucleotide sequence encoding the recombinant polymerase or variant PumA1 polymerase can be codon-optimized for the recombinant expression system.

[0033] In some aspects, the present invention provides a method for producing a recombinant polymerase or variant PumA1 polymerase as described in any of the foregoing embodiments, the method comprising culturing a cell population comprising a polynucleotide encoding the recombinant polymerase or the variant PumA1 polymerase, and isolating the recombinant polymerase or variant PumA1 polymerase from the cell population. In any of the foregoing embodiments, the method may include inducing the expression of the recombinant polymerase or the variant PumA1 polymerase. In any of the foregoing embodiments, the recombinant polymerase or variant PumA1 polymerase may be separated using an affinity tag. In any of the foregoing embodiments, the method may include cutting the heterologous sequence.

[0034] In some aspects, a method for amplifying DNA is provided herein, the method comprising contacting a biological sample containing DNA to be amplified with a recombinant polymerase, a variant PumA1 polymerase or a composition as described in any of the foregoing embodiments. In some embodiments, DNA is amplified by rolling circle amplification (RCA). In any embodiment herein, DNA can be amplified by in situ RCA in a biological sample (e.g., at a position in a biological sample). In some embodiments, the biological sample is on a substrate.

[0035] In some aspects, the present invention provides a method for performing rolling circle amplification (RCA), the method comprising contacting a biological sample containing DNA to be amplified with a recombinant polymerase, variant PumA1 polymerase or composition as described in any one of the preceding embodiments. In some embodiments, the method comprises incubating the recombinant polymerase or composition and the biological sample.

[0036] In any embodiment herein, the recombinant polymerase or composition and the biological sample can be incubated at a temperature of at least or about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 42°C, 45°C or higher. In any embodiment herein, the recombinant polymerase can comprise amino acid substitutions I545E, A574S and P575V, wherein the amino acid numbering corresponds to the position of the sequence shown in SEQ ID NO: 1, and RCA is performed at a temperature of at least 42°C. In any embodiment herein, the method can cause an increase in the signal intensity of the detected RCA product compared to a reference RCA product produced using a reference polymerase. In some embodiments, the reference polymerase is a wild-type Phi29 polymerase. In some embodiments, the reference polymerase is a wild-type PumA1 polymerase. In any embodiment herein, RCA can be performed for at least or about 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours or longer. In some embodiments of any of the embodiments herein, RCA can be performed for no more than about 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, or 3 hours.

[0037] In any embodiment herein, the method may include detecting the RCA product generated by RCA. In any embodiment herein, detection may include detecting the sequence in the RCA product. In any embodiment herein, the RCA product may be detected at a position in the biological sample. In any embodiment herein, detection may include: contacting the biological sample with one or more detectably labeled probes directly or indirectly combined with one or more barcode sequences or their complementary sequences, and detecting the signal associated with the one or more detectably labeled probes. In any embodiment herein, detection may include: contacting the biological sample with one or more intermediate probes directly or indirectly combined with the one or more barcode sequences or their complementary sequences, wherein the one or more intermediate probes can be detected using one or more detectably labeled probes, and detecting the signal associated with the one or more detectably labeled probes. In any embodiment herein, the method may also include removing the one or more intermediate probes and / or the one or more detectably labeled probes.

[0038] In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may exhibit an average processivity of at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, or at least 70 kb. In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may have strand displacement activity. In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may exhibit an average nucleotide polymerization rate of at least 1000 nucleotides (nt) / min, at least 1500 nt / min, at least 2000 nt / min, or at least 2280 nt / min. In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may exhibit an average processivity of less than 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, or at least 70 kb. In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may have strand displacement activity. In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may exhibit an average nucleotide polymerization rate of less than 10 -5 Errors / bp, less than 10 -6 Errors / bp or less than 10 -7 In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may have exonuclease activity. In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may have reverse transcriptase activity. In any embodiment herein, under the same reaction conditions, the recombinant polymerase or the variant PumA1 polymerase may produce at least 10%, at least 15%, at least 20%, at least 25% or at least 30% more rolling circle amplification products than the reference Phi29 polymerase.

[0039] In any embodiment herein, the method may include contacting the biological sample with a buffer. In any embodiment herein, the pH of the buffer may be about 8.5. In any embodiment herein, the buffer may contain about 5 mM to about 10 mM MgCl 2 In any embodiment herein, the buffer may comprise about 0.1 mM to about 0.2 mM dNTPs. In any embodiment herein, the buffer may comprise about 1 mM to about 4 mM DTT. In any embodiment herein, the buffer may comprise about 2 mg / mL BSA. In any embodiment herein, the buffer may comprise about 10 mM NH 4 SO 4 .

[0040] In some aspects, a kit is provided herein, comprising a recombinant polymerase, a variant PumA1 polymerase or a composition as described in any of the foregoing embodiments. In some embodiments, the kit comprises one or more of the following: (a) dNTPs; (b) one or more divalent-cations; (c) a reaction buffer; (d) a buffer for use with any one of (a)-(c); and (e) instructions for rolling circle amplification using a recombinant polymerase, a variant PumA1 polymerase or a composition. In any embodiment herein, the kit may also include one or more primers. In any embodiment herein, the kit may also include one or more circularizable probes. In any embodiment herein, the kit may be used for amplification of nucleic acids. In any embodiment herein, the kit may also include a detection probe for detecting amplified nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings illustrate certain features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

[0042] Figure 1 Depicted are the results of SDS-PAGE comparing the protein solubility of a recombinant PumA1 polypeptide having an amino-terminal polyhistidine / SUMO tag (N-His / Sumo) and a recombinant PumA1 polypeptide having a carboxyl-terminal polyhistidine / SET tag (C-His / SET).

[0043] Figure 2 Depicted are SDS-PAGE results comparing heparin purification of N-His / SUMO or C-His / SET recombinant PumA1 polypeptides.

[0044] Figure 3 Depicted are the results of an in situ rolling circle amplification (RCA) assay comparing the activity of wild-type (WT) Phi29 and an exemplary recombinant PumA1 polymerase. The graph shows the fluorescence intensity readout of the amount of rolling circle product (RCP) generated by amplification.

[0045] Figure 4 Depicted are the results of an in situ rolling circle amplification (RCA) assay comparing the activity of wild-type (WT) Phi29 and an exemplary recombinant PumA1 polymerase in tissue samples. The signal density and signal intensity of the rolling circle products (RCPs) generated by amplification are shown.

[0046] Figure 5A-Figure 5B Depicted are the results of optimized conditions for the RCA buffer specific for the C-His / SET recombinant PumA1 polymerase. Figure 5A Results showing the sensitivity of the detected RCPs for different RCA buffer formulations. Figure 5BThe results of the signal intensity of the detected RCP for different RCA buffer formulations are shown.

[0047] Figure 6 Depicted are the results of an in situ rolling circle amplification (RCA) assay comparing the signal intensity of the wild-type (WT) recombinant PumA1 polymerase and an exemplary variant PumA1 polymerase ("PumA1-ESV", comprising I545E, A574S, P575V substitutions, wherein the amino acid numbering is based on SEQ ID NO: 1) thermal stability. The RCA assay was performed at 37°C and 42°C using tissue samples and two concentrations of polymerase (0.2 μM or 0.5 μM).

[0048] Figure 7 Depicted are the results of an in situ rolling circle amplification (RCA) assay comparing the signal intensity and quantity of rolling circle products (RCPs) of wild-type ("PumA1-WT") recombinant PumA1 polymerase and an exemplary variant recombinant PumA1 polymerase "PumA1-ESV" (I545E, A574S, P575V). The RCA assay was performed using tissue samples at 37°C and 42°C. DETAILED DESCRIPTION

[0049] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If definitions set forth herein are contrary to or contradictory to definitions set forth in patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0050] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0051] I. Overview

[0052] In some aspects, the present invention provides a recombinant polymerase comprising an amino acid sequence of a PumA1 polymerase or a variant thereof (e.g., an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2). In some embodiments, the recombinant polymerase comprises a heterologous sequence. In some cases, the heterologous sequence comprises a cleavable sequence. In some cases, the heterologous sequence remains part of the recombinant polymerase after purification (e.g., during nucleic acid amplification using the recombinant polymerase). In some embodiments, the heterologous sequence comprises an affinity tag. Exemplary affinity tags include, but are not limited to, polyhistidine tags (His tags), glutathione-s-transferase (GST) tags, maltose binding protein (MBP) tags, mutated dehalogenase (Halo) tags, Fasciola hepatica 8-kDa antigen (Fh8) tags, streptavidin binding peptide (SBP) tags, and Tam avidin tags. In some cases, the affinity tag is a His tag. In some cases, the heterologous sequence comprises a solubility tag. Exemplary solubility tags include, but are not limited to, thioredoxin (Trx) tags, small ubiquitin modification (SUMO) tags, IgG domain B1 (GB1) tags of protein G, or solubility enhancement tags (SET). In some cases, the recombinant polymerase is at least 85%, at least 86%, at least 87%, at least 88%, at least 90% or at least 95% soluble (e.g., at 20°C, 30°C, 35°C, 40°C or 42°C). In some aspects, the recombinant polymerase is functional in nucleic acid amplification methods (e.g., rolling circle amplification). In some embodiments, the recombinant polymerase exhibits one or more features improved relative to Phi29 polymerase in a rolling circle amplification method, such as using a similar amount of starting template to produce a higher number of RCA products.

[0053] In some aspects, PumA1 polymerase (e.g., recombinant PumA1 polymerase and / or variant PumA1 polymerase) is provided herein. In some aspects, compared with wild-type PumA1 polymerase, the recombinant PumA1 polymerase or variant PumA1 polymerase provided comprises one or more mutations, such as amino acid substitutions. In some aspects, compared with wild-type Phi29 or PumA1 polymerase, the variant PumA1 polymerase provided exhibits improved properties, such as improved activity, thermostability and / or continuous synthesis ability. In some aspects, compared with wild-type Phi29 polymerase, the recombinant PumA1 polymerase provided exhibits improved properties, such as improved activity, thermostability and / or continuous synthesis ability. Related polynucleotides, recombinant nucleic acid molecules, vectors, recombinant expression systems, compositions, and methods and uses thereof are also provided.

[0054] In some aspects, compared with a reference Phi29 or PumA1 polymerase, such as a wild-type Phi29 or PumA1 polymerase, the variant PumA1 polymerase provided exhibits one or more improved features or characteristics, respectively. In some aspects, for example, compared with a reference Phi29 or PumA1 polymerase, such as a wild-type Phi29 or PumA1 polymerase, the variant PumA1 polymerase provided exhibits one or more features, such as improved thermostability and improved continuous synthesis capacity, respectively. In some aspects, compared with a reference Phi29 or PumA1 polymerase, such as a wild-type Phi29 or PumA1 polymerase, the variant PumA1 polymerase provided exhibits improved stability or activity, continuous synthesis capacity, increased thermostability and / or yield, respectively. In some aspects, compared with a reference Phi29 polymerase, such as a wild-type Phi29, the PumA1 polymerase provided (e.g., a recombinant wild-type PumA1 polymerase) exhibits improved stability or activity, continuous synthesis capacity, increased thermostability and / or yield.

[0055] In some aspects, provided embodiments are based on the observation that in different types of assays and contexts for nucleic acid amplification, such as rolling circle amplification (RCA), the exemplary PumA1 polymerase exhibits various improved properties of nucleic acid amplification compared to the wild-type Phi29 polymerase in both in vitro assays and in situ assays, including improved thermal stability at elevated temperatures (e.g., 37°C or 42°C), improved polymerization rates, and increased rolling circle amplification (RCA) product generation, as observed, for example, by higher RCA product signal density and / or signal intensity.

[0056] In some aspects, provided PumA1 polymerases can be used to improve nucleic acid amplification, such as in RCA, and lead to improved analysis of biological samples, such as in RCA-based assays performed in solution (e.g., in vitro) or in situ in biological samples such as tissue samples or matrix-embedded biological samples.

[0057] II. Recombinant polymerase

[0058] Provided herein are recombinant polymerases comprising the amino acid sequence of a PumA1 polymerase or a variant or derivative thereof (e.g., an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2). Expression, purification, and functional characterization of various exemplary recombinant polymerases comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 are provided in Examples 1-4. Provided herein are isolated and purified recombinant polymerases for performing amplification reactions.

[0059] In some aspects, provided herein are variant PumA1 polymerases, such as PumA1 polymerases comprising one or more mutations or modifications compared to wild-type or unmodified PumA1 polymerase. Characterization of exemplary variant PumA1 polymerases that exhibit improved properties, such as improved thermostability, is provided in Example 7.

[0060] A. Recombinant and / or variant polymerases

[0061] In some embodiments, provided herein is a recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.5%, or 99.6% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the recombinant polymerase comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant polymerase comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the recombinant polymerase comprises the amino acid sequence of a wild-type PumA1 polymerase. In some embodiments, the recombinant polymerase comprises the amino acid sequence of a variant PumA1 polymerase disclosed herein.

[0062] In some embodiments, the recombinant polymerase further comprises one or more heterologous sequences. In some embodiments, the heterologous sequence comprises 1 to 300, 2 to 250, 3 to 200, 4 to 150, 5 to 100, 10 to 90, 20 to 80, 30 to 70 or 40 to 60 amino acid residues. In some embodiments, the heterologous sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues, or an amino acid residue in a range limited by any of the foregoing. In some embodiments, the recombinant polymerase comprises two or more heterologous sequences. In some embodiments, the heterologous sequence is fused, optionally via a linker, to the N-terminus and / or C-terminus of an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.5%, or 99.6% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the heterologous sequence is fused, optionally via a linker, to the N-terminus of an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.5%, or 99.6% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the heterologous sequence is fused to the C-terminus of an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.5%, or 99.6% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, optionally via a linker. In some embodiments, the first heterologous sequence is fused to the N-terminus of an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.5%, or 99.6% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the second heterologous sequence is fused to the C-terminus of an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.5%, or 99.6% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (optionally wherein the first heterologous sequence and the second heterologous sequence are different, and wherein the first heterologous sequence and / or the second heterologous sequence are optionally fused to the amino acid sequence via a linker). In some embodiments, the heterologous sequence comprises a cleavable sequence. The cleavable sequence is typically cleaved, for example, by an enzymatic or reductive mechanism. Exemplary cleavable sequences include protease-sensitive sequences, cyclic peptide sequences, and / or disulfide-sensitive sequences.In some embodiments, the recombinant PumA1 polymerase (eg, a recombinant wild-type PumA1 polymerase comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2) or variant PumA1 polymerase comprises two or more heterologous sequences. In some embodiments, the cleavable sequence is a SUMO tag.

[0063] In some embodiments, variant PumA1 polymerases are provided herein. In some aspects, the wild-type PumA1 polymerase is modified. In some embodiments, the variant PumA1 polymerase comprises one or more variations or modifications, e.g., amino acid substitutions, mutations, deletions, insertions, and / or fusions of additional peptide or protein sequences (e.g., for fixing the polymerase on a surface or otherwise labeling the polymerase) compared to a reference PumA1 polymerase such as a sequence of a wild-type PumA1 polymerase. In some embodiments, a variant PumA1 polymerase is provided herein that exhibits improved thermal stability relative to a wild-type PumA1 polymerase.

[0064] Also provided herein are compositions comprising any recombinant (e.g., wild-type) or variant PumA1 polymerase, polynucleotides encoding any recombinant or variant PumA1 polymerase, recombinant nucleic acid molecules encoding any recombinant or variant PumA1 polymerase or comprising any polynucleotide, vectors encoding any recombinant or variant PumA1 polymerase or comprising any polynucleotide, recombinant expression systems encoding any recombinant or variant PumA1 polymerase or comprising any polynucleotide, and kits comprising or encoding any recombinant or variant PumA1 polymerase, as well as methods and uses of any of the foregoing.

[0065] In some aspects, provided herein are variant PumA1 polymerases comprising an amino acid substitution at one or more positions compared to a reference PumA1 polymerase. In some aspects, the reference PumA1 polymerase is a wild-type PumA1 polymerase. In some aspects, the reference PumA1 polymerase comprises the sequence shown in SEQ ID NO: 1.

[0066] In some aspects, the corresponding position of the one or more modifications (such as one or more substitutions) can be determined with reference to the position of a reference amino acid sequence or a reference nucleotide sequence. In some aspects, the amino acid position or nucleotide position corresponding to the amino acid position or nucleotide position in the disclosed sequence (such as the reference sequence shown in the sequence table) refers to the amino acid position or nucleotide position identified after alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm (such as the GAP algorithm or other available algorithms). In general, in order to identify the corresponding position, the amino acid sequence is aligned so as to obtain the highest order match. The alignment for determining the corresponding position can be obtained in various ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including any algorithm required to achieve maximum alignment over the full length of the compared sequence. For example, by an alignment method available to those skilled in the art, the reference sequence of the wild-type PumA1 polymerase shown in SEQ ID NO: 1 is aligned with the sequence of interest (e.g., the variant PumA1 polymerase sequence), and the corresponding residues can be determined. By aligning sequences (eg, a reference sequence and a variant PumA1 polymerase sequence), one skilled in the art can identify corresponding residues, for example, using conserved and / or identical amino acid residues as a guide.

[0067] In some aspects, the position (e.g., amino acid residue number) of the one or more amino acid substitutions present in the variant PumA1 polymerase is described with reference to the corresponding position number or the corresponding amino acid residue number of the reference PumA1 polymerase as shown in SEQ ID NO: 1. In some aspects, the one or more substituted positions are described with reference to the corresponding positions shown in SEQ ID NO: 1, including the initial methionine residue at position 1. In some aspects, the one or more substituted positions are described with reference to the positions shown in SEQ ID NO: 2, excluding the initial methionine residue. In some embodiments, position 545 (isoleucine, I545) of SEQ ID NO: 1 corresponds to position 544 (isoleucine, I544) of SEQ ID NO: 2. In some embodiments, position 574 (alanine, A574) of SEQ ID NO: 1 corresponds to position 573 (alanine, A573) of SEQ ID NO: 2. In some embodiments, position 575 (proline, P575) of SEQ ID NO: 1 corresponds to position 574 (proline, P574) of SEQ ID NO: 2. In some aspects, the corresponding positions of the one or more substitutions can be identified by available alignment methods, such as reference to substitutions at positions corresponding to positions I545, A574, and / or P575 of SEQ ID NO: 1. In some embodiments, the sequence shown in SEQ ID NO: 1 is the amino acid sequence of a wild-type PumA1 polymerase, including an initial methionine residue at position 1, and a substitution is present at a position corresponding to said position of SEQ ID NO: 1. In some aspects, the corresponding positions of the one or more substitutions can be identified by available alignment methods, such as reference to substitutions at positions corresponding to positions I545, A574, and / or P575 of SEQ ID NO: 2. In some embodiments, the sequence shown in SEQ ID NO: 2 is the amino acid sequence of a wild-type PumA1 polymerase, excluding an initial methionine residue, and a substitution is present at a position corresponding to said position of SEQ ID NO: 2.

[0068] In some aspects, the recombinant (eg, wild-type) or variant PumA1 polymerase comprises a deletion or truncation. In some aspects, the recombinant or variant PumA1 polymerase comprises an N-terminal deletion or truncation. In some aspects, the recombinant or variant PumA1 polymerase comprises a C-terminal deletion or truncation.

[0069] In some embodiments, the recombinant or variant PumA1 polymerase comprises one or more substitutions and an N-terminal deletion or truncation of a methionine (M) residue at position 1, corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant or variant PumA1 polymerase comprises one or more substitutions at positions corresponding to the positions of the sequence shown in SEQ ID NO: 2, excluding the initial methionine (M) residue. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions compared to the sequence shown in SEQ ID NO: 2, the positions of the one or more amino acid substitutions corresponding to positions I544, A573 and / or P574 with reference to the positions of SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions compared to the sequence shown in SEQ ID NO: 2, the positions of the one or more amino acid substitutions corresponding to positions I544, A573 and / or P574 with reference to the positions of SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions compared to a reference PumA1 polymerase, for example, wherein the reference sequence is the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises 2 amino acid substitutions compared to a reference PumA1 polymerase, for example, wherein the reference sequence is the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises 3 amino acid substitutions compared to a reference PumA1 polymerase, for example, wherein the reference sequence is the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises 4 amino acid substitutions compared to a reference PumA1 polymerase, for example, wherein the reference sequence is the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises 5 amino acid substitutions compared to a reference PumA1 polymerase, for example, wherein the reference sequence is the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase has at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.5% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0070] In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions at a position selected from 545, 574, and 575 of the positions corresponding to the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions at a position selected from 545 and 574 of the positions corresponding to the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions at a position selected from 545 and 575 of the positions corresponding to the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions at a position selected from 574 and 575 of the positions corresponding to the sequence shown in SEQ ID NO: 1.

[0071] In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions at a position selected from 544, 573, and 574 of the positions corresponding to the sequence shown in SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions at a position selected from 544 and 573 of the positions corresponding to the sequence shown in SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions at a position selected from 544 and 574 of the positions corresponding to the sequence shown in SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions at a position selected from 573 and 574 of the positions corresponding to the sequence shown in SEQ ID NO: 2.

[0072] In some aspects, where position numbers are described with reference to the sequence set forth in SEQ ID NO: 1, available alignment algorithms can be used to determine corresponding positions. In some aspects, where position numbers are described with reference to the sequence set forth in SEQ ID NO: 2, available alignment algorithms can be used to determine corresponding positions.

[0073] In some embodiments, variant PumA1 polymerases are provided herein, comprising one or more amino acid substitutions at positions corresponding to a reference PumA1 polymerase, such as the wild-type PumA1 polymerase sequence shown in SEQ ID NO: 1. In some embodiments, variant PumA1 polymerases are provided herein, comprising one or more amino acid substitutions at positions corresponding to a reference PumA1 polymerase, such as the wild-type PumA1 polymerase sequence shown in SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 545 corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 544 corresponding to the position of the sequence shown in SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 574 corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 573 corresponding to the position of the sequence shown in SEQ ID NO: 2. In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 575 corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 574 corresponding to the position of the sequence shown in SEQ ID NO:2.

[0074] In some embodiments, the variant PumA1 polymerase comprises two or more amino acid substitutions, including amino acid substitutions at positions 545 and 574 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises two or more amino acid substitutions, including amino acid substitutions at positions 545 and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises two or more amino acid substitutions, including amino acid substitutions at positions 574 and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises three or more amino acid substitutions, including amino acid substitutions at positions 545, 574, and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1.

[0075] In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 545 corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, in wild-type PumA1, the amino acid residue at position 545 corresponding to the position of the sequence shown in SEQ ID NO: 1 is isoleucine (I). In some aspects, in exemplary variant PumA1 polymerases, the isoleucine at position 545 is substituted with a different amino acid residue, wherein the numbering is based on position (I545) in SEQ ID NO: 1. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the amino acid substitution is a non-conservative substitution. In some embodiments, the amino acid substitution at position 545 is selected from I545E, I545D, I545Q, I545V, I545A, and I545G, wherein the numbering is based on the position in SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 545 is I545E or I545D. In some embodiments, the amino acid substitution at position 545 is I545E (wherein numbering is based on the position in SEQ ID NO: 1). In some embodiments, the variant PumA1 polymerase comprises an I545E amino acid substitution. In some embodiments, the amino acid substitution at position 545 is a conservative substitution of glutamic acid (E). Exemplary conservative substitutions of E include, for example, D, Q, V, A, or G. In some embodiments, the amino acid substitution at position 545 is I545D. In some embodiments, the variant PumA1 polymerase further comprises one or more amino acid substitutions other than position 545 corresponding to the position of the sequence shown in SEQ ID NO: 1, and / or one or more other modifications.

[0076] In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 574 corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, the sequence shown in SEQ ID NO: 1 is a wild-type PumA1 amino acid sequence. In some embodiments, in wild-type PumA1, the amino acid residue at position 574 corresponding to the position of the sequence shown in SEQ ID NO: 1 is alanine (A). In some aspects, in an exemplary variant PumA1 polymerase, the alanine at position 574 is substituted with a different amino acid residue, wherein the numbering is based on the position (A574) in SEQ ID NO: 1. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the amino acid substitution is a non-conservative substitution. In some embodiments, the amino acid substitution at position 574 is selected from A574S, A574C, A574N, A574P, A574T, A574W and A574Y. In some embodiments, the amino acid substitution at position 574 is A574S or A574T. In some embodiments, the amino acid substitution at position 574 is A574S. In some embodiments, the variant PumA1 polymerase comprises an A574S amino acid substitution. In some embodiments, the amino acid substitution at position 574 is a conservative substitution of serine (S). Exemplary conservative substitutions of S include, for example, A, C, N, P, T, W or Y. In some embodiments, the amino acid substitution at position 574 is A574T. In some embodiments, the variant PumA1 polymerase further comprises one or more amino acid substitutions other than position 574 corresponding to the position of the sequence shown in SEQ ID NO: 1, and / or one or more other modifications.

[0077] In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at position 575 corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, in wild-type PumA1, the amino acid residue at position 575 corresponding to the position of the sequence shown in SEQ ID NO: 1 is proline (P). In some aspects, in exemplary variant PumA1 polymerases, the proline at position 575 is substituted with a different amino acid residue, wherein the numbering is based on the position (P575) in SEQ ID NO: 1. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the amino acid substitution is a non-conservative substitution. In some embodiments, the amino acid substitution at position 575 is, for example, P575V, P575D, P575E, P575I, P575L, or P575M. In some embodiments, the amino acid substitution at position 575 is P575V, P575I, or P575L. In some embodiments, the amino acid substitution at position 575 is P575V. In some embodiments, the variant PumA1 polymerase comprises a P575V amino acid substitution. In some embodiments, the amino acid substitution at position 575 is a conservative substitution of valine (V). Exemplary conservative substitutions of V include, for example, D, E, I, L, or M. In some embodiments, the amino acid substitution at position 575 is P575I. In some embodiments, the amino acid substitution at position 575 is P575L. In some embodiments, the variant PumA1 polymerase further comprises one or more amino acid substitutions other than position 575 corresponding to the position of the sequence shown in SEQ ID NO: 1, and / or one or more other modifications.

[0078] In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at one or more positions selected from positions 545 and 574 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises an amino acid substitution at positions 545 and 574 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, in wild-type PumA1, the amino acid residue at position 545 corresponding to the position of the sequence shown in SEQ ID NO: 1 is isoleucine (I) and the amino acid residue at position 574 corresponding to the position of the sequence shown in SEQ ID NO: 1 is alanine (A). In some aspects, in exemplary variant PumA1 polymerases, the isoleucine at position 545 (I545) and the alanine at position 574 (A574) are substituted with different amino acid residues, such as any substitutions described herein. In some embodiments, the amino acid substitution at position 545 is, for example, I545E, I545D, I545Q, I545V, I545A or I545G, and the amino acid substitution at position 574 is, for example, A574S, A574A, A574C, A574N, A574P, A574T, A574W or A574Y. In some embodiments, the amino acid substitution at position 545 is I545E or I545D, and the amino acid substitution at position 574 is A574S or A574T. In some embodiments, the amino acid substitution at position 545 is I545E, and the amino acid substitution at position 574 is A574S. In some embodiments, the variant PumA1 polymerase comprises I545E and A574S amino acid substitutions. In some embodiments, the amino acid substitutions at positions 545 and 574 are I545E and A574S, respectively. In some embodiments, the variant PumA1 polymerase further comprises one or more amino acid substitutions other than positions 545 and 574 corresponding to the positions in the sequence shown in SEQ ID NO: 1, and / or one or more other modifications.

[0079] In some embodiments, the variant PumA1 polymerase further comprises an amino acid substitution at position 575 corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase comprises amino acid substitutions at positions 545 and 575 corresponding to the position of the sequence shown in SEQ ID NO: 1. In some embodiments, in wild-type PumA1, the amino acid residue at position 545 corresponding to the position of the sequence shown in SEQ ID NO: 1 is isoleucine (I) and the amino acid residue at position 575 corresponding to the position of the sequence shown in SEQ ID NO: 1 is proline (P). In some aspects, in exemplary variant PumA1 polymerases, the isoleucine at position 545 (I545) and the proline at position 575 (P575) are substituted with different amino acid residues, such as any substitutions described herein. In some embodiments, the amino acid substitution at position 545 is, for example, I545E, I545D, I545Q, I545V, I545A or I545G, and the amino acid substitution at position 575 is, for example, P575V, P575D, P575E, P575I, P575L or P575M. In some embodiments, the amino acid substitution at position 545 is I545E or I545D, and the amino acid substitution at position 575 is P575V, P575I or P575L. In some embodiments, the amino acid substitution at position 545 is I545E, and the amino acid substitution at position 575 is P575V. In some embodiments, the variant PumA1 polymerase comprises I545E and P575V amino acid substitutions. In some embodiments, the amino acid substitutions at positions 545 and 575 are I545E and P575V, respectively. In some embodiments, the variant PumA1 polymerase further comprises one or more amino acid substitutions other than positions 545 and 575 corresponding to the positions in the sequence shown in SEQ ID NO: 1, and / or one or more other modifications.

[0080] In some embodiments, the variant PumA1 polymerase comprises amino acid substitutions at positions 574 and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, in wild-type PumA1, the amino acid residue at position 574 corresponding to the position of the sequence shown in SEQ ID NO: 1 is alanine (A) and the amino acid residue at position 575 corresponding to the position of the sequence shown in SEQ ID NO: 1 is proline (P). In some aspects, in exemplary variant PumA1 polymerases, the alanine at position 574 (A574) and the proline at position 575 (P575) are substituted with different amino acid residues, such as any substitutions described herein. In some embodiments, the amino acid substitution at position 574 is, for example, A574S, A574A, A574C, A574N, A574P, A574T, A574W or A574Y, and the amino acid substitution at position 575 is, for example, P575V, P575D, P575E, P575I, P575L or P575M. In some embodiments, the amino acid substitution at position 574 is A574S or A574T, and the amino acid substitution at position 575 is P575V, P575I or P575L. In some embodiments, the amino acid substitution at position 574 is A574S, and the amino acid substitution at position 575 is P575V. In some embodiments, the variant PumA1 polymerase comprises A574S and P575V amino acid substitutions. In some embodiments, the amino acid substitutions at positions 574 and 575 are A574S and P575V, respectively. In some embodiments, the variant PumA1 polymerase further comprises one or more amino acid substitutions other than positions 574 and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1, and / or one or more other modifications. In some embodiments, the variant PumA1 polymerase has at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the amino acid sequence shown in SEQ ID NO: 10.

[0081] In some embodiments, the variant PumA1 polymerase comprises amino acid substitutions at positions 545, 574, and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, in wild-type PumA1, the amino acid residue at position 545 corresponding to the position of the sequence shown in SEQ ID NO: 1 is isoleucine (I), the amino acid residue at position 574 corresponding to the position of the sequence shown in SEQ ID NO: 1 is alanine (A), and the amino acid residue at position 575 corresponding to the position of the sequence shown in SEQ ID NO: 1 is proline (P). In some aspects, in exemplary variant PumA1 polymerases, the isoleucine at position 545 (I545), the alanine at position 574 (A574), and the proline at position 575 (P575) are substituted with different amino acid residues, such as any substitutions described herein. In some embodiments, the amino acid substitution at position 545 is, e.g., I545E, I545D, I545Q, I545V, I545A, or I545G, the amino acid substitution at position 574 is, e.g., A574S, A574A, A574C, A574N, A574P, A574T, A574W, or A574Y, and the amino acid substitution at position 575 is, e.g., P575V, P575D, P575E, P575I, P575L, or P575M. In some embodiments, the amino acid substitution at position 545 is I545E or I545D, the amino acid substitution at position 574 is A574S or A574T, and the amino acid substitution at position 575 is P575V, P575I, or P575L. In some embodiments, the amino acid substitution at position 545 is I545E, the amino acid substitution at position 574 is A574S, and the amino acid substitution at position 575 is P575V. In some embodiments, the variant PumA1 polymerase comprises I545E, A574S, and P575V amino acid substitutions. In some embodiments, the amino acid substitutions at positions 545, 574, and 575 are I545E, A574S, and P575V, respectively. In some embodiments, the variant PumA1 polymerase has a sequence identity of or about or at least about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% to the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the variant PumA1 polymerase comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the variant PumA1 polymerase consists of the amino acid sequence shown in SEQ ID NO: 10.

[0082] In some aspects, amino acid sequence variants of PumA1 polymerase can be prepared by introducing appropriate modifications into the nucleotide sequence encoding wild-type PumA1 polymerase or by peptide synthesis. Examples of modifications or variations include deletions and / or insertions and / or substitutions of residues in the amino acid sequence of wild-type PumA1 polymerase.

[0083] Variants of the PumA1 polymerase can also be prepared by inserting or deleting one or more amino acids with reference to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The variant PumA1 polymerase may include any combination of modifications or variations, such as insertions, deletions, and substitutions, including any combination of modifications or variations described herein or any known modifications or variations. In some aspects, the variant PumA1 polymerase has improved activity and / or properties, such as any activity and / or properties described herein (e.g., improved thermal stability). In some aspects, such modifications or variations can be used to improve aspects of protein production or to alter one or more properties of the polymerase, such as to achieve improved activity and / or properties.

[0084] Exemplary amino acid sequence insertions include N-terminal and / or C-terminal fusions in the range of one amino acid to a polypeptide containing one hundred or more amino acids, and internal insertions of single or multiple amino acids. Examples of terminal fusions include the addition of affinity tags, linkers, and / or solubility domains. Exemplary tags include purification, substrate binding or other tags, such as polyhistidine tags, His10 tags, His6 tags, alanine tags, Ala1O tags, Ala16 tags, biotin tags, biotin ligase recognition sequences or other biotin attachment sites (e.g., BiTag or Btag or variants thereof, e.g., BtagV1-11), GST tags, S tags, SNAP tags, hemagglutinin (HA) tags, DSB (Sso7D) tags, lysine tags, NanoTags, Cmyc tags. Exemplary linkers include polyamino acid linkers, such as linkers rich in amino acids glycine, serine, alanine, histidine, arginine, lysine, glutamine, and / or proline. Other polypeptide domains that may be fused at the N-terminus and / or C-terminus include domains from a polypeptide or protein, such as an antibody or antibody domain, antibody fragment, antigen, receptor, receptor domain, receptor fragment, binding protein or ligand.

[0085] In some aspects, the variant PumA1 polymerase comprises one or more deletions. In some aspects, the deletions can include deletions of N-terminal and / or C-terminal amino acids as well as internal amino acids, ranging from a single amino acid to a hundred or more amino acids.

[0086] In some aspects, the variant PumA1 polymerase, such as any variant comprising one or more amino acid substitutions described herein, further comprises one or more heterologous sequences. In some embodiments, the heterologous sequence comprises 1 to 300, 2 to 250, 3 to 200, 4 to 150, 5 to 100, 10 to 90, 20 to 80, 30 to 70 or 40 to 60 amino acid residues. In some embodiments, the heterologous sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues, or an amino acid residue in a range limited by any of the foregoing. In some embodiments, the variant PumA1 polymerase comprises two or more heterologous sequences.

[0087] In some embodiments, the heterologous sequence is fused to the N-terminus and / or C-terminus of the variant PumA1 polymerase. In some embodiments, the heterologous sequence is fused to the N-terminus of the variant PumA1 polymerase. In some embodiments, the heterologous sequence is fused to the C-terminus of the variant PumA1 polymerase. In some embodiments, the first heterologous sequence is fused to the N-terminus of the variant PumA1 polymerase, and the second heterologous sequence is fused to the C-terminus of the variant PumA1 polymerase (optionally wherein the first heterologous sequence and the second heterologous sequence are different). In some embodiments, the heterologous sequence comprises a cleavable sequence. The cleavable sequence is usually cleaved, for example, by an enzymatic or reduction mechanism. Exemplary cleavable sequences include protease-sensitive sequences, cyclic peptide sequences, and / or disulfide-sensitive sequences. In some embodiments, the variant PumA1 polymerase comprises two or more heterologous sequences. In some embodiments, the cleavable sequence is a SUMO tag.

[0088] In some embodiments, the recombinant polymerase or variant PumA1 polymerase comprises a heterologous sequence. In some cases, the heterologous sequence comprises a tag. In some aspects, the tag is a tag for purification, substrate binding or other tags. In some embodiments, the tag is selected from the following: polyhistidine (HIS) tag, solubility enhancement tag (SET), small ubiquitin modified (SUMO) tag, liver fluke 8-kDa antigen (Fh8) tag, thioredoxin (Trx) tag, glutathione-s-transferase (GST) tag, maltose binding protein (MBP) tag, protein G IgG domain B1 (GB1) tag, mutated dehalogenase (Halo) tag, streptavidin binding peptide (SBP) tag and Tamavidin tag. In some aspects, exemplary tags include those described in, for example, Kimple et al., Curr Protoc Protein Sci. 2013; 73: Unit-9.9. In some embodiments, the tag is a SUMO tag. In some embodiments, the tag comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the tag is a poly-HIS tag. In some embodiments, the tag comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0089] In some embodiments, the tag is 1 to 50, 2 to 40, 3 to 30, 5 to 20, 5 to 12, or 8 to 20 amino acid residues in length.

[0090] In some embodiments, the tag is a solubility enhancement tag (SET). In some embodiments, the tag has a net negative charge at pH 7. In some embodiments, the tag comprising a negative charge improves the solubility and / or stability of the variant PumA1 polymerase. In some embodiments, the tag has a net negative charge of at least or at least about -1, -2, -4, -6, -10, -12, -14, -15, -16, -17 or -18. In some embodiments, the tag has a net negative charge of -1, -2, -4, -6, -10, -12, -14, -15, -16, -17 or -18. In some embodiments, the tag comprises one or more acidic amino acid residues, for example, aspartic acid (Asp, D) and / or glutamic acid (Glu, E). In some embodiments, the tag comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more acidic amino acid residues. In some embodiments, the length of the tag is 1 to 50, 2 to 40, 3 to 30, 5 to 20, 5 to 12, or 8 to 20 amino acid residues. In some embodiments, the tag has the sequence S(E / D) n K m (E / D) pG (SEQ ID NO: 12), wherein S is serine, (E / D) is aspartic acid or glutamic acid, K is lysine, G is glycine, n is 5 or 6, m is 1 or 0, and p is an integer between 2 and 6. In some embodiments, the tag comprises the sequence shown in any one of SEQ ID NOs: 13-18. In some embodiments, the tag comprises the sequence shown in SEQ ID NO: 14. In some embodiments, the tag is operably linked to the C-terminus of the recombinant polymerase or variant PumA1 polymerase.

[0091] In some embodiments, the recombinant polymerase comprises, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and a heterologous sequence. In some embodiments, the recombinant polymerase comprises, from the N-terminus to the C-terminus, an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, an affinity tag, and a solubility tag. In some embodiments, the recombinant polymerase comprises, from the N-terminus to the C-terminus, an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, a polyhistidine (HIS) tag, and a solubility tag. In some embodiments, the polyhistidine tag comprises 4, 6, or 8 histidines. In some embodiments, the polyhistidine tag comprises 6 histidines. In some embodiments, the solubility tag comprises the sequence S(E / D) n K m (E / D) pG (SEQ ID NO: 12), wherein S is serine, (E / D) is aspartic acid or glutamic acid, K is lysine, G is glycine, n is 5 or 6, m is 1 or 0, and p is an integer between 2 and 6. In some embodiments, the solubility tag comprises the sequence shown in any one of SEQ ID NOs: 13-18. In some embodiments, the solubility tag comprises the sequence shown in SEQ ID NO: 14. In some cases, the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 comprises one or more amino acid substitutions at one or more positions selected from positions 545, 574, and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some cases, the plurality of amino acid substitutions are selected from the group consisting of I545E, A574S, and P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO: 1. In some cases, the one or more amino acid substitutions are I545E, A574S and / or P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant polymerase further comprises a linker sequence (e.g., a serine and / or glycine linker sequence), which is optionally positioned between the affinity tag and the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the recombinant polymerase comprises an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 7.

[0092] In some embodiments, the recombinant polymerase may comprise a heterologous sequence and an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 from the N-terminus to the C-terminus. In some embodiments, the recombinant polymerase comprises an affinity tag, a solubility tag, and an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 from the N-terminus to the C-terminus. In some embodiments, the solubility tag is a SUMO tag. In some cases, the solubility tag comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 comprises one or more amino acid substitutions at one or more positions selected from positions 545, 574, and 575 corresponding to positions of the sequence shown in SEQ ID NO: 1. In some cases, the plurality of amino acid substitutions are selected from the group consisting of I545E, A574S, and P575V, wherein the amino acid numbers correspond to positions of the sequence shown in SEQ ID NO: 1. In some cases, the one or more amino acid substitutions are I545E, A574S, and / or P575V, wherein the amino acid numbers correspond to positions of the sequence shown in SEQ ID NO: 1. In some cases, the heterologous sequence is removed after cleavage of the heterologous sequence (e.g., cleavage of the SUMO tag). In some cases, prior to cleavage, the recombinant polymerase comprises an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 3. In some cases, after cleavage, the recombinant polymerase comprises an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 2.

[0093] In some embodiments, the heterologous sequence comprises a linker. In some embodiments, the linker comprises one or more glycine, serine, alanine, histidine, arginine, lysine, glutamine and / or proline residues. In some embodiments, the linker comprises one or more glycine and / or serine residues. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues in length, or a range defined by any of the foregoing. In some embodiments, the linker is 1 to 30, 2 to 25, 3 to 20, 4 to 18, 5 to 16, 6 to 14, 7 to 12 or 8 to 10 amino acid residues in length. In some embodiments, the linker comprises the sequence shown in any one of SEQ ID NOs: 19-26. In some embodiments, the linker comprises the sequence SSG shown in SEQ ID NO: 4. In some embodiments, the linker comprises 2, 3, 4, 5, 6 or more repeats of any one of the sequences shown in SEQ ID NOs: 19-26.

[0094] In some embodiments, the recombinant polymerase or variant PumA1 polymerase comprises two or more joints. In some embodiments, the joint is fused to the N-terminus and / or C-terminus of the recombinant polymerase or variant PumA1 polymerase. In some embodiments, the joint sequence comprises a cleavable joint. The cleavable joint is usually cleaved, for example, by an enzymatic or reduction mechanism. Exemplary cleavable joints include protease-sensitive joints, cyclic peptide joints, and / or disulfide-sensitive joints. In some embodiments, the joint can be directly fused to the N-terminus and / or C-terminus of the recombinant polymerase or variant PumA1 polymerase (for example, fused to the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 10 or 11 with at least or about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.5% or 99% sequence identity of the amino acid sequence N-terminus and / or C-terminus).

[0095] In any of the embodiments herein, the recombinant polymerase or the variant PumA1 polymerase may comprise a sequence of amino acid residues 2-30, 2-50, 2-100, 2-200, 2-300, 2-400, or 2-500 of SEQ ID NO: 1. In any of the embodiments herein, the recombinant polymerase or the variant PumA1 polymerase may comprise a sequence of amino acid residues 2-150, 2-180, 2-190, or 2-200 of SEQ ID NO: 1. In some embodiments, the recombinant polymerase or the variant PumA1 polymerase comprises a sequence of amino acid residues 2-540 of SEQ ID NO: 1. In some embodiments, the recombinant polymerase or the variant PumA1 polymerase comprises a sequence of amino acid residues 2-100 of SEQ ID NO: 1.

[0096] In some aspects, compositions comprising recombinant polymerases or variant PumA1 polymerases disclosed herein are provided, such as those compositions comprising one or more amino acid substitutions, insertions and / or deletions, as described herein. In some aspects, the composition comprises a recombinant variant PumA1 polymerase, which comprises one or more heterologous sequences. In some embodiments, relative to the recombinant variant PumA1 polymerase, the composition has a purity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5%. In some aspects, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the biomolecules present in the composition are recombinant variant PumA1 polymerases. In some aspects, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the polypeptide or protein of the composition is a recombinant variant PumA1 polymerase. In some aspects, the composition comprising the recombinant variant PumA1 polymerase has a high level of purity and / or is close to pure relative to other biomolecules (such as other polypeptides, proteins, amino acids, nucleotides or polynucleotides) present in the composition. In some aspects, biomolecules except the recombinant variant PumA1 polymerase can be considered as pollutants in the composition. In some embodiments, the composition has less than or about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or 0.5% of pollutants.

[0097] B. Exemplary Features

[0098] In some aspects, the PumA1 polymerase provided, such as a wild-type polymerase or a polymerase comprising one or more amino acid substitutions, insertions and / or deletions, exhibits one or more improved features or characteristics. These features or characteristics relate to the activity and function of nucleic acid polymerases, such as but not limited to DNA amplification. Exemplary improved features may relate to polymerase activity, performance, robustness, utility, or a combination thereof. Exemplary improved features (e.g., improved thermostability) of the PumA1 polymerase provided relate to, for example, thermostability, persistence, and polymerization rate. In some cases, compared with a reference polymerase (e.g., wild-type Phi29), the improved polymerization rate may be a slower polymerization rate. In some cases, compared with a reference polymerase (e.g., wild-type Phi29), the improved polymerization rate may be a faster polymerization rate. Other exemplary features may relate to increased yield, altered cofactor selectivity, exonuclease lack, and increased resistance to light damage. In some aspects, the PumA1 polymerase provided exhibits improved thermal stability, continuous synthesis ability or polymerization rate, or otherwise exhibits improved ability to read through damaged, modified or other difficult fragments of nucleic acid templates, which can be used for precise amplification applications (e.g., RCA). In some embodiments, compared with a reference polymerase (e.g., wild-type Phi29), the improved activity can be to produce more amplified DNA. In some embodiments, compared with a reference polymerase (e.g., wild-type Phi29), the improved activity can be to produce less amplified DNA. In some aspects, the PumA1 polymerase has strand displacement activity. In some aspects, the PumA1 polymerase has exonuclease activity. In some aspects, the PumA1 polymerase has reverse transcriptase activity. In some embodiments, compared with a reference PumA1 polymerase (e.g., wild-type PumA1 polymerase), the one or more features of the variant PumA1 polymerase are improved. In some embodiments, compared with a reference polymerase (e.g., wild-type Phi29), the one or more features are recombinant wild-type PumA1 polymerase. In some embodiments, the one or more characteristics are determined using an equal amount of a reference polymerase under the same conditions. The improvement of one or more characteristics supports the expanded application and / or utility of the variant PumA1 polymerase, for example for DNA amplification and / or sequencing. In some embodiments, the provided PumA1 polymerase has one or more characteristics, resulting in improved nucleic acid amplification and / or nucleic acid amplification methods (such as rolling circle amplification (RCA)) of biological samples.

[0099] 1. Improved thermal stability

[0100] In some embodiments, the PumA1 polymerase described herein (e.g., recombinant wild-type PumA1 polymerase or variant PumA1 polymerase) exhibits improved thermal stability. In some aspects, improved thermal stability includes improved polymerase activity. In some embodiments, improved thermal stability includes improved, faster, more stable and / or more accurate polymerase activity, for example at elevated temperatures. In some embodiments, the PumA1 polymerase exhibiting improved thermal stability can be used for nucleic acid amplification reactions, such as DNA amplification reactions. In some embodiments, variant PumA1 polymerases are used for nucleic acid amplification performed at or about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 42°C, 45°C or higher temperatures. In some embodiments, the PumA1 polymerase is used in nucleic acid amplification performed at or about 5°C to 50°C, 10°C to 42°C, 10°C to 45°C, 25°C to 42°C, 25°C to 45°C, 30°C to 42°C, 30°C to 45°C, 35°C to 42°C, or 35°C to 45°C. In some embodiments, the PumA1 polymerase is used in nucleic acid amplification performed at elevated temperatures. In some embodiments, the PumA1 polymerase is used in nucleic acid amplification performed at or about 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C or higher. In some embodiments, the PumA1 polymerase is used in nucleic acid amplification performed at or about 30° C. to 50° C., 32° C. to 48° C., 34° C. to 46° C., 36° C. to 44° C., or 38° C. to 42° C. In some embodiments, the PumA1 polymerase is used in nucleic acid amplification performed at or about 42° C.

[0101] In some embodiments, the provided PumA1 polymerase (e.g., a recombinant wild-type PumA1 polymerase or a variant PumA1 polymerase), for example, those described in Section II.A, exhibit improved thermostability. In some aspects, the variant PumA1 polymerase exhibiting improved thermostability comprises one or more amino acid substitutions at one or more positions selected from positions 545, 574, and 575 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase exhibiting improved thermostability further comprises one or more amino acid substitutions other than positions 545, 574, and 575. In some embodiments, the variant PumA1 polymerase exhibiting improved thermostability has a sequence identity of or about or at least about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% with the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the variant PumA1 polymerase exhibiting improved thermostability comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the variant PumA1 polymerase exhibiting improved thermostability has a sequence identity of at or about or at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% to the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the variant PumA1 polymerase exhibiting improved thermostability comprises the amino acid sequence shown in SEQ ID NO: 11.

[0102] In some embodiments, the PumA1 polymerase provided (e.g., a recombinant wild-type PumA1 polymerase or a variant PumA1 polymerase) produces more amplified DNA compared to a reference polymerase. In some embodiments, the variant PumA1 polymerase provided produces more amplified DNA at elevated temperatures compared to a reference PumA1 polymerase. In some embodiments, the recombinant wild-type PumA1 polymerase provided produces more amplified DNA at elevated temperatures compared to a reference Phi29 polymerase. In some embodiments, a fluorescent dye is used to measure or assess the increase in nucleic acid amplification and the generation of amplified nucleic acid products. Exemplary fluorescent dyes for measuring the increase in nucleic acid amplification and the generation of amplified nucleic acid products such as in an RCA reaction include any fluorescent dye described herein. In some embodiments, the PumA1 polymerase produces at or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% more amplified DNA than a reference polymerase. In some embodiments, the PumA1 polymerase produces 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% more amplified DNA than a reference polymerase.

[0103] In some embodiments, compared to a reference polymerase, a PumA1 polymerase (e.g., a recombinant wild-type PumA1 polymerase or a variant PumA1 polymerase) produces more amplified DNA, such that the polymerization reaction time can be reduced, the amount of input template DNA can be reduced, the product signal from the increased DNA yield can be increased, or a combination thereof. In some embodiments, compared to a reference PumA1 polymerase, using a provided variant PumA1 polymerase, the polymerization reaction time can be reduced to or about or at least to or about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 75%. In some embodiments, compared to a reference Phi-29 polymerase, using a provided recombinant wild-type PumA1 polymerase, the polymerization reaction time can be reduced to or about or at least to or about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 75%. In some embodiments, the input template DNA required can be reduced by or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 75% using a provided variant PumA1 polymerase compared to a reference PumA1 polymerase, or by or about or at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 75% using a provided recombinant wild-type PumA1 polymerase compared to a reference PumA1 polymerase. In some embodiments, the signal from the increased DNA yield is increased by or about or at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% using a provided variant PumA1 polymerase compared to a reference Phi-29 polymerase, or by or about or at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% using a provided recombinant wild-type PumA1 polymerase compared to a reference PumA1 polymerase. In some embodiments, the improved thermostability of the provided PumA1 polymerase results in increased DNA amplification and DNA yield at a temperature including, but not limited to, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or higher. In some embodiments, the improved thermostability of the provided PumA1 polymerase results in increased DNA amplification and DNA yield at elevated temperatures including, but not limited to, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C.

[0104] In some embodiments, the provided PumA1 polymerase amplifies nucleic acids, such as DNA, faster than a reference polymerase. In some embodiments, the provided variant PumA1 polymerase amplifies nucleic acids faster at elevated temperatures compared to a reference PumA1 polymerase. In some embodiments, the provided recombinant wild-type PumA1 polymerase amplifies nucleic acids faster at elevated temperatures compared to a reference Phi29 polymerase. In some embodiments, a fluorescent dye is used to measure or assess the increase in nucleic acid amplification rate. Exemplary fluorescent dyes for measuring total products and nucleic acid amplification rates such as in RCA reactions include any fluorescent dye described herein. In some embodiments, the recombinant wild-type PumA1 polymerase amplifies nucleic acid, e.g., DNA, at a polymerization rate (e.g., nucleic acid, e.g., DNA, signal per unit time) that is higher than or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% as compared to a reference Phi29 polymerase. In some embodiments, the variant PumA1 polymerase amplifies nucleic acid, such as DNA, at a polymerization rate (e.g., nucleic acid, such as DNA, signal per unit time) that is at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% higher than a reference PumA1 polymerase. In some embodiments, the variant PumA1 polymerase produces 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% more amplified DNA per unit time than a reference PumA1 polymerase. For example, compared to a reference polymerase, a provided PumA1 polymerase produces 100% to 200% more amplified nucleic acid, e.g., DNA, within 1 hour. In some embodiments, a provided PumA1 polymerase amplifies nucleic acid (e.g., DNA) faster by at least 1, 2, 3, 4, 6, 8, 10, 12 hours or more compared to a reference polymerase.

[0105] In some aspects, the polymerization rate of a polymerase is the speed at which a polymerase can catalyze the incorporation of nucleotides into a growing polynucleotide chain during nucleic acid amplification, which can be described as the number of nucleotide bases incorporated per unit time (e.g., bases / second). The polymerization rate depends on different reaction conditions, including temperature, buffer composition, substrate, substrate concentration, and time. In some embodiments, the PumA1 polymerase provided as described herein (e.g., recombinant wild-type PumA1 polymerase or variant PumA1 polymerase) shows improved polymerization rate. In some embodiments, wild-type PumA1 polymerase or variant PumA1 polymerase respectively show improved polymerization rate compared to reference Phi29 polymerase or wild-type PumA1 polymerase. In some embodiments, the PumA1 polymerase provided (e.g., recombinant wild-type PumA1 polymerase or variant PumA1 polymerase) is used in nucleic acid amplification processes.

[0106] In some embodiments, the recombinant wild-type PumA1 polymerase provided produces more amplified nucleic acid compared to a reference Phi29 polymerase. In some embodiments, the nucleic acid amplification is an RCA reaction. In some embodiments, the recombinant wild-type polymerase provided generates a higher density of detected RCA products in the RCA reaction compared to a reference Phi29 polymerase. In some embodiments, the recombinant wild-type PumA1 polymerase provided generates a higher product signal intensity in the RCA reaction compared to a reference Phi29 polymerase.

[0107] In some embodiments, the variant PumA1 polymerase provided produces more amplified nucleic acid compared to the reference PumA1 polymerase. In some embodiments, the nucleic acid amplification is an RCA reaction. In some embodiments, the variant PumA1 polymerase provided generates a higher density of detected RCA products in the RCA reaction compared to the reference PumA1 polymerase. In some embodiments, the variant PumA1 polymerase provided generates a higher product signal intensity in the RCA reaction compared to the reference PumA1 polymerase.

[0108] In some embodiments, the provided PumA1 polymerase (e.g., a recombinant wild-type PumA1 polymerase or a variant PumA1 polymerase) produces amplified nucleic acids (e.g., DNA) at a faster rate than a reference polymerase, such that the polymerization reaction time can be reduced and / or the product signal from the increased nucleic acid (e.g., DNA) amplification is increased. In some embodiments, the polymerization reaction time can be reduced by or about or at least by or about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 75% using a provided variant PumA1 polymerase compared to a reference PumA1 polymerase, or by or about 5% by or at least by or about 10% by or at least ... In some embodiments, the amount of nucleic acid (e.g., DNA) produced or the yield of reaction products produced using the provided variant PumA1 polymerases compared to the reference PumA1 polymerases, or using the provided recombinant wild-type PumA1 polymerases compared to the reference Phi-29 polymerases, is increased by or about or at least by or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200%. In some embodiments, the improved thermal stability of the variant PumA1 polymerases results in an increased rate of nucleic acid amplification at a specific temperature, including but not limited to, being or about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or higher. In some embodiments, the improved thermostability of the variant PumA1 polymerase results in an increased rate of nucleic acid amplification at elevated temperatures, including but not limited to, at or about 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C.

[0109] In some embodiments, the provided PumA1 polymerase (e.g., a recombinant wild-type PumA1 polymerase or a variant PumA1 polymerase) is used to amplify nucleic acids, such as DNA, using a specific method, such as in a rolling circle amplification (RCA) reaction. In some embodiments, the provided variant PumA1 polymerase generates a higher density of detected rolling circle amplification (RCA) products in the RCA reaction compared to the reference PumA1 polymerase. In some embodiments, the provided recombinant wild-type PumA1 polymerase generates a higher density of detected rolling circle amplification (RCA) products in the RCA reaction compared to the reference Phi29 polymerase. In some embodiments, determining the density of detected RCA products (RCPs) involves the use and detection of specific fluorescent dyes, wherein the density of detected RCA products is measured in terms of fluorescence per μm 2In some embodiments, the RCA product counts of the sample area are measured. In some embodiments, the density of the detected RCPs is related to the total sample signal. In some embodiments, the PumA1 polymerase provided generates a higher density of detected RCPs in the RCA reaction compared to the reference polymerase. In some embodiments, the PumA1 polymerase provided generates a higher density of detected RCPs than the reference polymerase by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150% or 200% or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150% or 200% of the detected RCA product density. In some embodiments, a provided PumA1 polymerase produces a 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110% or 95% to 105% higher density of detected RCA products compared to a reference polymerase.

[0110] In some embodiments, the RCA product signal intensity generated by the RCA reaction is evaluated or assessed. In some aspects, the product signal intensity is calculated as the local signal intensity divided by the local background average intensity. In some embodiments, compared with the reference PumA1 polymerase, the variant PumA1 polymerase provided generates a higher product signal intensity in the RCA reaction. In some embodiments, compared with the reference polymerase, the PumA1 polymerase provided produces a high or about or at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150% or 200% RCA reaction product signal intensity. In some embodiments, compared with the reference polymerase, the PumA1 polymerase provided produces a high RCA reaction product signal intensity of at least 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110% or 95% to 105%.

[0111] In some embodiments, the RCA reaction using the variant PumA1 polymerase provided is carried out at a temperature of or about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or higher. In some embodiments, the RCA reaction is carried out at 5°C to 50°C, 10°C to 45°C, 25°C to 45°C, 30°C to 45°C or 35°C to 45°C. In some embodiments, the RCA reaction using the PumA1 polymerase provided (e.g., recombinant wild-type PumA1 polymerase or variant PumA1 polymerase) is carried out at an elevated temperature. In some embodiments, the RCA reaction is carried out at or about 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. In some embodiments, the RCA reaction is performed at 30°C to 50°C, 32°C to 48°C, 34°C to 46°C, 36°C to 44°C, or 38°C to 42°C.

[0112] 2. Other exemplary features

[0113] In some aspects, the recombinant and / or variant PumA1 polymerase provided exhibits other features or improvements, such as features or improvements related to polymerase activity and applications in analyzing or evaluating biological samples (such as in nucleic acid amplification and sequencing). Examples of other improved or changed properties and features include improved or changed continuous synthesis capacity, polymerization rate, product yield, increased half-life, cofactor selectivity, exonuclease activity and / or strand displacement activity. The recombinant and / or variant PumA1 polymerase provided can also combine and exhibit two or more changed features, or also have additional improved or changed features. In some aspects, the variant PumA1 polymerase has strand displacement activity. In some embodiments, the features or improvements exhibited by the recombinant and / or variant PumA1 polymerase provided are related to polymerase activity in biological samples (such as sections of tissue samples or cell sediments or cell blocks).

[0114] In some aspects, polymerase processivity is the ability of nucleic acid polymerase to perform continuous nucleic acid synthesis on template nucleic acid without frequent dissociation. Processivity can be measured by the average number of nucleotides incorporated by nucleic acid polymerase in a single association / dissociation event. In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase can show an average processivity of at least 10kb, at least 20kb, at least 30kb, at least 40kb, at least 50kb, at least 60kb or at least 70kb. In some embodiments, the recombinant and / or variant PumA1 polymerase described herein shows improved processivity relative to a reference polymerase, wherein the improved processivity improves polymerase activity. In some embodiments, compared with a reference polymerase (e.g., with reference to Phi29 polymerase), the recombinant and / or variant PumA1 polymerase shows improved processivity. In some cases, compared with a reference polymerase (e.g., with reference to Phi29 polymerase or wild-type PumA1 polymerase), the variant PumA1 polymerase shows improved processivity. In some embodiments, the recombinant and / or variant PumA1 polymerase provided produces more amplified nucleic acids compared to a reference polymerase. In some embodiments, the recombinant and / or variant PumA1 polymerase provided amplifies nucleic acids faster than a reference polymerase. In some embodiments, nucleic acid amplification is an RCA reaction. In some embodiments, the recombinant and / or variant PumA1 polymerase provided generates a higher density of detected RCA products in an RCA reaction compared to a reference polymerase. In some embodiments, the recombinant and / or variant PumA1 polymerase provided generates a higher product signal intensity in an RCA reaction compared to a reference polymerase.

[0115] In some embodiments, the variant PumA1 polymerases provided (e.g., those described in Section II.A) exhibit one or more altered or improved characteristics (e.g., thermostability, processivity, and / or polymerization rate). In some aspects, the variant PumA1 polymerase exhibiting altered or improved characteristics comprises one or more amino acid substitutions at one or more positions selected from positions 545 and 574 corresponding to the positions of the sequence shown in SEQ ID NO: 1. In some embodiments, the variant PumA1 polymerase exhibiting altered or improved characteristics further comprises an amino acid substitution at position 575. In some embodiments, the variant PumA1 polymerase exhibiting altered or improved characteristics further comprises one or more amino acid substitutions other than positions 545, 574, and 575. In some embodiments, the variant PumA1 polymerase exhibiting altered or improved characteristics has a sequence identity of or about or at least about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% with the amino acid sequence shown in any one of SEQ ID NOs: 10-11. In some embodiments, the variant PumA1 polymerase exhibiting altered or improved characteristics comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10-11. In some embodiments, the variant PumA1 polymerase exhibiting altered or improved characteristics has a sequence identity of or about or at least about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% to the amino acid sequence set forth in any one of SEQ ID NOs: 10. In some embodiments, the variant PumA1 polymerase exhibiting altered or improved characteristics comprises the amino acid sequence set forth in any one of SEQ ID NOs: 11.

[0116] In some embodiments, recombinant and / or variant PumA1 polymerases described herein that exhibit one or more altered characteristics (eg, thermostability, processivity, and / or polymerization rate) are used in nucleic acid amplification processes.

[0117] In some embodiments, under the same reaction conditions, the variant PumA1 polymerase produces at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% more rolling circle amplification products than the reference (wild type) PumA1 polymerase. In some embodiments, under the same reaction conditions, the variant PumA1 polymerase produces at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% more rolling circle amplification products than the reference (wild type) Phi29 polymerase. In some embodiments, under the same reaction conditions, the recombinant polymerase produces at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% more rolling circle amplification products than the reference (wild type) Phi29 polymerase.

[0118] In some embodiments, the variant PumA1 polymerase produces at or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% more amplified nucleic acids compared to a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase). In some embodiments, the variant PumA1 polymerase produces 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% more amplified nucleic acids compared to a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase). In some embodiments, the variant PumA1 polymerase produces more amplified nucleic acid compared to a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase), such that the polymerization reaction time can be reduced, the amount of input template nucleic acid can be reduced, the product signal from the increased nucleic acid yield is increased, or a combination thereof. In some embodiments, the variant PumA1 polymerase amplifies nucleic acid (e.g., product signal per unit time) faster than a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase) by or about or at least by or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200%. In some embodiments, the variant PumA1 polymerase amplifies 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% more nucleic acid per unit time compared to a reference polymerase (e.g., a reference wild-type PumA1 polymerase or a Phi29 polymerase). For example, the variant PumA1 polymerase produces 100% to 200% more amplified nucleic acid within 1 hour compared to a reference polymerase (e.g., a reference wild-type PumA1 polymerase or a Phi29 polymerase). In some embodiments, a variant PumA1 polymerase exhibiting improved thermal stability amplifies nucleic acid faster by at least 1, 2, 3, 4, 6, 8, 10, 12 hours or more compared to a reference polymerase (e.g., a reference wild-type PumA1 polymerase or a Phi29 polymerase). In some embodiments, the variant PumA1 polymerase produces amplified nucleic acids at a faster rate than a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase), such that polymerization reaction time can be reduced, product signal from the increased nucleic acid amplification rate is increased, or a combination thereof.

[0119] In some embodiments, a PumA1 polymerase (e.g., a recombinant wild-type PumA1 polymerase or a variant PumA1 polymerase) described herein that exhibits one or more altered characteristics (e.g., thermostability, processivity, and / or polymerization rate) is used in a nucleic acid amplification process, wherein the nucleic acid amplification is an RCA reaction. In some embodiments, the density of detected RCA products produced by the variant PumA1 polymerase is higher than or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% compared to a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase). In some embodiments, the density of detected RCA products produced by the variant PumA1 polymerase described herein is at least 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% higher than a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase). In some embodiments, the variant PumA1 polymerase described herein produces a product signal intensity that is or is about or is at least or is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% higher than a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase). In some embodiments, the variant PumA1 polymerase described herein produces a product signal intensity that is at least 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% higher compared to a reference polymerase (e.g., a reference wild-type PumA1 polymerase or Phi29 polymerase).

[0120] In some embodiments, the PumA1 polymerase provided as described herein (e.g., a recombinant wild-type PumA1 polymerase or a variant PumA1 polymerase) further exhibits a longer half-life in the absence of a substrate compared to a reference PumA1 polymerase. In some embodiments, the variant PumA1 polymerase provided as described herein further exhibits a longer half-life in the presence of a substrate compared to a reference PumA1 polymerase. In some embodiments, the variant PumA1 polymerase provided is more stable in the presence and absence of a substrate compared to a reference PumA1 polymerase. In some embodiments, the variant PumA1 polymerase substrate comprises a nucleotide, a nucleotide derivative, a polynucleotide, or a combination thereof. In some embodiments, the variant PumA1 polymerase exhibits an extended half-life of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500% as compared to a reference PumA1 polymerase in the presence or absence of a substrate. In some embodiments, the variant PumA1 polymerase provided as described herein exhibits an extended half-life of 5% to 500%, 15% to 300%, 50% to 400%, 30% to 100%, 20% to 200%, 40% to 150%, 75% to 400% or 50% and 300% as compared to a reference PumA1 polymerase in the presence or absence of a substrate.

[0121] In some embodiments, the recombinant polymerase (e.g., a recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2) produces a detected RCA product that is at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% higher than a reference polymerase (e.g., Phi29 polymerase). In some embodiments, the recombinant polymerase described herein produces a detected RCA product that is 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% higher than a reference polymerase (e.g., Phi29 polymerase). In some embodiments, the recombinant polymerase described herein produces a product signal intensity that is or is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% higher than a reference polymerase (e.g., Phi29 polymerase). In some embodiments, the recombinant polymerase described herein produces a product signal intensity that is at least 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% higher than a reference polymerase (e.g., Phi29 polymerase).

[0122] In some embodiments, the recombinant polymerase (e.g., a recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2) produces a smaller RCA product that is at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, or 100% smaller than a reference polymerase (e.g., Phi29 polymerase). In some embodiments, the recombinant polymerase described herein produces a smaller RCA product that is 10% to 100%, 25% to 75%, 50% to 100%, 75% to 100%, or 00% to 50% smaller than a reference polymerase (e.g., Phi29 polymerase). In some embodiments, under similar amplification reaction conditions (e.g., same reagent concentration and reaction time), the recombinant polymerase described herein produces smaller RCA products compared to a reference polymerase (e.g., Phi29 polymerase). In some embodiments, under similar amplification reaction conditions (e.g., same reagent concentration and reaction time), the recombinant polymerase described herein produces smaller RCA products due to slower activity compared to a reference polymerase (e.g., Phi29 polymerase).

[0123] In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may have strand displacement activity. In any embodiment herein, the recombinant polymerase or variant PumA1 polymerase may show an average nucleotide polymerization rate of at least 1000 nucleotides (nt) / minute, at least 1500nt / minute, at least 2000nt / minute, or at least 2280nt / minute. In some embodiments, the recombinant polymerase or variant PumA1 polymerase may show an average nucleotide polymerization rate of 1000 nucleotides (nt) / minute to 2500nt / minute, 1000nt / minute to 2280nt / minute, 1000nt / minute to 2000nt / minute, 2000nt / minute to 2500nt / minute, or 2000nt / minute to 2280nt / minute. In some cases, the average polymerization rate is measured at a temperature of at least or about 30°C. In some cases, the average polymerization rate is measured at a temperature of at least or about 37°C. In some cases, the average polymerization rate is measured at a temperature of at least or about 40° C. In some cases, the average polymerization rate is measured at a temperature of at least or about 42° C.

[0124] In any of the embodiments herein, the recombinant polymerase or variant PumA1 polymerase may exhibit less than 10 -5 Errors / bp, less than 10 -6 Errors / bp or less than 10-7 In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may have exonuclease activity. In any embodiment herein, the recombinant polymerase or the variant PumA1 polymerase may have reverse transcriptase activity.

[0125] In some aspects described herein, the reference polymerase is a wild-type PumA1 polymerase, such as any wild-type PumA1 polymerase described herein. In some aspects described herein, the reference polymerase is a PumA1 polymerase that does not contain the one or more amino acid substitutions, such as any wild-type PumA1 polymerase described herein. In some aspects, the reference polymerase is a Phi29 polymerase (e.g., a wild-type Phi29 polymerase), and the recombinant polymerase or variant polymerase exhibits one or more improved features relative to the Phi29 polymerase.

[0126] III. Nucleic Acids, Vectors and Expression Systems

[0127] Also provided herein are polynucleotides, vectors, and expression systems encoding any of the provided recombinant polymerases or variant PumA1 polymerases disclosed herein, such as any of those described in Section I.

[0128] Nucleic acid can include those nucleic acids comprising natural and / or non-naturally occurring nucleotides and bases, for example including nucleic acids with backbone modifications. The terms "nucleic acid molecule," "nucleic acid" and "polynucleotide" can be used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides can include natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA." Nucleic acid sequence" refers to a linear nucleotide sequence comprising a nucleic acid molecule or polynucleotide.

[0129] In some embodiments, the recombinant polymerase may comprise a heterologous sequence (e.g., one or more affinity tags and / or one or more solubility tags). In some embodiments, the recombinant polymerase comprises the sequence of a wild-type PumA1 polymerase (e.g., provided in SEQ ID NO: 1). In some embodiments, the recombinant polymerase comprises the sequence of a variant PumA1 polymerase. In some embodiments, the variant PumA1 polymerase may be a recombinantly produced variant of a natural protein in which one or more properties (such as thermal stability) have been altered. In some aspects, the nucleic acid is synthetic. In some aspects, the nucleic acid molecule may be modified (e.g., codon optimized) for use in the constructs described herein. In some cases, the sequence may be designed to contain a terminal restriction site sequence in order to be cloned into a vector.

[0130] A. Polynucleotides

[0131] In some embodiments, provided herein is a polynucleotide encoding a recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, provided herein is a polynucleotide encoding a recombinant polymerase comprising the sequence shown in SEQ ID NO: 1. In some embodiments, provided herein is a polynucleotide encoding a recombinant polymerase comprising the sequence shown in SEQ ID NO: 2. In some embodiments, provided herein is a polynucleotide encoding a recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, provided herein is a polynucleotide encoding a recombinant polymerase comprising the sequence shown in SEQ ID NO: 10. In some embodiments, provided herein is a polynucleotide encoding a recombinant polymerase comprising the sequence shown in SEQ ID NO: 11. In some embodiments, provided herein is a polynucleotide encoding a variant PumA1 polymerase. In some embodiments, the polynucleotide encodes a variant PumA1 polymerase comprising one or more amino acid modifications or variations (e.g., substitutions, insertions, deletions, and / or combinations thereof) compared to the sequence of the wild-type PumA1 polymerase. In some embodiments, provided herein is a polynucleotide encoding a variant PumA1 polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, provided herein is a polynucleotide encoding a variant PumA1 polymerase comprising the sequence shown in SEQ ID NO: 10. In some embodiments, provided herein is a polynucleotide encoding a variant PumA1 polymerase comprising the sequence shown in SEQ ID NO: 11. In some embodiments, the polynucleotide encodes a variant PumA1 polymerase provided herein, such as any of those described in Section II.A.

[0132] The amino acid sequence variants of PumA1 polymerase can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the wild-type PumA1 polymerase. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the nucleotide sequence encoding the wild-type PumA1 polymerase. The encoded variant PumA1 polymerase can include any combination of deletions, insertions and substitutions. In some aspects, the encoded variant PumA1 polymerase exhibits desired characteristics, for example, improved polymerase activity, characteristics and / or properties.

[0133] In some aspects, a polynucleotide sequence encoding any of the provided recombinant polymerases or variant PumA1 polymerases, such as any of those described in Section II.A, is provided. In some aspects, the encoded recombinant polymerase or variant PumA1 polymerase exhibits or has one or more of the exemplary features or properties, such as any of those described in Section II.B.

[0134] In some aspects, a polynucleotide sequence encoding any of the recombinant polymerases provided herein, such as any of those described in Section II.A, is provided. In some aspects, a polynucleotide sequence encoding any of the variant PumA1 polymerases provided, such as any of those described in Section II.A, is provided.

[0135] In some embodiments, the polynucleotide encodes a variant PumA1 polymerase comprising an amino acid substitution at positions 545, 574, and / or 575 corresponding to the positions of the wild-type PumA1 polymerase amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the polynucleotide encodes an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1. In some embodiments, the polynucleotide encodes a variant PumA1 polymerase comprising amino acid substitutions I545E, A574S, and / or P575V at positions corresponding to the wild-type PumA1 polymerase amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the polynucleotide encodes a variant PumA1 polymerase comprising an amino acid substitution at positions 544, 573, and / or 574 corresponding to the positions of the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the polynucleotide encodes an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 2. In some embodiments, the polynucleotide encodes a variant PumA1 polymerase comprising amino acid substitutions I544E, A573S, and / or P574V at positions corresponding to the amino acid sequence shown in SEQ ID NO: 2.

[0136] In some embodiments, a polynucleotide sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the nucleotide sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9 is provided. In some embodiments, a polynucleotide sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the nucleotide sequence shown in SEQ ID NO: 8 is provided. In some embodiments, a polynucleotide sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the nucleotide sequence shown in SEQ ID NO: 9 is provided. In some aspects, the polynucleotide sequence comprises the sequence shown in SEQ ID NO: 8. In some aspects, the polynucleotide sequence comprises the sequence shown in SEQ ID NO: 9.

[0137] In some embodiments, the polynucleotide sequence encoding the provided recombinant polymerase or variant PumA1 polymerase is codon-optimized for a host cell or a recombinant expression system to generate a variant PumA1 polymerase. Typically, codon optimization involves balancing the percentage of selected codons with the public abundance of transfer RNA suitable for the host cell or expression system so that none of them is overloaded or restrictive. This may be advantageous in some cases because most amino acids are encoded by more than one codon, and the use of codons varies from organism to organism. The difference in codon use between transfected genes and host cells may affect protein expression and immunogenicity of nucleic acid constructs. Typically, for codon optimization, codons are selected to select those codons that are balanced with the frequency of use of the host cell. Typically, the redundancy of the codons of an amino acid makes different codons encode an amino acid. In some embodiments, when selecting a codon for replacement, it may be desirable that the resulting mutation is a silent mutation so that the codon change does not affect the amino acid sequence. Generally speaking, the last nucleotide of a codon can remain unchanged without affecting the amino acid sequence.

[0138] B. Recombinant Nucleic Acids and Vectors

[0139] In some aspects, a recombinant nucleic acid is provided herein, comprising any polynucleotide described in Section III.A. In some aspects, a vector is provided herein, comprising any polynucleotide described in Section III.A. In some embodiments, the recombinant nucleic acid or vector comprises a polynucleotide encoding a recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the recombinant polymerase comprises a heterologous sequence (e.g., one or more affinity tags and / or one or more solubility tags). In some embodiments, the recombinant nucleic acid or vector comprises a polynucleotide encoding a variant PumA1 polymerase described herein, which comprises one or more amino acid modifications or variations, such as substitutions, deletions, insertions and / or mutations, compared to the sequence of the wild-type PumA1 polymerase.

[0140] In some embodiments, a recombinant nucleic acid molecule comprises a polynucleotide described in Section III.A.

[0141] In some embodiments, the recombinant nucleic acid molecule further comprises a transcriptional regulatory sequence operably connected to the polynucleotide. In some embodiments, the transcriptional regulatory sequence comprises a promoter selected from bacteria, viruses and mammalian promoters. In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a SV40 promoter, a RSV promoter and a promoter found in the long terminal repeat sequence of a mouse stem cell virus. Other promoters known to the technician are also considered.

[0142] In some embodiments, there is provided a polynucleotide comprising the polynucleotides described in Part III.A or a recombinant nucleic acid molecule as described herein, such as a vector encoding any recombinant polymerase or variant PumA1 polymerase as described herein. In some embodiments, the vector is a plasmid, a viral vector, a cosmid or a transposon. The expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. In some embodiments, the vector is an expression vector. Exemplary vectors can be pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (PharmaciaBiotech, Uppsala, Sweden) or pEX series (Clontech, Palo Alto, Calif.) vectors. In some cases, phage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. In some embodiments, plant expression vectors can be used, and include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). In some cases, the vector is a viral vector. In some such aspects, the viral vector is a retroviral vector, such as a lentiviral vector.

[0143] C. Host cells and expression systems

[0144] In some cases, provided herein are host cells or recombinant expression systems comprising any polynucleotides, recombinant nucleic acid molecules or vectors provided herein, such as any polynucleotides, recombinant nucleic acid molecules or vectors described in Part III. In some embodiments, polynucleotides, recombinant nucleic acid molecules and / or vectors are used to transform host cells or recombinant expression systems to generate transformed host cells or recombinant expression systems.

[0145] Polynucleotides, recombinant nucleic acid molecules and / or vectors can be introduced into host cells or recombinant expression systems using various methods for nucleic acid introduction or transfer, including by viruses (e.g., retroviruses or slow viruses), transduction, transposons and electroporation. In some aspects, the recombinant expression system comprises a host cell, such as any host cell described herein.

[0146] In some embodiments, the transformed host cell or recombinant expression system produces a recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the recombinant polymerase comprises a heterologous sequence. In some embodiments, the transformed host cell or recombinant expression system produces a variant PumA1 polymerase. In some embodiments, the host cell or recombinant expression system is used to produce a recombinant polymerase or a variant PumA1 polymerase. In some embodiments, the host cell or recombinant expression system is a cell system or a cell-free system. In some embodiments, the host cell or recombinant expression system comprises one or more cell systems selected from bacterial cells, fungal cells, insect cells and mammalian cells. In some embodiments, the cell system can be selected from the group including Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster and CHO cells. For example, Escherichia coli is a bacterial cell that can be transformed with the polynucleotides, recombinant nucleic acids and / or vectors described herein for effectively producing a recombinant polymerase or a variant PumA1 polymerase. In some aspects, in bacterial host cells or expression systems (such as E. coli), protein translation is initiated by N-formylmethionine (fMet), which is cleaved during translation initiation by fMet-tRNA fMet In some aspects, the formylated methionine at the N-terminus of a nascent bacterial protein is typically removed co-translationally by a peptide deformylase (PDF) shortly after the nascent protein emerges from the peptide exit tunnel of the ribosome, and in most cases, is followed by methionine excision by a methionine aminopeptidase (MAP). Thus, in some aspects, the bacterially produced protein does not include an initial Met residue.

[0147] In some embodiments, the polynucleotide sequence encoding the recombinant polymerase or variant PumA1 polymerase is codon-optimized for the host cell or recombinant expression system, for example, as described in Section III.A.

[0148] IV. Methods of use, uses, and applications of recombinant PUMA1 polymerase, variant PUMA1 polymerase, and compositions thereof

[0149] Also provided herein are methods involving or employing any of the provided variants and / or recombinant polymerases (including PumA1 polymerases or variants thereof), compositions or kits comprising the recombinant polymerases or variant PumA1 polymerases disclosed herein, or nucleic acids or vectors encoding the recombinant polymerases or variant PumA1 polymerases, and the uses of any of the foregoing, such as for nucleic acid amplification and / or sequencing. In some aspects, the provided recombinant polymerases, variant PumA1 polymerases, or compositions comprising recombinant polymerases or variant PumA1 polymerases can be used for a variety of analysis or evaluation methods or experiments (e.g., sequencing, genotyping, and amplification reactions) for evaluating biological samples. In some aspects, the provided variant PumA1 polymerases are used to amplify nucleic acids, such as DNA or RNA, including in applications such as rolling circle amplification (RCA).

[0150] In some aspects, methods are provided for amplifying nucleic acids such as DNA, the methods involving contacting a biological sample containing the DNA to be amplified with any of the provided recombinant polymerases, variant PumA1 polymerases, or compositions disclosed herein.

[0151] In some aspects, methods for performing rolling circle amplification (RCA) are provided, which methods involve using any of the provided recombinant polymerase or variant PumA1 polymerase, a composition or kit comprising a recombinant polymerase or variant PumA1 polymerase disclosed herein, or a nucleic acid or vector encoding a variant PumA1 polymerase. In some aspects, the method for performing RCA involves contacting a biological sample containing a nucleic acid (e.g., DNA) to be amplified with a provided recombinant polymerase, a variant PumA1 polymerase, or a composition comprising any of the recombinant polymerase or variant PumA1 polymerase disclosed herein. In some aspects, the method thereby amplifies nucleic acids by RCA.

[0152] In some embodiments, the recombinant polymerase or variant PumA1 polymerase provided is used in a method involving amplifying a nucleic acid probe by rolling circle amplification. In some embodiments, RCA involves the use of a circular probe or a circularizable probe that recognizes a specific target nucleic acid sequence, and can be amplified via RCA (e.g., amplified after circularization in the case of a circularizable probe) for detection. In some embodiments, a circularizable probe or a probe set (e.g., a padlock probe) contains one or more barcodes. In some embodiments, the barcode is combined by a detection probe, and the detection of a primary probe does not need to be fluorescent, but includes a target binding portion (e.g., for hybridization with one or more primary probes) and a plurality of other barcodes (e.g., a secondary barcode, relative to a primary barcode on a primary probe). In some embodiments, the barcode of the detection probe is targeted by a detectably labeled detection oligonucleotide (such as a fluorescently labeled oligonucleotide). In some embodiments, one or more decoding schemes are used to decode a signal (such as fluorescence) for sequence determination. Exemplary decoding schemes are described in Eng et al., Nature 2019; 568(7751): 235-239; Chen et al., Science; 2015; 348(6233): aaa6090; US10,457,980 B2; US 2016 / 0369329 A1; WO 2018 / 026873 A1; and US 2017 / 0220733 A1, all of which are incorporated by reference in their entirety. In some embodiments, these assays enable simultaneous signal amplification, combined decoding, and error correction schemes.

[0153] In some embodiments, the recombinant polymerase, variant PumA1 polymerase or composition provided is used for methods involving the use of a circular or cyclizable construct hybridized with a nucleic acid of interest to generate a circular nucleic acid. In some embodiments, RCA includes linear RCA. In some embodiments, RCA includes branched RCA. In some embodiments, RCA includes dendritic RCA. In some embodiments, RCA includes any combination of the foregoing. In some embodiments, the circular nucleic acid is a construct formed using a connection. In some embodiments, a circular construct is formed by extending a template primer and then connecting. In some embodiments, a circular construct is formed by providing an insert between the ends to be connected. In some embodiments, a circular construct is formed using any of the foregoing combinations. In some embodiments, the connection is a DNA-DNA templated connection. In some embodiments, the connection is an RNA-RNA templated connection. Exemplary RNA templated connection probes and methods are described in US2020 / 0224244, which is incorporated herein by reference in its entirety. In some embodiments, the connection is an RNA-DNA templated connection. In some embodiments, a splint is provided as a template for connection.

[0154] In some embodiments, the method comprises contacting the biological sample with the amplification primer added. In some cases, the amplification primer is added together with one or more other probes, such as with a circular probe or a circularizable probe or a probe group. In some cases, the amplification primer can also be complementary to the target nucleic acid and a padlock probe (for example, a SNAIL probe). In some embodiments, washing steps are carried out to remove any unbound probe, primer, etc. In some embodiments, washing is a strict washing. Washing steps can be carried out at any time point during the process to remove non-specifically bound probes, connected probes, etc.

[0155] In some cases, after adding a recombinant polymerase, variant PumA1 polymerase or composition according to the present disclosure in the presence of appropriate dNTP precursors and other cofactors, the amplification primer is extended by replication of multiple copies of the template. The amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after forming a hybridization complex and any subsequent circularization (such as connecting a circularizable probe or probe set to generate a circularized probe), the circular or circularized probe is subjected to rolling circle amplification to generate an RCA product (RCP) containing multiple copies of the RCP.

[0156] After amplification, the sequence of an amplicon (e.g., RCA product) or a portion thereof is determined or otherwise analyzed, for example, by using a detectably labeled probe and imaging. The sequencing or analysis of the amplified product may include sequencing while hybridizing, sequencing while connecting and / or fluorescence in situ sequencing, and / or wherein in situ hybridization includes sequential fluorescence in situ hybridization. In some cases, the sequence of the RCA product is detected using, for example, secondary and higher level probes and detection oligonucleotides described herein.

[0157] In some cases, the provided recombinant polymerase or variant PumA1 polymerase can be used in a method for amplifying DNA, such as a sequencing method, a genotyping method, or an amplification method. In some embodiments, the method involves contacting a biological sample containing DNA to be amplified with any of the provided recombinant polymerase, variant PumA1 polymerase, or compositions. In some embodiments, the method also involves incubating the biological sample and the recombinant polymerase, variant PumA1 polymerase, or composition.

[0158] In some embodiments, the biological sample containing DNA to be amplified by a recombinant polymerase or variant PumA1 polymerase comprises isolated or synthesized nucleic acids. In some embodiments, the biological sample is tested and / or analyzed in solution. In some embodiments, the biological sample is tested and / or analyzed in vitro. In some embodiments, the biological sample is an organ or tissue sample. In some embodiments, the biological sample is tested in situ, for example, wherein the variant PumA1 polymerase is added to a fresh or previously prepared tissue sample for target analyte (e.g., gene or mRNA) analysis in a tissue section. In some embodiments, DNA is amplified by rolling circle amplification (RCA) as described in this section. In some embodiments, DNA is amplified by in situ RCA in a biological sample.

[0159] In some cases, the recombinant polymerase or variant PumA1 polymerase provided can be used in a method for rolling circle amplification (RCA). In some embodiments, these methods involve contacting a biological sample containing DNA to be amplified with a recombinant polymerase, variant PumA1 polymerase or composition. In some embodiments, the method further involves incubating a biological sample and a recombinant polymerase, variant PumA1 polymerase or composition provided herein. In some embodiments, the recombinant polymerase, variant PumA1 polymerase or composition used in the method of RCA can be any one of those described in Part II.

[0160] In some embodiments, the method of RCA includes incubating a biological sample and a recombinant polymerase, a variant PumA1 polymerase or a composition at a specific temperature. In some embodiments, the temperature is optimal for maximizing the activity of the PumA1 polymerase or its variant. In some embodiments, the PumA1 polymerase or its variant and the biological sample are incubated at a temperature of or about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or higher. In some embodiments, the PumA1 polymerase or its variant and the biological sample are incubated at a temperature of 5°C to 50°C, 10°C to 45°C, 25°C to 45°C, 30°C to 45°C or 35°C to 45°C. In some embodiments, the PumA1 polymerase or its variant and the biological sample are incubated at an elevated temperature. In some embodiments, the PumA1 polymerase or variant thereof and the biological sample are incubated at or about 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., or 50° C. In some embodiments, the PumA1 polymerase or variant thereof and the biological sample are incubated at 30° C. to 50° C., 32° C. to 48° C., 34° C. to 46° C., 36° C. to 44° C., or 38° C. to 42° C.

[0161] In some embodiments, RCA is carried out during the incubation of variant PumA1 polymerase and biological sample. In some embodiments, RCA is carried out for about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours. In some embodiments, RCA is carried out for 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours. In some embodiments, RCA is carried out for or is about 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours or longer. In some embodiments, RCA is carried out for more than 24 hours. In some embodiments, RCA is performed for 1 hour to 8 hours, 0.25 hours to 12 hours, 4 hours to 24 hours, 2 hours to 16 hours, 3 hours to 10 hours, 4 hours to 18 hours, 5 hours to 14 hours, or 0.5 hours to 20 hours. In some embodiments, RCA is performed for no more than 12 hours. In some embodiments, RCA is performed for no more than 8 hours. In some embodiments, RCA is performed for no more than 6 hours. In some embodiments, the incubation time used is sufficient to generate a detectable amount of RCA product.

[0162] In some embodiments, the method for performing RCA also includes detecting amplification products. In some embodiments, the amplification products are RCA products (RCP). In some embodiments, the amplification products are detected by contacting the biological sample with a fluorescent dye or an affinity molecule. In some embodiments, before starting the RCA reaction, the biological sample is contacted with a fluorescent dye or an affinity molecule. In some embodiments, the amplification products are detected by contacting the biological sample with SYBR gold or SYBR green. SYBR dye is an asymmetric cyanine dye for dyeing double-stranded DNA, single-stranded DNA and RNA molecules. In some embodiments, the amplification products are detected by contacting the biological sample with ATTO-532 (Rhodamine 6G). In some embodiments, the amplification products are detected in real time during amplification.

[0163] In some embodiments, provided methods employ a recombinant polymerase (eg, comprising a PumA1 polymerase or a variant thereof) or a variant PumA1 polymerase that exhibits one or more of any of the characteristics described herein, such as those described in Section II.B.

[0164] Provided herein is a method for analysis, the method comprising: contacting a sample with a nucleic acid probe that hybridizes to a target nucleic acid (e.g., RNA) in the sample, wherein the nucleic acid probe is a circular probe or a circularizable probe comprising one or more barcodes; generating an amplification product by contacting the sample with an isolated recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof; and detecting the sequence of the amplification product. In some embodiments, detection of the sequence of the amplification product comprises contacting the biological sample with one or more detectably labeled probes that bind directly or indirectly to one or more barcode sequences or their complements in the amplification product, and detecting a signal associated with the one or more detectably labeled probes. In some embodiments, the one or more detectably labeled probes bind indirectly to the one or more barcode sequences or their complements via one or more intermediate probes that bind to the one or more barcode sequences or their complements, wherein the one or more intermediate probes can be detected using the one or more detectably labeled probes.

[0165] A.Sample

[0166] The sample (such as a biological sample) that can be evaluated or analyzed using any of a recombinant polymerase, a variant PumA1 polymerase, a composition or kit including a recombinant polymerase or a variant PumA1 polymerase disclosed herein, or a nucleic acid or vector encoding a recombinant polymerase or a variant PumA1 polymerase as provided herein can be or is derived from any biological sample. The methods and compositions disclosed herein can be used to analyze biological samples, which can be obtained from a subject using any of a variety of techniques (including but not limited to biopsy, surgery, and laser capture microscopy (LCM)), and generally include cells and / or other biological materials from a subject. In addition to the subjects described above, biological samples can be obtained from prokaryotes (such as bacteria, archaea, viruses, or viroids). Biological samples can also be obtained from non-mammalian organisms (e.g., plants, insects, arthropods, nematodes, fungi, or amphibians). Biological samples can also be obtained from eukaryotic organisms, such as tissue samples, patient-derived organoids (PDOs), or patient-derived xenografts (PDXs). Biological samples from organisms can contain one or more other organisms or components thereof. For example, in addition to mammalian cells and non-cellular tissue components, mammalian tissue sections can contain prions, viroids, viruses, bacteria, fungi, or components from other organisms. The subject from which the biological sample can be obtained can be a healthy or asymptomatic individual, an individual suffering from or suspected of suffering from a disease (e.g., a patient suffering from a disease such as cancer) or susceptible to a disease, and / or an individual in need of therapy or suspected of needing therapy.

[0167] Biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (for example, mitochondria and nucleus). Biological sample can include nucleic acids (such as DNA or RNA), proteins / polypeptides, carbohydrates and / or lipids. Biological sample can be obtained as tissue sample (such as tissue section, biopsy sample, core needle biopsy sample, cell precipitation, cell block, needle aspirate or fine needle aspirate). Sample can be a fluid sample, such as a blood sample, urine sample or saliva sample. Sample can be a skin sample, colon sample, cheek swab, histological sample, histopathological sample, plasma or serum sample, tumor sample, living cell, cultured cell, clinical sample (for example, whole blood or blood-derived products, hemocytes or cultured tissue or cells, including cell suspension). In some embodiments, biological sample can include cells deposited on the surface. In some embodiments, biological sample is a tissue sample. In some embodiments, tissue sample is a complete tissue sample or a non-homogenized tissue sample.

[0168] The biological sample may be derived from a homogenous culture or population of the subjects or organisms mentioned herein, or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0169] Biological samples can include one or more diseased cells. Diseased cells may have altered metabolic properties, gene expression, protein expression and / or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells.

[0170] The biological sample may include an analyte (e.g., protein, RNA, and / or DNA) embedded in a 3D matrix. In some embodiments, an amplicon (e.g., rolling circle amplification product) derived from an analyte (e.g., protein, RNA, and / or DNA) or associated with an analyte may be embedded in a 3D matrix. In some embodiments, the 3D matrix may include natural molecules and / or synthetic molecular networks chemically and / or enzymatically connected (e.g., by crosslinking). In some embodiments, the 3D matrix may include a synthetic polymer. In some embodiments, the 3D matrix includes a hydrogel.

[0171] In some embodiments, the substrate herein can be any support that is insoluble in aqueous liquids, and allows positioning biological samples, analytes, features and / or reagents (e.g., probes) on the support. In some embodiments, biological samples can be attached to substrates. The attachment of biological samples can be irreversible or reversible, depending on the subsequent steps in the properties of the sample and the analytical method. In certain embodiments, by applying a suitable polymer coating to substrates and contacting the sample with the polymer coating, the sample can be reversibly attached to the substrate. Then, for example, using an organic solvent that at least partially dissolves the polymer coating, the sample can be separated from the substrate. Hydrogels are examples of polymers suitable for this purpose.

[0172] In some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0173] Various steps may be performed to prepare or process biological samples for and / or during an assay. Unless otherwise indicated, the preparation or processing steps described below may generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and / or analysis.

[0174] B. Analyte

[0175] Can use the method and composition provided herein to detect and analyze a variety of different analytes.In some aspects, analyte can include any biological substance, structure, part or component to be analyzed.In some aspects, target disclosed herein can similarly include any analyte of interest.In some instances, target or analyte can be detected directly or indirectly.

[0176] The analyte can be derived from a particular type of cell and / or a particular subcellular region. For example, the analyte can be derived from the cytosol, from the nucleus, from the mitochondria, from the microsomes, and more generally, from any other compartment, organelle or part of the cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release the analyte from the cell for analysis, and / or allow one or more reagents (e.g., probes for analyte detection) to approach the analyte in the cell or cell compartment or organelle.

[0177] Analyte can comprise any biomolecule or chemical compound, including macromolecule (such as protein or peptide, lipid or nucleic acid molecule) or small molecule (including organic or inorganic molecule). Analyte can be cell or microorganism, including virus or its fragment or product. Analyte can be any substance or entity, can develop specific binding partner (such as affinity binding partner) for it. Such specific binding partner can be nucleic acid probe (for nucleic acid analyte) and can directly lead to the generation of RCA template (such as circularizable probe or circular probe). Alternatively, specific binding partner can be coupled to nucleic acid, and described nucleic acid can be detected using RCA strategy, for example, in the determination of circular nucleic acid molecule that can be used as RCA template using or producing.

[0178] Analytes of particular interest may include nucleic acid molecules such as DNA (e.g., genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g., mRNA, microRNA, rRNA, snRNA, viral RNA, etc.); and synthetic and / or modified nucleic acid molecules (e.g., including nucleic acid domains comprising synthetic or modified nucleotides such as LNA, PNA, morpholino, etc. or consisting of these substances); protein molecules such as peptides, polypeptides, proteins or prions, or any molecule comprising a protein or polypeptide component, etc., or a fragment thereof; or lipid or carbohydrate molecules or any molecule comprising a lipid or carbohydrate component. The analyte may be a single molecule or a complex containing two or more molecular subunits, for example, including but not limited to protein-DNA complexes, which may or may not be covalently bound to each other and which may be the same or different. Therefore, in addition to cells or microorganisms, such a complex analyte may also be a protein complex or a protein interactor. Therefore, such a complex or interaction may be a homopolymer or a heteropolymer. Aggregates of molecules (e.g., proteins) can also be target analytes, such as aggregates of the same protein or different proteins. Analytes can also be complexes between proteins or peptides and nucleic acid molecules (such as DNA or RNA), such as interactors between proteins and nucleic acids (e.g., regulatory factors (such as transcription factors) and DNA or RNA).

[0179] 1. Endogenous analytes

[0180] In some embodiments, the analyte herein is endogenous to the biological sample, and may include nucleic acid analytes and non-nucleic acid analytes. The methods and compositions disclosed herein may be used in any suitable combination to analyze nucleic acid analytes (e.g., using nucleic acid probes or probe groups that are directly or indirectly hybridized with nucleic acid analytes) and / or non-nucleic acid analytes (e.g., using a labeling agent that includes a reporter oligonucleotide and is directly or indirectly combined with a non-nucleic acid analyte).

[0181] Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-connected or O-connected), lipoproteins, phosphoproteins, specific phosphorylation or acetylation variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitination variants of proteins, sulfated variants of proteins, viral capsid proteins, extracellular proteins and intracellular proteins, antibodies and antigen binding fragments. In some embodiments, the analyte is inside the cell or on the cell surface, such as a transmembrane analyte or an analyte attached to the cell membrane. In some embodiments, the analyte can be an organelle (e.g., a nucleus or mitochondria). In some embodiments, the analyte is an extracellular analyte, such as a secreted analyte. Exemplary analytes include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, clusters of differentiation proteins, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presentation complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, the post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation) state of cell surface proteins, nick junctions, or adherens junctions.

[0182] Examples of nucleic acid analytes include DNA analytes, such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, PCR products synthesized in situ, and RNA / DNA hybrids. A DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA (such as mRNA)) present in a tissue sample.

[0183] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Examples of different types of RNA analytes include messenger RNA (mRNA), including nascent RNA, pre-mRNA, primary transcription RNA, and processed RNA (such as capped mRNA (e.g., with a 5'7-methylguanosine cap), polyadenylated mRNA (poly A tail at the 3' end), and spliced ​​mRNA with one or more introns removed). Uncapped mRNA, unpolyadenylated mRNA, and unspliced ​​mRNA are also included in the analytes disclosed herein. RNA analytes can be transcripts of another nucleic acid molecule (e.g., DNA or RNA (such as viral RNA)) present in a tissue sample. Examples of non-coding RNA (ncRNA) that are not translated into proteins include transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as small non-coding RNAs, such as microRNA (miRNA), small interfering RNA (siRNA), Piwi interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), small Cajal body specific RNA (scaRNA) and long ncRNA (such as Xist and HOTAIR). RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA with a length greater than 200 nucleic acid bases). Examples of small RNA include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNA, piRNA, tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). RNA can be double-stranded RNA or single-stranded RNA. RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

[0184] In some embodiments described herein, the analyte can be a denatured nucleic acid, wherein the resulting denatured nucleic acid is single-stranded. The nucleic acid can be denatured, for example, optionally denatured using formamide, heat, or both formamide and heat. In some embodiments, the nucleic acid is not denatured and is used in the methods disclosed herein.

[0185] The methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in one region of a sample or in a separate feature of a substrate.

[0186] 2. Labeling Reagents

[0187] In some embodiments, the methods and compositions provided herein can be used to analyze endogenous analytes (e.g., RNA, ssDNA, cell surface or intracellular proteins and / or metabolites) in a sample using one or more labeling agents. In some embodiments, an analyte labeling agent may include a reagent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the labeling agent may include a reporter oligonucleotide indicating an analyte or a portion thereof that interacts with the labeling agent. For example, the reporter oligonucleotide may include a barcode sequence that allows identification of the labeling agent. In some cases, the sample contacted by the labeling agent may be further contacted with a probe (e.g., a single-stranded probe sequence), which is hybridized with the reporter oligonucleotide of the labeling agent to identify the analyte associated with the labeling agent. In some embodiments, the analyte labeling agent includes an analyte binding portion and a labeling agent barcode domain, and the labeling agent barcode domain includes one or more barcode sequences, such as a barcode sequence corresponding to an analyte binding portion and / or an analyte. The analyte binding portion barcode includes a barcode that associates with the analyte binding portion or otherwise identifies the analyte binding portion. In some embodiments, the analyte binding moiety is identified by identifying its associated analyte binding moiety barcode, and the analyte to which the analyte binding moiety binds can also be identified. The analyte binding moiety barcode can be a nucleic acid sequence of a given length and / or a sequence associated with the analyte binding moiety. The analyte binding moiety barcode can generally include any of the aspects of the barcode described herein.

[0188] In some embodiments, the methods include one or more post-fixation steps after contacting the sample with one or more labeling agents.

[0189] In the methods and systems described herein, one or more labeling agents that can be combined with one or more features or otherwise coupled can be used to characterize analyte, cell and / or cell feature. In some cases, cell feature includes cell surface feature. Analyte can include but is not limited to protein, receptor, antigen, surface protein, transmembrane protein, differentiation protein cluster, protein channel, protein pump, carrier protein, phospholipid, glycoprotein, glycolipid, cell-cell interaction protein complex, antigen presentation complex, major histocompatibility complex, engineered T cell receptor, T cell receptor, B cell receptor, chimeric antigen receptor, gap connection, adhesion connection or any combination thereof. In some cases, cell feature can include intracellular analyte, such as protein, protein modification (for example, phosphorylation state or other post-translational modifications), nucleoprotein, nuclear membrane protein or any combination thereof.

[0190] In some embodiments, the analyte binding moiety may include any molecule or part capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular component). Labeling agents may include, but are not limited to, proteins, peptides, antibodies (or their epitope binding fragments), lipophilic moieties (such as cholesterol), cell surface receptor binding molecules, receptor ligands, small molecules, bispecific antibodies, bispecific T cell adapters, T cell receptor adapters, B cell receptor adapters, antibody prodrugs, aptamers, monomers, affimers, darpins, and protein scaffolds or any combination thereof. Labeling agents may include (e.g., connected to) a reporter oligonucleotide that indicates the cell surface features to which the binding group is bound. For example, a reporter oligonucleotide may include a barcode sequence that allows identification of a labeling agent. For example, a labeling agent specific to a type of cell feature (e.g., a first cell surface feature) may have a first reporter oligonucleotide coupled thereto, and a labeling agent specific to different cell features (e.g., a second cell surface feature) may have different reporter oligonucleotides coupled thereto. For a description of exemplary labeling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Patent 10,550,429; U.S. Patent Publication 20190177800; and U.S. Patent Publication 20190367969, each of which is incorporated herein by reference in its entirety.

[0191] In some embodiments, the analyte binding moiety includes one or more antibodies or antigen binding fragments thereof. Antibodies or antigen binding fragments including analyte binding moieties can specifically bind to target analytes. In some embodiments, the analyte is a protein (e.g., a protein on the surface of a biological sample (e.g., a cell) or an intracellular protein). In some embodiments, a plurality of analyte labeling agents comprising a plurality of analyte binding moieties bind to a plurality of analytes present in a biological sample. In some embodiments, a plurality of analytes include a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which a plurality of analytes include a single species of analyte, the analyte binding moieties of a plurality of analyte labeling agents are the same. In some embodiments in which a plurality of analytes include a single species of analyte, the analyte binding moieties of a plurality of analyte labeling agents are different (e.g., a member of a plurality of analyte labeling agents can have two or more species of analyte binding moieties, wherein each species in the two or more species of analyte binding moieties binds to a single species of analyte, such as in different binding sites). In some embodiments, a plurality of analytes include a plurality of different species of analyte (e.g., a plurality of different species of polypeptide).

[0192] In other cases, for example to facilitate multiplex analysis of samples, labeling agents specific for particular cellular characteristics can have a first plurality of labeling agents (e.g., antibodies or lipophilic moieties) coupled to a first reporter oligonucleotide and a second plurality of labeling agents coupled to a second reporter oligonucleotide.

[0193] In some aspects, these reporter oligonucleotides may comprise a nucleic acid barcode sequence that allows identification of the labeling agent to which the reporter oligonucleotide is coupled. Selecting an oligonucleotide as a reporter can provide the advantages of being able to generate significant diversity in sequence while also being easily attached to most biomolecules (e.g., antibodies, etc.) and being easily detected (e.g., using in situ detection techniques described herein).

[0194] The reporter oligonucleotide can be linked (coupled) to the labeling agent by any of a variety of direct or indirect, covalent or non-covalent associations or linkages. For example, the oligonucleotide can be linked (coupled) using chemical conjugation techniques (e.g., available from InnovaBiosciences). Antibody labeling kits) are used to covalently link a portion of a labeling agent (such as a protein, e.g., an antibody or antibody fragment), as well as using other non-covalent linking mechanisms, such as using a biotinylated antibody and an oligonucleotide with an avidin or streptavidin linker (or beads comprising one or more biotinylated linkers coupled to an oligonucleotide). Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang et al., "Fluoride-Cleavable Biotinylation Phosphoramidite for 5′-end-Labelling and Affinity Purification of Synthetic Oligonucleotides," Nucleic Acids Res. 2003 Jan 15; 31(2):708-715, which is incorporated herein by reference in its entirety for all purposes. Similarly, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552, which is incorporated herein by reference for all purposes. In addition, click reaction chemistry can be used to couple the reporter oligonucleotide to a labeling agent.Commercially available kits (such as those from Thunderlink and Abcam) and common techniques in the art can be used to couple the reporter oligonucleotide to a labeling agent when appropriate.In another example, the labeling agent is indirectly (e.g., via hybridization) coupled to the reporter oligonucleotide, and the reporter oligonucleotide includes a barcode sequence for identifying the labeling agent.For example, the labeling agent can be directly coupled (e.g., covalently bound) to a hybrid oligonucleotide, and the hybrid oligonucleotide includes a sequence hybridized with the sequence of the reporter oligonucleotide.The hybridization of the hybrid oligonucleotide and the reporter oligonucleotide couples the labeling agent to the reporter oligonucleotide.In some embodiments, such as when applying stimulation, the reporter oligonucleotide can be released from the labeling agent.For example, the reporter oligonucleotide can be connected to the labeling agent by an unstable bond (e.g., chemically unstable, light unstable, thermally unstable, etc.), as generally described elsewhere herein for releasing molecules from a support.

[0195] In some cases, the labeling agent can include a reporter oligonucleotide and a label. The label can be a fluorophore, a radioisotope, a molecule capable of colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be directly or indirectly conjugated to a labeling agent (or reporter oligonucleotide) (for example, the label can be conjugated to a molecule that can be combined with a labeling agent or a reporter oligonucleotide). In some cases, the label is conjugated to a first oligonucleotide complementary to the sequence of the reporter oligonucleotide (for example, hybridization).

[0196] In some embodiments, a variety of different species of analytes (e.g., polypeptides) from biological samples can be subsequently associated with one or more physical properties of the biological sample. For example, a variety of different species of analytes can be associated with the position of the analyte in the biological sample. Such information (e.g., proteome information when the analyte binding moiety recognizes the polypeptide) can be used in combination with other spatial information (e.g., genetic information from biological samples, such as DNA sequence information, transcriptome information (e.g., transcript sequence) or both). For example, the cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., the shape, size, activity or type of the cell). The one or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte labeling agent, the analyte labeling agent comprising an analyte binding moiety that binds to a cell surface protein and an analyte binding moiety barcode that identifies the analyte binding moiety. The protein analysis results in a sample (e.g., a tissue sample or a cell) can be associated with DNA and / or RNA analysis in the sample.

[0197] 3. Products of endogenous analytes and / or labeling agents

[0198] In some embodiments, provided herein are methods and compositions for generating and / or analyzing one or more products of endogenous analytes and / or labeling agents in biological samples. In some embodiments, endogenous analytes (e.g., viral or cellular DNA or RNA) or products (e.g., amplification products or extension products, for example, produced by a recombinant polymerase comprising PumA1 polymerase or its variants), replication products, transcription / reverse transcription products, and / or amplification products (such as rolling circle amplification (RCA product)) are analyzed. In some embodiments, the labeling agent directly or indirectly combined with the analyte in the biological sample is analyzed. In some embodiments, the product (e.g., amplification product, such as rolling circle amplification (RCA product), extension product (e.g., by nucleic acid polymerase), hybridization product, connection product, replication product, transcription / reverse transcription product), and / or the labeling agent directly or indirectly combined with the analyte in the biological sample.

[0199] C. Detection and Analysis

[0200] In some aspects, provided herein are methods including in situ assays, such as in situ assays involving the use of any of the recombinant polymerases or variant PumA1 polymerases provided herein. In some aspects, in situ assays involve in situ generation of amplified products, such as using any of the recombinant polymerases or variant PumA1 polymerases or compositions provided herein, and in situ detection of amplified products, such as using a microscope or other detection or assay methods involving optical readings. In other aspects, nucleic acid amplification and / or detection is performed in vitro.

[0201] In some aspects, detection or determination of the sequence of a target nucleic acid or its amplified product is performed in situ in cells in intact tissue. In some aspects, the method involves generating a nucleic acid amplification product, such as a rolling circle amplification (RCA) product, in situ using any of the recombinant polymerase, variant PumA1 polymerase, or compositions provided herein, and detecting the amplified product.

[0202] In some embodiments, the method involves imaging the sample to detect a signal associated with a probe (e.g., a circular probe or a circularizable probe) hybridized with a target nucleic acid in the sample. In some embodiments, the probe to be hybridized with the target nucleic acid comprises one or more barcode sequences for detection and / or a component for signal amplification (e.g., RCA). In some embodiments, the signal can be amplified in situ in the sample. In some embodiments, the signal associated with the probe is amplified in the sample (e.g., using any of the recombinant polymerase or variant PumA1 polymerase or compositions provided herein). In some embodiments, an amplification signal associated with the probe and a related RCA product can be generated in the sample (e.g., using a detection probe such as a fluorescently labeled probe).

[0203] In some embodiments, the method involves imaging a biological sample to detect a specific nucleic acid or its amplified product. In some embodiments, the sequence of a rolling circle amplification product or other generated product can be analyzed in situ in a biological sample. In some embodiments of any embodiment, imaging can include detecting a signal associated with a fluorescently labeled probe, which directly or indirectly binds to the rolling circle amplification product of a circularized probe. In some embodiments, the sequence of a rolling circle amplification product, an extension product, or other generated product can be analyzed by sequential hybridization, sequencing while hybridizing, sequencing while connecting, sequencing while synthesizing, sequencing while combining, or a combination thereof. In some embodiments, any suitable method or technique (including those described herein, such as sequencing while synthesizing (SBS), sequencing while connecting (SBL), or sequencing while hybridizing (SBH)) can be used to analyze (e.g., detect or sequence) a barcode or its complementary sequence (e.g., a barcode sequence or its complementary sequence contained in a probe disclosed herein or its product). In some cases, the barcode scheme and / or barcode detection scheme described in RNA sequential detection of targets (RNA SPOT), single molecule fluorescence in situ hybridization (smFISH), multiple error stable fluorescence in situ hybridization (MERFISH) or sequential fluorescence in situ hybridization (seqFISH+) can be used. In any of the aforementioned embodiments, the method provided herein may include analyzing the barcode by sequential hybridization and detection with multiple labeled probes (e.g., detection probes (e.g., detection oligomers) or barcode probes). In some cases, the barcode detection step can be performed as described in hybridization-based in situ sequencing (HybISS). In some cases, the probe can be detected and analyzed (e.g., detected or sequenced), as performed in fluorescence in situ sequencing (FISSEQ), or as performed in the detection step of spatial resolution transcription amplicon readout mapping (STARmap) method. In some cases, the signal associated with the analyte can be detected as performed in sequential fluorescence in situ hybridization (seqFISH).

[0204] In some aspects, in situ hybridization-based assays are used to locate and analyze nucleic acid sequences (e.g., probes comprising one or more specific barcode sequences of interest) in a natural biological sample (e.g., a portion or section of a tissue). In some embodiments, in situ assays are used to analyze the presence, absence, amount, or level of probes that hybridize to a nucleic acid substrate in a biological sample while retaining spatial context.

[0205] In some embodiments, the method involves in situ hybridization using directly or indirectly labeled molecules (e.g., complementary DNA or RNA or modified nucleic acids) as probes that bind or hybridize to an RNA substrate in a biological sample of interest. In some instances, the nucleic acid probe or probe set can be labeled with a radioisotope, epitope, hapten, biotin, or fluorophore to enable detection of the location of a specific nucleic acid probe or probe set in a tissue.

[0206] In some embodiments, the barcode sequence of the circular or circularizable probe (or its product, such as amplification, extension, connection or hybridization product) is targeted by a detectably labeled detection probe (such as a fluorescently labeled oligonucleotide). In some embodiments, one or more decoding schemes are used to decode the signal (such as fluorescence) for sequence determination. In any embodiment herein, any suitable method or technique can be used to analyze (e.g., detect or sequence) barcodes (e.g., primary and / or secondary barcode sequences), including those described herein, such as RNA target sequential detection (RNA SPOT), sequential fluorescence in situ hybridization (seqFISH), single molecule fluorescence in situ hybridization (smFISH), multiple error robust fluorescence in situ hybridization (MERFISH), hybridization-based in situ sequencing (HybISS), in situ sequencing, targeted in situ sequencing, fluorescence in situ sequencing (FISSEQ) or spatial resolution transcript amplicon read mapping (STARmap). In some embodiments, the method provided herein includes analyzing barcodes by sequential hybridization and detection with multiple labeled probes (e.g., detection oligonucleotides). Exemplary decoding schemes are described in Eng et al., "Transcriptome-scale Super-Resolved Imaging in Tissues by RNA SeqFISH+," Nature 568(7751):235-239 (2019); Chen et al., "Spatially resolved, highly multiplexed RNA profiling in single cells," Science; 348(6233):aaa6090 (2015); Gyllborg et al., Nucleic Acids Res (2020) 48(19):e112; US10,457,980B2; US 2016 / 0369329A1; WO 2018 / 026873A1; and US2017 / 0220733A1, all of which are incorporated by reference in their entirety. In some embodiments, these assays enable simultaneous signal amplification, combined decoding, and error correction schemes.

[0207] In some embodiments, the barcode sequence in the probes herein (e.g., circular probes or circularizable probes) or their products can be analyzed by in situ sequencing. In some embodiments, the analysis and / or sequence determination includes sequencing and / or in situ hybridization with all or a portion of the amplified product or probe. In some embodiments, the sequencing step involves sequencing while hybridizing, sequencing while ligating, and / or fluorescent in situ sequencing while synthesizing.

[0208] In some embodiments, in situ hybridization includes sequential fluorescence in situ hybridization. In some embodiments, analysis and / or sequence determination includes detecting the product generated by amplification using any one of the recombinant polymerase or variant PumA1 polymerase or compositions provided herein. In some embodiments, detection or determination includes hybridization of a detection oligonucleotide labeled with a fluorophore, isotope, mass label or a combination thereof to the amplified product. In some embodiments, detection or determination includes imaging the amplified product. In some embodiments, detection or determination is performed when the RNA substrate is in situ in a tissue sample.

[0209] In some respects, in situ determination uses microscopy as readout, such as nucleic acid sequencing, hybridization or other detection or determination methods related to optical readout. In some respects, in situ detection or determination of the sequence of one, two, three, four, five or more nucleotides of target nucleic acid is carried out in the cell of intact tissue. In some respects, detection or determination is relevant to target nucleic acid or indicates the sequence of target nucleic acid. In some respects, detection or determination of sequence is carried out, so as to detect the location of target nucleic acid (or product or derivative thereof relevant to target nucleic acid) in original sample. In some embodiments, the determination includes the presence or absence of detection amplification product or a part thereof (for example, RCA product). In some embodiments, the method provided is quantitative, and retains the spatial information in tissue sample, without physically separating cells or using homogenate.

[0210] In some aspects, these methods also involve imaging one or more portions of an amplification product (e.g., an amplicon) and / or a polynucleotide, for example, by binding of a detectably labeled detection probe (e.g., binding to a circular probe or a circularizable probe or its complementary sequence) and detecting a detectable label. In some embodiments, the detectably labeled probe comprises a detectable label that can be measured and quantified. Labels and detectable labels can include directly or indirectly detectable portions associated (e.g., conjugated) with a molecule to be detected, such as a detectable probe, including but not limited to a fluorophore, a radioisotope, a fluorescent agent, a chemiluminescent agent, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a chromophore, a dye, a metal ion, a metal sol, a ligand (e.g., biotin or a hapten), etc.

[0211] Fluorophores can include substances or a portion thereof that can display fluorescence within a detectable range. Specific examples of labels that can be used according to the provided embodiments include, but are not limited to, phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade Blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acridinium esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, β-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urease.

[0212] Fluorescence detection in tissue samples may be hindered by the presence of strong background fluorescence. Autofluorescence may include background fluorescence (which may be caused by a variety of sources (including aldehyde fixation, extracellular matrix components, erythrocytes, lipofuscin, etc.), which is different from the expected immunofluorescence from fluorescently labeled antibodies or probes. Tissue autofluorescence may cause difficulty in distinguishing the signal caused by fluorescent antibodies or probes from general background. In some embodiments, methods disclosed herein utilize one or more reagents to reduce tissue autofluorescence, such as autofluorescence eliminator (Sigma / EMD Millipore), TrueBlack lipofuscin autofluorescence quencher (Biotium), MaxBlock autofluorescence reduction kit (MaxVisionBiosciences) and / or very strong black dyes (e.g., Sudan black or suitable dark chromophores).

[0213] In some embodiments, a detectable probe containing a detectable label can be used to detect one or more polynucleotides and / or amplification products (e.g., amplicons) described herein (e.g., probes hybridized to an RNA substrate). In some embodiments, the method involves incubating a detectable probe containing a detectable label with a sample, washing unbound detectable probes, and detecting the label, e.g., by imaging.

[0214] Examples of detectable labels include, but are not limited to, various radioactive moieties, enzymes, prosthetic groups, fluorescent labels, luminescent labels, bioluminescent labels, metal particles, protein-protein binding pairs, and protein-antibody binding pairs. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin.

[0215] Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacteria, fireflies, and click beetles), luciferin, aequorin, and the like. Examples of enzyme systems with visually detectable signals include, but are not limited to, beta-galactosidase, glucuronidase, phosphatase, peroxidase, and cholinesterase. Identifiable markers also include radioactive compounds, such as 125I, 35S, 14C, or 3H. Identifiable markers are commercially available from a variety of sources.

[0216] Examples of fluorescent labels and nucleotides and / or polynucleotides conjugated to such fluorescent labels include those described in, for example, Hoagland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, Second Edition (Stockton Press, New York, 1993); Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259 (1991). In some embodiments, exemplary techniques and methods suitable for use with the provided embodiments include, for example, those described in US 4,757,141, US 5,151,507, and US 5,091,519. In some embodiments, one or more fluorescent dyes are used as labels for labeled target sequences, for example, as described in: US 5,188,934 (4,7-dichlorofluorescein dye); US 5,366,860 (spectrally resolvable rhodamine dye); US 5,847,162 (4,7-dichlororhodamine dye); US 4,318,846 (ether-substituted fluorescein dye); US 5,800,996 (energy transfer dye); US 5,066,580 (xanthine dye); and US 5,688,648 (energy transfer dye). Quantum dots can also be used for labeling, as described in the following documents: US 6,322,901, US 6,576,291, US 6,423,551, US 6,251,303, US 6,319,426, US 6,426,513, US 6,444,143, US 5,990,479, US 6,207,392, US 2002 / 0045045 and US 2003 / 0017264. Fluorescent labels can include signal transduction moieties that transmit information through the fluorescence absorption and / or emission characteristics of one or more molecules. Exemplary fluorescence characteristics include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer.

[0217] Examples of commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and / or polynucleotide sequences include, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, Texas Red TM -5-dUTP, CASCADE BLUE TM -7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHOD AMINE GREEN TM -5-dUTP、OREGON GREENR TM 488-5-dUTP, Texas Red TM -l2-dUTP, BODIPY TM 630 / 650-14-dUTP, BODIPY TM 650 / 665-14-dUTP、ALEXA FLUOR TM 488-5-dUTP、ALEXA FLUOR TM 532-5-dUTP、ALEXA FLUOR TM 568-5-dUTP、ALEXA FLUOR TM 594-5-dUTP、ALEXA FLUOR TM 546-14-dUTP, fluorescein-12-UTP, tetramethylrhodamine-6-UTP, Texas Red TM -5-UTP, mCherry, CASCADE BLUE TM -7-UTP, BODIPY TM FL-14-UTP, BODIPY TMR-14-UTP, BODIPY TM TR-14-UTP、RHODAMINE GREEN TM -5-UTP、ALEXA FLUOR TM 488-5-UTP and ALEXA FLUOR TM 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.) Nucleotides with other fluorophores can also be synthesized (see Henegariu et al. (2000) Nature Biotechnol. 18:345).

[0218] Other fluorophores available for post-synthetic attachment include, but are not limited to, ALEXA FLUOR TM 350、ALEXAFLUOR TM 532. ALEXA FLUOR TM 546. ALEXA FLUOR TM 568. ALEXA FLUOR TM 594、ALEXA FLUOR TM 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, Lissamine Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Biology, Inc. Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, NJ). FRET tandem fluorophores can also be used, including but not limited to PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), and APC-Alexa dyes.

[0219] In some cases, metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or polynucleotide sequences (Lakowicz et al. (2003) Bio Techniques 34:62).

[0220] Biotin or its derivatives can also be used as labels on nucleotides and / or polynucleotide sequences, and are subsequently bound by detectably labeled avidin / streptavidin derivatives (e.g., phycoerythrin-conjugated streptavidin) or detectably labeled anti-biotin antibodies. Digoxigenin can be incorporated as a label, and is subsequently bound by detectably labeled anti-digoxigenin antibodies (e.g., fluoresceinized anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into polynucleotide sequences, and subsequently coupled to fluorescent dyes derived from N-hydroxysuccinimide (NHS). In general, any member of the conjugate pair can be incorporated into the detection polynucleotide as long as the detectably labeled conjugation partner can be combined to allow detection. In some embodiments, the antibody comprises any class of antibody molecules or any subfragment thereof, such as Fab.

[0221] Other suitable labels for polynucleotide sequences can include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis) and phosphor-amino acids (e.g., P-tyr, P-ser, P-thr). In some embodiments, the following hapten / antibody pairs are used for detection, wherein each antibody is derivatized with a detectable label: biotin / a-biotin, digoxigenin / a-digoxigenin, dinitrophenol (DNP) / a-DNP, 5-carboxyfluorescein (FAM) / a-FAM.

[0222] In some embodiments, the nucleotide and / or polynucleotide sequence can be indirectly labeled, particularly with a hapten, which is then bound by a capture agent, for example, as disclosed in US 5,344,757, US 5,702,888, US 5,354,657, US 5,198,537 and US 4,849,336 and US 5,192,782. Many different hapten-capture agent pairs are available for use. Exemplary haptens include, but are not limited to, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, Cy5 and digoxigenin. For biotin, the capture agent can be avidin, streptavidin or an antibody. Antibodies can be used as capture agents for other haptens (many dye-antibody pairs are commercially available, for example, Molecular Probes, Eugene, Oreg.).

[0223] In some aspects, detection includes determining a signal, such as a fluorescent signal. In some aspects, detection (including imaging) is performed using any of a number of different types of microscopy, such as confocal microscopy, two-photon microscopy, light field microscopy, intact tissue expansion microscopy, and / or CLARITY TMOptimized light sheet microscopy (COLM).

[0224] In some embodiments, fluorescence microscopy is used to detect and image the detection probe. In some aspects, a fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of or in addition to reflection and absorption to study the properties of organic or inorganic substances. In fluorescence microscopy, the sample is illuminated with light of a wavelength that excites fluorescence in the sample. The fluorescence is then imaged by a microscope objective, and the fluorescence wavelength is usually longer than the illumination wavelength. Two filters can be used in this technology; an illumination (or excitation) filter that ensures that the illumination is close to monochromatic and at the correct wavelength, and a second emission (or barrier) filter that ensures that no excitation light source reaches the detector. Alternatively, these functions can all be achieved by a single dichroic filter. A "fluorescence microscope" can be any microscope that uses fluorescence to produce an image, whether a simpler setup such as an epifluorescence microscope, or a more complex design such as a confocal microscope that uses optical sectioning to obtain better resolution of fluorescent images.

[0225] In some embodiments, confocal microscopy is used for detection and imaging of the detection probe. Confocal microscopy uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signals. Since only light generated by fluorescence very close to the focal plane can be detected, the optical resolution of the image, especially in the sample depth direction, is much better than that of a wide-field microscope. However, since much of the light from the sample fluorescence is blocked at the pinhole, this increase in resolution comes at the expense of reduced signal intensity - so long exposure times are usually required. Since only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning in the specimen with a regular raster (e.g., a rectangular pattern of parallel scan lines). The achievable thickness of the focal plane is primarily limited by the wavelength of the light used divided by the numerical aperture of the objective, but is also limited by the optical properties of the specimen. Thin optical sections make these types of microscopes particularly good at 3D imaging and surface profile analysis of samples. CLARITY TM - Optimized Light Sheet Microscopy (COLM) provides an alternative microscopy technique for rapid 3D imaging of large clarified samples. COLM interrogates large immunostained tissues, allowing for increased acquisition speeds and producing higher quality generated data.

[0226] Other types of microscopy that may be employed include bright field microscopy, oblique illumination microscopy, dark field microscopy, phase contrast microscopy, differential interference contrast (DIC) microscopy, reflection interference microscopy (also known as reflected interference contrast or RIC), single plane illumination microscopy (SPIM), ultra-high resolution microscopy, laser microscopy, electron microscopy (EM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), reflection electron microscopy (REM), scanning transmission electron microscopy (STEM), and low voltage electron microscopy. (LVEM), scanning probe microscopy (SPM), atomic force microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive atomic force microscopy (C-AFM), electrochemical scanning tunneling microscopy (ECSTM), electrostatic force microscopy (EFM), fluid force microscopy (FluidFM), force modulation microscopy (FMM), feature-oriented scanning probe microscopy (FOSPM), Kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM ), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or SNOM, scanning near-field optical microscopy, SNOM, piezoelectric response force microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS, photothermal microspectroscopy / microscopy (PTMS), SCM, scanning capacitance microscopy (SCM), SECM, scanning electrochemical microscopy (SECM), SGM, scanning gate microscopy (SGM), SHPM, scanning Hall probe microscopy ( The invention relates to the present invention to a method for the treatment of tissue damage and tissue structure. The invention relates to a method for the treatment of tissue damage and tissue structure. The invention relates to a method for the treatment of tissue damage and tissue structure. The invention relates to a method for the treatment of tissue damage and tissue structure.

[0227] In some embodiments, the sequence can be isolated in situ, for example, by incorporating labeled nucleotides (e.g., fluorescently labeled mononucleotides or dinucleotides) or hybridizing labeled primers (e.g., labeled random hexamers) to a nucleic acid template in a sequential, template-dependent manner, so that the identity (e.g., nucleotide sequence) of the incorporated nucleotides or labeled primer extension products can be determined, thereby determining the nucleotide sequence of the corresponding template nucleic acid. Aspects of in situ analysis are described in, for example, Mitra et al., (2003) Anal. Biochem. 320, 55-65; and Lee et al., (2014) Science, 343 (6177), 1360-1363; US 2016 / 0024555; US 2019 / 0194709; US 10,138,509; US 10,494,662; US 10,179,932.

[0228] In some cases, sequencing can be performed after the analyte is released from the biological sample. In some embodiments, sequencing can be performed by sequencing by synthesis (SBS). In some embodiments, the sequencing primer is complementary to the sequence at or near one or more barcodes. In such embodiments, sequencing by synthesis can include reverse transcription and / or amplification to generate a template sequence to which the primer sequence can bind. Exemplary SBS methods include, for example, but are not limited to, those described in US 2007 / 0166705, US 2006 / 0188901, US 7,057,026, US 2006 / 0240439, US 2006 / 0281109, US 2011 / 005986, US 2005 / 0100900, US 9,217,178, US 2009 / 0118128, US 2012 / 0270305, US 2013 / 0260372, and US 2013 / 0079232.

[0229] In some embodiments, the method involves contacting a biological sample containing an amplification product with one or more detectably labeled probes that are directly or indirectly hybridized with one or more barcode sequences, detecting a signal associated with one or more detectably labeled probes, and dehybridizing one or more detectably labeled probes to detect one or more barcode sequences in a ring-shaped or circularizable probe or its product. In some embodiments, one or more detectably labeled probes and / or one or more other detectably labeled probes that are directly or indirectly hybridized with one or more barcode sequences are repeated to contact, detect, and dehybridize. In some embodiments, the detectably labeled probe includes a detectable label (e.g., conjugated with a detectable label). In some embodiments, the detectably labeled probe is labeled with a sequence that can hybridize with a detection probe, wherein the detection probe includes a detectable label (e.g., conjugated with a detectable label). The method for detecting and / or analyzing a sequence by sequential hybridization of a probe has been described in, for example, U.S. Patent Publication 20210340618, the contents of which are incorporated herein by reference in their entirety.

[0230] In some embodiments, sequencing can be performed using single molecule sequencing by ligation. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. Oligonucleotides typically have different tags associated with the identity of specific nucleotides in the sequence to which the oligonucleotide hybridizes. Aspects and features involved in sequencing by ligation are described in, for example, Shendure et al., Science (2005), 309: 1728-1732, and US 5,599,675; US 5,750,341; US ​​6,969,488; US 6,172,218; and US 6,306,597.

[0231] In some embodiments, nucleic acid hybridization can be used for sequence detection. These methods utilize labeled nucleic acid decoder probes that are complementary to at least a portion of the barcode sequence. Multiple decoding can be performed with a pool of many different probes with distinguishable labels. Non-limiting examples of nucleic acid hybridization sequencing are described in, for example, US 8,460,865 and Gunderson et al., Genome Research 14: 870-877 (2004).

[0232] In some embodiments, real-time monitoring of DNA polymerase activity can be used during sequencing. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET), as described in, for example, Levene et al., Science (2003), 299, 682-686, Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al., Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181.

[0233] In some aspects, analysis and / or sequence determination can be performed at room temperature to best preserve tissue morphology with low background noise and reduced errors. In some embodiments, analysis and / or sequence determination includes eliminating error accumulation as sequencing proceeds.

[0234] V. Kits, Compositions, and Articles of Manufacture

[0235] Also provided herein are kits, compositions, and articles of manufacture, e.g., comprising a recombinant polymerase described in Section II or a variant PumA1 polymerase described in Section II and one or more additional components. In some embodiments, the kit comprises an isolated and purified recombinant polymerase or variant PumA1 polymerase as described in Section II.

[0236] In some embodiments, provided herein is a composition comprising a recombinant polymerase disclosed herein (e.g., a recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2). In some embodiments, provided herein is a composition comprising a variant PumA1 polymerase disclosed herein (e.g., a variant PumA1 polymerase comprising one or more amino acid substitutions at one or more positions selected from positions 545, 574, and 575 corresponding to the sequence shown in SEQ ID NO: 1). In some embodiments, the variant PumA1 polymerase comprises one or more amino acid substitutions selected from the group consisting of I545E, A574S, and P575V.

[0237] In some embodiments, the pH of the composition is about 8.5. In some cases, the composition comprises about 5 mM to about 10 mM MgCl 2In some cases, the composition comprises about 0.1 mM to about 0.2 mM dNTPs. In some cases, the composition comprises about 1 mM to about 4 mM DTT. In some cases, the composition comprises 1 mg / mL to 3 mg / mL BSA (e.g., about 2 mg / mL BSA). In some cases, the composition comprises 5 mM to 15 mM NH 4 SO 4 (e.g., about 10 mM NH 4 SO 4 ).

[0238] In some embodiments, the composition comprises a recombinant polymerase or variant PumA1 polymerase of about 0.05 μM to about 1 μM. In some cases, the composition comprises a recombinant polymerase or variant PumA1 polymerase of about 0.5 μM to about 1 μM, about 0.2 μM to about 1 μM, about 0.8 μM to about 1 μM or about 0.05 μM to about 0.5 μM. In some cases, the composition comprises at least 1 μM, 10 μM, 50 μM or 100 μM of a recombinant polymerase or variant PumA1 polymerase. In some embodiments, the composition comprises 10 to 50, 10 to 100, 50 to 100, 25 to 50 or 25 to 100 μM of a recombinant polymerase or variant PumA1 polymerase. In any embodiment herein, the recombinant polymerase or variant PumA1 polymerase can be at least 85%, at least 90% or at least 95% soluble in the composition. In any of the embodiments herein, the recombinant polymerase or variant PumAl polymerase may be at least 85%, at least 90%, or at least 95% soluble in the composition at 20°C, 30°C, 37°C, 40°C, or 42°C.

[0239] In some embodiments, the composition has a pH of about 8.5 and comprises 10 mM MgCl 2 , 10 mM NH 4 SO 4 , 4 mM DTT and 0.2 mg / mL BSA. In some embodiments, the composition further comprises 0.2 mM dNTPs.

[0240] In some embodiments, the composition has a pH of about 8.5 and comprises 10 mM MgCl 2 , 10 mM NH 4 SO 4 , 1 mM DTT and 0.2 mg / mL BSA. In some embodiments, the composition further comprises 0.1 mM dNTPs.

[0241] In some embodiments, the composition has a pH of about 8.5 and comprises 5 mM MgCl 2, 10 mM NH 4 SO 4 , 1 mM DTT and 0.2 mg / mL BSA. In some embodiments, the composition further comprises 0.2 mM dNTPs.

[0242] In some embodiments, the kit comprises one or more additional reagents for performing the methods provided herein, such as reagents required for one or more steps of amplification, detection, sequencing and / or sample preparation described herein. In some embodiments, the kit includes a recombinant polymerase or variant PumA1 polymerase, for example, any polymerase described in Part II herein, and / or a polynucleotide, a recombinant nucleic acid molecule or a vector, for example, as described in Part III. In some cases, the kit includes any composition disclosed herein.

[0243] In some embodiments, the kit includes a buffer or a buffer stock solution (e.g., a solution that can be diluted to form a working buffer. In some embodiments, the kit includes a buffer stock solution that can be diluted 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold to produce a buffer having a pH of about 8.5 and containing 10 mM MgCl 2 , 10 mM NH 4 SO 4 , 4 mM DTT and 0.2 mg / mL BSA working buffer. In some embodiments, the kit includes a buffer stock solution that can be diluted 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold to produce a pH of about 8.5 and containing 10 mM MgCl 2 , 10 mM NH 4 SO 4 , 1 mM DTT and 0.2 mg / mL BSA working buffer. In some embodiments, the kit includes a buffer stock solution that can be diluted 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold to produce a pH of about 8.5 and containing 5 mM MgCl 2 , 10 mM NH 4 SO 4 In some embodiments, the composition further comprises 0.2 mM dNTPs.

[0244] In some embodiments, the kit further comprises one or more components, such as dNTPs, divalent-cations, and a reaction buffer. In some embodiments, the composition comprises MgCl 2 , dNTP, DTT, BSA, NH 4In some embodiments, the kit further comprises primers, dNTPs, divalent cations, and reaction buffer. In some embodiments, the kit further comprises instructions for use, e.g., instructions for amplifying nucleic acids, e.g., instructions for amplifying using any of the recombinant polymerase or variant PumA1 polymerase or compositions provided herein.

[0245] In some embodiments, the kit further comprises a detection probe (e.g., a plurality of detectably labeled probes) for detecting a nucleic acid (e.g., DNA or RNA) as described in Section IV. In some embodiments, the probe is a circular probe or a circularizable probe (e.g., a padlock probe) for detecting a target nucleic acid (e.g., DNA or RNA).

[0246] The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined into a single container. In some embodiments, the kit also contains instructions for using the kit components to perform the provided methods.

[0247] In some embodiments, the test kit may contain reagents and / or consumables required for one or more steps of the method provided. In some embodiments, the test kit contains reagents for fixing, embedding and / or permeabilizing biological samples. In some embodiments, the test kit contains reagents, such as enzymes and buffers for connection and / or amplification, such as ligases and / or polymerases. In some respects, the test kit may also include any reagent as described herein, such as wash buffer and connection buffer. In some embodiments, the test kit contains reagents for detection, such as barcode detection probes or detectable labels. In some embodiments, the test kit optionally contains other components, such as nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, reagents for other determinations.

[0248] In some respects, the embodiments provided can be applied to the in situ methods for analyzing nucleic acid sequences, such as in situ transcriptome analysis or in situ sequencing, such as nucleic acid sequences from intact tissues or samples in which spatial information has been retained. In some respects, the embodiments can be applied to imaging or detection methods for multiple nucleic acid analysis. In some respects, the embodiments provided can be used to identify or detect regions of interest in target nucleic acids.

[0249] In some respects, the embodiments can be applied to research and / or diagnostic applications, for example, to characterize or evaluate specific cells or tissues from a subject. The application of the provided method can include biomedical research and clinical diagnosis. For example, in biomedical research, applications include but are not limited to spatially resolved gene expression analysis for biological research or drug screening. In clinical diagnosis, applications include but are not limited to detecting gene markers such as disease, immune response, bacteria or viral DNA / RNA of patient samples.

[0250] VI. Terminology

[0251] Specific terminology is used throughout this disclosure to explain various aspects of the described devices, systems, methods, and compositions.

[0252] Some illustrative embodiments of the present disclosure have been described, but it will be apparent to those skilled in the art that the foregoing is illustrative and non-restrictive and has been presented only by way of example. Many modifications and other illustrative embodiments are within the scope of those of ordinary skill in the art and are considered to fall within the scope of the present disclosure. Specifically, although many of the examples presented herein relate to specific combinations of method actions or system elements, it will be understood that these actions and these elements can be combined in some ways to achieve the same goal.

[0253] It should be understood that the various aspects and embodiments of the present invention described herein, in addition to embodiments that "include" certain aspects and features, may also include "consisting of" and / or "consisting essentially of" various aspects and embodiments. As used herein, unless otherwise indicated, the singular forms "a", "an", and "the" include plural referents. As used herein, unless the context clearly dictates otherwise, the singular forms "a / an" and "the" include plural referents. For example, "a" or "a" means "at least one" or "one or more".

[0254] As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments involving that value or parameter per se.

[0255] Throughout this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that the description of the range format is only for convenience and simplicity, and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, in the case of providing a range of values, it should be understood that each intermediate value between the upper and lower limits of the range and any other stated or intermediate values ​​in the stated range are all included in the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the claimed subject matter, but are subject to any specific exclusion limits in the stated range. In the case where the stated range includes one or two limits in these limits, the range excluding any one or two limits in those included limits is also included in the claimed subject matter. Regardless of the breadth of the range, this applies.

[0256] The use of ordinal terms (such as "first", "second", "third", etc.) in the claims to modify claim elements does not by itself imply any priority, precedence, or order of one claim element over another sequential or chronological order of the acts of performing the method, but is merely used as a marker to distinguish one claim element with a particular name from another element with the same name (but using an ordinal term) to distinguish these claim elements. Similarly, the use of a), b), etc. or i), ii), etc. in the claims does not by itself imply any priority, precedence, or order of steps. Similarly, the use of these terms in the specification does not by itself imply any required priority, precedence, or order.

[0257] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, such as an alpha carbon bound to a hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid.

[0258] In some aspects, conservative amino acid substitutions can often be made in proteins without changing the conformation or function of the protein. The protein of the present invention can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include replacing any of these hydrophobic amino acids with any one of isoleucine (I), valine (V) and leucine (L); replacing glutamic acid (E) with aspartic acid (D), and vice versa; replacing asparagine (N) with glutamine (Q), and vice versa; and replacing threonine (T) with serine (S), and vice versa. Depending on the environment of the specific amino acid and its role in the three-dimensional structure of the protein, other substitutions can also be considered conservative. For example, glycine (G) and alanine (A) can often be interchanged, and alanine (A) and valine (V) can also be interchanged. Relatively hydrophobic methionine (M) can often be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable at positions where the distinguishing feature of the amino acid residue is its charge and the different pKs of the two amino acid residues are not significant. Other changes may be considered "conservative" in certain circumstances (see, e.g., US8562989, pp. 13-15 "Biochemistry" 2nd edition, edited by Lubert Stryer (Stanford University); Henikoff et al., PNAS 1992 Vol. 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270 (20): 11882-6, the contents of each reference are incorporated herein by reference in their entirety).

[0259] Amino acid substitutions may include replacing one amino acid in a polypeptide with another amino acid.As described herein, amino acid substitutions may be introduced to generate variant PumA1 polymerases.

[0260] Amino acids can generally be grouped according to the following common side chain properties:

[0261] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0262] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0263] (3) Acidic: Asp, Glu;

[0264] (4) Basic: His, Lys, Arg;

[0265] (5) Residues that affect chain orientation: Gly, Pro;

[0266] (6) Aromatic: Trp, Tyr, Phe.

[0267] In some cases, conservative substitutions may involve replacing a member of one of these classes with another member of the same class. In some cases, non-conservative amino acid substitutions may involve replacing a member of one of these classes with another class. In some cases, a particular substitution may be considered "conservative" or "non-conservative" depending on the stringency and context and environment of the particular residue in the primary, secondary, and / or tertiary structure of the protein.

[0268] As used herein, the term "peptide" refers to a plurality of amino acids joined together, such as in the form of a linear or cyclic chain. The term oligopeptide is generally used to describe peptides having 2 to about 50 or more amino acids. Peptides greater than about 50 are generally referred to as polypeptides or proteins.

[0269] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the antibodies and antibody chains provided and other peptides, such as linkers, can include amino acid residues, including natural and / or non-natural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, the polypeptide may contain modifications relative to the native or native sequence, as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein or due to errors in PCR amplification.

[0270] As used herein, "percentage (%) of amino acid sequence identity" and "percentage of identity" are defined as the percentage of amino acid residues in a candidate sequence (e.g., a test antibody or fragment) that are identical to the amino acid residues in the reference polypeptide sequence when used relative to an amino acid sequence (reference polypeptide sequence), after aligning the sequences and introducing gaps (if necessary) to achieve maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0271] An "isolated" protein, polypeptide, or nucleic acid refers to a protein, polypeptide, or nucleic acid molecule that has been separated from a component of its natural environment. An isolated protein or polypeptide may refer to a purified protein or polypeptide. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An isolated protein or polypeptide (e.g., an isolated polymerase) includes a polymerase present in a composition comprising other molecules (including other proteins and / or nucleic acids), wherein the isolated protein or polypeptide has been separated from a component of its natural environment.

[0272] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0273] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of these cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, without regard to the number of passages. The nucleic acid content of progeny cells may not be exactly the same as that of the parental cells, but may contain mutations. Included herein are mutant progeny with the same function or biological activity that have been screened or selected in the original transformed cells. In some aspects, a "recombinant expression system" comprises a host cell.

[0274] The terms "nucleic acid" and "nucleotide" are intended to comply with their uses in the art and include naturally occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids can hybridize with nucleic acids in a sequence-specific manner (e.g., can hybridize with two nucleic acids so that connection can occur between the two hybridized nucleic acids) or can be used as a replication template for a specific nucleotide sequence. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. Analog structures can have alternative backbone bonds, including any of a variety of backbone bonds known in the art. Naturally occurring nucleic acids generally have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

[0275] Nucleic acids can contain nucleotides of any of the various analogs of these sugar moieties known in the art. Nucleic acids can contain natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acids can have one or more bases selected from adenine (A), thymine (T), cytosine (C) or guanine (G), and ribonucleic acids can have one or more bases selected from uracil (U), adenine (A), cytosine (C) or guanine (G). Useful non-natural bases that can be included in nucleic acids or nucleotides are known in the art.

[0276] "PCR amplification" refers to the use of polymerase chain reaction (PCR) to generate copies of genetic material, including DNA and RNA sequences. Suitable reagents and conditions for implementing PCR are described in, for example, U.S. Patents 4,683,202, 4,683,195, 4,800,159, 4,965,188 and 5,512,462, the entire contents of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture includes genetic material to be amplified, an enzyme, one or more primers for primer extension reactions and reagents for the reaction. Oligonucleotide primers have sufficient length to provide hybridization with complementary genetic material under annealing conditions. The length of the primer is usually dependent on the length of the amplification domain, but will usually be at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 base pairs (bp), at least 11bp, at least 12bp, at least 13bp, at least 14bp, at least 15bp, at least 16bp, at least 17bp, at least 18bp, at least 19bp, at least 20bp, at least 25bp, at least 30bp, at least 35bp, and can be up to 40bp or longer, wherein the length of the primer will usually be in the range of 18 to 50bp. The genetic material can be contacted with a single primer or a set of two primers (forward primer and reverse primer), depending on whether primer extension, linear or exponential amplification of the genetic material is desired.

[0277] The term "polymerase" includes not only naturally occurring enzymes, but also all modified derivatives thereof, and also derivatives of naturally occurring polymerases. For example, in some embodiments, the polymerase may have been modified to remove 5'-3' exonuclease activity. Sequence-modified derivatives or mutants of polymerases that can be used include, but are not limited to, mutants that retain at least some functions (e.g., polymerase activity) of the wild-type sequence. Mutations can affect the activity distribution of the enzyme under different reaction conditions (e.g., temperature, template concentration, primer concentration, etc.), such as increasing or decreasing the polymerization rate. Mutations or sequence modifications may also affect exonuclease activity and / or the thermal stability of the enzyme.

[0278] In some embodiments, PCR amplification can include reactions such as, but not limited to, strand displacement amplification reactions, rolling circle amplification reactions, ligase chain reactions, transcription-mediated amplification reactions, isothermal amplification reactions, and / or loop-mediated amplification reactions.

[0279] In some embodiments, PCR amplification uses a single primer that is complementary to the 3' tag of the target DNA fragment. In some embodiments, PCR amplification uses a first primer and a second primer, wherein at least the 3' terminal portion of the first primer is complementary to at least a portion of the 3' tag of the target nucleic acid fragment, and wherein at least the 3' terminal portion of the second primer displays the sequence of at least a portion of the 5' tag of the target nucleic acid fragment. In some embodiments, the 5' terminal portion of the first primer is not complementary to the 3' tag of the target nucleic acid fragment, and the 5' terminal portion of the second primer does not display the sequence of at least a portion of the 5' tag of the target nucleic acid fragment. In some embodiments, the first primer comprises a first universal sequence and / or the second primer comprises a second universal sequence.

[0280] In some embodiments, DNA ligase can be used to connect the PCR amplification product to the additional sequence. DNA ligase activity can be provided by one or more different DNA ligases. In some embodiments, the DNA ligase is from bacteria, for example, the DNA ligase is a bacterial DNA ligase. In some embodiments, the DNA ligase is from a virus (e.g., a bacteriophage). For example, the DNA ligase can be T4 DNA ligase. Other enzymes suitable for the ligation step include, but are not limited to, TthDNA ligase, Taq DNA ligase, Thermococcus (strain 9oN) DNA ligase (9oNTM DNA ligase, available from NewEngland Biolabs, Ipswich, MA) and Ampligase TM (Available from Epicentre Biotechnologies, Madison, WI.) Derivatives (eg, sequence-modified derivatives) and / or mutants thereof may also be used.

[0281] In some embodiments, the genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR). The required reverse transcriptase activity can be provided by one or more different reverse transcriptases, suitable examples of which include, but are not limited to, M-MLV, MuLV, AMV, HIV, ArrayScript TM 、MultiScribe TM 、ThermoScript TM and I, II, III and IV enzymes. "Reverse transcriptase" includes not only the naturally occurring enzyme, but also all such modified derivatives thereof, and also includes derivatives of the naturally occurring reverse transcriptase.

[0282] In addition, reverse transcription can be performed using sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases, including mutants that retain at least some functional activity (e.g., reverse transcriptase activity) of the wild-type sequence. The reverse transcriptase can be provided as part of a composition that includes other components, such as a stabilizing component that enhances or improves reverse transcriptase activity, such as an RNase inhibitor, a DNA-dependent DNA synthesis inhibitor, such as actinomycin D. Many sequence-modified derivatives or mutants of reverse transcriptases (e.g., M-MLV), as well as compositions comprising unmodified and modified enzymes, are commercially available, such as ArrayScript TM 、MultiScribe TM 、ThermoScript TM ,as well as I, II, III and IV enzymes.

[0283] Certain reverse transcriptases (e.g., avian myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase) can use both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as templates to synthesize complementary DNA strands. Therefore, in some embodiments, the reverse transcription reaction can use an enzyme (reverse transcriptase) that can use both RNA and ssDNA as templates for extension reactions, such as AMV or MMLV reverse transcriptase.

[0284] In some embodiments, techniques well known in the art are used, such as but not limited to "TAQMAN TM "or or in a capillary (“ In some embodiments, the quantification of the genetic material is determined by optical absorbance and real-time PCR. In some embodiments, the quantification of the genetic material is determined by digital PCR. In some embodiments, the analyzed gene can be compared to reference nucleic acid extracts (DNA and RNA) corresponding to expression (mRNA) and quantity (DNA) in order to compare the expression level of the target nucleic acid.

[0285] The terms "detectable label", "optical label" and "label" are used interchangeably herein and refer to a directly or indirectly detectable portion associated with (e.g., conjugated to) a molecule to be detected, such as a probe or analyte for in situ determination. The detectable label can be directly detectable per se (e.g., radioisotope labeling or fluorescent labeling), or in the case of an enzymatic label, can be indirectly detectable, for example, by catalyzing a chemical change of a substrate compound or composition, which is directly detectable. Detectable labels can be suitable for small-scale detection and / or for high-throughput screening. Therefore, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds and dyes.

[0286] Detectable labels can be detected qualitatively (e.g., optically or spectrally), or can be quantitative. Qualitative detection generally includes detection methods in which the presence or occurrence of detectable labels is confirmed, while quantifiable detection generally includes detection methods with quantifiable (e.g., numerically reportable) values ​​such as intensity, duration, polarization and / or other properties. In some embodiments, detectable labels are combined with a feature or a probe associated with a feature. For example, a detectably labeled feature can include a fluorescent, colorimetric or chemiluminescent label attached to a bead (see, e.g., Rajeswari et al., J. Microbiol Methods 139: 22-28, 2017 and Forcucci et al., J. Biomed Opt. 10: 105010, 2015, the entire contents of each of these documents are incorporated herein by reference).

[0287] In some embodiments, multiple detectable labels can be combined with the feature, probe, or composition to be detected. For example, detectable labels (e.g., Labeled nucleotides, such as Any suitable detectable label may be used. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore may be from the group consisting of: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), Alexa Fluor 350. Alexa 430. Alexa 488. Alexa 532. Alexa 546. Alexa 555. Alexa 568. Alexa 594. Alexa 633. Alexa 647、Alexa 660、Alexa 680、Alexa 700、Alexa 750, allophycocyanin (APC), AMCA / AMCA-X, 7-aminoactinomycin D (7-AAD), 7-amino-4-methylcoumarin, 6-aminoquinoline, aniline blue, ANS, APC-Cy7, ATTO-TAG TM CBQCA, ATTO-TAG TM FQ, Auramine O-Folgen, BCECF (high pH), BFP (blue fluorescent protein), BFP / GFP FRET, BOBO TM -1 / BO-PRO TM -1.BOBO TM -3 / BO-PRO TM -3. FL, TMR, TR-X, 530 / 550, 558 / 568, 564 / 570, 581 / 591, 630 / 650-X, 650-665-X, BTC, Calcium Crimson, Calcium Crimson Blue TM 、CalciumGreen-1 TM Calcium Orange TM , White, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthylfluorescein, 6-carboxyrhodamine 6G, 5-carboxytetramethylrhodamine (5-TAMRA), carboxy-X-rhodamine (5-ROX), Cascade Cascade Yellow TM 、CCF2(GeneBLAzer TM ), CFP (cyan fluorescent protein), CFP / YFP FRET, Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC formazan, Cychrome (PE-Cy5), Dansylamide, Dansylcadaverine, Dansyl chloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18 (5)), DIDS, Dil (DilC18 (3)), DiO (DiOC18 (3)), DiR (DilC18 (7)), Di-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, -97 alcohol, eosin, erythrosine, ethidium bromide, ethidium homodimer-1 (EthD-1), europium (III) chloride, 5-FAM (5-carboxyfluorescein), fast blue, fluorescein-dT phosphoramidite, FITC, Fluo-3, Fluo-4, Fluoro-Gold TM (High pH), Fluoro-Gold TM (Low pH), Fluoro-Jade, 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red TM (High calcium), Fura Red TM / Fluo-3、GeneBLAzer TM (CCF2), red-shifted GFP (rsGFP), wild-type GFP, GFP / BFPFRET, GFP / DsRed FRET, Hoechst 33342 and 33258, 7-hydroxy-4-methylcoumarin (pH 9), 1,5 IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), indodicarbocyanine, indotricarbocyanine, JC-1, 6-JOE, JOJO TM -1 / JO-PRO TM -1, LDS 751(+DNA), LDS 751(+RNA), LOLO TM -1 / LO-PRO TM -1. Fluorescent yellow, LysoSensor TM Blue(pH 5), LysoSensor TM Green(pH5), LysoSensor TM Yellow / Blue(pH 4.2), Green, Red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium GreenTM Marina 4-Methylumbelliferone, mithramycin, Green, Orange, Red, NBD (amine), Nile Red, Oregon 488. Oregon 500. Oregon 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinyl Chlorophyll Protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (propidium iodide), PKH26, PKH67, POPO TM -1 / PO-PRO TM -1. P0PO TM -3 / P0-PRO TM -3, propidium iodide (PI), PyMP0, Pyrene, Pyronine Y, Quantam Red (PE-Cy5), nitrogen mustard quinacrine, R670 (PE-Cy5), Red 613 (PE-Texas Red), red fluorescent protein (DsRed), resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine green TM , Rhodamine Red TM , rhodamine-labeled phalloidin, rhodamine 110, rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, (High pH), (High pH), (Low pH), Sodium Green TM , #1, #2, 11. 13. 17. 45. Blue, Green, Orange, 5-TAMRA (5-carboxytetramethylrhodamine), tetramethylrhodamine (TRITC), (NHS ester), thiodicarbocyanine, thiazole orange, TO- Tri-color (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein), 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE665, TYE 705, 5' 700, 5' 800, 5' 800CW (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye, 640 (NHS ester) and Dy 750 (NHS ester).

[0288] As mentioned above, in some embodiments, the detectable label is a luminescent or chemiluminescent portion or includes a luminescent or chemiluminescent portion. Common luminescent / chemiluminescent portions include, but are not limited to, peroxidases, such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. Given an appropriate substrate (e.g., an oxidizing agent plus a chemiluminescent compound), these protein moieties can catalyze a chemiluminescent reaction. Many families of compounds are known to provide chemiluminescence under various conditions. Non-limiting examples of chemiluminescent compound families include 5-amino-6,7,8-trimethoxy-2,3-dihydro-1,4-phthalazinedione luminol and dimethylamino [ca] benzo analogs. These compounds can emit light in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of chemiluminescent compound families include, for example, 2,4,5-triphenylimidazole, p-dimethylamino and -methoxy substituents, oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinium esters, luciferin, lucigenin, or acridinium esters. In some embodiments, the detectable label is or includes a metal-based or mass-based label. For example, a small cluster of metal ions, metals, or semiconductors can serve as mass codes. In some instances, the metal can be selected from Groups 3-15 of the periodic table, such as Y, La, Ag, Au, Pt, Ni, Pd, Rh, Ir, Co, Cu, Bi, or a combination thereof.

[0289] VII. Examples

[0290] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0291] Example 1: Protein solubility of recombinant PumA1 polypeptide

[0292] This example demonstrates the protein solubility of two different recombinant PumA1 constructs: (1) a recombinant PumA1 polypeptide with an amino-terminal polyhistidine / SUMO tag (N-His / Sumo), and (2) a recombinant PumA1 polypeptide with a carboxyl-terminal polyhistidine / SET tag (C-His / SET).

[0293] Escherichia coli was transformed with a plasmid vector encoding N-His / SUMO or C-His / SET recombinant PumA1, and the expression of the recombinant polymerase was induced. The sequences of the N-His / SUMO and C-His / SET recombinant PumA1 constructs are shown in Table E1 below.

[0294] Table E1. DNA and amino acid sequences of N-His / SUMO and C-His / SET

[0295]

[0296]

[0297]

[0298]

[0299] The culture was centrifuged and the cell pellet was resuspended in lysis buffer. The lysate was centrifuged at high speed to obtain the soluble and insoluble fractions. Figure 1 Samples were run on SDS-PAGE for protein solubility analysis as indicated. Figure 1 As shown, N-His / SUMO recombinant PumA1 produced a doublet with the first band being the expected size of approximately 80 kDa and the smaller band being approximately 75 kDa. C-His / SET recombinant PumA1 produced a single band of the expected size of approximately 70 kDa. Figure 1 As shown, N-His / SUMO and C-His / SET recombinant PumA1 were at least about 90% soluble. In addition, the majority of both recombinant PumA1 polypeptides were retained in the PEI supernatant (lane 5 on both blots) but not in the PEI pellet, indicating that most of PumA1 is stable.

[0300] Example 2: Heparin purification of recombinant PumA1

[0301] This example demonstrates the purification of N-His / SUMO or C-His / SET recombinant PumA1.

[0302] N-His / SUMO or C-His / SET recombinant PumA1 polypeptides were expressed as described in Example 1. Soluble fractions were collected and run on a heparin column. Fractions were collected for preloading (after cleavage of the N-His / SUMO vector), flow-through, washing, and obtaining six elution fractions, as Figure 2 As shown. Each elution fraction was collected as a 1 mL aliquot using a gradient of 80%-100% 1 M NaCl. Figure 2 As shown, the eluted fraction of the cleaved N-His / SUMO recombinant PumA1 produced a doublet band of approximately 68 kDa, indicating that the doublet band was retained by heparin purification. The eluted fraction of the C-His / SET recombinant PumA1 produced a single band of approximately 70 kDa.

[0303] Minor impurities of lower molecular weight are present in low concentrations and can be removed by an additional nickel chromatography step or by using a 50 kDa MWCO concentrator.

[0304] Example 3: Characterization of recombinant PumA1 polymerase activity in an in vitro rolling circle amplification assay

[0305] This example demonstrates that in an in vitro rolling circle amplification (RCA) assay, recombinant PumA1 polypeptide has a slower amplification rate but produces more rolling circle products (RCPs) compared to a Phi29 polymerase control. An RCA reaction mixture containing polymerase, polymerase reaction buffer, dNTPs, BSA, and SYBR Gold was prepared. The reaction was performed under Phi29 optimized conditions (NEBPhi29 buffer containing 50 mM Tris-Cl (pH 7.5), 10 mM MgCl 2 , 10 mM NH 4 SO 4 , 4 mM DTT, 0.2 mM dNTP, 0.2 mg / mL BSA; the reaction was carried out at 37°C).

[0306] After the start of RCA, the incorporation of SYBR Gold was monitored in real time for 5 hours, followed by polymerase inactivation. Different concentrations of Phi29 or recombinant PumA1 (C-His / SET PumA1) were used from 0.5U / μL to 5U / μL (the same unit definition was used for the polymerase). Figure 3As shown, the exponential phase of amplification of all concentrations of recombinant PumA1 polypeptide was later than that of Phi29, indicating that recombinant PumA1 had a slower RCA rate. However, the fluorescence intensity of all concentrations of recombinant PumA1 stabilized at a higher fluorescence intensity reading than that of Phi29, indicating that recombinant PumA1 produced more RCP.

[0307] Example 4: Characterization of recombinant PumA1 activity in tissue samples

[0308] Next, exemplary recombinant PumA1 polypeptides were tested in an in situ rolling circle amplification assay in tissue samples. This example demonstrates that in an RCA assay performed in tissue samples, recombinant PumA1 polymerase produces a greater number of detectable RCPs than Phi29, and that the detectable spots of RCPs are smaller and slightly darker than those produced by Phi29.

[0309] Tissue samples were obtained and processed for frozen sections on slides. Tissues were fixed and permeabilized by incubation in 3.7% paraformaldehyde (PFA). Washing buffer was added to tissue sections in preparation for probe hybridization. The circularizable probe hybridized with the target nucleic acid in the tissue sample and ligated to form a circular template for RCA. Rolling circle amplification was then performed using primers and one of two different recombinant PumA1 polypeptides (recombinant PumA1 without a heterologous tag (referred to as "PumA1") and C-His / SET PumA1) or Phi29. The samples were contacted with the respective polymerases and incubated under conditions optimized for Phi29, as described in Example 2 above. A fluorescently labeled probe capable of hybridizing to a common sequence in RCP was added to the sample, and the sample was washed to remove unbound probe. The sample was then imaged to determine the size, intensity, and number of detectable spots (corresponding to detectable RCPs).

[0310] like Figure 4 As shown in the left panel, the detected target density is higher for PumA1 and C-His / SET PumA1 compared to Phi29, indicating that recombinant PumA1 provides higher sensitivity in RCA assays performed in situ in tissue samples. The average detected target signal intensity is lower for both PumA1 and C-his / SET PumA1, indicating that the use of recombinant PumA1 polypeptides produces more ambiguous RCPs compared to Phi29-dependent RCA products ( Figure 4 Left). Figure 4 As shown in the right panel, the recombinant PumA1 polypeptide also produces smaller RCPs compared to Phi29. In some aspects, smaller RCP size may be advantageous for RCA-based assays (eg, reducing optical crowding and facilitating resolution of individual RCP spots).

[0311] Example 5: Improved buffer for PumA1 polymerase nucleic acid amplification

[0312] This example demonstrates the development of an RCA buffer with improved characteristics for nucleic acid amplification using recombinant PumA1 polymerase polypeptide. NEB's Phi29 buffer (50 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM NH 4 SO 4 , 4 mM DTT, 0.2 mM dNTPs, 0.2 mg / mL BSA) was used as a starting point for developing improved buffers. In addition to the original recipe of NEB Phi29 buffer, the buffers tested also included PumA1 enzyme and KCl.

[0313] According to NEB's Phi29 buffer, by changing a single variable (dNTP concentration, MgCl 2 concentration, enzyme concentration, KCl concentration, DTT concentration, and NH 4 SO 4 concentration) while keeping other variables constant to test different buffer conditions.

[0314] Each buffer was tested in an in situ RCA assay in 3 wells of a 96-well plate inoculated with NIH 3T3 cells. Before RCA, the circularizable probe hybridized with the target nucleic acid in the cell and ligated to form a circular template for RCA. RCA was then performed using PumA1 polymerase under each condition shown in Table E2. Three different fluorescently labeled probes hybridized with the common sequence in the RCP, and the RCP density detected under each experimental condition (reflecting the sensitivity of the RCA-based assay) and the signal intensity of the dye detected in three different channels (488nm, 532nm and 647nm) were measured. Figure 5A The sensitivity (detected RCP density) of the RCA assay in different buffers is shown. Figure 5B The signal intensity of RCP detected under each condition. Figure 5A-Figure 5B Based on the results shown, three modified buffers were selected for further characterization (Table E2).

[0315] Table E2. Modified buffers for PumA1 RCA

[0316] Buffer 1 Buffer 2 Buffer 3 50 mM Tris-Cl, pH 8.5 50 mM Tris-Cl, pH 8.5 50 mM Tris-Cl, pH 8.5 10mM MgCl2 10mM MgCl2 5mM MgCl2 10mM NH4S04 10mM NH4S04 10mM NH4S04 4mM DTT 1mM DTT 1mM DTT 0.2mM dNTPs 0.1mM dNTPs 0.2mM dNTPs 0.2mg / mL BSA 0.2mg / mL BSA 0.2mg / mL BSA

[0317] Example 6: In situ detection of nucleic acids using a modified buffer of PumA1

[0318] This example demonstrates that three improved buffers (buffer 1, buffer 2, and buffer 3) of the recombinant PumA1 polymerase polypeptide described in Example 5 can be used in an RCA assay to detect nucleic acids comparable to or better than an RCA assay performed with Phi29 and its standard buffer (Phi29_SOP). An in situ RCA assay was performed on fresh frozen mouse brain tissue to compare nucleic acid detection by PumA1 and the optimized buffers compared to a Phi29 control. Recombinant PumA1 polymerase in buffer 2 and buffer 3 produced a higher percentage of targets decoded with a quality score of >= 20 and assigned to cells, while buffer 1 was half the percentage of the Phi29 control, as shown in Table E3 below. While the recombinant PumA1 polymerase in buffer 1 produced a similar number of median transcripts per non-null cell as the Phi29 control, the recombinant PumA1 polymerase in buffer 2 or buffer 3 produced more median transcripts. In addition, all three optimized buffers resulted in a higher total number of targets detected compared to the control.

[0319] Table E3. Characterization of PumA1 RCA activity in tissue samples using optimized buffers

[0320]

[0321] Example 7: Variant PumA1 I545E / A574S / P575V polymerase shows higher thermostability, density and signal intensity

[0322] This example demonstrates that PumA1 polymerase comprising amino acid substitutions I545E, A574S, P575V (abbreviated as PumA1-ESV, where numbering is based on SEQ ID NO: 1) shows higher thermostability in an in vitro RCA assay and produces denser and brighter RCPs in an RCA assay performed in tissue samples compared to wild-type PumA1 polymerase.

[0323] In the in vitro RCA assay, PumA1-ESV showed better signal intensity at 37°C during the initial stage of amplification and was generally similar to the wild-type PumA1 (PumA1-WT) control, whereas at 42°C, PumA1-ESV signal intensity was similar to the PumA1-WT control during the exponential amplification step, but significantly improved once amplification plateaued (Figure 5—figure supplement 1A). Figure 6 ). These results demonstrate that PumA1-ESV has better thermal stability than PumA1-WT at 42°C.

[0324] In the RCA assay performed in tissue sections, at 37°C, PumA1-ESV had slightly higher signal intensity as shown in the box plots and produced increased RCP density. At 42°C, PumA1-ESV had significantly higher signal intensity and an equal number of RCPs compared to PumA1-WT ( Figure 7 ).

[0325] The scope of the present disclosure is not intended to be limited to the specific embodiments disclosed, which are provided, for example, to illustrate the various aspects of the present disclosure. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such changes may be practiced without departing from the true scope and essence of the present disclosure, and such changes are intended to fall within the scope of the present disclosure.

[0326] sequence

[0327]

[0328]

[0329]

[0330]

Claims

1. An isolated recombinant polymerase comprising an amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The isolated recombinant polymerase of claim 1, comprising one or more amino acid substitutions at one or more positions selected from positions 545, 574 and 575 corresponding to the positions in the sequence shown in SEQ ID NO:

1.

3. The isolated recombinant polymerase of claim 1, comprising one or more amino acid substitutions selected from the group consisting of I545E, A574S and P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO:

1.

4. The isolated recombinant polymerase of claim 1, comprising amino acid substitutions I545E, A574S and P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO:

1.

5. The isolated recombinant polymerase of any one of claims 1 to 4, comprising the sequence of amino acid residues 2-30 of SEQ ID NO:

1.

6. The isolated recombinant polymerase of any one of claims 1 to 5, comprising an N-terminal deletion of a methionine residue at position 1 corresponding to the position of the sequence shown in SEQ ID NO:

1.

7. The isolated recombinant polymerase according to claim 1, wherein the amino acid sequence is the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

8. The isolated recombinant polymerase according to any one of claims 1 to 6, wherein the amino acid sequence is the sequence shown in SEQ ID NO: 10 or SEQ ID NO:

11.

9. The isolated recombinant polymerase of any one of claims 1 to 8, wherein the recombinant polymerase further comprises a heterologous sequence.

10. The isolated recombinant polymerase of claim 9, wherein the recombinant polymerase comprises, from the amino terminus (N terminus) to the carboxyl terminus (C terminus), the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the heterologous sequence.

11. The isolated recombinant polymerase of claim 9, wherein the recombinant polymerase comprises, from N-terminus to C-terminus, the heterologous sequence and the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

12. The isolated recombinant polymerase of any one of claims 9 to 11, wherein the heterologous sequence comprises a cleavable sequence.

13. The isolated recombinant polymerase of any one of claims 9 to 12, wherein the heterologous sequence comprises an affinity tag selected from the group consisting of a polyhistidine tag (His tag), a glutathione-s-transferase (GST) tag, a maltose binding protein (MBP) tag, a mutated dehalogenase (Halo) tag, a Fasciola hepatica 8-kDa antigen (Fh8) tag, a streptavidin binding peptide (SBP) tag, and a Tam avidin tag.

14. The isolated recombinant polymerase of claim 13, wherein the affinity tag is a His tag.

15. The isolated recombinant polymerase of any one of claims 1 to 14, wherein the heterologous sequence comprises a solubility tag selected from the group consisting of a thioredoxin (Trx) tag, a small ubiquitin modification (SUMO) tag, a protein G IgG domain B1 (GB1) tag, and a solubility enhancing tag (SET).

16. The isolated recombinant polymerase of claim 15, wherein the solubility tag is located between the affinity tag and the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

17. An isolated recombinant polymerase as described in claim 15 or 16, wherein the solubility tag is attached to the N-terminus of the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

18. The isolated recombinant polymerase of claim 15, wherein the affinity tag is located between the solubility tag and the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

19. The isolated recombinant polymerase of any one of claims 15 to 18, wherein the solubility tag is a SUMO tag.

20. The isolated recombinant polymerase of claim 19, wherein the SUMO tag comprises the sequence shown in SEQ ID NO:

6.

21. The isolated recombinant polymerase of any one of claims 15 to 18, wherein the solubility tag has a net negative charge at pH 7.

22. The isolated recombinant polymerase of claim 21, wherein the solubility tag has a net negative charge of at least or at least about -6, -10, -12, -14, -15, -16, -17 or -18 at pH 7.

23. The isolated recombinant polymerase of claim 21 or 22, wherein the solubility tag comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more acidic amino acid residues.

24. The isolated recombinant polymerase of any one of claims 21 to 23, wherein the solubility tag is between 5 and 20 amino acid residues in length, optionally wherein the solubility tag is between 5 and 12 amino acid residues in length.

25. The isolated recombinant polymerase of any one of claims 21 to 24, wherein the solubility tag has the sequence S(E / D) n K m (E / D) p G (SEQ ID NO: 12), wherein S is serine, (E / D) is aspartic acid or glutamic acid, K is lysine, G is glycine, n is 5 or 6, m is 1 or 0, and p is an integer between 2 and 6.

26. The isolated recombinant polymerase of any one of claims 21 to 25, wherein the solubility tag has a sequence as shown in any one of SEQ ID NOs: 13-18.

27. The isolated recombinant polymerase of claim 26, wherein the solubility tag has the sequence shown in SEQ ID NO:

14.

28. The isolated recombinant polymerase of any one of claims 9 to 27, wherein the heterologous sequence comprises a linker.

29. The isolated recombinant polymerase of claim 28, wherein the linker comprises one or more glycine and / or serine residues.

30. The isolated recombinant polymerase of claim 29, wherein the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 19-26.

31. The isolated recombinant polymerase of any one of claims 1 to 9, wherein the recombinant polymerase comprises, from N-terminus to C-terminus, a polyhistidine tag (His tag), a linker, a SUMO tag, and the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

32. The isolated recombinant polymerase of any one of claims 1 to 9, wherein the recombinant polymerase comprises, from N-terminus to C-terminus, the amino acid sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, a linker, a polyhistidine tag (His tag), and a solubility tag.

33. The isolated recombinant polymerase of any one of claims 1 to 32, wherein the isolated recombinant polymerase is at least 85%, at least 90% or at least 95% soluble.

34. A variant PumA1 polymerase comprising one or more amino acid substitutions at one or more positions selected from positions 545, 574 and 575 corresponding to the positions in the sequence shown in SEQ ID NO:

1.

35. The variant PumA1 polymerase of claim 34, comprising one or more amino acid substitutions selected from the group consisting of I545E, A574S, and P575V, wherein the amino acid numbers correspond to positions of the sequence shown in SEQ ID NO:

1.

36. The variant PumA1 polymerase of claim 34, comprising amino acid substitutions I545E, A574S, and P575V, wherein the amino acid numbers correspond to positions in the sequence set forth in SEQ ID NO:

1.

37. The variant PumA1 polymerase of any one of claims 34 to 36, comprising the sequence of amino acid residues 2-30 of SEQ ID NO:

1.

38. The variant PumA1 polymerase of any one of claims 34 to 37, comprising an N-terminal deletion of a methionine residue at position 1 corresponding to the position of the sequence shown in SEQ ID NO:

1.

39. The variant PumA1 polymerase of any one of claims 34 to 38, comprising the amino acid sequence of SEQ ID NO:

10.

40. The variant PumA1 polymerase of any one of claims 34 to 39, comprising the amino acid sequence of SEQ ID NO:

11.

41. The variant PumA1 polymerase of any one of claims 34 to 40, wherein the variant PumA1 polymerase exhibits an improved polymerization rate at 42°C compared to a reference PumA1 polymerase.

42. The variant PumA1 polymerase of any one of claims 34 to 41, wherein the variant PumA1 polymerase generates an equivalent density of detected rolling circle amplification (RCA) products in a RCA reaction compared to a reference Phi29 polymerase.

43. The variant PumA1 polymerase of any one of claims 34 to 42, wherein the variant PumA1 polymerase produces a higher signal intensity in an RCA reaction at 42°C compared to a reference PumA1 polymerase.

44. The variant PumA1 polymerase of any one of claims 34 to 43, wherein the variant PumA1 polymerase is at least 85%, at least 90%, or at least 95% soluble.

45. A composition comprising the recombinant polymerase of any one of claims 1 to 33 or the variant PumA1 polymerase of any one of claims 34 to 44.

46. ​​The composition of claim 45, wherein the composition comprises no more than 10 5 No more than 10 4 No more than 10 3 No more than 10 2 or no more than 10 genome equivalents of bacterial or bacteriophage DNA.

47. The composition of claim 45 or 46, comprising about 0.05 μM to about 1 μM of the recombinant polymerase or variant PumA1 polymerase.

48. The composition of claim 45 or 46, comprising at least 1 μM, 10 μM, 50 μM or 100 μM of the recombinant polymerase or variant PumA1 polymerase.

49. The composition of any one of claims 45 to 48, wherein the recombinant polymerase or variant PumA1 polymerase is at least 85%, at least 90% or at least 95% soluble.

50. The composition of claim 49, wherein the recombinant polymerase or variant PumA1 polymerase is at least 85%, at least 90%, or at least 95% soluble at 20°C.

51. The composition of any one of claims 45 to 50, wherein the pH of the composition is about 8.

5.

52. The composition of any one of claims 45 to 51, wherein the composition comprises about 5 mM to about 10 mM MgCl2.

53. The composition of any one of claims 45 to 52, wherein the composition comprises about 0.1 mM to about 0.2 mM dNTPs.

54. The composition of any one of claims 45 to 53, wherein the composition comprises about 1 mM to about 4 mM DTT.

55. The composition of any one of claims 45 to 54, wherein the composition comprises about 2 mg / mL BSA.

56. The composition of any one of claims 45 to 55, wherein the composition comprises about 10 mM NH4SO4.

57. The isolated recombinant polymerase of any one of claims 1 to 33, the variant PumA1 polymerase of any one of claims 34 to 44, or the composition of any one of claims 45 to 56, wherein the apparent molecular weight of the recombinant polymerase or variant PumA1 polymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is about 55 kDa to about 80 kDa, or about 60 kDa to about 70 kDa.

58. A polynucleotide encoding the recombinant polymerase of any one of claims 2 to 33 or the variant PumA1 polymerase of any one of claims 34 to 44.

59. The polynucleotide of claim 58, wherein the polynucleotide has at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO:

9.

60. A recombinant nucleic acid molecule comprising the polynucleotide of claim 58 or 59.

61. The recombinant nucleic acid molecule of claim 60, further comprising a transcriptional regulatory sequence operably linked to the polynucleotide.

62. The recombinant nucleic acid molecule of claim 61, wherein the transcriptional regulatory sequence comprises a promoter selected from the group consisting of a bacterial promoter, a viral promoter, and a mammalian promoter.

63. A vector comprising the polynucleotide of claim 58 or claim 59 or the recombinant nucleic acid molecule of any one of claims 60 to 62.

64. The vector of claim 63, wherein the vector is a plasmid, a phagemid, a viral vector, a cosmid or a transposon.

65. A recombinant expression system comprising the polynucleotide of claim 58 or claim 59, the recombinant nucleic acid molecule of any one of claims 60 to 62, or the vector of claim 63 or claim 64.

66. The recombinant expression system of claim 65, wherein the recombinant expression system produces the recombinant polymerase of any one of claims 1 to 33 or the variant PumA1 polymerase of any one of claims 34 to 44.

67. The recombinant expression system of claim 65 or 66, wherein the recombinant expression system is a cell system or a cell-free system.

68. The recombinant expression system of any one of claims 65 to 67, wherein the recombinant expression system comprises one or more cell systems selected from the group consisting of bacterial cells, fungal cells, insect cells, and mammalian cells.

69. The recombinant expression system of any one of claims 65 to 68, wherein the polynucleotide sequence encoding the recombinant polymerase or variant PumA1 polymerase is codon-optimized for the recombinant expression system.

70. A method for producing a recombinant polymerase as described in any one of claims 1 to 33 or a variant PumA1 polymerase as described in any one of claims 34 to 44, the method comprising culturing a cell population comprising a polynucleotide encoding the recombinant polymerase or the variant PumA1 polymerase, and isolating the recombinant polymerase or the variant PumA1 polymerase from the cell population.

71. The method of claim 70, wherein the method comprises inducing expression of the recombinant polymerase or the variant PumA1 polymerase.

72. The method of claim 70 or claim 71, wherein the recombinant polymerase or variant PumA 1 polymerase is isolated using the affinity tag.

73. The method of any one of claims 70 to 72, further comprising cleaving the heterologous sequence.

74. A method for amplifying DNA, the method comprising contacting a biological sample containing the DNA to be amplified with a recombinant polymerase as claimed in any one of claims 1 to 33, a variant PumA1 polymerase as claimed in any one of claims 34 to 44, or a composition as claimed in any one of claims 45 to 56.

75. The method of claim 74, wherein the DNA is amplified by rolling circle amplification (RCA).

76. A method as claimed in claim 74 or claim 75, wherein the DNA is amplified by in situ RCA in the biological sample.

77. A method for performing rolling circle amplification (RCA), the method comprising contacting a biological sample containing DNA to be amplified with a recombinant polymerase as claimed in any one of claims 1 to 33, a variant PumA1 polymerase as claimed in any one of claims 34 to 44, or a composition as claimed in any one of claims 45 to 56.

78. The method of any one of claims 74 to 77, comprising incubating the recombinant polymerase or composition with the biological sample.

79. The method of claim 78, wherein the recombinant polymerase or composition and the biological sample are incubated at a temperature of at least or about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 42°C, 45°C or more.

80. The method of claim 79, wherein the recombinant polymerase comprises amino acid substitutions I545E, A574S and P575V, wherein the amino acid numbers correspond to the positions of the sequence shown in SEQ ID NO: 1, and the RCA is performed at a temperature of at least 42°C.

81. A method as claimed in claim 80, wherein the method causes an increase in the signal intensity of the detected RCA product compared to a reference RCA product produced using a reference polymerase.

82. The method of claim 81, wherein the reference polymerase is wild-type Phi29 polymerase.

83. The method of claim 81, wherein the reference polymerase is wild-type PumA1 polymerase.

84. The method of any one of claims 74 to 83, wherein the RCA is performed for at least or about 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours or longer.

85. A method as claimed in any one of claims 77 to 84, wherein the method comprises detecting an RCA product generated by said RCA.

86. The method of claim 85, wherein the detecting comprises detecting a sequence in the RCA product.

87. The method of claim 85 or claim 86, wherein the RCA product is detected at one location in the biological sample.

88. The method of any one of claims 85 to 86, wherein the detecting comprises: contacting the biological sample with one or more detectably labeled probes that bind directly or indirectly to the one or more barcode sequences or their complements, and A signal associated with the one or more detectably labeled probes is detected.

89. The method of any one of claims 85 to 88, wherein the detecting comprises: contacting the biological sample with one or more intermediate probes that bind directly or indirectly to the one or more barcode sequences or their complements, wherein the one or more intermediate probes are detectable using one or more detectably labeled probes, and A signal associated with the one or more detectably labeled probes is detected.

90. The method of claim 88 or 89, further comprising removing the one or more intermediate probes and / or the one or more detectably labeled probes.

91. The recombinant polymerase of any one of claims 1 to 33, the variant PumA1 polymerase of any one of claims 34 to 44, or the method of any one of claims 74 to 90, wherein the recombinant polymerase or the variant PumA1 polymerase exhibits an average processivity of at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, or at least 70 kb.

92. The recombinant polymerase of any one of claims 1 to 33, the variant PumA1 polymerase of any one of claims 34 to 44, or the method of any one of claims 74 to 90, wherein the recombinant polymerase or the variant PumA1 polymerase has strand displacement activity.

93. The recombinant polymerase of any one of claims 1 to 33, the variant PumA1 polymerase of any one of claims 34 to 44, or the method of any one of claims 74 to 90, wherein the recombinant polymerase or the variant PumA1 polymerase, wherein the recombinant polymerase or the variant PumA1 polymerase exhibits an average nucleotide polymerization rate of at least 1000 nucleotides (nt) / minute, at least 1500 nt / minute, at least 2000 nt / minute, or at least 2280 nt / minute.

94. The recombinant polymerase of any one of claims 1 to 33, the variant PumA1 polymerase of any one of claims 34 to 44, or the method of any one of claims 74 to 90, wherein the recombinant polymerase or variant PumA1 polymerase exhibits less than 10 -5 Errors / bp, less than 10 -6 Errors / bp or less than 10 -7 The nucleic acid replication error rate is 1 error / bp.

95. The recombinant polymerase of any one of claims 1 to 33, the variant PumA1 polymerase of any one of claims 34 to 44, or the method of any one of claims 74 to 90, wherein the recombinant polymerase or the variant PumA1 polymerase has exonuclease activity.

96. The recombinant polymerase of any one of claims 1 to 33, the variant PumA1 polymerase of any one of claims 34 to 44, or the method of any one of claims 74 to 90, wherein the recombinant polymerase or the variant PumA1 polymerase has reverse transcriptase activity.

97. The method of any one of claims 74 to 96, wherein the recombinant polymerase or the variant PumA1 polymerase produces at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% more rolling circle amplification products than the reference Phi29 polymerase under identical reaction conditions.

98. The method of any one of claims 74 to 97, wherein the method comprises contacting the biological sample with a buffer.

99. The method of claim 98, wherein the pH of the buffer is about 8.

5.

100. The method of claim 98 or 99, wherein the buffer comprises about 5 mM to about 10 mM MgCl2.

101. The method of any one of claims 98 to 100, wherein the buffer comprises about 0.1 mM to about 0.2 mM dNTPs.

102. The method of any one of claims 98 to 101, wherein the buffer comprises about 1 mM to about 4 mM DTT.

103. The method of any one of claims 98 to 102, wherein the buffer comprises about 2 mg / mL BSA.

104. The method of any one of claims 98 to 103, wherein the buffer comprises about 10 mM NH4SO4.

105. A kit comprising the recombinant polymerase of any one of claims 1 to 33, the variant PumA1 polymerase of any one of claims 34 to 44, or the composition of any one of claims 45 to 56.

106. The kit of claim 105, wherein the kit comprises one or more of: (a) dNTPs; (b) one or more divalent cations; (c) reaction buffer; (d) a buffer for use with any one of (a) to (c); and (e) instructions for performing rolling circle amplification using the recombinant polymerase, the variant PumA1 polymerase or the composition.

107. The kit of claim 105 or 106, further comprising one or more primers.

108. The kit of any one of claims 105 to 106, further comprising one or more circularizable probes.

109. The kit of any one of claims 105 to 108, wherein the kit is for nucleic acid amplification.

110. The kit as described in claim 109 further comprises a detection probe for detecting the amplified nucleic acid.

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