AP50 polymerases and uses thereof

Abstract

The present disclosure relates, in some aspects, to recombinant Bacillus bacteriophage AP50 polymerases, modified AP50 polymerases, and compositions thereof. Also provided are methods for nucleic acid amplification (e.g., rolling circle amplification) using the recombinant and / or modified AP50 polymerases. 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 / 398,507, filed on August 16, 2022, entitled “AP50 POLYMERASES AND USESTHEREOF,” which is incorporated herein by reference in its entirety for all purposes.

[0003] Citation of reference sequence listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named 202412016040SeqList.xml created on August 10, 2023, which is 78,540 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 for performing DNA amplification reactions, such as rolling circle amplification (RCA), using recombinant nucleic acid polymerases. Background Art

[0006] Polymerases such as Phi29 DNA polymerase are used in methods for amplifying nucleic acids, such as rolling circle amplification (RCA). However, Phi29 DNA polymerases have certain limitations, which limit their applications. New and improved polymerases are needed. Compositions and methods that address such needs and other needs are provided herein. Summary of the invention

[0007] Provided herein is a recombinant nucleic acid polymerase from a bacillus phage. In some aspects, a recombinant bacillus phage AP50 polymerase (APol) is provided, comprising anthrax bacillus phage AP50 polymerase or a modified AP50 polymerase. Also provided are compositions and kits comprising recombinant AP50 polymerases and related methods and uses of recombinant AP50 polymerases, such as in amplified nucleic acids, such as in rolling circle amplification (RCA) reactions, and methods for producing any recombinant AP50 polymerases. Also provided herein are polynucleotides encoding any provided recombinant AP50 polymerases, recombinant nucleic acid molecules, vectors, recombinant expression systems, and kits. In some embodiments of any embodiment, recombinant AP50 polymerases can be used to improve nucleic acid amplification, such as RCA, and result in improved analysis of biological samples, such as in vitro and in situ analysis.

[0008] In some embodiments of any of the provided embodiments, the recombinant AP50 polymerase is a modified AP50 polymerase, such as those comprising one or more amino acid deletions, insertions, substitutions, truncations, or combinations thereof. In some embodiments of any of the provided embodiments, the recombinant AP50 polymerase is an isolated, recombinantly produced wild-type AP50 polymerase. In some cases, the recombinant polymerase AP50 polymerase exhibits one or more characteristics, such as improved thermal stability, processivity, sensitivity, specificity, solubility, and / or polymerization rate, for example, relative to a reference polymerase (e.g., wild-type Phi29 polymerase). In some cases, the recombinant AP50 polymerase is thermostable, produces more amplification products, and / or can be used in a wider range of nucleic acid amplification methods or samples.

[0009] Provided herein are methods for nucleic acid amplification comprising contacting a biological sample comprising a nucleic acid to be amplified with a recombinant Bacillus phage AP50 polymerase.

[0010] In some embodiments, the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 200 amino acids in length, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the foregoing sequences. In some embodiments of any embodiment, the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the foregoing sequences. In some embodiments of any embodiment, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 6, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 6. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 6. In some embodiments of any of the embodiments, the recombinant polymerase consists of the sequence shown in SEQ ID NO:6.

[0011] In some embodiments of any of the provided embodiments, the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 1 that is at least 640 amino acids in length, or a sequence that has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the foregoing sequences. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 1, or a sequence that has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 1. In some embodiments of any of the embodiments, the recombinant polymerase consists of the sequence shown in SEQ ID NO: 1.

[0012] In some embodiments of any of the provided embodiments, the recombinant polymerase comprises one or more amino acid substitutions, deletions, or additions at one or more positions corresponding to the positions of the sequence shown in SEQ ID NO:1.

[0013] In some embodiments of any of the embodiments, the recombinant polymerase further comprises a heterologous sequence. In some embodiments of any of the embodiments, the heterologous sequence comprises one or more tags, linkers, and / or polypeptide domains.

[0014] In some embodiments of any of the provided embodiments, the heterologous sequence comprises one or more tags. In some embodiments of any of the embodiments, the tag is selected from the group consisting of a polyhistidine (HIS) tag, a solubility enhancing tag (SET), a small ubiquitin-modified (SUMO) tag, a Fasciola hepatica 8-kDa antigen (Fh8) tag, a thioredoxin (Trx) tag, a glutathione-s-transferase (GST) tag, a maltose binding protein (MBP) tag, an IgG domain B1 (GB1) tag of protein G, a mutated dehalogenase (Halo) tag, a streptavidin binding peptide (SBP) tag, and a Tamavidin tag. In some embodiments of any of the embodiments, the tag comprises the sequence shown in any one of SEQ ID NOs: 7, 8, and 26. In some embodiments of any of the embodiments, the tag comprises the sequence shown in SEQ ID NO: 7. In some embodiments of any of the embodiments, the tag comprises the sequence shown in SEQ ID NO: 8. In some embodiments of any of the embodiments, the tag comprises the sequence shown in SEQ ID NO: 26.

[0015] In some embodiments of any of the provided embodiments, the heterologous sequence comprises one or more polypeptide domains. In some embodiments of any of the embodiments, the polypeptide domain is selected from the group consisting of: a helix-hairpin-helix (HhH) DNA binding motif, a helix-hairpin-helix fusion [(HhH) 2 ]DNA binding motif, single-stranded DNA binding domain, SD07DNA binding motif, SS07 DNA binding motif and Escherichia coli HU domain.

[0016] In some embodiments of any of the provided embodiments, the heterologous sequence comprises one or more linkers. In some embodiments of any of the embodiments, the linker is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 amino acids in length. In some embodiments of any of the embodiments, the linker comprises the sequence shown in any one of SEQ ID NOs: 10-16. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 6, a linker, and a polyhistidine (HIS) tag.

[0017] In some embodiments of any of the provided embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO:6, a linker, a poly-histidine (HIS) tag, and a solubility enhancing tag (SET).

[0018] In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in any of SEQ ID NOs: 2-4, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NOs: 2-4. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 2. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 3. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 4.

[0019] In some embodiments of any of the provided embodiments, the recombinant polymerase is 900 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 800 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 732 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 638 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 600 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 500 amino acids or less in length.

[0020] In some embodiments of any of the provided embodiments, the nucleic acid comprises deoxyribonucleotide residues. In some embodiments of any of the embodiments, the nucleic acid comprises one or more ribonucleotide residues. In some embodiments of any of the embodiments, the nucleic acid is a circular probe or a circularizable probe.

[0021] In some embodiments of any provided embodiment, nucleic acid is amplified by rolling circle amplification (RCA) using recombinant polymerase. In some embodiments of any embodiment, nucleic acid is amplified in situ in biological sample using recombinant polymerase. In some embodiments of any embodiment, the method also includes incubating recombinant polymerase and biological sample with a primer hybridized with nucleic acid. In some embodiments of any embodiment, primer comprises synthetic oligonucleotide. In some embodiments of any embodiment, primer comprises cellular nucleic acid or its part in biological sample.

[0022] In some embodiments of any provided embodiment, the recombinant polymerase has DNA synthesis activity. In some embodiments of any embodiment, the recombinant polymerase can polymerize DNA based on a circular nucleic acid template. In some embodiments of any embodiment, the recombinant polymerase has RNA synthesis activity. In some embodiments of any embodiment, the recombinant polymerase has strand displacement activity. In some embodiments of any embodiment, the recombinant polymerase has reverse transcriptase activity. In some embodiments of any embodiment, the recombinant polymerase has exonuclease activity.

[0023] In some embodiments of any provided embodiment, the recombinant polymerase exhibits one or more features selected from improved processivity, improved polymerization rate, and improved thermostability compared to a reference polymerase. In some embodiments of any embodiment, the recombinant polymerase exhibits improved processivity compared to a reference polymerase. In some embodiments of any embodiment, the recombinant polymerase exhibits improved kinetics compared to a reference polymerase. In some embodiments of any embodiment, the recombinant polymerase exhibits improved polymerization rate compared to a reference polymerase. In some embodiments of any embodiment, the recombinant polymerase exhibits improved thermostability compared to a reference polymerase.

[0024] In some embodiments of any of the provided embodiments, the recombinant polymerase is capable of generating an RCA product in a rolling circle amplification (RCA) reaction. In some embodiments of any of the embodiments, the recombinant polymerase 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 of any of the embodiments, the RCA reaction is performed for or about 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours or longer.

[0025] In some embodiments of any of the provided embodiments, the method further comprises detecting or analyzing the amplified products. In some embodiments of any of the embodiments, the amplified products are detected or analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. In some embodiments of any of the embodiments, the nucleic acids and / or amplified products comprise one or more barcode sequences or their complements. In some embodiments of any of the embodiments, the one or more barcode sequences or their complements correspond to a target nucleic acid or an endogenous analyte. In some embodiments of any of the embodiments, the one or more barcode sequences or their complements are detected by the following steps: 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; detecting signals associated with the one or more detectably labeled probes; and removing the one or more detectably labeled probes. In some embodiments of any of the embodiments, the one or more barcode sequences or their complements are detected by 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; detecting signals associated with the one or more detectably labeled probes; and removing the one or more intermediate probes and / or the one or more detectably labeled probes.

[0026] In some embodiments of any of the provided embodiments, the recombinant polymerase generates a higher density of detected rolling circle amplification (RCA) products in RCA compared to a reference polymerase. In some embodiments of any of the embodiments, the recombinant polymerase generates a higher signal intensity in RCA compared to a reference polymerase. In some embodiments of any of the embodiments, the RCA reaction is performed 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 more.

[0027] In some embodiments of any of the provided embodiments, the reference polymerase is the Phi29 polymerase set forth in SEQ ID NO:5.

[0028] In some embodiments of any provided embodiment, the biological sample is a cell sample or a tissue sample. In some embodiments of any embodiment, the biological sample is non-homogenized. In some embodiments of any embodiment, the biological sample is selected from the following: formalin-fixed paraffin-embedded (FFPE) samples, frozen tissue samples and fresh tissue samples.

[0029] Also provided are recombinant Bacillus phage AP50 polymerases comprising i) a contiguous portion of SEQ ID NO: 6 that is at least 200 amino acids in length, or a sequence that has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the foregoing sequences; and ii) a heterologous sequence. In some embodiments of any of the provided embodiments, the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length, or a sequence that has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the foregoing sequences.

[0030] Also provided herein is a recombinant Bacillus phage AP50 polymerase comprising i) the sequence set forth in SEQ ID NO:6, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence set forth in SEQ ID NO:6; and ii) a heterologous sequence.

[0031] In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 6. In some embodiments of any of the provided embodiments, the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 1 that is at least 637 amino acids in length, or a sequence that has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the foregoing sequences. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 1, or a sequence that has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 1. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 1. In some embodiments of any of the embodiments, the recombinant polymerase consists of the sequence shown in SEQ ID NO: 1 and a heterologous sequence.

[0032] Provided herein are recombinant Bacillus phage AP50 polymerases consisting of a contiguous portion of the sequence set forth in SEQ ID NO: 6 that is at least 200 amino acids in length, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the foregoing sequences. In some embodiments of any of the embodiments, the recombinant polymerase consists of the sequence set forth in SEQ ID NO: 6, or a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of the foregoing sequences. In some embodiments of any of the embodiments, the recombinant polymerase consists of a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO: 6. In some embodiments of any of the embodiments, the recombinant polymerase consists of the sequence shown in SEQ ID NO: 6.

[0033] Provided herein are recombinant Bacillus phage AP50 polymerases comprising one or more amino acid substitutions, deletions or additions at one or more positions corresponding to the positions of the sequence set forth in SEQ ID NO:1.

[0034] In some embodiments of any of the provided embodiments, the recombinant polymerase has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NO:1.

[0035] In some embodiments of any of the provided embodiments, the recombinant polymerase further comprises a heterologous sequence.

[0036] In some embodiments of any of the provided embodiments, the heterologous sequence comprises one or more tags, linkers, and / or polypeptide domains. In some embodiments of any of the embodiments, the heterologous sequence comprises one or more tags. In some embodiments of any of the embodiments, the tag is selected from the group consisting of a polyhistidine (HIS) tag, a solubility enhancing tag (SET), a small ubiquitin-modified (SUMO) tag, a Fasciola hepatica 8-kDa antigen (Fh8) tag, a thioredoxin (Trx) tag, a glutathione-s-transferase (GST) tag, a maltose binding protein (MBP) tag, an IgG domain B1 (GB1) tag of protein G, a mutated dehalogenase (Halo) tag, a streptavidin binding peptide (SBP) tag, and a Tamavidin tag. In some embodiments of any of the embodiments, the tag comprises the sequence shown in any one of SEQ ID NOs: 7, 8, and 26. In some embodiments of any of the embodiments, the tag comprises the sequence shown in SEQ ID NO: 7. In some embodiments of any of the embodiments, the tag comprises the sequence shown in SEQ ID NO: 8. In some embodiments of any of the embodiments, the tag comprises the sequence shown in SEQ ID NO:26.

[0037] In some embodiments of any of the provided embodiments, the heterologous sequence comprises one or more polypeptide domains. In some embodiments of any of the embodiments, the polypeptide domain is selected from the group consisting of: a helix-hairpin-helix (HhH) DNA binding motif, a helix-hairpin-helix fusion [(HhH) 2 ]DNA binding motif, single-stranded DNA binding domain, SD07DNA binding motif, SS07 DNA binding motif and Escherichia coli HU domain.

[0038] In some embodiments of any of the provided embodiments, the heterologous sequence comprises one or more linkers. In some embodiments of any of the embodiments, the linker is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 amino acids in length. In some embodiments of any of the embodiments, the linker comprises the sequence shown in any one of SEQ ID NOs: 10-16.

[0039] In some embodiments of any of the provided embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO:6, a linker, and a polyhistidine (HIS) tag. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO:6, a linker, a polyhistidine (HIS) tag, and a solubility enhancing tag (SET).

[0040] In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in any of SEQ ID NOs: 2-4, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence shown in SEQ ID NOs: 2-4. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 2. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 3. In some embodiments of any of the embodiments, the recombinant polymerase comprises the sequence shown in SEQ ID NO: 4.

[0041] In some embodiments of any of the provided embodiments, the recombinant polymerase is 900 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 800 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 732 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 638 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 600 amino acids or less in length. In some embodiments of any of the embodiments, the recombinant polymerase is 500 amino acids or less in length.

[0042] In some embodiments of any provided embodiment, the recombinant polymerase has DNA synthesis activity. In some embodiments of any embodiment, the recombinant polymerase can polymerize DNA based on a circular nucleic acid template. In some embodiments of any embodiment, the recombinant polymerase has RNA synthesis activity. In some embodiments of any embodiment, the recombinant polymerase has strand displacement activity. In some embodiments of any embodiment, the recombinant polymerase has reverse transcriptase activity. In some embodiments of any embodiment, the recombinant polymerase has exonuclease activity.

[0043] In some embodiments of any provided embodiment, the recombinant polymerase exhibits one or more features selected from improved processivity, improved polymerization rate, and improved thermostability compared to a reference polymerase. In some embodiments of any embodiment, the recombinant polymerase exhibits improved processivity compared to a reference polymerase. In some embodiments of any embodiment, the recombinant polymerase exhibits improved kinetics compared to a reference polymerase. In some embodiments of any embodiment, the recombinant polymerase exhibits improved polymerization rate compared to a reference polymerase. In some embodiments of any embodiment, the recombinant polymerase exhibits improved thermostability compared to a reference polymerase.

[0044] In some embodiments of any of the provided embodiments, the recombinant polymerase is capable of generating RCA products in a rolling circle amplification (RCA) reaction. In some embodiments of any of the embodiments, the RCA reaction is performed 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 of any of the embodiments, the recombinant polymerase generates a higher density of detected rolling circle amplification (RCA) products in RCA compared to a reference polymerase. In some embodiments of any of the embodiments, the recombinant polymerase generates a higher signal intensity in RCA compared to a reference polymerase. In some embodiments of any of the embodiments, the reference polymerase is the Phi29 polymerase shown in SEQ ID NO: 5.

[0045] Also provided are compositions comprising any of the recombinant Bacillus phage AP50 polymerases described herein.

[0046] Also provided are polynucleotides encoding any of the recombinant Bacillus phage AP50 polymerases described herein.

[0047] In some embodiments of any embodiment, the polynucleotide comprises the sequence shown in any one of SEQ ID NOs: 17-21, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or about 99%.5% sequence identity to the sequence shown in any one of SEQ ID NOs: 17-21. In some embodiments of any embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 17. In some embodiments of any embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 18. In some embodiments of any embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 19. In some embodiments of any embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 20. In some embodiments of any embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 21.

[0048] In some embodiments of any of the embodiments, the polynucleotide is codon optimized for a recombinant expression system to produce a recombinant polymerase.

[0049] Also provided are recombinant nucleic acid molecules comprising any of the polynucleotides described herein, such as any polynucleotide encoding any of the recombinant polymerases described herein.

[0050] Also provided are recombinant nucleic acid molecules comprising a polynucleotide encoding a recombinant Bacillus phage AP50 polymerase comprising the sequence set forth in SEQ ID NO:6, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% .5% sequence identity to the sequence set forth in SEQ ID NO:6. In some embodiments of any of the embodiments, the encoded recombinant polymerase comprises the sequence set forth in SEQ ID NO:6. In some embodiments of any of the embodiments, the encoded recombinant polymerase comprises the sequence set forth in SEQ ID NO:1, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% .5% sequence identity to the sequence set forth in SEQ ID NO:1. In some embodiments of any of the embodiments, the encoded recombinant polymerase comprises the sequence set forth in SEQ ID NO:1. In some embodiments of any of the embodiments, the encoded recombinant polymerase consists of the sequence set forth in SEQ ID NO:1.

[0051] In some embodiments of any embodiment, the encoded recombinant nucleic acid molecule further comprises a transcriptional regulatory sequence operably linked to the polynucleotide. In some embodiments of any embodiment, the transcriptional regulatory sequence comprises a promoter selected from bacteria, viruses, and mammalian promoters.

[0052] Also provided are vectors comprising any of the polynucleotides or recombinant nucleic acid molecules described herein.In some embodiments of any of the embodiments, the vector comprises a plasmid, a phagemid, a viral vector, a cosmid, or a transposon.

[0053] Also provided herein are recombinant expression systems comprising any polynucleotide described herein, such as any polynucleotide encoding any recombinant polymerase described herein, any recombinant nucleic acid molecule described herein, or any vector described herein.

[0054] In some embodiments of any embodiment, polynucleotides, recombinant nucleic acid molecules or vectors are used to transform recombinant expression systems to generate transformed recombinant expression systems. In some embodiments of any embodiment, the transformed recombinant expression system generates recombinant polymerase. In some embodiments of any embodiment, the recombinant expression system is a cell system or a cell-free system. In some embodiments of any embodiment, the recombinant expression system comprises one or more cell systems selected from bacterial cells, fungal cells, insect cells and mammalian cells.

[0055] In some embodiments of any of the embodiments, the polynucleotide sequence encoding the recombinant polymerase is codon-optimized for a recombinant expression system.

[0056] In some embodiments of any of the embodiments, the recombinant expression system produces a recombinant polymerase having improved solubility, improved processivity, improved polymerization rate, improved thermal stability, and improved homogeneity compared to a reference polymerase.

[0057] Also provided are kits comprising (1) any recombinant Bacillus phage AP50 polymerase described herein, any polynucleotide described herein, any recombinant nucleic acid molecule described herein, or any vector described herein, and (2) 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 of (a)-(c); and (e) instructions for use.

[0058] In some embodiments of any of the embodiments, the nucleic acid comprises deoxyribonucleotide residues. In some embodiments of any of the embodiments, the nucleic acid comprises one or more ribonucleotide residues.

[0059] In some embodiments of any embodiment, the kit further comprises one or more primers. In some embodiments of any embodiment, the kit further comprises one or more circularizable probes. In some embodiments of any embodiment, the kit is used for nucleic acid amplification.

[0060] In some embodiments of any embodiment, nucleic acid is amplified by rolling circle amplification (RCA) using a recombinant polymerase. In some embodiments of any embodiment, nucleic acid is amplified in situ in a biological sample using a recombinant polymerase. In some embodiments of any embodiment, the kit also includes a probe for detecting amplified nucleic acid.

[0061] Also provided is a kit comprising (1) any polynucleotide described herein, any recombinant nucleic acid molecule described herein, or any vector described herein, and (2) a recombinant expression system. In some embodiments of any embodiment, the kit further comprises instructions for use. In some embodiments of any embodiment, the recombinant expression system is a cell system or a cell-free system. In some embodiments of any embodiment, the recombinant expression system comprises one or more cell systems selected from bacterial cells, fungal cells, insect cells, and mammalian cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] 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.

[0063] Figure 1 Depicted are the results of an in vitro rolling circle amplification (RCA) assay comparing the activity of wild-type Phi29 polymerase and an exemplary modified Bacillus phage AP50 polymerase (APolTr).

[0064] Figure 2A-2D Depicted is a comparison of different concentrations of an exemplary modified Bacillus phage AP50 polymerase (APolTr) at 30°C ( Figure 2A )、37℃( Figure 2B )、42℃( Figure 2C ) and 50℃( Figure 2D ) under the results of in vitro rolling circle amplification (RCA) assay of activity.

[0065] Figure 3 Depicted are the results of an in vitro rolling circle amplification (RCA) assay using the Cassini workflow to compare the activity of wild-type Phi29 polymerase and an exemplary modified Bacillus phage AP50 polymerase (APolTr). DETAILED DESCRIPTION

[0066] 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.

[0067] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0068] I. Overview

[0069] Provided herein are Bacillus anthracis phage AP50 polymerases (APols) and related methods and uses thereof, such as methods and uses in amplifying nucleic acids. Also provided are recombinant and / or modified AP50 polymerases, for example, modified AP50 polymerases comprising one or more amino acid deletions, insertions, substitutions, truncations, or combinations thereof. In some aspects, the provided AP50 polymerases exhibit robust or improved properties, functions, or activities, such as thermostability, processivity, or polymerization rate. In some aspects, the provided AP50 polymerases exhibit improved properties, functions, or activities, such as improved thermostability, processivity, sensitivity, specificity, solubility, or polymerization rate, compared to a reference polymerase. Also provided are related polynucleotides, recombinant nucleic acid molecules, vectors, recombinant expression systems, and kits, as well as methods and uses thereof.

[0070] Polymerases (such as Phi29 polymerase) can be used for specific nucleic acid amplification methods, such as rolling circle amplification (RCA). However, available polymerases have certain limitations, which may limit their use under specific reactions or conditions, such as for in situ or high temperature reactions. Rolling circle amplification and other methods that benefit from high nucleic acid continuous synthesis ability (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 a spatially localized position in a biological sample. 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, with an optimal reaction temperature of 30°C, which results in slower reaction kinetics, fewer amplified products, and greater heterogeneity and amplification bias, particularly for DNA with a high guanine / cytosine (G / C) content. For example, wild-type Phi29 has limited thermal stability because the typical RCA reaction temperature for wild-type Phi29 is about 30°C, and wild-type Phi29 polymerase may exhibit reduced stability at higher temperatures, resulting in extended reaction times and / or reduced amplified nucleic acid signals. In some cases, wild-type Phi29 polymerase may result in slower RCA or weaker RCA efficiency. In some aspects, a nucleic acid polymerase with improved properties is needed, for example, for use in specific RCA conditions, such as higher temperatures, in situ experiments, or more complex RCA methods. A polymerase that meets such needs is provided.

[0071] In some aspects, a recombinant and / or modified AP50 polymerase (APol) is provided. In some aspects, the AP50 polymerase provided exhibits robust nucleic acid amplification activity, such as RCA activity. In some aspects, compared with a reference polymerase, such as a wild-type Phi29 polymerase, the AP50 polymerase provided exhibits one or more improved features or characteristics. In some aspects, for example, compared with a reference polymerase, such as a wild-type Phi29 polymerase, the AP50 polymerase provided exhibits one or more features, such as improved thermal stability, improved continuous synthesis ability, improved sensitivity, improved specificity, improved solubility and improved polymerization rate. In some aspects, compared with a reference polymerase, such as a wild-type Phi29 polymerase, the AP50 polymerase provided exhibits improved stability or continuous synthesis ability, increased thermal stability and / or product yield, improved specificity, improved retention time, improved surface stability, changed cofactor selectivity, improved accuracy, speed and / or read length and / or the kinetic properties of the change. In some aspects, the recombinant AP50 polymerase is soluble and well expressed. In addition, the present application provides data demonstrating that recombinant and / or modified polymerases comprising AP50 polymerase sequences have features that are conducive to rolling circle amplification. Notably, the recombinant and / or modified AP50 polymerase polypeptides provided herein exhibit higher polymerization rates and produce higher amounts of detectable rolling circle amplification products under the same conditions as Phi29 (e.g., 30°C and elevated temperatures) in rolling circle amplification assays.

[0072] In some aspects, the embodiments provided are based on the observation that exemplary recombinant AP50 polymerases and modified AP50 polymerases exhibit various improved properties for nucleic acid amplification, including improved thermostability at elevated temperatures (e.g., 42° C.), improved polymerization rates, and increased rolling circle amplification (RCA) product generation, as observed, for example, by higher polymerization rates and / or shorter reaction times, compared to wild-type Phi29 polymerase. In some embodiments, the increased activity of the AP50 polymerase is compared to wild-type Phi29 polymerase in an assay using the same or lower concentration of AP50 polymerase.

[0073] In some aspects, the provided AP50 polymerase can lead to improvements in different types of assays and environments for nucleic acid amplification (such as rolling circle amplification (RCA)) in in vitro assays and in situ assays and using different types of tissue samples or target genes. In some aspects, the provided AP50 polymerase can be used to improve nucleic acid amplification, such as RCA, and lead to improvements in biological sample analysis, such as in vitro and in situ analysis.

[0074] II. Bacillus anthracis phage AP50 polymerase (APOL)

[0075] Provided herein are Bacillus anthracis phage AP50 polymerases, recombinant AP50 polymerases, and modified AP50 polymerases, such as AP50 polymerases comprising one or more variations or modifications compared to a wild-type, full-length or unmodified AP50 polymerase.

[0076] Bacillus anthracis phage AP50 polymerase (APol) is an uncharacterized phage protein that is predicted to be a soluble DNA polymerase (e.g., Sozhamannan et al., Applied and Environmental Microbiology, 74(21):6792-6796). It is predicted to be a polymerase and is annotated as being encoded by Bacillus anthracis phage AP50 ORF 5. The full-length AP50 polymerase is 732 amino acids (shown in SEQ ID NO:27; 731 amino acids without the initial methionine or formylmethionine (fMet) residues, as shown in SEQ ID NO:1) and shares 47% sequence identity with the Bacillus thuringiensis Bam35 phage protein. However, the function of the predicted AP50 polymerase has not been characterized, for example, it has not been demonstrated to have nucleic acid polymerase activity.

[0077] Phi29 is a mesophilic DNA polymerase derived from Bacillus phage Phi29 of rice straw, which has 3'-5' exonuclease activity and nucleic acid polymerization activity. Phi29 can be used for specific methods of amplifying and / or sequencing DNA and RNA, including isothermal rolling circle amplification (RCA) reactions. RCA reactions are important for single cell analysis and / or multiple analysis, for example, multiple endogenous analyte detection and single cell multi-omics analysis (e.g., genome, transcriptome and proteome analysis). 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, wild-type Phi29 has certain limitations, including limited thermal stability, with an optimal reaction temperature of 30°C, which results in slower reaction kinetics, fewer amplification products, and greater heterogeneity and amplification bias, particularly for DNA with high guanine / cytosine (G / C) content. In some aspects, the utility of wild-type Phi29 polymerase is limited, and the provided thermostable modified AP50 polymerase can polymerize substrates faster, produce more amplification products and / or provide comparable assay sensitivity. In some aspects, the provided modified AP50 polymerase can be used in a wider range of nucleic acid amplification methods or samples, for example, compared to the wild-type Phi29 polymerase.

[0078] A. Recombinant and / or modified AP50 polymerase

[0079] In some embodiments, recombinant AP50 polymerases and modified AP50 polymerases are provided herein. In some aspects, recombinant AP50 polymerases are provided, e.g., recombinantly produced and / or purified AP50 polymerases. In some aspects, modified AP50 polymerases are provided, which are modified compared to wild-type, full-length or unmodified AP50 polymerases. In some embodiments, the modified AP50 polymerase comprises one or more variations or modifications, e.g., amino acid deletions, insertions, substitutions, truncations and / or fusions of additional peptide or protein sequences, compared to the sequence of a reference polymerase (such as a wild-type, full-length AP50 polymerase, such as the wild-type, full-length AP50 polymerase shown in SEQ ID NO: 1 or SEQ ID NO: 27). In some aspects, the modified AP50 polymerase is a variant of a wild-type AP50 polymerase. In some aspects, the modified AP50 polymerase is recombinantly produced and / or purified, or is used for recombinant production and / or purification. In some aspects, reference to a "modified AP50 polymerase" can be understood to include that it is a recombinant AP50 polymerase. Provided herein are isolated and purified recombinant AP50 polymerases for use in performing amplification reactions.

[0080] In some aspects, the modified AP50 polymerase comprises a heterologous sequence, for example, a modified AP50 polymerase or a portion thereof fused to a heterologous amino acid sequence. Exemplary modified AP50 polymerases include those designed to have robust thermostability, processivity, sensitivity, specificity, solubility, polymerization rate, increased closed complex stability, reduced branching fraction and / or other biological properties of the polymerase. In some aspects, exemplary modified AP50 polymerases exhibit improved polymerase properties, such as thermostability, processivity, sensitivity, specificity, solubility, polymerization rate, increased closed complex stability, reduced branching fraction and / or other biological properties of the polymerase. Other exemplary modifications include modifications for fixing the polymerase on a surface or otherwise labeling the polymerase. Recombinant AP50 polymerases or modified AP50 polymerases are provided that exhibit one or more such properties or features.

[0081] Also provided herein are compositions comprising any recombinant AP50 polymerase or modified AP50 polymerase, polynucleotides encoding any recombinant AP50 polymerase or modified AP50 polymerase, recombinant nucleic acid molecules encoding any recombinant AP50 polymerase or modified AP50 polymerase or comprising any polynucleotide, vectors encoding any modified AP50 polymerase or comprising any polynucleotide, recombinant expression systems encoding any recombinant AP50 polymerase or modified AP50 polymerase or comprising any polynucleotide, and kits comprising or encoding any recombinant AP50 polymerase or modified AP50 polymerase, as well as methods and uses of any of the foregoing.

[0082] In some aspects, provided herein are modified AP50 polymerases comprising amino acid deletions at one or more positions compared to wild-type AP50 polymerase. In some aspects, provided herein are modified AP50 polymerases comprising amino acid truncations compared to wild-type AP50 polymerase. In some aspects, the wild-type AP50 polymerase comprises the sequence shown in SEQ ID NO: 1.

[0083] In some aspects, with reference to SEQ ID NO: 1, the AP50 polymerase provided comprises a deletion at one or more positions. In some aspects, with reference to SEQ ID NO: 1, the modified AP50 polymerase comprises a truncation. In some aspects, with reference to SEQ ID NO: 1, one or more deletions or truncations are located at the N-terminus of the AP50 polymerase. In some aspects, with reference to SEQ ID NO: 1, the AP50 polymerase comprises a deletion of one amino residue or two amino residues located at the N-terminus of the AP50 polymerase. In some aspects, with reference to SEQ ID NO: 1, one or more deletions or truncations are located at the C-terminus of the AP50 polymerase.

[0084] In some aspects, the modified AP50 polymerase comprises a deletion of at least one amino acid residue compared to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprises a deletion of 1 to 350, 5 to 300, 10 to 250, 25 to 150, 50 to 125, or 75 to 100 amino acid residues (such as consecutive amino acid residues) compared to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprises a deletion of at least 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, or 350 amino acid residues (such as consecutive amino acid residues) compared to SEQ ID NO: 1. In some aspects, with reference to SEQ ID NO: 1, the modified AP50 polymerase comprises a deletion of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 consecutive amino acids, or a deletion within a range defined by any of the foregoing. In some aspects, with reference to SEQ ID NO: 1, the modified AP50 polymerase comprises a deletion of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 consecutive amino acids from the N-terminus, or a deletion within a range defined by any of the foregoing. In some aspects, with reference to SEQ ID NO: 1, the modified AP50 polymerase comprises a deletion of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 consecutive amino acids from the C-terminus, or a deletion within a range defined by any of the foregoing.

[0085] In some aspects, the modified AP50 polymerase comprises a deletion of at least 25 amino acid residues (such as consecutive amino acid residues) compared to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprises a deletion of at least 50 amino acid residues (such as consecutive amino acid residues) compared to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprises a deletion of at least 75 amino acid residues (such as consecutive amino acid residues) compared to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprises a deletion of at least 100 amino acid residues (such as consecutive amino acid residues) compared to SEQ ID NO: 1. In some aspects, with reference to SEQ ID NO: 1, the modified AP50 polymerase comprises a deletion of 94 consecutive amino acids from the C-terminus. In some aspects, with reference to SEQ ID NO: 1, the modified AP50 polymerase comprises a truncation of 94 consecutive amino acids from the C-terminus.

[0086] In some aspects, provided herein are modified AP50 polymerases comprising two or more non-contiguous amino acid deletions at positions referenced to SEQ ID NO: 1.

[0087] In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 80% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 85% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 90% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 95% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 96% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 97% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 98% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 99% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprising one or more deletions has at least or about 99.5% sequence identity to SEQ ID NO: 1.

[0088] In some aspects, an exemplary wild-type, full-length AP50 polymerase amino acid sequence is set forth in SEQ ID NO: 1, which does not include the initial fMet residue. In some aspects, an exemplary wild-type, full-length AP50 polymerase amino acid sequence is set forth in SEQ ID NO: 27, which includes the initial fMet residue. In some embodiments of any of the provided embodiments, reference to the description of the amino acid sequence and amino acid residues of SEQ ID NO: 1 is also understood to include a description of the corresponding amino acid sequence and amino acid residues from the corresponding sequence including the initial fMet residue, i.e., reference to SEQ ID NO: 27. In some aspects, reference to amino acid residue 1 of SEQ ID NO: 1 corresponds to reference to amino acid residue 2 of SEQ ID NO: 27. In some aspects, reference to amino acid residue 731 of SEQ ID NO: 1 corresponds to reference to amino acid residue 732 of SEQ ID NO: 27.

[0089] In some aspects, an exemplary truncated AP50 polymerase (e.g., a C-terminal truncation or C-terminal deletion with reference to 94 amino acids of SEQ ID NO: 1; i.e., a truncation with reference to amino acid residues 638-731 of SEQ ID NO: 1) amino acid sequence is set forth in SEQ ID NO: 6, which does not include the initial fMet residue. In some aspects, an exemplary truncated AP50 polymerase (e.g., a C-terminal truncation or C-terminal deletion with reference to 94 amino acids of SEQ ID NO: 27; i.e., a truncation with reference to amino acid residues 639-732 of SEQ ID NO: 27) amino acid sequence is set forth in SEQ ID NO: 32, which includes the initial fMet residue. In some embodiments of any of the provided embodiments, a description of an amino acid sequence and amino acid residues with reference to SEQ ID NO: 6 is also understood to include a description of the corresponding amino acid sequence and amino acid residues from the corresponding sequence including the initial fMet residue, i.e., with reference to SEQ ID NO: 32. In some aspects, amino acid residue 1 with reference to SEQ ID NO: 6 corresponds to amino acid residue 2 with reference to SEQ ID NO: 32. In some aspects, amino acid residue 637 of reference SEQ ID NO:1 corresponds to amino acid residue 638 of reference SEQ ID NO:27.

[0090] In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 200, 300, 400, 500, or 600 amino acids in length, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 200, 300, 400, 500, or 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 6 that is 100 to 600, 200 to 500, or 300 to 400 amino acids in length.

[0091] In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 200 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 300 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 6 that is at least 200 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 6 that is at least 300 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length.

[0092] In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 80% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 85% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 90% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 95% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 96% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 98% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99.5% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 400 amino acids in length.

[0093] In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 80% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 85% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 90% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 95% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 96% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 98% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99.5% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 500 amino acids in length.

[0094] In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 80% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 85% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 90% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 95% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 96% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 98% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99.5% sequence identity to a contiguous portion of SEQ ID NO: 6 that is at least 600 amino acids in length.

[0095] In some aspects, the modified AP50 polymerase comprises SEQ ID NO: 6. In some aspects, the modified AP50 polymerase comprises a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 80% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 85% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 90% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 95% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 96% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 97% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 98% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 99% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase has at least or about 99.5% sequence identity to SEQ ID NO: 6. In some aspects, the modified AP50 polymerase consists of SEQ ID NO: 6.

[0096] In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 1 that is at least 200, 300, 400, 500, 600, or 700 amino acids in length, or a sequence that has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 200, 300, 400, 500, 600, or 700 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 1 that is 100 to 700, 200 to 600, 300 to 500, or 350 to 450 amino acids in length.

[0097] In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 1 that is at least 200 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 1 that is at least 300 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 1 that is at least 200 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 1 that is at least 300 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length.

[0098] In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 80% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 85% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 90% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 95% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 96% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 98% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99.5% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 400 amino acids in length.

[0099] In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 80% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 85% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 90% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 95% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 96% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 98% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99.5% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 500 amino acids in length.

[0100] In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 80% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 85% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 90% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 95% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 96% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 98% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99.5% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 600 amino acids in length.

[0101] In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 80% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 85% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 90% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 95% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 96% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 98% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length. In some aspects, the modified AP50 polymerase comprises or consists of a sequence having at least or about 99.5% sequence identity to a contiguous portion of SEQ ID NO: 1 that is at least 700 amino acids in length.

[0102] In some aspects, the modified AP50 polymerase comprises SEQ ID NO: 1. In some aspects, the modified AP50 polymerase comprises a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 80% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 85% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 90% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 95% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 96% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 97% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 98% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 99% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has at least or about 99.5% sequence identity to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase consists of SEQ ID NO: 1.

[0103] In some aspects, exemplary modified AP50 polymerases comprise one or more deletions, insertions and / or substitutions, including any combination of modifications or variations described herein with reference to SEQ ID NO: 1. In some aspects, such modifications or variations can be used to improve aspects of polymerase activity and / or other properties. In some aspects, the modified AP50 polymerase comprises an amino acid deletion of 50 to 100 amino acids compared to SEQ ID NO: 1. In some aspects, the length of the modified AP50 polymerase does not exceed 732 amino acids. In some aspects, the modified AP50 polymerase comprises a full-length AP50 polymerase or a contiguous portion thereof comprising no more than 732 amino acid residues. In some aspects, the modified AP50 polymerase consists of a contiguous portion of SEQ ID NO: 6 having a length of at least 400 amino acids, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with any of the foregoing sequences. In some aspects, the modified AP50 polymerase comprises SEQ ID NO: 6. In some aspects, the modified AP50 polymerase consists of SEQ ID NO:6.

[0104] In some aspects, a modified AP50 polymerase (e.g., a full-length AP50 polymerase or an AP50 polymerase containing a modification (such as a deletion or truncation) or a contiguous portion thereof) comprising an AP50 polymerase domain has a certain amino acid length. In some aspects, the length of the modified AP50 polymerase is no more than 732, 731, 700, 650, 638, 637, 600, 550, 500, 450, or 400 amino acids, or within the range defined by any of the foregoing. In some aspects, the length of the modified AP50 polymerase is 732 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 731 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 638 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 637 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 600 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 500 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 732 amino acids. In some aspects, the length of the modified AP50 polymerase is 731 amino acids. In some aspects, the length of the modified AP50 polymerase is 638 amino acids. In some aspects, the length of the modified AP50 polymerase is 637 amino acids. In some aspects, the length of the modified AP50 polymerase is 600 amino acids. In some aspects, the length of the modified AP50 polymerase is 500 amino acids.

[0105] In some aspects, the modified AP50 polymerase is a functional nucleic acid polymerase, eg, having DNA or RNA polymerase activity.

[0106] In some aspects, provided herein are modified AP50 polymerases comprising an amino acid substitution at one or more positions compared to a wild-type AP50 polymerase, e.g., the AP50 polymerase sequence set forth in SEQ ID NO: 1. In some aspects, provided herein are modified AP50 polymerases comprising an amino acid insertion at one or more positions compared to a wild-type AP50 polymerase.

[0107] In some aspects, provided herein are modified AP50 polymerases that further comprise a heterologous amino acid sequence fused to the N-terminus and / or C-terminus compared to a wild-type AP50 polymerase.

[0108] Exemplary modified AP50 polymerases provided include one or more amino acid substitutions at one or more positions. In some aspects, with reference to SEQ ID NO: 6, the modified AP50 polymerase includes one or more amino acid substitutions. In some aspects, with reference to SEQ ID NO: 6, the modified AP50 polymerase includes two or more amino acid substitutions. In some aspects, the modified AP50 polymerase including one or more amino acid substitutions has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 6. In some aspects, with reference to SEQ ID NO: 1, the AP50 polymerase includes one or more amino acid substitutions. In some aspects, with reference to SEQ ID NO: 1, the modified AP50 polymerase includes two or more amino acid substitutions. In some aspects, the modified AP50 polymerase comprising one or more amino acid substitutions has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 1. Exemplary modified AP50 polymerases comprise conservative or non-conservative amino acid substitutions. Exemplary modified AP50 polymerases may also comprise non-natural and / or synthetic amino acid substitutions.

[0109] In some aspects, the modified AP50 polymerase provided herein comprises one or more amino acid insertions. Examples of amino acid insertions include N-terminal insertions, C-terminal insertions, and internal insertions of single or multiple amino acids. In some aspects, with reference to SEQ ID NO: 6, the modified AP50 polymerase comprises one or more amino acid insertions. In some aspects, with reference to SEQ ID NO: 6, the modified AP50 polymerase comprises two or more amino acid insertions. In some aspects, the modified AP50 polymerase comprising one or more amino acid insertions has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 6. In some aspects, with reference to SEQ ID NO: 1, the AP50 polymerase comprises one or more amino acid insertions. In some aspects, with reference to SEQ ID NO: 1, the modified AP50 polymerase comprises two or more amino acid insertions. In some aspects, the modified AP50 polymerase comprising one or more amino acid insertions has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 1. Exemplary modified AP50 polymerases comprise conservative or non-conservative amino acid insertions. In some instances, the AP50 polymerase may also comprise non-natural and / or synthetic amino acid insertions.

[0110] In some aspects, the modified AP50 polymerase further comprises one or more heterologous sequences. In some embodiments of any of the embodiments herein, the recombinant AP50 polymerase comprises an affinity tag, a linker, and / or a solubility tag. In some aspects, the provided AP50 polymerases (such as those comprising one or more amino acid deletions and / or substitutions) further comprise one or more heterologous sequences. In some aspects, the modified AP50 polymerase comprises a deletion or truncation, and one or more heterologous sequences, compared to the full-length wild-type AP50 polymerase.

[0111] In some aspects, the length of the heterologous sequence is 1 to 200, 2 to 180, 3 to 160, 5 to 140, 6120, 7 to 100, 8 to 80, 960, 10 to 40, 1530, 512, or 8 to 20 amino acid residues. In some aspects, the heterologous sequence is 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 a range of amino acid residues defined by any of the foregoing. In some aspects, the length of the heterologous sequence does not exceed 200, 180, 160, 140, 120, 100, 80, 60, 40, 30, 20, or 10 amino acids. In some aspects, the length of the heterologous sequence does not exceed 200 amino acids. In some aspects, the length of the heterologous sequence does not exceed 150 amino acids. In some aspects, the heterologous sequence is no more than 100 amino acids in length.

[0112] Exemplary heterologous sequences include N-terminal and / or C-terminal fusions ranging in length from one amino acid to a polypeptide containing one hundred or more amino acids, and internal insertions of heterologous sequences. Examples of heterologous sequences include the addition of affinity tags, linkers, and / or solubility domains. Other polypeptide domains that can be fused at the N-terminus and / or C-terminus include domains from polypeptides or proteins, such as antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, binding proteins, or ligands. In some aspects, such heterologous sequences can change or improve one or more properties of a polymerase.

[0113] In some aspects, the modified AP50 polymerase comprises one or more heterologous sequences, such as tags and / or joints. In some aspects, the modified AP50 polymerase comprises two or more heterologous sequences, such as tags and / or joints. In some aspects, the modified AP50 polymerase comprises three or more heterologous sequences, such as tags and / or joints. In some aspects, the modified AP50 polymerase (e.g., including one or more heterologous sequences) has a certain amino acid length. In some aspects, the length of the modified AP50 polymerase (e.g., including one or more heterologous sequences) does not exceed 900, 800, 742, 741, 732, 731, 700, 650, 659, 658, 638, 637, 600, 550, 500, 450 or 400 amino acids, or within the range defined by any of the foregoing. In some aspects, the length of the modified AP50 polymerase is 742 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 741 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 732 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 731 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 659 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 658 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 638 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 637 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 600 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 500 amino acids or less. In some aspects, the length of the modified AP50 polymerase is 742 amino acids. In some aspects, the length of the modified AP50 polymerase is 741 amino acids. In some aspects, the length of the modified AP50 polymerase is 732 amino acids. In some aspects, the length of the modified AP50 polymerase is 731 amino acids. In some aspects, the length of the modified AP50 polymerase is 659 amino acids. In some aspects, the length of the modified AP50 polymerase is 658 amino acids. In some aspects, the length of the modified AP50 polymerase is 638 amino acids. In some aspects, the length of the modified AP50 polymerase is 637 amino acids. In some aspects, the length of the modified AP50 polymerase is 600 amino acids. In some aspects, the length of the modified AP50 polymerase is 500 amino acids.

[0114] In some aspects, the heterologous sequence is fused to the N-terminus and / or C-terminus of an AP50 polymerase domain (e.g., a full-length AP50 polymerase or an AP50 polymerase containing a modification (such as a deletion or truncation). In some aspects, the heterologous sequence is fused to the N-terminus of the AP50 polymerase domain. In some aspects, the heterologous sequence is fused to the C-terminus of the AP50 polymerase domain. In some aspects, the heterologous sequence is fused to the N-terminus of the AP50 polymerase domain and another heterologous sequence is fused to the C-terminus of the modified AP50 polymerase domain.

[0115] In some aspects, the modified AP50 polymerase comprises two or more heterologous sequences. In some aspects, the modified AP50 polymerase comprises an affinity tag, such as a polyhistidine (His) tag. In some aspects, the modified AP50 polymerase comprises a solubility tag, such as a solubility enhancement tag (SET). In some aspects, the modified AP50 polymerase comprises an amino acid linker that connects an AP50 polymerase domain or a continuous portion thereof (e.g., a full-length AP50 polymerase or an AP50 polymerase containing a modification (e.g., a deletion or truncation)) to a tag. In some aspects, two or more heterologous sequences are adjacent to each other. In some aspects, two or more heterologous sequences are indirectly fused, such as separated by one or more amino acid residues.

[0116] In some aspects, the heterologous sequence includes a tag, such as an affinity tag and / or a solubility tag. In some aspects, the tag is a tag for recombinant purification, substrate binding or adding other tags. Exemplary tags include purification, substrate binding or other tags, such as polyhistidine (His) tags, His10 tags, His6 tags, alanine tags, Ala10 tags, Ala16 tags, biotin tags, biotin ligase recognition sequences or other biotin attachment sites (e.g., BiTag or Btag or variants thereof, such as BtagV1-11), GST tags, S tags, SNAP tags, hemagglutinin (HA) tags, DSB (Sso7D) tags, lysine tags, NanoTags, Cmyc tags. In some aspects, the tag is selected from the following: polyhistidine (HIS) tag, solubility enhancement tag (SET), small ubiquitin-modified (SUMO) tag, Fasciola hepatica 8-kDa antigen (Fh8) tag, thioredoxin (Trx) tag, glutathione-s-transferase (GST) tag, maltose binding protein (MBP) tag, IgG domain B1 (GB1) tag of protein G, 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 aspects, the tag is a polyHIS tag. In some aspects, the tag comprises the sequence shown in SEQ ID NO: 7. In some aspects, the tag is a SET tag. In some aspects, the tag comprises the sequence shown in SEQ ID NO: 8. In some aspects, the tag is a polyHIS tag and a SET tag. In some aspects, the heterologous sequence comprises the sequence set forth in SEQ ID NO: 9. In some aspects, the tag is a SUMO tag. In some aspects, the tag comprises the sequence set forth in SEQ ID NO: 26.

[0117] For example, during recombinant expression, purification and / or post-purification, an exemplary heterologous sequence can be included to improve the solubility of the modified AP50 polymerase. In some aspects, the heterologous sequence comprises a tag for improving the solubility of the modified AP50 polymerase. In some aspects, the tag is a solubility enhancement tag (SET). In some aspects, the tag has a net negative charge at pH 7. In some aspects, the tag comprising a negative charge improves the solubility and / or stability of the AP50 polymerase. In some aspects, 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 aspects, the tag has a net negative charge of -1, -2, -4, -6, -10, -12, -14, -15, -16, -17 or -18. In some aspects, the tag comprises one or more acidic amino acid residues, for example, aspartic acid (Asp, D) and / or glutamic acid (Glu, E). In some aspects, the tag comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more acidic amino acid residues. In some aspects, 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. In some aspects, the tag comprises the sequence shown in SEQ ID NO: 8 or 10. In some aspects, the tag comprises SEQ ID NO: 8. In some aspects, the tag comprises SEQ ID NO: 10.

[0118] In some aspects, the heterologous sequence comprising the tag is directly fused to the AP50 polymerase domain. In some aspects, the tag is directly fused to the N-terminus and / or C-terminus of the AP50 polymerase domain. In some aspects, the tag is indirectly fused to the AP50 polymerase domain. In some aspects, the tag is fused to a second heterologous sequence, such as a linker or another tag. In some aspects, the tag is inserted into the interior of the AP50 polymerase domain. In some embodiments of any of the embodiments herein, the recombinant AP50 polymerase is directly fused to a solubility tag located at the C-terminus of the AP50 polymerase domain. In some embodiments of any of the embodiments herein, the recombinant AP50 polymerase is indirectly fused to a solubility tag located at the C-terminus of the AP50 polymerase domain via a linker.

[0119] In some aspects, the heterologous sequence comprises one or more polypeptide domains, such as a fusion domain. In some aspects, the tag is a tag for recombinant expression and purification, substrate binding, or improving polymerase performance. In some aspects, the polypeptide domain comprises a domain selected from the group consisting of a helix-hairpin-helix (HhH) DNA binding motif, a helix-hairpin-helix fusion [(HhH) 2 ]DNA binding motif, single-stranded DNA binding domain, SD07 DNA binding motif, SS07 DNA binding motif and Escherichia coli HU domain.

[0120] In some aspects, the polypeptide domain is a HhH domain. In some aspects, exemplary tags include, for example, those described in Ordonez et al., Sci Rep. 2020; 10: 15046 and Pavlov et al., PNAS 2002; 99(21): 13510-5. In some aspects, an exemplary polypeptide domain comprises an HhH domain or motif from any of the following proteins: Accession No. Q93H64 from Streptomyces avermitilis; Accession No. Q9CEH6 from Lactococcus lactis; Accession No. 000847 from Trypanosoma brucei; Accession No. Q8Y7P2 from Listeria monocytogenes; Accession No. RL32_HALN1 from Halobacterium; Accession No. Q99YR8 from Streptococcus pyogenes; Accession No. Q55769 from Synechocystis; Accession No. YACK_BACSU from Bacillus subtilis; Accession No. Q91VP3 from Mus musculus; Accession No. Q91VP3 from Caenorhabditis elegans. elegans, accession number P91153; from Thermus aquaticus, accession number P77987; from Bacillushalodurans, accession number Q9KGG0; from Methanosarcina mazei goel, accession number AAM31590; from Myxococcus xanthus, accession number Q9APNO; from Clostridium perfringens, accession number Q8XHQ0; from Homo sapiens, accession number Q9C0F7; from Salmonella typhimurium, accession number Q8XGV0; from Methanobacterium thermoautotrophicum, accession number O26650; from Methanopyrus kandleri)av19, accession number AAM02649; from Mus musculus, accession number Q9D7J3; from Xanthomonas axonopodis, accession number AAM38234; from Clostridium difficile, accession number P94624; from Pyrococcus horikoshii, accession number O74103;Accession number AAK99660 from Streptococcus pneumoniae r6; Accession number AAM02248 from Methanopyrus kandleri av19; Accession number AAH28155 from Homo sapiens; Accession number P71728 from Mycobacterium tuberculosis; Accession number AAM06574 from Methanosarcina acetivorans str; Accession number Q9YC15 from Aeropyrum pernix; Accession number AAK99347 from Streptococcus pneumoniae r6; Accession number AAL93832 from nucleatum; Accession number AAH08930 from Homo sapiens; Accession number EX9_ECOLI from Escherichia coli; Accession number Pyrococcus furiosus dsm 3638, accession number AAL81517; accession number Q8Y3B2 from Ralstonia solanacearum; accession number Q8XBD5 from Escherichia coli o157:h7; accession number Q8XIJ9 from Clostridium perfringens; accession number Q9PJA0 from Campylobacter jejuni; accession number Q9RA49 from Thermus aquaticus; accession number AAM25145 from Thermoanaerobacterium tengchongensis; accession number BAB95406 from Staphylococcus aureus ...XBD5 from kandleri)av19, accession number AAM01328; from Tengchong Thermoanaerobacterium tengchong (Tengchong Thermoanaerobacterium tengchong), accession number AAM25645; from nucleatum, accession number AAL95150; from Bacillus licheniformis, accession number Q8VQ77; from Methanococcusjannaschii, accession number HELS_METJA; from Listeria innocua, accession number Q92D76; from nucleatum, accession number AAL95286; from Escherichia coli o157:h7, accession number Q8XBL5; from Pseudomonas aeruginosa, accession number Q9HZ87; from Hydra oligactis, accession number Q9GRA1; from Rhizobium loti) accession number Q98AC3; from Spirogyra maxima accession number RPOA_SPIMX;Accession number AAL91594 from Homo sapiens; Accession number Q8Y8F9 from Listeria monocytogenes; Accession number Q99LC7 from Mus musculus; Accession number P72464 from Streptomyces lividans; Accession number Q96K64 from Homo sapiens; Accession number Q974S1 from Sulfolobus tokodaii; Accession number Q9HMU5 from Halobacterium; Accession number Q8XE54 from Escherichia coli o157:h7; Accession number Q96NY9 from Homo sapiens; Accession number AAM34796 from Homo sapiens; Accession number AAL98145 from Streptococcus pyogenes mgas8232; Accession number AAL98145 from Methanobacterium thermoautotrophicum thermoautotrophicum, accession number 027790; from Dictyostelium discoideum, accession number AAL92998; from Methanosarcina acetivorans str, accession number AAM05700; from Methanococcus maripaludis, accession number RADA_METMP; from Streptococcus pneumoniae r6, accession number AAK99292; from Methanosarcina acetivorans str, accession number AAM04167; from Xenopus laevis, accession number Q9I869; from Methanosarcina acetivorans acetivorans)str, accession number AAM06907; accession number Q94K65 from Arabidopsis thaliana; accession number Q8XIE6 from Clostridium perfringens; accession number AAH27384 from Mus musculus; accession number Q97EC6 from Clostridium acetobutylicum; accession number AAM23354 from Thermoanaerobacterium tengchongensis; accession number Q9FF89 from Arabidopsis thaliana; accession number AAM02891 from Methanopyrus kandleri av19; accession number HELS_METTH from Methanobacterium thermoautotrophicum;Accession number AAL82139 from Pyrococcus furiosus dsm 3638; Accession number 030253 from Archaeoglobus fulgidus; Accession number Q9HSD6 from Halobacterium; Accession number Q9WY43 from Thermotoga maritima; Accession number AAK84937 from Drosophila melanogaster; Accession number AAL98006 from Streptococcus pyogenes mgas8232; Accession number 085197 from Streptococcus pneumoniae; Accession number 085197 from Methanosarcina mazei mazei)goel accession number AAM31083; from Tengchong Thermoanaerobacterium (Tengchong Thermoanaerobacterium) accession number AAM24182; from Halobacterium (Halobacterium) accession number Q9HMW5; from Bacillus subtilis accession number RECR_BACSU; from Homo sapiens accession number Q9BTN8; from Aeropyrum pernix accession number Q8X233; and from Drosophila melanogaster accession number NOD_DROME. In some aspects, the polypeptide domain comprises [(HhH); 2 ] domain, e.g., as described in Ordonez et al., SciRep. 2020; 10: 15046. In some aspects, the polypeptide domain comprises [(HhH) 2 ] domain, which comprises one or more of any HhH domains described herein. In some aspects, the polypeptide domain comprises [(HhH) 2 ] domain, which comprises a fusion of any two or more HhH domains described herein. In some aspects, [(HhH) 2 ] domain is a fusion of the H and I DNA binding domains of topoisomerase V from the thermophilic bacterium Methanothermus cannelligensis.

[0121] In some aspects, the heterologous sequence comprises one or more joints. In some aspects, the modified AP50 polymerase comprises two or more joints. In some aspects, the joint comprises one or more glycine, serine, alanine, histidine, arginine, lysine, glutamine and / or proline residues. In some aspects, the joint comprises one or more glycine and / or serine residues. In some aspects, the length of the joint is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues, or a range limited by any of the foregoing. In some aspects, the length of the joint 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.

[0122] In some aspects, the linker is a flexible linker or a rigid linker. In some aspects, the linker comprises a cleavable linker sequence. The cleavable linker is usually cleaved, for example, by an enzymatic or reduction mechanism. Exemplary cleavable linkers include protease-sensitive linkers, cyclic peptide linkers, and / or disulfide-sensitive linkers. In some aspects, 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 aspects, the modified AP50 polymerase comprises a heterologous sequence containing a cleavable sequence. In some aspects, the cleavable sequence is contained in a linker. In some aspects, the cleavable sequence is contained in a non-linker sequence. For example, after recombinant expression and / or purification, an exemplary cleavable sequence can be included to remove one or more heterologous sequences from the modified AP50 polymerase. In some aspects, the linker comprises any one of SEQ ID NOs: 10-16. In some aspects, the linker comprises SEQ ID NO: 10. In some aspects, the linker comprises SEQ ID NO: 11. In some aspects, the linker comprises SEQ ID NO: 12. In some aspects, the linker comprises SEQ ID NO: 13. In some aspects, the linker comprises SEQ ID NO: 14. In some aspects, the linker comprises SEQ ID NO: 15. In some aspects, the linker comprises SEQ ID NO: 16.

[0123] In some aspects, the linker is fused to the N-terminus and / or C-terminus of an AP50 polymerase domain (e.g., a full-length AP50 polymerase or an AP50 polymerase containing modifications such as deletions or truncations). In some aspects, the linker is fused directly to the N-terminus and / or C-terminus of the AP50 polymerase domain. In some aspects, the linker is fused indirectly to the N-terminus and / or C-terminus of the AP50 polymerase domain. In some aspects, the linker is fused to the C-terminus of the AP50 polymerase domain.

[0124] In some aspects, the modified AP50 polymerase comprises two or more heterologous sequences, such as tags and / or linkers. In some aspects, the modified AP50 polymerase comprises three or more heterologous sequences, such as tags and / or linkers. In some aspects, the modified AP50 polymerase comprises one or more sequences selected from any one of SEQ ID NOs: 7, 8, 10, and 26. In some aspects, the modified AP50 polymerase comprises one or more sequences selected from any one of SEQ ID NOs: 10-16. In some aspects, the modified AP50 polymerase comprises a heterologous sequence comprising one or more polyhistidine (His) tags and a SET tag. In some aspects, the modified AP50 polymerase comprises a heterologous sequence containing a linker.

[0125] In some aspects, the modified AP50 polymerase comprises, in N-terminal to C-terminal order: SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 7. In some aspects, the modified AP50 polymerase comprises, in N-terminal to C-terminal order: SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 7, and SEQ ID NO: 8. In some aspects, the modified AP50 polymerase comprises, in N-terminal to C-terminal order: SEQ ID NO: 6 and SEQ ID NO: 9. In some aspects, the modified AP50 polymerase comprises, in N-terminal to C-terminal order: SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 7. In some aspects, the modified AP50 polymerase comprises, in N-terminal to C-terminal order: SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 7, and SEQ ID NO: 8. In some aspects, the modified AP50 polymerase comprises any one of SEQ ID NOs: 2-4. In some aspects, the modified AP50 polymerase comprises SEQ ID NO: 2. In some aspects, the modified AP50 polymerase comprises SEQ ID NO: 3. In some aspects, the modified AP50 polymerase comprises SEQ ID NO: 4. In some aspects, the modified AP50 polymerase consists of any one of SEQ ID NOs: 2-4. In some aspects, the modified AP50 polymerase consists of SEQ ID NO: 2. In some aspects, the modified AP50 polymerase consists of SEQ ID NO: 3. In some aspects, the modified AP50 polymerase consists of SEQ ID NO: 4.

[0126] In some aspects, the modified AP50 polymerases provided herein can be used as part of a chimeric polymerase, such as a polymerase made from chimeras of different sources or origins. Chimeras can be generated, for example, using regions of similarity between polymerases to define a consensus sequence used in the chimera, or using gene shuffling techniques, such as where multiple AP50 polymerases or related polymerases are randomly or semi-randomly shuffled via available gene shuffling techniques (e.g., via "family gene shuffling"; see Crameri et al. (1998) Nature 391: 288-291; Clackson et al. (1991) Nature 352: 624-628; Gibbs et al. (2001) Gene 271: 13-20; and Hiraga and Arnold (2003) J. Mol. Biol. 330: 287-296). In certain aspects, to generate chimeric polymerases, recombination points can be predetermined so that gene fragments are assembled in the correct order or randomly. In some aspects, any modification or variant described herein can be introduced into a chimera to achieve improvement in a desired property or characteristic.

[0127] Exemplary modified AP50 polymerases include any combination of modifications or variations, such as deletions, insertions, and substitutions, including any combination of modifications or variations described herein, or with any known modification or variation with reference to SEQ ID NO: 1. In some aspects, the modified AP50 polymerase has improved activity and / or properties, such as any of the properties described herein. 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.

[0128] In some aspects, amino acid sequence variants of modified AP50 polymerases can be prepared by introducing appropriate modifications into a nucleotide sequence encoding a wild-type AP50 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 a wild-type AP50 polymerase. The modified AP50 polymerases described herein can be recombinantly produced and / or purified as recombinant polymerases from one or more suitable expression systems (e.g., cell systems or cell-free systems).

[0129] In some aspects, compositions comprising recombinant AP50 polymerases are provided, e.g., recombinantly produced and / or purified modified AP50 polymerases, such as those comprising one or more amino acid substitutions, insertions and / or deletions, as described herein. In some aspects, the compositions have a purity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% relative to the recombinant AP50 polymerase. 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 AP50 polymerases. In some aspects, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptides or proteins of the composition are recombinant AP50 polymerases. In some aspects, the composition comprising the recombinant AP50 polymerase has a high level of purity and / or is nearly pure relative to other biomolecules (e.g., other polypeptides, proteins, amino acids, nucleotides, or polynucleotides) present in the composition. In some aspects, biomolecules other than the recombinant AP50 polymerase can be considered contaminants in the composition. In some aspects, the composition has less than or about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of contaminants.

[0130] B. Exemplary Features of Modified AP50 Polymerases

[0131] In some aspects, the recombinant and / or modified AP50 polymerase provided can exhibit 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 nucleic acid amplification. Exemplary improved features can relate to modified AP50 polymerase activity, function, performance, robustness, utility or a combination thereof. Exemplary improved features of the AP50 polymerase provided relate to, for example, thermal stability, processivity, sensitivity, specificity, solubility and polymerization rate. Other exemplary features can relate to increased yield, altered cofactor selectivity, exonuclease deficiency and / or increased resistance to light damage. In some aspects, the modified AP50 polymerase provided exhibits improved thermal stability, processivity, sensitivity, specificity, solubility and / or polymerization rate, and / 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 aspects, the modified AP50 polymerase has strand displacement activity. In some aspects, the modified AP50 polymerase has exonuclease activity. In some aspects, the modified AP50 polymerase has reverse transcriptase activity. In some embodiments, one or more features are improved compared to a reference polymerase, such as a reference polymerase used for similar applications (e.g., nucleic acid amplification) or known to have similar functions (e.g., nucleic acid polymerization). In some embodiments, one or more features are improved compared to a reference AP50 polymerase (e.g., a wild-type AP50 polymerase). In some embodiments, one or more features are improved compared to a reference Phi29 polymerase (e.g., a wild-type Phi29 polymerase) shown in SEQ ID NO: 5. The improvement in one or more features supports the expanded application and / or utility of recombinant and / or modified AP50 polymerases, for example for nucleic acid amplification and / or sequencing. In some embodiments, the modified AP50 polymerase provided has one or more features that result in improved nucleic acid amplification and / or nucleic acid amplification methods (such as rolling circle amplification (RCA)) of biological samples.

[0132] 1. Improved thermal stability

[0133] In some aspects, the thermal stability of a polymerase (e.g., wild-type Phi29 polymerase) used for a specific nucleic acid amplification method (e.g., RCA) may be limited. In some cases, the typical reaction temperature for wild-type Phi29 polymerase activity is about 30°C. In some aspects, many biological or chemical experiments, reactions, and / or analyses require higher temperatures. For example, specific steps of nucleic acid amplification and / or sequencing methods (e.g., probe connection) are performed at higher temperatures or require higher temperatures, for example, for maximum efficiency. In some instances, most tissue culture growth, manipulations, and experiments need to be performed at higher temperatures. Compared to polymerase activity at 30°C, the use of wild-type Phi29 polymerase at elevated temperatures may result in reduced polymerization rates, yields, and polymerase stability. In some cases, the use of Phi29 polymerase at temperatures such as 30°C may complicate the experimental method by introducing the need for multiple temperatures and temperature switching, making the method applicable or suitable for high-throughput and high-capacity applications in some cases.

[0134] In some embodiments, the recombinant and / or modified AP50 polymerase described herein exhibits improved thermostability. In some aspects, improved thermostability includes improved polymerase activity. In some embodiments, improved thermostability includes improved, faster, more stable and / or more accurate polymerase activity, for example, at elevated temperatures. In some embodiments, the modified AP50 polymerase provided (for example, those described in Part II.A) exhibits improved thermostability. In some embodiments, the modified AP50 polymerase exhibiting improved thermostability can be used for nucleic acid amplification reactions, such as DNA amplification reactions. In some embodiments, the modified AP50 polymerase is 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, 45°C, 50°C or higher temperatures. In some embodiments, the modified AP50 polymerase is used for nucleic acid amplification performed at or about 5°C to 55°C, 10°C to 50°C, 25°C to 45°C or 30°C to 40°C. In some embodiments, the modified AP50 polymerase is used in nucleic acid amplification performed at elevated temperatures. In some embodiments, the modified AP50 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, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C or more. In some embodiments, the modified AP50 polymerase is used in nucleic acid amplification performed at or about 30°C to 55°C, 32°C to 53°C, 34°C to 51°C, 36°C to 49°C, 38°C to 47°C, or 40°C to 45°C.

[0135] In some embodiments, the AP50 polymerase provided is more active (e.g., produces more amplified DNA) at the same concentration or lower concentration compared to a reference polymerase (e.g., Phi29 polymerase). In some embodiments, the recombinant and / or modified AP50 polymerase provided produces more amplified DNA compared to a reference polymerase (e.g., the wild-type Phi29 polymerase of SEQ ID NO: 5). In some embodiments, the recombinant and / or modified AP50 polymerase provided produces more amplified DNA at elevated temperatures compared to a reference polymerase. In some embodiments, the recombinant and / or modified AP50 polymerase provided produces more amplified DNA compared to the wild-type AP50 polymerase of SEQ ID NO: 1. In some embodiments, fluorescent dyes are 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 dyes described herein. In some embodiments, the modified AP50 polymerase produces at or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% more amplified DNA compared to a reference polymerase. In some embodiments, the modified AP50 polymerase produces 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% more amplified DNA compared to a reference polymerase.

[0136] In some embodiments, the modified AP50 polymerase produces more amplified DNA compared to a reference polymerase, such that polymerization reaction time can be reduced, the amount of input template DNA can be reduced, product signal from increased DNA yield can be increased, or a combination thereof. In some embodiments, polymerization reaction time can be reduced by at least or about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 75% using the provided recombinant and / or modified AP50 polymerases compared to a reference polymerase. In some embodiments, the input template DNA required can be reduced by at least or about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 75% using the provided recombinant and / or modified AP50 polymerases compared to a reference polymerase. In some embodiments, the signal from the increased DNA yield using the provided recombinant and / or modified AP50 polymerases is increased by at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% compared to a reference polymerase. In some embodiments, the improved thermal stability of the modified AP50 polymerases results in increased DNA amplification and DNA yield at temperatures including, but not limited to, temperatures of or about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, or more. In some embodiments, the improved thermostability of the modified AP50 polymerase results in increased DNA amplification and DNA yield at elevated temperatures, including but not limited to temperatures 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., 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C. In some embodiments, provided AP50 polymerases exhibit improved kinetics at a temperature of at least about 42° C. compared to a reference polymerase.

[0137] In some embodiments, the recombinant and / or modified AP50 polymerase provided amplifies nucleic acids, such as DNA, faster than a reference polymerase. In some embodiments, the recombinant and / or modified AP50 polymerase provided amplifies nucleic acids faster at elevated temperatures compared to a reference 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 dyes described herein. In some embodiments, the modified AP50 polymerase amplifies nucleic acids, such as DNA, at a polymerization rate (e.g., nucleic acids, 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 polymerase. In some embodiments, the modified AP50 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 compared to a reference polymerase. For example, a provided recombinant and / or modified AP50 polymerase produces 100% to 200% more amplified nucleic acid, e.g., DNA, within 1 hour compared to a reference polymerase. In some embodiments, a modified AP50 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.

[0138] 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 modified AP50 polymerase described herein exhibits an improved polymerization rate. In some embodiments, the modified AP50 polymerase exhibits an improved polymerization rate compared to a reference polymerase. In some embodiments, the recombinant and / or modified AP50 polymerase provided is used in a nucleic acid amplification process. In some embodiments, the recombinant and / or modified AP50 polymerase provided produces more amplified nucleic acids compared to a reference polymerase. In some embodiments, nucleic acid amplification is an RCA reaction. In some embodiments, the recombinant and / or modified AP50 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 modified AP50 polymerase provided generates a higher product signal intensity in an RCA reaction compared to a reference polymerase.

[0139] In some embodiments, the recombinant and / or modified AP50 polymerases provided produce amplified nucleic acids, such as DNA, at a faster rate than a reference polymerase, such that polymerization reaction times can be reduced and / or product signals from increased amplification of nucleic acids, such as DNA, can be increased. In some embodiments, the polymerization reaction time can be reduced by at least or about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 75% using the recombinant and / or modified AP50 polymerases provided compared to a reference polymerase. In some embodiments, the amount of nucleic acid, such as DNA, produced or the yield of reaction products is increased by at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% using the recombinant and / or modified AP50 polymerases provided compared to a reference polymerase. In some embodiments, the improved thermostability of the modified AP50 polymerase results in an increased rate of nucleic acid amplification at a particular temperature, including but not limited to, at or about 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C or more. In some embodiments, the improved thermostability of the modified AP50 polymerase results in an increased rate of nucleic acid amplification at an elevated temperature, 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, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C.

[0140] In some embodiments, the recombinant and / or modified AP50 polymerase provided 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 recombinant and / or modified AP50 polymerase provided generates a higher density of detected rolling circle amplification (RCA) products in an RCA reaction compared to a reference 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 density of detected RCPs is measured by counting the RCA products of the sample area. In some embodiments, the density of detected RCPs is related to the total sample signal. In some embodiments, compared with a reference polymerase, the recombinant and / or modified AP50 polymerase provided generates a higher density of detected RCPs in the RCA reaction. In some embodiments, compared with a reference polymerase, the recombinant and / or modified AP50 polymerase provided produces a density of detected RCA products that is at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150% or 200% higher. In some embodiments, compared with a reference polymerase, the recombinant and / or modified AP50 polymerase provided produces a density of detected RCA products that is 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110% or 95% to 105% higher.

[0141] 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 a reference polymerase, the recombinant and / or modified AP50 polymerase provided generates a higher product signal intensity in the RCA reaction. In some embodiments, compared with a reference polymerase, the recombinant and / or modified AP50 polymerase provided produces a high RCA reaction product signal intensity of at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150% or 200%. In some embodiments, compared with a reference polymerase, the recombinant and / or modified AP50 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%.

[0142] In some embodiments, the RCA reaction using the provided recombinant and / or modified AP50 polymerase is performed at a temperature of or about 5° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C. or more. In some embodiments, the RCA reaction is performed at 5° C. to 55° C., 10° C. to 50° C., 25° C. to 45° C., 30° C. to 40° C., or 35° C. to 45° C. In some embodiments, the RCA reaction using the provided recombinant and / or modified AP50 polymerase is performed 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., 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C. In some embodiments, the RCA reaction is carried out at 30° C. to 55° C., 32° C. to 53° C., 34° C. to 51° C., 36° C. to 49° C., 38° C. to 47° C., or 40° C. to 45° C.

[0143] 2. Other exemplary features

[0144] In some aspects, the recombinant and / or modified AP50 polymerases provided exhibit other features or improvements, such as features or improvements related to polymerase activity and use in analyzing or evaluating biological samples (such as in nucleic acid amplification and sequencing). Examples of other improved or altered properties and characteristics include improved or altered processivity, sensitivity, specificity, solubility, polymerization rate, product yield, increased half-life, cofactor selectivity, exonuclease activity, resistance to light damage, strand displacement activity, exonuclease activity, or reverse transcriptase activity. In some embodiments, the recombinant and / or modified AP50 polymerases provided (such as those described in Section II.A) exhibit one or more altered or improved characteristics (e.g., thermal stability, processivity, sensitivity, specificity, solubility, and / or polymerization rate). The recombinant and / or modified AP50 polymerases provided may also exhibit two or more altered features in combination, or have additional improved or altered properties. In some aspects, the modified AP50 polymerase has strand displacement activity. In some embodiments, provided recombinant and / or variant AP50 polymerases exhibit characteristics or improvements associated with polymerase activity in biological samples, such as tissue samples or sections of cell pellets or cell blocks.

[0145] In some aspects, polymerase processivity is the ability of a nucleic acid polymerase to perform continuous nucleic acid synthesis on a template nucleic acid without frequent dissociation. Processivity can be measured by the average number of nucleotides incorporated by a nucleic acid polymerase in a single association / dissociation event. In some embodiments, the modified AP50 polymerase described herein shows improved processivity, wherein the improved processivity improves polymerase activity. In some embodiments, the modified AP50 polymerase shows improved processivity compared to a reference polymerase. In some embodiments, the recombinant and / or modified AP50 polymerase provided produces more amplified nucleic acids compared to a reference polymerase. In some embodiments, the recombinant and / or modified AP50 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 modified AP50 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 modified AP50 polymerase provided generates a higher product signal intensity in an RCA reaction compared to a reference polymerase.

[0146] In some embodiments, the recombinant and / or modified AP50 polymerase produces at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% more rolling circle amplification product than a reference polymerase under the same reaction conditions.

[0147] In some embodiments, the recombinant and / or modified AP50 polymerase produces at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% more amplified nucleic acid compared to a reference polymerase. In some embodiments, the recombinant and / or modified AP50 polymerase produces 10% to 200%, 25% to 175%, 50% to 150%, 75% to 125%, 90% to 110%, or 95% to 105% more amplified nucleic acid compared to a reference polymerase. In some embodiments, the recombinant and / or modified AP50 polymerase produces more amplified nucleic acid compared to a reference polymerase, such that polymerization reaction time can be reduced, the amount of input template nucleic acid can be reduced, product signal from increased nucleic acid yield is increased, or a combination thereof. In some embodiments, a recombinant and / or modified AP50 polymerase amplifies nucleic acid (e.g., product signal per unit time) at least or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, or 200% faster than a reference polymerase. In some embodiments, a recombinant and / or modified AP50 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 than a reference polymerase. For example, a recombinant and / or modified AP50 polymerase produces 100% to 200% more amplified nucleic acid in 1 hour than a reference polymerase. In some embodiments, a recombinant and / or modified AP50 polymerase that exhibits improved thermostability amplifies nucleic acids faster by at least 1, 2, 3, 4, 6, 8, 10, 12 hours or more compared to a reference polymerase. In some embodiments, a recombinant and / or modified AP50 polymerase produces amplified nucleic acids at a faster rate than a reference polymerase, such that polymerization reaction times can be reduced, product signals from increased nucleic acid amplification rates are increased, or a combination thereof.

[0148] In some embodiments, the recombinant and / or modified AP50 polymerase described herein that exhibits one or more altered characteristics (e.g., thermostability, processivity, sensitivity, specificity, solubility 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 recombinant and / or modified AP50 polymerase produces a density of detected RCA products 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. In some embodiments, the recombinant and / or modified AP50 polymerase described herein produces a density of detected RCA products 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. In some embodiments, the recombinant and / or modified AP50 polymerase described herein produces a product signal intensity 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. In some embodiments, the recombinant and / or modified AP50 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.

[0149] In some embodiments, the recombinant and / or modified AP50 polymerases provided herein further exhibit a longer half-life in the absence of a substrate compared to a reference polymerase. In some embodiments, the recombinant and / or modified AP50 polymerases provided herein further exhibit a longer half-life in the absence of a substrate compared to a wild-type AP50 polymerase. In some embodiments, the recombinant and / or modified AP50 polymerases provided herein further exhibit a longer half-life in the presence of a substrate compared to a reference polymerase. In some embodiments, the recombinant and / or modified AP50 polymerases provided herein are more stable in the presence and absence of a substrate compared to a reference polymerase. In some embodiments, the modified AP50 polymerase substrate comprises a nucleotide, a nucleotide derivative, a polynucleotide, or a combination thereof. In some embodiments, the modified AP50 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 polymerase in the presence or absence of a substrate. In some embodiments, the recombinant and / or modified AP50 polymerases provided herein exhibit 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 polymerase in the presence or absence of a substrate.

[0150] In some aspects described herein, the reference polymerase is the wild-type Phi29 polymerase of SEQ ID NO: 5. In some aspects described herein, the reference polymerase is the wild-type AP50 polymerase of SEQ ID NO: 1.

[0151] III. Nucleic Acids, Vectors and Expression Systems

[0152] Also provided herein are polynucleotides, vectors, and expression systems encoding any of the provided recombinant AP50 polymerases and modified AP50 polymerases (e.g., those described in Section II). In some aspects, the provided polynucleotides, vectors, and expression systems can be used to generate the provided recombinant AP50 polymerases and modified AP50 polymerases.

[0153] 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.

[0154] In some embodiments, the modified AP50 polymerase can be a recombinant variant of a native protein in which one or more properties (such as thermostability) have been altered. In some aspects, the nucleic acid is synthetic. In some aspects, the nucleic acid molecule can be modified (e.g., codon optimized) for use in constructs described herein. In some cases, the sequence can be designed to contain terminal restriction site sequences in order to be cloned into a vector.

[0155] A. Polynucleotides

[0156] In some embodiments, polynucleotides encoding recombinant AP50 polymerases are provided herein. In some embodiments, polynucleotides encoding modified AP50 polymerases are provided herein. In some embodiments, the polynucleotides encoding modified AP50 polymerases comprise one or more amino acid modifications or variations (e.g., deletions, insertions, substitutions, and / or combinations thereof) compared to the sequence of the wild-type AP50 polymerase. In some embodiments, the polynucleotides encode any of the provided recombinant and / or modified AP50 polymerases described herein, such as those described in Part II.

[0157] The amino acid sequence variants of modified and / or recombinant AP50 polymerase can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the wild-type AP50 polymerase. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the nucleotide sequence encoding the wild-type AP50 polymerase. The encoded modified AP50 polymerase can comprise any combination of deletions, insertions and substitutions. In some aspects, the encoded modified AP50 polymerase exhibits desired characteristics, for example, improved polymerase activity, characteristics and / or properties.

[0158] In some aspects, provided herein are polynucleotides encoding a modified AP50 polymerase comprising SEQ ID NO: 6 (excluding the initial fMet residue). In some aspects, provided herein are polynucleotides encoding an AP50 polymerase comprising SEQ ID NO: 1 (excluding the initial fMet residue). In some aspects, provided herein are polynucleotides encoding a modified AP50 polymerase comprising an amino acid deletion of at least 1, 5, 10, 25, 50, 75, or 100 amino acids with reference to the positions of SEQ ID NO: 1. In some embodiments, the encoded modified AP50 polymerase has at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 6. In some embodiments, the encoded modified AP50 polymerase has at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 1. In some aspects, provided herein is a polynucleotide encoding a modified AP50 polymerase, further comprising one or more heterologous sequences, such as a tag and / or a linker.

[0159] In some aspects, provided herein is a polynucleotide encoding a modified AP50 polymerase comprising SEQ ID NO: 32 (including the initial fMet residue). In some aspects, provided herein is a polynucleotide encoding an AP50 polymerase comprising SEQ ID NO: 27 (including the initial fMet residue). In some aspects, provided herein is a polynucleotide encoding a modified AP50 polymerase comprising an amino acid deletion of at least 1, 5, 10, 25, 50, 75, or 100 amino acids with reference to the positions of SEQ ID NO: 27. In some embodiments, the encoded modified AP50 polymerase has at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 32. In some embodiments, the encoded modified AP50 polymerase has at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 27. In some aspects, provided herein is a polynucleotide encoding a modified AP50 polymerase, further comprising one or more heterologous sequences, such as a tag and / or a linker.

[0160] In some aspects, polynucleotide sequences encoding any of the provided modified AP50 polymerases (such as any of the polymerases described in Section II) are provided. In some aspects, the encoded modified AP50 polymerase exhibits or has one or more exemplary features or properties, such as any of the features or properties described in Section II.B.

[0161] In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in any one of SEQ ID NOs: 1-4 and 6 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 1 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 2 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 3 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 4 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 6 is provided.

[0162] In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in any one of SEQ ID NOs: 27-30 and 32 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 27 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 28 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 29 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 30 is provided. In some embodiments, a polynucleotide sequence encoding a modified AP50 polymerase set forth in SEQ ID NO: 32 is provided.

[0163] In some embodiments, the polynucleotide comprises any one of SEQ ID NOs: 17-21, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one of SEQ ID NOs: 17-21. In some embodiments, the polynucleotide comprises SEQ ID NO: 17, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 17. In some embodiments, the polynucleotide comprises SEQ ID NO: 17. In some embodiments, the polynucleotide comprises SEQ ID NO: 18, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 18. In some embodiments, the polynucleotide comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises SEQ ID NO: 19, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 19. In some embodiments, the polynucleotide comprises SEQ ID NO: 19. In some embodiments, the polynucleotide comprises SEQ ID NO: 20, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 20. In some embodiments, the polynucleotide comprises SEQ ID NO: 20. In some embodiments, the polynucleotide comprises SEQ ID NO: 21, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 21. In some embodiments, the polynucleotide comprises SEQ ID NO: 21.

[0164] In some embodiments, the polynucleotide comprises any one of SEQ ID NOs: 33-37, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any one of SEQ ID NOs: 33-37. In some embodiments, the polynucleotide comprises SEQ ID NO: 33, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 33. In some embodiments, the polynucleotide comprises SEQ ID NO: 33. In some embodiments, the polynucleotide comprises SEQ ID NO: 34, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 34. In some embodiments, the polynucleotide comprises SEQ ID NO: 34. In some embodiments, the polynucleotide comprises SEQ ID NO: 35, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 35. In some embodiments, the polynucleotide comprises SEQ ID NO: 35. In some embodiments, the polynucleotide comprises SEQ ID NO: 36, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 36. In some embodiments, the polynucleotide comprises SEQ ID NO: 36. In some embodiments, the polynucleotide comprises SEQ ID NO: 37, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 37. In some embodiments, the polynucleotide comprises SEQ ID NO: 37.

[0165] In some embodiments, the polynucleotide sequence encoding the modified AP50 polymerase provided is codon optimized for a host cell or a recombinant expression system to generate a modified AP50 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 necessary 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 the transfected gene and the host cell may affect protein expression and immunogenicity of the nucleic acid construct. 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.

[0166] B. Recombinant Nucleic Acids and Vectors

[0167] In some aspects, provided herein are recombinant nucleic acids comprising a polynucleotide encoding any of the described recombinant and / or modified AP50 polymerases, such as those described in Section II, or any of the described polynucleotides, such as those described in Section III.A. In some aspects, provided herein are vectors comprising a polynucleotide encoding any of the described recombinant and / or modified AP50 polymerases, such as those described in Section II, or any of the described polynucleotides, such as those described in Section III.A. In some embodiments, recombinant nucleic acids and vectors comprise a polynucleotide encoding any of the described recombinant and / or modified AP50 polymerases described herein, e.g., the polynucleotide comprises one or more amino acid modifications or variations, e.g., deletions, insertions, and / or substitutions, compared to the sequence of a wild-type AP50 polymerase.

[0168] In some embodiments, a recombinant nucleic acid molecule comprises a polynucleotide described in Section III.A.

[0169] 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 T7 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.

[0170] In some embodiments, a vector is provided, comprising a polynucleotide encoding any of the described recombinant and / or modified AP50 polymerases (such as those described in Part II), or any of the described polynucleotides (such as those described in Part III.A) or a recombinant nucleic acid molecule described herein (such as those encoding any modified AP50 polymerase described herein). In some embodiments, the vector is a plasmid, a phagemid, 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 proliferation 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 vectors of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.). 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, p BI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, 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.

[0171] C. Host cells and expression systems

[0172] 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.

[0173] 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.

[0174] In some embodiments, the transformed host cell or recombinant expression system produces a recombinant AP50 polymerase or a modified AP50 polymerase. In some embodiments, the host cell or recombinant expression system is used to produce a recombinant AP50 polymerase or a modified AP50 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 modified AP50 polymerases. In some aspects, in a bacterial host cell or expression system (such as Escherichia coli), protein translation is initiated by N-formylmethionine (fMet), which is tRNA-mediated during translation initiation. 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.

[0175] In some embodiments, the polynucleotide sequence encoding the recombinant AP50 polymerase or modified AP50 polymerase is codon-optimized for the host cell or recombinant expression system, for example, as described in Section III.A.

[0176] IV. Methods of use and uses and applications of recombinant and / or modified AP50 polymerases

[0177] Also provided herein are methods involving or using any of the provided recombinant and / or modified AP50 polymerases, compositions or kits comprising the recombinant and / or modified AP50 polymerases, or nucleic acids or vectors encoding the recombinant and / or modified AP50 polymerases, and uses of any of the foregoing, such as for nucleic acid amplification and / or sequencing. In some aspects, the provided recombinant and / or modified AP50 polymerases are used in a variety of analytical or evaluation methods or experiments (e.g., sequencing, genotyping, and amplification reactions) for evaluating biological samples. In some aspects, the provided recombinant and / or modified AP50 polymerases are used to amplify nucleic acids, such as DNA or RNA, including in applications such as rolling circle amplification (RCA).

[0178] In some aspects, methods for amplifying nucleic acids (such as DNA) are provided, comprising contacting a biological sample containing the DNA to be amplified with any of the provided recombinant and / or modified AP50 polymerases and uses thereof, compositions or kits comprising recombinant and / or modified AP50 polymerases, or nucleic acids or vectors encoding recombinant and / or modified AP50 polymerases.

[0179] In some aspects, a method for performing rolling circle amplification (RCA) is provided, comprising using any provided recombinant and / or modified AP50 polymerase, a composition or kit comprising a recombinant and / or modified AP50 polymerase, or a nucleic acid or vector encoding a recombinant and / or modified AP50 polymerase. In some aspects, the method for performing RCA comprises contacting a biological sample containing a nucleic acid (e.g., DNA) to be amplified with any provided recombinant and / or modified AP50 polymerase, a composition or kit comprising a recombinant and / or modified AP50 polymerase, or a nucleic acid or vector encoding a recombinant and / or modified AP50 polymerase. In some aspects, the method thereby amplifies nucleic acids by RCA.

[0180] In some embodiments, the recombinant and / or modified AP50 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 primary probe that recognizes a specific target nucleic acid sequence, and can be amplified via RCA for detection. In some embodiments, a primary probe, such as a circularizable probe or a probe set, can include one or more barcodes. In some embodiments, the barcode is combined by a detection probe, and the detection of the 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 the primary barcode on the 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 the 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; US 10,457,980 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.

[0181] In some embodiments, the recombinant and / or modified AP50 polymerase provided is used in a method involving the use of a circular or circularizable 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 US 2020 / 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.

[0182] In some embodiments, the recombinant and / or modified AP50 polymerase provided is used in a method involving the use of a probe (e.g., a circularizable probe, such as a padlock probe) comprising a 5' flank that can be recognized by a structure-specific cutting enzyme, such as an enzyme that can recognize the junction between a single-stranded 5' overhang and a DNA duplex and cut the single-stranded overhang. It should be understood that a branched triple-stranded structure that is a substrate for a structure-specific cutting enzyme can be formed by the 5' end of one probe portion and the 3' end of another probe portion (when both have hybridized to a target nucleic acid molecule) as well as by the 5' and 3' ends of a single-part probe. Enzymes suitable for such cutting include flanking endonucleases (FENS), which are a class of enzymes that have endonucleolytic activity and can catalyze the hydrolytic cleavage of phosphodiester bonds at the junction of single-stranded and double-stranded DNA. Therefore, in some embodiments, cutting of the additional sequence at 5' of the first target-specific binding site is performed by a structure-specific cutting enzyme (e.g., a flanking endonuclease). Suitable flanking endonucleases are described in Ma et al., JBC 2000; 275: 24693-24700 and US 2020 / 0224244, and may include P. furiosus (Pfu), A. fulgidus (Afu), M. jannaschii (Mja) or M. thermoautotrophicum (Mth). In some embodiments, enzymes capable of recognizing and degrading single-stranded oligonucleotides with free 5' ends can be used to cut additional sequences (e.g., 5' flanks) from structures as described above. Therefore, enzymes with 5' nuclease activity can be used to cut 5' additional sequences. Such 5' nuclease activity can be 5' exonuclease activity and / or 5' endonuclease activity. 5' nucleases can recognize the free 5' end of single-stranded oligonucleotides and degrade the single-stranded oligonucleotides. 5' exonucleases degrade single-stranded oligonucleotides with free 5' ends by degrading the oligonucleotides from their 5' ends into constituent mononucleotides. 5' endonuclease activity can internally cut 5' flanking sequences at one or more nucleotides. In addition, once the enzyme recognizes the free 5' end, the enzyme will pass through the single-stranded oligonucleotide to the duplex region and cut the single-stranded region into larger constituent nucleotides (e.g., dinucleotides or trinucleotides), or cut the entire 5' single-stranded region, thereby causing 5' nuclease activity, for example, as described in Lyamichev et al., PNAS 1999; 96, 6143-6148 for Taq DNA polymerase and its 5' nuclease. Preferred enzymes with 5' nuclease activity include exonucleases VIII, or natural or recombinant DNA polymerases from Thermus aquaticus (Taq), Thermus thermophilus or Thermus flavus, or nuclease domains thereof.

[0183] In some cases, after forming circular nucleic acid, add amplification primer. In some cases, amplification primer is added together with primary and / or secondary probe. In some cases, amplification primer can also be complementary to target nucleic acid and padlock probe (for example, SNAIL probe). In some embodiments, wash step is carried out to remove any unbound probe, primer etc. In some embodiments, washing is strict washing. Washing step can be carried out at any time point during process to remove non-specifically bound probe, connected probe etc.

[0184] In some cases, after adding DNA polymerase 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 a copy of multiple RCPs.

[0185] 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.

[0186] In some cases, the provided recombinant and / or modified AP50 polymerases can be used in methods for amplifying DNA, such as sequencing methods, genotyping methods, or amplification methods. In some embodiments, the method comprises contacting a biological sample containing the DNA to be amplified with any provided recombinant and / or modified AP50 polymerase, any composition or kit comprising a recombinant and / or modified AP50 polymerase, or a nucleic acid or vector encoding a recombinant and / or modified AP50 polymerase. In some embodiments, the method further comprises incubating the biological sample and the recombinant and / or modified AP50 polymerase, any composition or kit comprising a recombinant and / or modified AP50 polymerase, or a nucleic acid or vector encoding a recombinant and / or modified AP50 polymerase.

[0187] In some embodiments, the biological sample containing the DNA to be amplified by the recombinant and / or modified AP50 polymerase is an isolated or synthesized nucleic acid. In some embodiments, the biological sample is tested 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 recombinant and / or modified AP50 polymerase is added to a fresh or previously prepared tissue sample for target gene analysis within 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.

[0188] In some cases, the recombinant and / or modified AP50 polymerase provided can be used in a method for performing rolling circle amplification (RCA). In some embodiments, the method includes contacting a biological sample containing the DNA to be amplified with a recombinant and / or modified AP50 polymerase, any composition or kit comprising a recombinant and / or modified AP50 polymerase, or a nucleic acid or vector encoding a recombinant and / or modified AP50 polymerase. In some embodiments, the method also includes incubating the biological sample and the recombinant and / or modified AP50 polymerase, any composition or kit comprising a recombinant and / or modified AP50 polymerase, or a nucleic acid or vector encoding a recombinant and / or modified AP50 polymerase. In some embodiments, the recombinant and / or modified AP50 polymerase used in the method of RCA can be any one of those described in Part II.

[0189] In some embodiments, the method of RCA includes incubating the biological sample and the recombinant and / or modified AP50 polymerase at a specific temperature. In some embodiments, the temperature is optimal for maximizing the activity of the recombinant and / or modified AP50 polymerase. In some embodiments, the recombinant and / or modified AP50 polymerase 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, 50°C or higher. In some embodiments, the recombinant and / or modified AP50 polymerase and the biological sample are incubated at a temperature of 5°C to 55°C, 10°C to 50°C, 25°C to 45°C, 30°C to 40°C or 35°C to 42°C. In some embodiments, the recombinant and / or modified AP50 polymerase and the biological sample are incubated at an elevated temperature. In some embodiments, the recombinant and / or modified AP50 polymerase 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., 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C. In some embodiments, the recombinant and / or modified AP50 polymerase and the biological sample are incubated at 30° C. to 55° C., 32° C. to 53° C., 34° C. to 51° C., 36° C. to 49° C., 38° C. to 47° C., or 40° C. to 45° C.

[0190] In some embodiments, RCA is carried out during the incubation of the recombinant and / or modified AP50 polymerase and the 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 of any embodiment herein, RCA can be performed for no more than about 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours or 3 hours. In some embodiments, the incubation time used is sufficient to generate a detectable amount of RCA product.

[0191] 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.

[0192] In some embodiments, the methods provided use recombinant and / or modified AP50 polymerases that exhibit one or more of any of the characteristics described herein, such as those described in Section II.B.

[0193] Provided herein is a method for analysis, comprising: contacting a sample with a nucleic acid probe that hybridizes to a target nucleic acid (e.g., RNA), 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 a recombinant Bacillus phage AP50 polymerase; 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 signals associated with one or more detectably labeled probes. In some embodiments, one or more detectably labeled probes are indirectly bound to one or more barcode sequences or their complements via one or more intermediate probes that bind to one or more barcode sequences or their complements, wherein the one or more intermediate probes can be detected using one or more detectably labeled probes.

[0194] A.Sample

[0195] The sample (such as a biological sample) that can be evaluated or analyzed using any recombinant and / or modified AP50 polymerase, a composition or kit comprising a recombinant and / or modified AP50 polymerase, or a nucleic acid or vector encoding a recombinant and / or modified AP50 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 the 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.

[0196] 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 includes 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.

[0197] 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.

[0198] 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.

[0199] 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 comprises a natural molecule and / or a synthetic molecule network chemically and / or enzymatically connected (e.g., by cross-linking). In some embodiments, the 3D matrix comprises a synthetic polymer. In some embodiments, the 3D matrix comprises a hydrogel.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] B. Analyte

[0204] 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.

[0205] 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.

[0206] Analytes can include any biomolecule or chemical compound, including macromolecules (such as proteins or peptides, lipids or nucleic acid molecules) or small molecules (including organic or inorganic molecules). Analytes can be cells or microorganisms, including viruses or fragments or products thereof. Analytes can be any substance or entity for which specific binding partners (e.g., affinity binding partners) can be developed. Such specific binding partners can be nucleic acid probes (for nucleic acid analytes) and can directly lead to the generation of RCA templates using any AP50 polymerase described herein (e.g., circularizable probes). Alternatively, specific binding partners can be coupled to nucleic acids, which can be detected using RCA strategies, such as in assays using or generating circular nucleic acid molecules that can serve as RCA templates.

[0207] 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).

[0208] 1. Endogenous analytes

[0209] 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).

[0210] 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.

[0211] 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.

[0212] 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 23srRNA).

[0213] 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.

[0214] 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.

[0215] 2. Labeling Reagents

[0216] 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 includes 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.

[0217] In some embodiments, the methods include one or more post-fixation steps after contacting the sample with one or more labeling agents.

[0218] 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.

[0219] 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, which indicates the cell surface features to which the binding group is combined. For example, the reporter oligonucleotide includes a barcode sequence allowing identification of the labeling agent. For example, a labeling agent having specificity for a type of cell feature (e.g., a first cell surface feature) may have a first reporter oligonucleotide coupled thereto, and a labeling agent having specificity for 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, all of which are incorporated herein by reference in their entirety.

[0220] 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).

[0221] 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.

[0222] In some aspects, these reporter oligonucleotides contain certain nucleic acid barcode sequences that allow identification of the labeling agent to which the reporter oligonucleotide is coupled. Selecting an oligonucleotide as a reporter can provide the following advantages: the ability 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).

[0223] 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., Lightning-Tran available from Innova Biosciences). 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.

[0224] 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).

[0225] 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.

[0226] 3. Products of endogenous analytes and / or labeling agents

[0227] In some embodiments, provided herein are methods and compositions for 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 their products (e.g., amplification products or extension products, e.g., by nucleic acid polymerase), 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 products generated using any AP50 polymerase provided in Part II, such as rolling circle amplification (RCA product), extension products (e.g., by nucleic acid polymerase), hybridization products, connection products, replication products, transcription / reverse transcription products), and / or the labeling agent directly or indirectly combined with the analyte in the biological sample.

[0228] C. Detection and Analysis

[0229] In some aspects, provided herein are methods comprising in situ assays, such as in situ assays involving use of any recombinant and / or modified AP50 polymerase provided herein. In some aspects, in situ assays involve in situ generation of amplification products, such as use of any recombinant and / or modified AP50 polymerase or composition provided herein, and in situ detection of amplification products, such as use of a microscope or other detection or assay methods involving optical readouts. In other aspects, nucleic acid amplification and / or detection is performed in vitro.

[0230] In some aspects, the detection or determination of the sequence of the target nucleic acid or its amplified product is carried out in situ in cells in intact tissues. In some aspects, the method relates to generating nucleic acid amplification products, such as rolling circle amplification (RCA) products, in situ using the recombinant and / or modified AP50 polymerase provided herein, and detecting the amplified products.

[0231] In some embodiments, the method includes imaging the sample to detect a signal associated with a detection probe (e.g., a detectably labeled detection oligonucleotide or a detectably labeled probe, such as a fluorescently labeled oligonucleotide) hybridized in the sample. In some embodiments, the probe (e.g., a primary probe or an amplification product generated using a primary probe) comprises one or more barcode sequences for detection. In some embodiments, the signal can be amplified in situ in the sample. In some embodiments, an amplification signal associated with a primary probe and a related RCA product can be generated in the sample (e.g., using a detection probe such as a fluorescently labeled oligonucleotide).

[0232] 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).

[0233] In some aspects, in situ hybridization-based assays are used to locate and analyze nucleic acid sequences in natural biological samples (e.g., a portion or slice of a tissue). In some embodiments, in situ assays are used to analyze the presence, absence, amount or level of a probe (e.g., a primary probe) hybridized to a nucleic acid substrate in a biological sample while retaining spatial context.

[0234] 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.

[0235] In some embodiments, the barcode sequence of the probe (or its product, such as amplification, extension, connection or hybridization product) 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 the signal (such as fluorescence) for sequence determination. In some embodiments of 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 continuous detection of targets (RNA SPOT), continuous 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 methods provided herein include 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 Seq FISH+," 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; US 10,457,980 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.

[0236] 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.

[0237] In some aspects, the method also includes imaging one or more parts of the amplified product (e.g., amplicon) and / or polynucleotide, for example, by combining and detecting a detectable label with a detectably labeled probe. In some embodiments, the detectably labeled probe comprises a detectable label that can be measured and quantified. Labels and detectable labels can include a directly or indirectly detectable portion associated with a molecule to be detected (e.g., conjugated), 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.

[0238] 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.

[0239] 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).

[0240] In some embodiments, a detectable probe containing a detectable label can be used to detect one or more probes and / or amplification products (e.g., amplicons) described herein. 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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、ALEXAFLUOR TM 532-5-dUTP、ALEXA FLUOR TM 568-5-dUTP、ALEXA FLUOR TM 594-5-dUTP、ALEXAFLUOR 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、RHOD AMINE 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).

[0245] 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 RGG, 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 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.

[0246] 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).

[0247] 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.

[0248] Other suitable labels for polynucleotide sequences 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.

[0249] 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.).

[0250] 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).

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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 of the probe or its product. In some embodiments, contact, detection, and dehybridization steps are repeated with 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. In some embodiments, the detectably labeled probe includes a detectable label (for example, 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 (for example, 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.

[0257] In some embodiments of any embodiment herein, the detection of specific nucleic acid or its amplification product may include contacting biological sample with one or more intermediate probes directly or indirectly combined with rolling circle amplification product, wherein one or more intermediate probes can be detected using one or more detectably labeled probes. In some embodiments of any embodiment herein, the detection step may also include removing (e.g., dehybridizing) one or more intermediate probes and / or one or more detectably labeled probes from rolling circle amplification product. In some embodiments of any embodiment herein, contact and removal (e.g., dehybridizing) steps may be repeated with one or more intermediate probes, one or more detectably labeled probes, one or more other intermediate probes and / or one or more other detectably labeled probes.

[0258] In some embodiments, the one or more intermediate probes comprise one or more overhang regions (e.g., 5' and / or 3' ends of the probe that do not hybridize with the rolling circle amplification product). A probe comprising a single overhang region may be referred to as an "L-shaped probe", and a probe comprising two overhangs may be referred to as a "U-shaped probe". In some cases, the overhang region comprises a binding region for binding to one or more detectably labeled probes. In some embodiments, detection comprises contacting a biological sample with an intermediate probe pool corresponding to different barcode sequences or portions thereof and a detectably labeled probe pool corresponding to different detectable labels. In some embodiments, the biological sample is contacted with different intermediate probe pools in sequence. In some cases, a common or universal detectably labeled probe pool is used in multiple sequential hybridization steps (e.g., with different intermediate probe pools).

[0259] 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 a specific nucleotide in the sequence to which the oligonucleotide hybridizes. Aspects and features involved in sequencing by ligation are described, for example, in 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.

[0260] 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).

[0261] 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.

[0262] 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.

[0263] V. Kit

[0264] Also provided herein are kits, for example, comprising recombinant and / or modified AP50 polymerases and one or more additional components, and reagents for carrying out the methods provided herein, such as reagents required for one or more steps of amplification, detection, sequencing and / or sample preparation as described herein. In some embodiments, the kit comprises a recombinant and / or modified AP50 polymerase separated and purified as described in Part II. In some embodiments, the kit comprises a recombinant and / or modified AP50 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 embodiments, the recombinant nucleic acid comprises deoxyribonucleotides. In some embodiments, the recombinant nucleic acid comprises one or more ribonucleotide residues. In some embodiments, the kit also comprises a detection probe (e.g., a plurality of detectably labeled probes) for detecting nucleic acids (e.g., DNA or RNA) as described in Part IV.

[0265] In some embodiments, the test kit also includes one or more components, such as dNTP, divalent-cation and reaction buffer. In some embodiments, the test kit also includes one or more primers. In some embodiments, the one or more primers included in the test kit may include one or more circular or circularizable probes (e.g., padlock probes). In some embodiments, the circularizable probe is a circularizable probe group. In some embodiments, the test kit also includes instructions for use, for example, instructions for amplifying nucleic acid (e.g., DNA). In some embodiments, the test kit also includes a probe for detecting amplified nucleic acid (e.g., DNA). In some embodiments, the probe is a circular or circularizable probe or probe group (e.g., padlock probe) for detecting amplified nucleic acid (e.g., DNA). In some embodiments, the test kit is used for nucleic acid amplification. In some embodiments, nucleic acid is amplified by rolling circle amplification (RCA) using recombinant and / or modified AP50 polymerase. In some embodiments, nucleic acid is amplified in situ in a biological sample using a recombinant polymerase.

[0266] In some aspects, the kit includes a provided polynucleotide (e.g., those described in Section III.A), a provided recombinant nucleic acid molecule or vector (e.g., those described in Section III.B), and a provided recombinant expression system (e.g., the recombinant expression system described in Section III.C). In some embodiments, the kit also includes instructions for use. In some embodiments, the recombinant expression system is a cell system or a cell-free system. In some embodiments, the recombinant expression system comprises one or more cell systems selected from bacterial cells, fungal cells, insect cells, and mammalian cells. In some embodiments, the kit is used to improve the processivity, sensitivity, specificity, solubility, polymerization rate, thermal stability, and uniformity of the recombinant polymerase activity compared to the reference polymerase. In some embodiments, the reference polymerase is the wild-type AP50 polymerase shown in SEQ ID NO: 1. In some embodiments, the reference polymerase is the wild-type Phi29 polymerase shown in SEQ ID NO: 5.

[0267] 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.

[0268] 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 and / or sequencing, 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, recombinant and / or modified nucleotides, reagents for other determinations.

[0269] 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.

[0270] 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.

[0271] VI. Terminology

[0272] Specific terminology is used throughout this disclosure to explain various aspects of the described devices, systems, methods, and compositions.

[0273] 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.

[0274] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more".

[0275] 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.

[0276] 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.

[0277] 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.

[0278] "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.

[0279] 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 specific 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).

[0280] 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 modified AP50 polymerases.

[0281] Amino acids can generally be grouped according to the following common side chain properties:

[0282] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0283] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0284] (3) Acidic: Asp, Glu;

[0285] (4) Basic: His, Lys, Arg;

[0286] (5) Residues that affect chain orientation: Gly, Pro;

[0287] (6) Aromatic: Trp, Tyr, Phe.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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."

[0294] 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.

[0295] 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)).

[0296] 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.

[0297] "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 reaction and a reagent 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.

[0298] 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 enzymes under different reaction conditions (e.g., temperature, template concentration, primer concentration, etc.), such as increasing or decreasing polymerization rates. Mutations or sequence modifications may also affect exonuclease activity and / or the thermostability of the enzyme.

[0299] In some aspects, the AP50 polymerase is referred to as a modified AP50 polymerase. The term "modified AP50 polymerase" includes any AP50 polymerase variant that is different from the wild-type AP50 polymerase shown in the sequence SEQ ID NO: 1. For example, it includes any amino acid deletion, insertion and / or substitution that changes the amino acid sequence of the AP50 polymerase. The modified AP50 polymerase may also be referred to as a variant AP50 polymerase, a mutant AP50 polymerase and / or a derivative AP50 polymerase.

[0300] 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.

[0301] 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.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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).

[0309] 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 -dCTP). 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 0-Feulgen, BCECF (high pH), BFP (blue fluorescent protein), BFP / GFP FRET, BOBO TM -1 / B0-PRO TM -1.BOBO TM -3 / B0-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 、Calcium Green-1 TM Calcium Orange TM ,

[0310] White, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthylfluorescein, 6-carboxyrhodamine 6G, 5-carboxytetramethylrhodamine (5-TAMRA), carboxy-X-rhodamine (5-ROX), Cascade CascadeYellow TM 、CCF2(GeneBLAzer TM ), CFP (cyan fluorescent protein), CFP / YFP FRET, Chromomycin A3, C1-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-4ANEPPS, Di-8ANEPPS, DM-NERF (4.5-6.5pH), 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), 1,5IAEDANS, 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(pH 5), LysoSensor TM Yellow / Blue(pH4.2), 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. POPO TM -3 / PO-PRO TM -3, propidium iodide (PI), PyMPO, 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, -1 (high pH), -2. -1 (high pH), -1 (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, 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, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, 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).

[0311] 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 chemiluminescent reactions. 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.

[0312] VII. Examples

[0313] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0314] Example 1: Comparison of Bacillus phagocytosis bacteria AP50 polymerase and Phi29 polymerase In vitro rolling circle amplification (RCA) assay

[0315] This example describes the generation and use of an exemplary Bacillus phage AP50 polymerase (APol) in rolling circle amplification (RCA), demonstrating improved in vitro activity compared to wild-type Phi29 polymerase.

[0316] An exemplary modified AP50 polymerase, a truncated Bacillus phage AP50 polymerase (APolTr; as shown in SEQ ID NO: 6, containing a truncation of 94 consecutive amino acids starting from the C-terminus, refer to SEQ ID NO: 1) fused to a linker and a poly-histidine tag, was recombinantly produced and tested in an in vitro RCA assay.

[0317] Briefly, 50 nM circularizable probe was mixed with 100 nM RNA template and 50 nM RCA primer and incubated at 37 ° C for 1 hour for probe-template hybridization. Then 1 μM ligase was added and incubated at 37 ° C for 1 hour to connect the circularizable probe. The ligation product, RCA polymerase (truncated AP50 polymerase (APolTr) or wild-type Phi29 polymerase (150 nM) at various concentrations (30, 100, 300 and 1000 nM)) and dNTP were incubated at different temperatures to generate RCA products (RCPs) at 30 ° C, and the SYBR Gold label of RCPs was monitored in real time based on fluorescence changes (at an excitation wavelength of 495 nm and an emission wavelength of 537 nm) using a plate reader.

[0318] like Figure 1 As shown, the results of the in vitro RCA assay at 30°C show that the exemplary AP50 polymerase (APolTr) generates more RCPs compared to wild-type Phi 29, as shown by the increased fluorescence intensity of SYBR Gold-labeled RCPs. The exemplary AP50 polymerase generates more RCPs than wild-type Phi29 even at lower polymerase concentrations (e.g., 30 nM). The results indicate that the exemplary AP50 polymerase exhibits improved properties, including increased polymerization rates and increased RCP generation at 30°C, compared to the wild-type Phi29 polymerase, which may be related to the improved thermal stability, processivity, and / or polymerization rate of the AP50 polymerase. The results support the utility and benefits of the exemplary AP50 polymerase in nucleic acid amplification methods such as RCA.

[0319] Example 2: Use of Bacillus phage AP50 polymerase at different temperatures and concentrations In vitro rolling circle amplification (RCA) assay

[0320] This example describes the generation and use of an exemplary Bacillus phage AP50 polymerase in rolling circle amplification (RCA) at different temperatures and concentrations, demonstrating robust in vitro RCA activity.

[0321] Exemplary modified AP50 polymerases, such as the truncated APol (APolTr) described in Example 1, were recombinantly produced and tested in an in vitro RCA assay, generally as described in Example 1 above, at 30°C, 37°C, 42°C or 50°C and at concentrations of 3, 10, 30, 100, 300, 1000, 3000 and 10000 nM APolTr polymerase.

[0322] like Figure 2A-2D As shown, at 30℃( Figure 2A )、37℃( Figure 2B )、42℃( Figure 2C ) and 50℃( Figure 2D The results of the in vitro RCA assay under ) showed that the exemplary modified AP50 polymerase was highly expressed at all temperatures (including at 50°C ( Figure 2D ) showed robust RCA activity and generated a large amount of RCP, as shown by the fluorescence intensity of SYBR Gold-labeled RCP. Improved polymerization kinetics were also observed at 50 °C compared to the kinetics at lower temperatures. Figure 2B ) compared to the activity of AP50 polymerase at 42°C ( Figure 2C ) exhibited improved RCA activity at elevated temperatures, indicating improved thermostability. In contrast, other RCA polymerases (such as wild-type Phi29 polymerase) exhibited reduced activity and thermostability at certain elevated temperatures (such as 42°C). The results indicate that the exemplary AP50 polymerase exhibits improved properties, including improved polymerization rates and increased RCP generation at elevated temperatures (e.g., 42°C and 50°C), which may be related to the improved thermostability, processivity, and / or polymerization rate of the exemplary AP50 polymerase. The results support the utility and benefits of the exemplary AP50 polymerase in nucleic acid amplification methods (such as RCA).

[0323] Example 3: Evaluation of Bacillus phage AP50 polymerase and Phi29 polymerase in in situ rolling circle amplification (RCA) Enzyme sensitivity

[0324] This example describes the generation and use of an exemplary Bacillus phage AP50 polymerase in rolling circle amplification (RCA), demonstrating comparable in vitro RCA sensitivity compared to wild-type Phi29 polymerase.

[0325] Recombinantly produce exemplary modified AP50 polymerases, such as truncated APol (APolTr) described in Example 1, and test in an in situ RCA assay. In brief, APolTr or wild-type Phi29 polymerase is used to prepare tissue samples for in situ RCA for gene expression analysis. A circularizable probe is added to each tissue sample and hybridized. After probe hybridization, the tissue sample is washed to remove unbound probes. Circularizable probe ligation is performed by adding ligase and ligase buffer to the sample. For RCA, the sample is incubated at 30°C or 42°C in a reaction mixture containing polymerase buffer, dNTPs and various concentrations (250, 500 and 1000nM) of APolTr or 5U wild-type Phi29 polymerase. RCP is detected by in situ hybridization of the labeled probe with RCP and imaging the sample with a fluorescence microscope. The median value of the transcript of each cell is evaluated.

[0326] like Figure 3 As shown, the results of in situ RCA assays at 30°C and 42°C show that the exemplary AP50 polymerase produces more comparable moderate numbers of transcripts per cell than 5U of wild-type Phi29 polymerase. At a concentration of 1000 nM, the exemplary AP50 polymerase produces more transcripts per cell at 30°C than the wild-type Phi29 polymerase.

[0327] The results demonstrate that the exemplary AP50 polymerase exhibits comparable sensitivity to amplification in in situ RCA compared to high concentrations of wild-type Phi29 polymerase, supporting the utility and benefits of the exemplary AP50 polymerase in in situ analytical methods involving nucleic acid amplification, such as in situ RCA.

[0328] 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.

[0329] sequence

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

Claims

1. A method for nucleic acid amplification, the method comprising contacting a biological sample containing a nucleic acid to be amplified with a recombinant Bacillus phage AP50 polymerase.

2. The method of claim 1, wherein the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 6 having a length of at least 200 amino acids, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to any of the foregoing sequences.

3. The method of claim 1 or 2, wherein the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 6 having a length of at least 600 amino acids, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to any of the foregoing sequences.

4. The method of any one of claims 1 to 3, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:6 or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO:

6.

5. The method of any one of claims 1 to 4, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

6.

6. The method of any one of claims 1 to 5, wherein the recombinant polymerase consists of the sequence shown in SEQ ID NO:

6.

7. The method of any one of claims 1 to 5, wherein the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 1 having a length of at least 637 amino acids, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to any of the foregoing sequences.

8. The method of any one of claims 1 to 5 and 7, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO: 1 or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO:

1.

9. The method of any one of claims 1 to 5, 7 and 8, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

1.

10. The method of any one of claims 1 to 5 and 7 to 9, wherein the recombinant polymerase consists of the sequence shown in SEQ ID NO:

1.

11. The method of any one of claims 1 to 10, wherein the recombinant polymerase comprises one or more amino acid substitutions, deletions or additions at one or more positions corresponding to the positions of the sequence shown in SEQ ID NO:

1.

12. The method of any one of claims 1 to 11, wherein the recombinant polymerase further comprises a heterologous sequence.

13. The method of claim 12, wherein the heterologous sequence comprises one or more tags, linkers and / or polypeptide domains.

14. The method of claim 12 or 13, wherein the heterologous sequence comprises one or more tags.

15. The method of claim 13 or 14, wherein the tag is selected from the group consisting of a polyhistidine (HIS) tag, a solubility enhancing tag (SET), a small ubiquitin-modified (SUMO) tag, a Fasciola hepatica 8-kDa antigen (Fh8) tag, a thioredoxin (Trx) tag, a glutathione-s-transferase (GST) tag, a maltose binding protein (MBP) tag, a protein G IgG domain B1 (GB1) tag, a mutated dehalogenase (Halo) tag, a streptavidin binding peptide (SBP) tag, and a Tamavidin tag.

16. The method of any one of claims 13 to 15, wherein the tag comprises the sequence shown in any one of SEQ ID NOs: 7, 8 and 26.

17. The method of any one of claims 13 to 16, wherein the tag comprises the sequence shown in SEQ ID NO:

7.

18. The method of any one of claims 13 to 16, wherein the tag comprises the sequence shown in SEQ ID NO:

8.

19. The method of any one of claims 13 to 16, wherein the tag comprises the sequence shown in SEQ ID NO:

26.

20. The method of any one of claims 12 to 19, wherein the heterologous sequence comprises one or more polypeptide domains.

21. The method of claim 20, wherein the polypeptide domain is selected from the group consisting of a helix-hairpin-helix (HhH) DNA binding motif, a helix-hairpin-helix fusion [(HhH)2] DNA binding motif, a single-stranded DNA binding domain, an SD07 DNA binding motif, an SS07 DNA binding motif, and an E. coli HU domain.

22. The method of any one of claims 12 to 21, wherein the heterologous sequence comprises one or more linkers.

23. The method of claim 22, wherein the linker is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 amino acids in length.

24. The method of claim 22 or 23, wherein the linker comprises the sequence shown in any one of SEQ ID NOs: 10-16.

25. The method of any one of claims 1 to 24, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO: 6, a linker and a poly-histidine (HIS) tag.

26. The method of any one of claims 1 to 25, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO: 6, a linker, a poly-histidine (HIS) tag, and a solubility enhancing tag (SET).

27. The method of any one of claims 1 to 26, wherein the recombinant polymerase comprises a sequence as set forth in any one of SEQ ID NOs: 2-4, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence as set forth in SEQ ID NOs: 2-4.

28. The method of claim 27, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

2.

29. The method of claim 27, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

3.

30. The method of claim 27, wherein the recombinant polymerase comprises the sequence set forth in SEQ ID NO:

4.

31. The method of any one of claims 1 to 30, wherein the recombinant polymerase is 900 amino acids or less in length.

32. The method of any one of claims 1 to 31, wherein the recombinant polymerase is 800 amino acids or less in length.

33. The method of any one of claims 1 to 32, wherein the recombinant polymerase is 732 amino acids or less in length.

34. The method of any one of claims 1 to 6 and 11 to 30, wherein the recombinant polymerase is 638 amino acids or less in length.

35. The method of any one of claims 1 to 3, 11 to 30 and 34, wherein the recombinant polymerase is 600 amino acids or less in length.

36. The method of any one of claims 1, 2, 11 to 30, 34 and 35, wherein the recombinant polymerase is 500 amino acids or less in length.

37. The method of any one of claims 1 to 36, wherein the nucleic acid comprises deoxyribonucleotide residues.

38. The method of any one of claims 1 to 37, wherein the nucleic acid comprises one or more ribonucleotide residues.

39. The method of any one of claims 1 to 38, wherein the nucleic acid is a circular probe or a circularizable probe.

40. The method of any one of claims 1 to 39, wherein the nucleic acid is amplified by rolling circle amplification (RCA) using the recombinant polymerase.

41. The method of any one of claims 1 to 40, wherein the nucleic acid is amplified in situ in the biological sample using the recombinant polymerase.

42. The method of any one of claims 1 to 41, further comprising incubating the recombinant polymerase and the biological sample with a primer that hybridizes to the nucleic acid.

43. The method of claim 42, wherein the primer comprises a synthetic oligonucleotide.

44. The method of claim 42, wherein the primers comprise cellular nucleic acid or a portion thereof in the biological sample.

45. The method of any one of claims 1 to 44, wherein the recombinant polymerase has DNA synthesis activity.

46. ​​The method of any one of claims 1 to 45, wherein the recombinant polymerase is capable of polymerizing DNA based on a circular nucleic acid template.

47. The method of any one of claims 1 to 46, wherein the recombinant polymerase has RNA synthesis activity.

48. The method of any one of claims 1 to 47, wherein the recombinant polymerase has strand displacement activity.

49. The method of any one of claims 1 to 48, wherein the recombinant polymerase has reverse transcriptase activity.

50. The method of any one of claims 1 to 49, wherein the recombinant polymerase has exonuclease activity.

51. The method of any one of claims 1 to 50, wherein the recombinant polymerase exhibits one or more characteristics selected from improved processivity, improved polymerization rate, and improved thermal stability compared to a reference polymerase.

52. The method of any one of claims 1 to 51, wherein the recombinant polymerase exhibits improved processivity compared to a reference polymerase.

53. The method of any one of claims 1 to 52, wherein the recombinant polymerase exhibits improved kinetics compared to a reference polymerase.

54. The method of any one of claims 1 to 53, wherein the recombinant polymerase exhibits an improved polymerization rate compared to a reference polymerase.

55. The method of any one of claims 1 to 54, wherein the recombinant polymerase exhibits improved thermostability compared to a reference polymerase.

56. The method of any one of claims 1 to 55, wherein the recombinant polymerase is capable of generating a rolling circle amplification (RCA) product in an RCA reaction.

57. The method of claim 56, wherein the recombinant polymerase 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 more.

58. The method of claim 56 or 57, wherein the RCA reaction is carried out for or about 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours or longer.

59. The method of any one of claims 1 to 58, further comprising detecting or analyzing the amplification product.

60. The method of any one of claims 56 to 59, wherein the amplification product is detected or analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by ligation, or a combination thereof.

61. The method of any one of claims 1 to 60, wherein the nucleic acid and / or the amplification product comprises one or more barcode sequences or their complements.

62. The method of claim 61, wherein the one or more barcode sequences or their complements correspond to a target nucleic acid or an endogenous analyte.

63. The method of claim 61 or 62, wherein the one or more barcode sequences or their complements are detected by: 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, detecting a signal associated with the one or more detectably labeled probes, and The one or more detectably labeled probes are removed.

64. The method of claim 61 or 62, wherein the one or more barcode sequences or their complements are detected by: 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, detecting a signal associated with the one or more detectably labeled probes, and The one or more intermediate probes and / or the one or more detectably labeled probes are removed.

65. The method of any one of claims 1 to 64, wherein the recombinant polymerase generates a higher density of detected rolling circle amplification (RCA) products in RCA compared to a reference polymerase.

66. The method of claim 65, wherein the recombinant polymerase generates a higher signal intensity in RCA compared to a reference polymerase.

67. The method of claim 65 or 66, wherein the RCA reaction 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 more.

68. The method of any one of claims 51 to 67, wherein the reference polymerase is the Phi 29 polymerase set forth in SEQ ID NO:

5.

69. The method of any one of claims 1 to 68, wherein the biological sample is a cell sample or a tissue sample.

70. The method of any one of claims 1 to 69, wherein the biological sample is non-homogenized and is optionally selected from the group consisting of formalin-fixed paraffin-embedded (FFPE) samples, frozen tissue samples, and fresh tissue samples.

71. A recombinant Bacillus phage AP50 polymerase, comprising: i) a contiguous portion of SEQ ID NO: 6 that is at least 200 amino acids in length, or a sequence that has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to any of the preceding sequences; and ii) Heterologous sequences.

72. A recombinant polymerase as described in claim 71, wherein the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 6 having a length of at least 600 amino acids, or a sequence having a sequence identity of at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% to any of the aforementioned sequences.

73. A recombinant Bacillus phage AP50 polymerase, comprising: i) the sequence set forth in SEQ ID NO:6, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence set forth in SEQ ID NO:6; and ii) Heterologous sequences.

74. The recombinant polymerase of any one of claims 71 to 73, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

6.

75. The recombinant polymerase of any one of claims 71 to 74, wherein the recombinant polymerase comprises a contiguous portion of SEQ ID NO: 1 having a length of at least 637 amino acids, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to any of the aforementioned sequences.

76. The recombinant polymerase of any one of claims 71 to 75, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO: 1 or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO:

1.

77. The recombinant polymerase of any one of claims 71 to 76, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

1.

78. The recombinant polymerase of any one of claims 71 to 77, wherein the recombinant polymerase consists of the sequence shown in SEQ ID NO: 1 and the heterologous sequence.

79. A recombinant Bacillus phage AP50 polymerase consisting of a contiguous portion of the sequence shown in SEQ ID NO: 6 having a length of at least 200 amino acids, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to any of the foregoing sequences.

80. The recombinant polymerase of claim 79, wherein the recombinant polymerase consists of a contiguous portion of SEQ ID NO: 6 having a length of at least 600 amino acids, or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to any of the foregoing sequences.

81. The recombinant polymerase of claim 79 or 80, wherein the recombinant polymerase consists of the sequence shown in SEQ ID NO:6 or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO:

6.

82. The recombinant polymerase of any one of claims 79 to 81, wherein the recombinant polymerase consists of the sequence shown in SEQ ID NO:

6.

83. A Bacillus phage AP50 polymerase comprising one or more amino acid substitutions, deletions or additions at one or more positions corresponding to the positions of the sequence shown in SEQ ID NO:

1.

84. The recombinant polymerase of claim 83, wherein the recombinant polymerase has at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO:

1.

85. The recombinant polymerase of claim 83 or 84, further comprising a heterologous sequence.

86. The recombinant polymerase of any one of claims 71 to 78 and 85, wherein the heterologous sequence comprises one or more tags, linkers and / or polypeptide domains.

87. The recombinant polymerase of any one of claims 71 to 78, 85 and 86, wherein the heterologous sequence comprises one or more tags.

88. The recombinant polymerase of claim 86 or 87, wherein the tag is selected from the group consisting of a polyhistidine (HIS) tag, a solubility enhancing tag (SET), a small ubiquitin-modified (SUMO) tag, a Fasciola hepatica 8-kDa antigen (Fh8) tag, a thioredoxin (Trx) tag, a glutathione-s-transferase (GST) tag, a maltose binding protein (MBP) tag, a protein G IgG domain B1 (GB1) tag, a mutated dehalogenase (Halo) tag, a streptavidin binding peptide (SBP) tag, and a Tamavidin tag.

89. The recombinant polymerase of any one of claims 86 to 88, wherein the tag comprises the sequence shown in any one of SEQ ID NOs: 7, 8, and 26.

90. The recombinant polymerase of any one of claims 86 to 89, wherein the tag comprises the sequence shown in SEQ ID NO:

7.

91. The recombinant polymerase of any one of claims 86 to 89, wherein the tag comprises the sequence shown in SEQ ID NO:

8.

92. The recombinant polymerase of any one of claims 86 to 89, wherein the tag comprises the sequence shown in SEQ ID NO:

26.

93. The recombinant polymerase of any one of claims 71 to 78, 85 and 86, wherein the heterologous sequence comprises one or more polypeptide domains.

94. A recombinant polymerase as described in claim 93, wherein the polypeptide domain is selected from the following: a helix-hairpin-helix (HhH) DNA binding motif, a helix-hairpin-helix fusion [(HhH)2] DNA binding motif, a single-stranded DNA binding domain, an SD07 DNA binding motif, an SS07 DNA binding motif and an Escherichia coli HU domain.

95. The recombinant polymerase of any one of claims 71 to 78, 85 and 86, wherein the heterologous sequence comprises one or more linkers.

96. The recombinant polymerase of claim 95, wherein the linker is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 amino acids in length.

97. The recombinant polymerase of claim 95 or 96, wherein the linker comprises the sequence set forth in any one of SEQ ID NOs: 10-16.

98. The recombinant polymerase of any one of claims 71 to 78 and 86 to 97, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO: 6, a linker and a poly-histidine (HIS) tag.

99. The recombinant polymerase of any one of claims 71 to 78 and 86 to 98, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO: 6, a linker, a poly-histidine (HIS) tag, and a solubility enhancing tag (SET).

100. The recombinant polymerase of any one of claims 71 to 77 and 83 to 99, wherein the recombinant polymerase comprises a sequence as set forth in any one of SEQ ID NOs: 2-4 or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence as set forth in SEQ ID NOs: 2-4.

101. The recombinant polymerase of any one of claims 71 to 77 and 83 to 100, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

2.

102. The recombinant polymerase of any one of claims 71 to 77 and 83 to 100, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

3.

103. The recombinant polymerase of any one of claims 71 to 77 and 83 to 100, wherein the recombinant polymerase comprises the sequence shown in SEQ ID NO:

4.

104. The recombinant polymerase of any one of claims 71 to 103, wherein the recombinant polymerase is 900 amino acids or less in length.

105. The recombinant polymerase of any one of claims 71 to 104, wherein the recombinant polymerase is 800 amino acids or less in length.

106. The recombinant polymerase of any one of claims 71 to 105, wherein the recombinant polymerase is 732 amino acids or less in length.

107. The recombinant polymerase of any one of claims 71 to 75 and 79 to 106, wherein the recombinant polymerase is 638 amino acids or less in length.

108. The recombinant polymerase of any one of claims 71, 72, 79, 80, and 86 to 107, wherein the recombinant polymerase is 600 amino acids or less in length.

109. The recombinant polymerase of any one of claims 71, 79, and 86 to 108, wherein the recombinant polymerase is 500 amino acids or less in length.

110. The recombinant polymerase of any one of claims 71 to 109, wherein the recombinant polymerase has DNA synthesis activity.

111. The recombinant polymerase of any one of claims 71 to 110, wherein the recombinant polymerase is capable of polymerizing DNA based on a circular nucleic acid template.

112. The recombinant polymerase of any one of claims 71 to 111, wherein the recombinant polymerase has RNA synthesis activity.

113. The recombinant polymerase of any one of claims 71 to 112, wherein the recombinant polymerase has strand displacement activity.

114. The recombinant polymerase of any one of claims 71 to 113, wherein the recombinant polymerase has reverse transcriptase activity.

115. The recombinant polymerase of any one of claims 71 to 114, wherein the recombinant polymerase has exonuclease activity.

116. The recombinant polymerase of any one of claims 71 to 115, wherein the recombinant polymerase exhibits one or more characteristics selected from improved processivity, improved polymerization rate, and improved thermal stability compared to a reference polymerase.

117. The recombinant polymerase of any one of claims 71 to 116, wherein the recombinant polymerase exhibits improved processivity compared to a reference polymerase.

118. The recombinant polymerase of any one of claims 71 to 117, wherein the recombinant polymerase exhibits improved kinetics compared to a reference polymerase.

119. The recombinant polymerase of any one of claims 71 to 118, wherein the recombinant polymerase exhibits an improved polymerization rate compared to a reference polymerase.

120. The recombinant polymerase of any one of claims 71 to 119, wherein the recombinant polymerase exhibits improved thermal stability compared to a reference polymerase.

121. The recombinant polymerase of any one of claims 71 to 120, wherein the recombinant polymerase is capable of generating a rolling circle amplification (RCA) product in an RCA reaction.

122. The recombinant polymerase of claim 121, wherein the RCA reaction is performed 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 more.

123. A recombinant polymerase as described in any one of claims 71 to 122, wherein the recombinant polymerase generates a higher density of detected rolling circle amplification (RCA) products in RCA compared to a reference polymerase.

124. The recombinant polymerase of any one of claims 71 to 123, wherein the recombinant polymerase generates a higher signal intensity in RCA compared to a reference polymerase.

125. The recombinant polymerase of any one of claims 116 to 124, wherein the reference polymerase is the Phi29 polymerase shown in SEQ ID NO:

5.

126. A composition comprising the recombinant Bacillus phage AP50 polymerase of any one of claims 71 to 125.

127. A polynucleotide encoding the recombinant Bacillus phage AP50 polymerase of any one of claims 71 to 125.

128. The polynucleotide of claim 124, wherein the polynucleotide comprises the sequence shown in any one of SEQ ID NOs: 17-21, or a sequence having at least or about 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in any one of SEQ ID NOs: 17-21.

129. The polynucleotide of claim 127 or 128, wherein the polynucleotide comprises the sequence shown in SEQ ID NO:

17.

130. The polynucleotide of claim 127 or 128, wherein the polynucleotide comprises the sequence shown in SEQ ID NO:

18.

131. The polynucleotide of claim 127 or 128, wherein the polynucleotide comprises the sequence shown in SEQ ID NO:

19.

132. The polynucleotide of claim 127 or 128, wherein the polynucleotide comprises the sequence shown in SEQ ID NO:

20.

133. The polynucleotide of claim 127 or 128, wherein the polynucleotide comprises the sequence shown in SEQ ID NO:

21.

134. The polynucleotide of any one of claims 127 to 133, wherein the polynucleotide is codon-optimized for a recombinant expression system to produce the recombinant polymerase.

135. A recombinant nucleic acid molecule comprising the polynucleotide of any one of claims 127 to 134.

136. A recombinant nucleic acid molecule comprising a polynucleotide encoding a recombinant Bacillus phage AP50 polymerase, wherein the recombinant Bacillus phage AP50 polymerase comprises the sequence shown in SEQ ID NO:6 or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO:

6.

137. The encoded recombinant nucleic acid molecule of claim 136, wherein the encoded recombinant polymerase comprises the sequence shown in SEQ ID NO:

6.

138. The encoded recombinant nucleic acid molecule of claim 136 or 137, wherein the encoded recombinant polymerase comprises the sequence shown in SEQ ID NO: 1 or a sequence having at least or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence shown in SEQ ID NO:

1.

139. The encoded recombinant nucleic acid molecule of any one of claims 136 to 138, wherein the encoded recombinant polymerase comprises the sequence shown in SEQ ID NO:

1.

140. The encoded recombinant nucleic acid molecule of any one of claims 136 to 139, wherein the encoded recombinant polymerase consists of the sequence shown in SEQ ID NO:

1.

141. The recombinant nucleic acid molecule of any one of claims 135 to 140, further comprising a transcriptional regulatory sequence operably linked to the polynucleotide.

142. The recombinant nucleic acid molecule of claim 141, wherein the transcriptional regulatory sequence comprises a promoter selected from the group consisting of bacterial, viral, and mammalian promoters.

143. A vector comprising the polynucleotide of any one of claims 127 to 134 or the recombinant nucleic acid molecule of any one of claims 135 to 142.

144. The vector of claim 143, wherein the vector comprises a plasmid, a phagemid, a viral vector, a cosmid, or a transposon.

145. A recombinant expression system comprising the polynucleotide of any one of claims 127 to 134, the recombinant nucleic acid molecule of any one of claims 135 to 142, or the vector of claim 143 or 144.

146. The recombinant expression system of claim 145, wherein the polynucleotide, the recombinant nucleic acid molecule or the vector is used to transform the recombinant expression system to generate a transformed recombinant expression system.

147. The recombinant expression system of claim 146, wherein the transformed recombinant expression system produces the recombinant polymerase.

148. The recombinant expression system of any one of claims 145 to 147, wherein the recombinant expression system is a cell system or a cell-free system.

149. The recombinant expression system of any one of claims 145 to 148, wherein the recombinant expression system comprises one or more cell systems selected from bacterial cells, fungal cells, insect cells, and mammalian cells.

150. The recombinant expression system of any one of claims 145 to 149, wherein the polynucleotide sequence encoding the recombinant polymerase is codon-optimized for the recombinant expression system.

151. The recombinant expression system of any one of claims 145 to 150, wherein the recombinant expression system produces the recombinant polymerase having improved solubility, improved processivity, improved polymerization rate, improved thermal stability, and improved uniformity compared to a reference polymerase.

152. A kit comprising: (1) the recombinant Bacillus phage AP50 polymerase according to any one of claims 71 to 125, the polynucleotide according to any one of claims 127 to 134, the recombinant nucleic acid molecule according to any one of claims 135 to 142, or the vector according to claim 143 or 144, and (2) One or more of the following: (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 use.

153. A kit as described in claim 152, wherein the nucleic acid comprises deoxyribonucleotide residues.

154. A kit as described in claim 152 or 153, wherein the nucleic acid comprises one or more ribonucleotide residues.

155. The kit of any one of claims 152 to 154, further comprising one or more primers.

156. The kit of claim 155, further comprising one or more circularizable probes.

157. A kit as described in any one of claims 152 to 156, wherein the kit is for nucleic acid amplification.

158. The kit of claim 157, wherein the nucleic acid is amplified by rolling circle amplification (RCA) using the recombinant polymerase.

159. The kit of claim 157 or 158, wherein the nucleic acid is amplified in situ in a biological sample using the recombinant polymerase.

160. The kit of claim 159, further comprising a probe for detecting the amplified nucleic acid.

161. A kit comprising: (1) the polynucleotide according to any one of claims 127 to 134, the recombinant nucleic acid molecule according to any one of claims 135 to 142, or the vector according to claim 143 or 144, and (2) Recombinant expression system.

162. The kit of claim 161 further comprising instructions for use.

163. The kit of claim 161 or 162, wherein the recombinant expression system is a cell system or a cell-free system.

164. The kit of any one of claims 161 to 163, wherein the recombinant expression system comprises one or more cell systems selected from bacterial cells, fungal cells, insect cells, and mammalian cells.

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