CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of United States Provisional Patent Application No. 63/194,727, filed May 28, 2021, the disclosure of which is incorporated herein by references in its entirety.

SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via EFS_Web ans is hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 2202402_ST25.txt. The size of the text file is 272,032 bytes, and the text file was created on May 24, 2022.

BACKGROUND

[0003] Considerable research and development have been directed towards nucleic acid modifying enzymes (e.g., polymerase enzymes, more particularly DNA polymerase enzymes). These efforts have provided a number of polymerase enzymes that can be broadly categorized as either: (1) mutant and chimeric polymerase variants; or (2) domain- augmented polymerases. The recent polymerase termed “RTX” represents an example of a mutant polymerase variant as it is a mutant form of naturally-occurring KOD DNA polymerase (“KOD”) (Science 24 Jun 2016: Vol. 352, Issue 6293, pp. 1590-1593. KOD is the post-intein spliced form (PDB: 4K8Z_A) of poll from Thermococcus kodakarensis KOD1 [NCBI: DPOL_THEKO], which is capable of performing thermocycling or polymerase chain reaction (PCR). As a mutant form of KOD, RTX has a relaxed substrate specificity permitting amplification from either DNA or RNA template. Regarding domain- augmented polymerases, Phusion® polymerase (described in U.S. Patent No. 6,627,424, the contents of which are herein incorporated by reference in its entirety) is a commercially successful example of a domain-augmented polymerase which enhances the function of a given DNA polymerase by creating a fusion protein of the DNA polymerase (e.g. Pfu DNA polymerase) joined to a DNA-binding protein (e.g. Sul7).

[0004] The efficiency or performance of a nucleic acid modifying enzyme (e.g., a DNA polymerase) can be enhanced by increasing the stability of the macromolecular complex formed between the nucleic acid modifying enzyme (e.g., the DNA polymerase) and its nucleic acid substrate, which includes primer/template dsDNA before and during initiation of DNA replication as well as product dsDNA during elongation. As described in U.S. Patent No. 6,627,424, the efficiency or performance of the DNA polymerase can be enhanced not by mutating or altering the DNA polymerase, but rather by the DNA polymerase becoming the catalytic or polymerase domain of a two-domain protein ( e.g ., a fusion polymerase) with enhanced DNA-binding granted through the second domain, a DNA-binding protein.

[0005] With regards to polymerase chain reaction (PCR), the above-mentioned fusion polymerase can interact throughout the thermocycling process given temperature constraints. More specifically, during each cycle, the fusion polymerase enhances performance by increasing the binding affinity of the fusion polymerase to the initial template-primer double- stranded DNA (dsDNA) duplex during oligo/primer annealing, and also to the growing dsDNA amplicon during extension. First, enhanced affinity to the dsDNA template-primer duplex effectively increases the association (k _on ) of unbound, free polymerase improving initiation. The fusion polymerase also raises the effective template-primer annealing temperature, as its presence additionally enhances the association between the two nucleic acids. Second, during elongation, increased affinity to the growing dsDNA amplicon effectively decreases dissociation (k _0ff ) of the fusion polymerase, which increases processivity with gains observable in product yield and a faster extension rate. Increased association or affinity for dsDNA can be conceptualized as enhanced grip or traction.

[0006] For a PCR-based environment, one could consider a long piece or fragment of single- stranded DNA (ssDNA) generated following a melt step as the template for amplification. This ssDNA is conceptualized as a helical track on which the DNA polymerase will travel. Given temperature and concentration constraints, next, a short, ssDNA oligonucleotide known as a primer will anneal or hydrogen bond with the template sequence that is its reverse complement. Finally, a DNA polymerase binds to the dsDNA primer/template complex and can be thought of as the engine or locomotive for DNA replication. Following successful initiation and during elongation, a DNA polymerase seemingly spirals along the helical track that is the ssDNA template while leaving in its wake and gripping the dsDNA product that exits its posterior. Polymerases have evolved protein structures to assist in better gripping that dsDNA product such as the ‘thumb domain’ from the right hand model of polymerase. In nature, organisms further assemble complexes of accessory proteins (e.g. DNA clamps) that physically orient and secure ssDNA template and product dsDNA with DNA polymerases.

[0007] While Phusion® polymerase has increased processivity, along with increased fidelity and speed, resulting from increasing its binding affinity to its dsDNA product, the Phusion® polymerase does not address PCR stutter which arises from repeat amplification. Moreover, no commercially available DNA polymerase provides consistent stutter-resistant PCR performance. [0008] In theory, domain- augmented polymerases may also employ the use of DNA clamps ( e.g ., proliferating cell nuclear antigen (PCNA) or b-clamps) that could be used to enhance polymerase performance. DNA clamps are oligomeric, toroidal proteins that assemble from six clamp domains, which function to encircle dsDNA offering a protein system with perfect grip or traction. Once assembled or loaded, and until perturbed, a DNA clamp has no effective dissociation (k _0ff ) and instead slides along a DNA helix. Despite the expected gains to performance, adapting or employing DNA clamp technology for the purpose of domain- augmented polymerases has numerous disadvantages and problems. In nature, DNA clamps represent one component of a larger multi-protein complex that binds and forms the replication fork complex during DNA replication. Accordingly, and given that it is a discrete element of a multiprotein replication fork, adapting a DNA clamp to another protein (e.g., a DNA polymerase) is non-trivial, as the DNA clamp normally functions within the context of numerous proteins found in the replication fork complex and with the assistance of ATP-driven enzymes such as a DNA clamp loader. This complexity grows when seeking to adapt DNA clamp technology to be compatible with thermocycling (e.g. PCR) and/or a form of isothermal amplification, such as loop-mediated isothermal amplification (LAMP). While the adaptation of DNA clamp technology to a tractable polymerase may greatly enhance its function, the coordination of these two macromolecules is also non-trivial. A protein-based suspension system is necessary to secure a DNA clamp encircling dsDNA and the catalytically-active polymerase driving the production of dsDNA from ssDNA template. It is further necessary to coordinate the one or more protomers (i.e., monomers) that compose the oligomeric DNA clamp through a linker or connection sequence that permits assembly of the DNA clamp. Finally, the polymerase system must assembly and function within the context of a changing temperature and without the assistance of a secondary or auxiliary enzyme.

SUMMARY

[0009] It is an object of the invention herein to adapt DNA clamps to nucleic acid modifying enzymes (e.g., a DNA polymerase) to enhance the nucleic acid modifying enzyme performance and/or activity. It is also an object of the invention to adapt DNA clamps to nucleic acid modifying enzymes (e.g., a DNA polymerase) to provide a stutter-resistant DNA polymerase. Accordingly, provided herein is one or more DNA clamps that can be adapted for use with a nucleic acid modifying enzyme (e.g., a DNA polymerase), thereby enhancing the nucleic acid modifying enzyme performance, activity, processivity, or stutter-resistant capabilities. The novel protein complex adapting DNA clamp technology to a DNA polymerase is called Impala (e.g., Impala system, Impala polymerase system, Impala polymerase complex). The technology described herein is adaptable to a range of DNA polymerases ( e.g . Taq, KOD, Pfu, etc.) resulting in a series of enhanced or DNA clamp -augmented polymerases (e.g. Impala-Taq, Impala-KOD, Impala-Pfu, etc.).

[0010] Accordingly, provided herein is a protein complex including a heterologous protein, having a polymerase domain having a DNA polymerase, and a chassis linker domain, the polymerase domain joined by one or more bonds of the chassis linker domain to a DNA clamp protein having from one to six clamp domains joined by one or more bonds of an interdomain connector loop and/or a protomer connector sequence, the DNA polymerase configured for reversible association and dissociation in solution, wherein the chassis linker domain is an artificial protein sequence, optionally including 30 or more amino acids, and is configured to provide mechanical suspension between the polymerase domain and DNA clamp protein, wherein the interdomain connector loop is a linear protein sequence, optionally including 13 or more amino acids, that joins adjacent clamp domains within individual protomers and/or joins clamp domains between cognate protomers, and wherein the protomer connector sequence is a globular protein sequence comprising 30 or more amino acids that joins adjacent clamp domains from a DNA clamp protein and is configured to provide flexible association and dissociation of adjacent clamp domains.

[0011] Also provided herein is a kit for amplifying one or more nucleic acids, the kit including a first tube holding a heterologous protein as described herein, a second tube holding one or more deoxyribose nucleoside triphosphates (dNTPs), and a reaction buffer.

[0012] Also provided herein is a method of amplifying a nucleic acid sequence, including steps of providing a heterologous protein as described herein and amplifying the nucleic acid sequence by a polymerase chain reaction (PCR).

[0013] Also provided herein is an isolated nucleic acid molecule having a nucleotide sequence encoding or transcribing a heterologous protein as described herein.

[0014] Also provided herein is a cDNA encoding a nucleic acid molecule having a nucleotide sequence encoding or transcribing a heterologous protein as described herein. [0015] Also provided herein is an expression vector including a cDNA encoding a nucleic acid molecule having a nucleotide sequence encoding or transcribing a heterologous protein as described herein.

[0016] Various aspects of the present disclosure may be further characterized by one or more of the following clauses:

[0017] Clause 1: A protein complex comprising: a heterologous protein, comprising a polymerase domain comprising a DNA polymerase, and a chassis linker domain, the polymerase domain joined by one or more bonds of the chassis linker domain to a DNA clamp protein, comprising from one to six clamp domains joined by one or more bonds of an interdomain connector loop and/or a protomer connector sequence, the DNA clamp protein configured for reversible association and dissociation in solution, wherein the chassis linker domain is an artificial protein sequence, optionally comprising 30 or more amino acids, and is configured to provide mechanical suspension between the polymerase domain and DNA clamp protein, wherein the interdomain connector loop is a linear protein sequence, optionally comprising 13 or more amino acids, that joins adjacent clamp domains within individual protomers and/or joins clamp domains between cognate protomers, wherein the protomer connector sequence is a globular protein sequence comprising 30 or more amino acids that joins adjacent clamp domains from a DNA clamp protein and is configured to provide flexible association and dissociation of adjacent clamp domains.

[0018] Clause 2: The protein complex of clause 1 wherein the DNA polymerase of the heterologous protein has at least 80% sequence identity with one or more sequences selected from SEQ ID NOS:l-16.

[0019] Clause 3: The protein complex of clause 1 or clause 2 wherein the chassis linker domain comprises 30 or more amino acids, optionally 40 or more amino acids, or optionally 50 or more amino acids.

[0020] Clause 4: The protein complex of any of clauses 1-3, wherein the chassis linker domain has at least 80% sequence identity with one or more sequences selected from SEQ ID NOS: 152-157.

[0021] Clause 5: The protein complex of any of clauses 1-4, wherein the DNA clamp protein derives from a trimeric proliferating cell-nuclear antigen (PCNA), comprising three protomers, each protomer comprising two clamp domains, optionally a thermostable PCNA, or derives from a dimeric beta-clamp, comprising two protomers, each protomer comprising three clamp domains, optionally a thermostable beta-clamp.

[0022] Clause 6: The protein complex of any of clauses 1-5, wherein the clamp domains of the DNA clamp protein have at least 80% sequence identity with one or more sequences selected from SEQ ID NOS: 17-99.

[0023] Clause 7: protein complex of any of clauses 1-6, wherein the clamp domains are joined by one or more bonds of an interdomain connector loop configured to form rigid associations between adjacent clamp domains. [0024] Clause 8: The protein complex of any of clauses 1-7, wherein the interdomain connector loop has at least 80% sequence identity with one or more sequences selected from SEQ ID NOS: 100-130.

[0025] Clause 9: The protein complex of any of clauses 1-8, wherein the clamp domains are joined by one or more bonds of a protomer connector sequence configured to form flexible associations between adjacent clamp domains.

[0026] Clause 10: The protein complex of any of clauses 1-9, wherein the protomer connector sequence has at least 80% sequence identity with one or more sequences selected from SEQ ID NOS: 158-161.

[0027] Clause 11: The protein complex of any of clauses 1-10, wherein the DNA clamp reduces the DNA polymerase dissociation rate (koff) and/or increases the DNA polymerase association rate (kon).

[0028] Clause 12: The protein complex of any of clauses 1-11, wherein the one or more bonds is one or more of a peptide bond, an ionic bond, a disulfide bond, a hydrogen bond, or a hydrophobic/hydrophilic interaction .

[0029] Clause 13: The protein complex of any of clauses 1-12, wherein the one or more bonds is a covalent bond.

[0030] Clause 14: The protein complex of any of clauses 1-13, wherein the protein complex comprises a single polypeptide chain.

[0031] Clause 15: The protein complex of any of clauses 1-14, wherein the DNA clamp protein comprises two distinct polypeptide chains: a heterologous protein comprising a polymerase domain joined by one or more bonds of a chassis linker domain to a DNA binding domain, comprising between one and five clamp domains; and a secondary protein comprising one or more clamp domains of the DNA clamp protein that do not form part of the heterologous protein, wherein the clamp domains within a given polypeptide chain are joined by one or more bonds of an interdomain connector loop or a protomer connector sequence.

[0032] Clause 16: A kit for amplifying one or more nucleic acids, the kit comprising: a first tube comprising the heterologous protein of any of clauses 1-15; a second tube comprising deoxyribose nucleoside triphosphates (dNTPs); and a reaction buffer.

[0033] Clausel7: A method of amplifying a nucleic acid sequence, comprising: providing the heterologous protein of any of clauses 1-15; and amplifying the nucleic acid sequence by a polymerase chain reaction (PCR).

[0034] Clause 18: The method of clause 17, wherein amplifying the nucleic acid sequence comprises reducing PCR stutter, increasing processivity, or increasing extension rate. [0035] Clause 19: The method of clause 17 or clause 18, wherein the nucleic acid sequence is DNA.

[0036] Clause 20: An isolated nucleic acid molecule comprising a nucleotide sequence encoding or transcribing the heterologous protein of any of clauses 1-15.

[0037] Clause 21: A cDNA molecule encoding the nucleic acid molecule of clause 20. [0038] Clause 22: An expression vector comprising the cDNA molecule of clause 20.

BRIEF DESCRIPTION OF THE DRAWINGS [0039] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0040] Figure 1 : A lateral view of a model of an embodiment of the Impala protein complex, comprising a DNA polymerase (green) and the three protomers (blue, light green, purple) that form a trimeric DNA clamp, bound to dsDNA. The space in between the DNA clamp protein and the polymerase is referred to as the chassis gap. Labels also demonstrate the direction of polymerization with the polymerase seemingly moving along or in the direction of ssDNA template while producing a dsDNA product in its wake.

[0041] Figures 2A-2B: Representation of a protein equilibria resulting from a two- component system for the formation of Impala protein complex in accordance with certain embodiments. In this two-component system, the heterologous protein results from fusion of one trimeric DNA clamp protomer to a DNA polymerase (mPCNA-pol), which is cognate to and assembles with a secondary protein resulting from fusion of the remaining two PCNA protomers (dPCNA). In another embodiment of this Impala polymerase system, the heterologous protein results from fusion of two trimeric DNA clam protomers to another and a DNA polymerase (dPCNA-pol), which is cognate to and assembles with a secondary protein that is the remaining PCNA protomer (mPCNA). FIG. 2A demonstrates the equilibrium reactions for both embodiments (mPCNA-pokdPCNA and dPCNA-pokmPCNA) when employing a homotrimeric DNA clamp. For this system utilizing a homotrimeric DNA clamp, single protomers - either from mPCNA-pol or mPCNA) can assemble as a side-reaction to form a complete DNA clamp, but the incorrect protein complex. In order to drive the formation of the correct Impala-pol complex, one may add an excess of the dPCNA component, relative to mPCNA component. FIG. 2B demonstrates the equilibrium reactions for both embodiments (mPCNA-pokdPCNA and dPCNA-pokmPCNA) when employing a heterotrimeric DNA clamp. For this system utilizing a heterotrimeric DNA clamp, single protomers are incapable of self-assembling and no side reactions are possible.

[0042] Figures 3A-3C: FIG. 3A is a front view of a homotrimeric PCNA from P. abyssi bound to DNA. FIG. 3B is a perspective view of the PCNA from P. abyssi depicting the interdomain interface. FIG. 3C is a perspective view of the PCNA from P. abyssi depicting the interface between protomers. Color-coding identifies the three identical protomers (pink, blue, and green) which are further shaded to indicate the individual clamp domains (N-terminal domain “N”, darker; C-terminal domain “C”, lighter) for each protomer as well as the interdomain connector loop (IDCL, grey).

[0043] Figure 4: A front view of a b-clamp from D. radiodurans (PDB: 4TRT) bound to dsDNA from PDB: 6T8H. Color-coding identifies the two identical protomers which are identified by their individual clamp domains (N-terminal domain “N”, blue; Internal domain “I”, green; C-terminal domain “C”, yellow) as well as each interdomain connector loop (IDCL, grey).

[0044] Figure 5A-5B: A perspective view of a cyro-EM structure of PCNA from P. abyssi bound to DNA in association with the DNA polymerase (PolD) forming a complex of dsDNA, polymerase, and PCNA (PolD:PCNA:dsDNA) from PDB: 6T8H. The PCNA protomers (yellow, chartreuse and brown) form a DNA clamp homotrimer that interact with PolD, which is composed of subunits such as a proofreading exonuclease subunit (DPI; green) and a polymerase catalytic subunit (DP2; pink). FIG. 5A shows a lateral view of the PolD:PCNA:dsDNA complex wherease FIG. 5B presents a posterior view of the complex. [0045] Figures 6A-6C: Representations of different models for some embodiments of two- component Impala polymerase system. Here, the models further represent the mPCNA- pokdPCNA embodiment of the two-component Impala polymerase system employing a homotrimeric PCNA protein. The proteins are color-coded as follows: mPCNA-pol protein (green), dPCNA protein (blue), and a Cren7 DNA-binding protein (red). FIG. 6A is a lateral view of a variant Impala protein complex formed by a dPCNA protein complexed with the standard mPCNA-pol protein termed “Compact”. FIG. 6B and FIG. 6C are a lateral views of Impala protein complex design variants formed by dPCNA complexed with a mPCNA-pol protein that is further fused to a Cren7 DNA-binding protein. FIG. 6B represents the design variant or model termed “Coupe” For the Coupe model of mPCNA-pol from the two- component Impala system, the DNA clamp protein encircles dsDNA and separates polymerase on one face and Cren7 protein on the opposite face of the DNA clamp. In this manner, the Cren7 DNA-bind protein lies posterior to both polymerase and DNA clamp functioning to assist in further binding the dsDNA product exiting the Impala complex. FIG. 6C is a lateral view of the design variant of Impala protein complex termed “Coupe”. For the Coupe model of mPCNA-pol from the two-component Impala system, the Cren7 protein is central and lies in the chassis gap between the polymerase and the DNA clamp. For the Coupe design variant, the Cren7 DNA-binding protein is inserted into the chassis linker domain of the Impala protein complex.

[0046] Figures 7A-7D: Various views of a representative model of the Impala protein complex in accordance with some embodiments. FIG. 7A is a perspective (right side) view. FIG. 7B is a perspective (left side) view. FIG. 7C is a perspective top view. FIG. 7D is a perspective bottom view. The model demonstrates a mPCNA-pokdPCNA embodiment of the two-component Impala protein complex with the secondary protein (dPCNA) represented by two blue protomers of the trimeric DNA clamp connected by a protomer connector sequence (not shown). Peptides for the heterologous protein (mPCNA-pol) are color-coded: Taq polymerase (green) [PDB: 1TAU], mPCNA (green), and the chassis linker domain (yellow). Without additional DNA-binding proteins in the chassis gap or posterior to the DNA clamp protein, this model represents a Compact model of the heterologous protein. As its N- and C- termini face away from the polymerase, the homotrimeric DNA clamp protein [taken from PDB: 6T8H] is in the elaborate design configuration of the Impala protein complex.

[0047] Figures 8A-8B: FIG. 8 A and FIG. 8B illustrate various perspective views (anterior, posterior, dorsal, and ventral where indicated) of a representative model of the Impala protein complex represented in FIG. 7 in accordance with some embodiments. The arrow in FIGS. 8A- 8B illustrate the direction of DNA synthesis.

[0048] Figures 9A-9B: Lateral views of two representative models of the minimalist design configuration for the Impala protein complex in accordance with some embodiments. The models represent the dPCNA-pokmPCNA embodiment of the two-component Impala protein complex with the secondary protein (mPCNA) represented by the blue DNA clamp protomer. That DNA clamp is a PCNA123 heterotrimer [PDB: 2NTI]. Peptides for the heterologous protein (dPCNA-pol) are color-coded: Taq polymerase (white) [PDB: 1TAU], dPCNA (dark and light green), protomer connector sequence represented by a tryptophan cage (pink) [PDB: 1L2Y], and the chassis linker domain represented in two forms. FIG. 9A is a lateral view of a dPCNA-pokmPCNA embodiment of the minimalist design configuration for Impala-Taq with a chassis linker domain represented by a single elemental peptide; a tryptophan cage (yellow) [PDB: 2JOF]. FIG. 9B is a lateral view of a dPCNA-pokmPCNA embodiment of the minimalist design configuration for Impala-Taq with a chassis linker domain represented by two elemental peptides; the engrailed homeodomain (orange) [PDB: 3HDD] and a tryptophan cage (yellow) [PDB: 2JOF].

DETAILED DESCRIPTION

[0049] The following description is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. While the description is designed to permit one of ordinary skill in the art to make and use the invention, and specific examples are provided to that end, they should in no way be considered limiting. It will be apparent to one of ordinary skill in the art that various modifications to the following will fall within the scope of the appended claims. The present invention should not be considered limited to the presently disclosed aspects, whether provided in the examples or elsewhere herein.

[0050] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases. As used herein “a” and “an” refer to one or more. Patent publications cited below are hereby incorporated herein by reference in their entirety to the extent of their technical disclosure and consistency with the present specification.

[0051] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, are open ended and do not exclude the presence of other elements not identified. In contrast, the term “consisting of’ and variations thereof is intended to be closed and excludes additional elements in anything but trace amounts.

[0052] An elemental peptide is a discrete sequence of amino acids that exists with defined or undefined ( e.g . intrinsically disordered peptides) structure that remains discrete when joined through one or more bonds to another protein or sequence of amino acids. In this manner, elemental peptides may interact with one another, but do so while retaining their individual structure and/or fold.

[0053] DNA clamps (also referred to as sliding clamps, or sliding DNA clamps) are oligomeric toroidal protein complexes found throughout the spectrum of life including, but not limited to eukarya, archaea, bacteria, and in some viruses (Protein Sci. 2001 Jan; 10(1): 17- 23.)· DNA clamps comprise six structural clamp domains wherein each clamp domain adopts an a+b fold where two four-stranded b-sheets meet at an outer point while sandwiching two internal a-helices that contact the encircled double stranded DNA (Acta Cryst. (2008). D64, 941-949). The six clamp domains assemble into a toroidal structure with the capacity to encircle and slide along double-stranded DNA to enhance the processivity of a DNA polymerase during DNA replication. The six clamp domains may exist as six individual protomers or be organized into multidomain peptide subunits, which then assemble a complete DNA clamp as either a trimer of two-domain subunits or a dimer of three-domain subunits. [0054] DNA clamps in eukarya and archaea are termed proliferating cell nuclear antigen (PCNA), while DNA clamps in bacteria are termed b-clamps. DNA clamps are generally either dimeric or trimeric (in some instances hexameric), and while some DNA clamps exist as heterodimers or heterotrimers, most DNA clamps are either homodimeric or homo trimeric. DNA clamps exist as trimers in eukarya and archaea, whereas DNA clamps exist as dimers in bacteria. For whichever oligomeric arrangement, the protomer is defined as the basic structural unit of the oligomeric protein, the DNA clamp. Hence, each individual protein (i.e., distinct peptide) of the DNA clamp is defined as a protomer. For example, heterotrimeric DNA clamps are DNA clamps composed of three distinct protomers whereas homotrimeric DNA clamps are those composed of three identical protomers. Similarly, homodimeric DNA clamps are those composed of two identical protomers. Finally, the term ‘cognate’ is borrowed from linguistics to denote a correspondence between biological molecules that interact with one another. For example, the fidelity of the second genetic code is maintained due to the relationship between cognate tRNAs and tRNA-synthetases, which interact with one another ensuring a given tRNA is aminoacylated with the correct amino acid. With respect to DNA clamps, individual proteins or protomers are cognate if they interact with one another thus forming a toroidal, six domained DNA clamp. The individual protomer of a homotrimer or homodimer is said to be cognate with itself.

[0055] Referring to PCNA clamps, the trimeric DNA clamps found in eukarya and archaea, each protomer comprises an N-terminal clamp domain and a C-terminal clamp domain which are linked together by an interdomain connector loop. That trimeric DNA clamp assembles when three protomers interact and associate with one another thus forming the toroidal protein complex defined by six clamp domains.

[0056] Referring to b-clamps, the dimeric DNA clamps found in bacteria, each protomer of the dimeric DNA clamp comprises three clamp domains (N-terminal clamp domain, internal clamp domain, and C-terminal clamp domain) where adjacent clamp domains are linked together by two interdomain connector loops. This dimeric DNA clamp assembles when two protomers interact and associate with one another thus forming the toroidal protein complex defined by six clamp domains.

[0057] An interdomain connector loop (IDCL) is a linear amino acid linker sequence that connects adjacent clamp domains within the protomer of a DNA clamp. The interdomain connector loop functions to hold clamp domains together in a more rigid configuration thus creating multidomain protomers, which further assemble into the complete DNA clamp with all six clamp domains. It has been contemplated by others that the interdomain connector loop may function as a binding site for protein-protein interaction between the clamp domain and one or additional proteins ( e.g ., DNA polymerase). The interdomain connector loop can be and is defined using structural information to determine the flexible linker sequence between adjacent clamp domains. When structural data is missing for a given PCNA or b-clamp, it is possible to model the structure of the unknown clamp using homology modeling tools such as SWISS-MODEL Workspace (NAR, Vol 46, (2018) Pg W296-W303, Electrophoresis (2009)). Furthermore, it is possible to vary rigidity and flexibility between adjacent clamp domains by swapping interdomain connector loops or increasing their length. Finally, it is further possible to replace interdomain connector loops completely with artificial sequences bearing more complex structures and functions.

[0058] A protomer connector sequence is an artificial amino acid linker sequence that links one or more protomers of a DNA clamp. The protomer connector sequence biomimetically functions similar to the naturally-occurring interdomain connector loop by linking or fusing adjacent clamp domains. However, the protomer connector sequence differs by its larger size, which accommodates flexible movement between the linked protomers of the DNA clamp. In contrast to the linear structure of the interdomain connector loop, a protomer connector sequence has a globular nature to its protein structure, which affords the protomer connector sequence enhanced utility over a simple linear sequence (e.g., interdomain connector loop). [0059] More specifically, the protomer connector sequence affords temperature-based transition states for the Impala protein complex. Assuming the continued thermostability of two adjacent, cognate clamp domains above a given temperature, an interdomain connector loop can do little more than hold the two clamp domains together with varying flexibility determined by the its length and rigidity. Given those same thermostable clamp domains, the larger protomer connector sequence can afford two structural states. Below the transition temperature, a protomer connector sequence maintains its folded, globular form and so holds the two adjacent, cognate clamp domains together in a rigid state. However, above the transition temperature, a protomer connector sequence melts and elongates, and effectively linearizes thus creating a relaxed, or flexible state. Perhaps obviously, the desired transition temperature and overall performance of the protomer connector sequence depends on its amino acid and/or elemental peptide composition. It is contemplated that fast-folding proteins such as the villin headpiece (SEQ ID NO: 143) or the engrailed homeodomain (SEQ ID NO: 145) and their mutant variants would function as excellent elemental peptides in the design of effective protomer connector sequences. It is further contemplated that even smaller fast-folding proteins such as the tryptophan zipper and the tryptophan cage (SEQ ID NO: 138) would also prove effective in the design of protomer connector sequences.

[0060] Alternatively, a protomer connector sequence could replace one or more interdomain connector loops from a naturally-occurring DNA clamp protomer. It is contemplated herein that the protomer connector sequence may also provide a means for inclusion of a non-clamp, DNA-binding protein (e.g. Sul7d or Cren7) between DNA clamp protomers (or even between clamp domains) in order to further increase binding to and/or traction with dsDNA.

[0061] Provided herein is a protein complex comprising a DNA clamp protein having one or more clamp domains linked to a DNA polymerase in a manner that improves performance, activity, processivity, extension rate, or stutter-resistant capabilities of the DNA polymerase allowing for more complete or longer amplicon products while reducing the DNA polymerase error rate.

[0062] In some embodiments, the DNA clamp protein has one or more clamp domains. In some embodiments, the DNA clamp protein has two or more clamp domains. In some embodiments, the DNA clamp protein has one, two, three, four, five, or six clamp domains. In some embodiments, the DNA clamp protein has one clamp domain. In some embodiments, the DNA clamp protein has two clamp domains. In some embodiments, the DNA clamp protein in has three clamp domains. In some embodiments, the DNA clamp protein has four clamp domains. In some embodiments, the DNA clamp protein has five clamp domains. In some embodiments, the DNA clamp protein has six clamp domains.

[0063] In some embodiments, the protomer of the DNA clamp protein and/or the individual clamp domain has at least 75% sequence identity, optionally at least 80% sequence identity, optionally at least 85% sequence identity, optionally at least 90% sequence identity, optionally at least 95% sequence identity, optionally at least 99% sequence identity, optionally 100% sequence identity with one or more sequences of SEQ ID NO: 17-99, all values and subranges therebetween inclusive. [0064] In some embodiments, two or more clamp domains are joined by one or more bonds of an interdomain connector loop. In some embodiments, the interdomain connector loop comprises 13 or more amino acids. In some embodiments, the interdomain connector loop comprises 13-20 amino acids. In some embodiments, the interdomain connector loop comprises 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the interdomain connector loop has at least 75% sequence identity, optionally at least 80% sequence identity, optionally at least 85% sequence identity, optionally at least 90% sequence identity, optionally at least 95% sequence identity, optionally at least 99% sequence identity, optionally 100% sequence identity with one or more sequences of SEQ ID NO: 100-130, all values and subranges therebetween inclusive. In some embodiments, the interdomain connector loop comprises more than 20 amino acids with a flexible nature and no defined structure.

[0065] In some embodiments, the DNA clamp protein comprises a protomer of a PCNA (mPCNA), the protomer having 2 clamp domains. In some embodiments, the DNA clamp protein comprises two protomers of a PCNA (dPCNA), each protomer having 2 clamp domains. In some embodiments, the DNA clamp protein comprises three protomers of a PCNA, each having 2 DNA clamps.

[0066] In some embodiments, the DNA clamp protein comprises a protomer of a b-clamp, the protomer having 3 clamp domains. In some embodiments, the DNA binding domain comprises two protomers of a b-clamp, each monomer having 3 clamp domains.

[0067] In some embodiments, the individual protomers of a DNA clamp protein are joined by one or more bonds of a protomer connector sequence. In some embodiments, the protomer connector sequence comprises 30 or more amino acids. In some embodiments, protomer connector sequence has at least 75% sequence identity, optionally at least 80% sequence identity, optionally at least 85% sequence identity, optionally at least 90% sequence identity, optionally at least 95% sequence identity, optionally at least 99% sequence identity, optionally 100% sequence identity with one or more sequences of SEQ ID NO: 158- 162, all values and subranges therebetween inclusive. In some embodiments, the protomer connector sequence is an artificial sequence, an artificial synthesized sequence, an artificial peptide, or an artificial synthesized peptide that may provide improved performance, activity, processivity, extension rate, or stutter-resistant capabilities of the DNA polymerase. In some embodiments, the protomer connector sequence may include a DNA-binding protein such as Cren7 (SEQ ID NO: 149) or Sul7d (SEQ ID NO: 150) or GR (Sul7d-Cren7 chimera, SEQ ID NO: 151) that is configured to provide additional traction or grip to the dsDNA encircled by the DNA clamp. [0068] Further provided herein is a protein complex comprising a DNA clamp protein comprising one or more clamp domains, the DNA clamp protein joined by one or more bonds of a chassis linker domain to a polymerase domain comprising a DNA polymerase.

[0069] As will be understood by those of skill in the art, a polymerase is a nucleic acid modifying enzyme that performs template-directed synthesis of polynucleotides such as RNA or DNA. A DNA polymerase refers to an enzyme that performs template-directed DNA synthesis from a DNA template.

[0070] A chassis linker domain is a sequence of amino acids that joins a DNA clamp protein to a DNA polymerase. Whereas the DNA clamp protein functions to increase association with and so grip or traction to dsDNA, the chassis linker domain functions to provide mechanical suspension between (1) the DNA polymerase of the heterologous protein which catalyzes the synthesis of dsDNA and (2) the toroidal DNA clamp which encircles and secures newly synthesized dsDNA. The chassis linker domain is a designed assembly of elemental peptides including but not limited to short flexible linker sequences, fast-folding globular proteins, and other structural protein motifs. It is contemplated that multiple elemental peptides connected by flexibles linkers would function as the protein equivalent of solid or rigid bodies for the chassis linker domain thus granting increased degrees of freedom between DNA clamp protein and DNA polymerase within the context of a structured suspension. The chassis linker domain further functions to separate DNA clamp and DNA polymerase with the distance between them termed the chassis gap. In some embodiments, the chassis gap is filled with a DNA-binding protein such as Cren7 (SEQ ID NO: 149) or Sul7d (SEQ ID NO: 150) that is configured to provide additional traction or grip to the dsDNA between DNA polymerase and the DNA clamp.

[0071] In some embodiments, the chassis linker domain comprises 30 or more amino acids, 40 or more amino acids, or 50 or more amino acids.

[0072] In some embodiments, the chassis linker domain has at least 75% sequence identity, optionally at least 80% sequence identity, optionally at least 85% sequence identity, optionally at least 90% sequence identity, optionally at least 95% sequence identity, optionally at least 99% sequence identity, optionally 100% sequence identity with one or more sequences of SEQ ID NO: 152- 157, all values and subranges therebetween inclusive. In some embodiments, the chassis linker domain is an artificial sequence, an artificial synthesized sequence, an artificial peptide, or an artificial synthesized peptide that may provide improved performance, activity, processivity, extension rate, or stutter-resistant capabilities of the DNA polymerase. [0073] In some embodiments, the DNA polymerase has at least 75% sequence identity, optionally at least 80% sequence identity, optionally at least 85% sequence identity, optionally at least 90% sequence identity, optionally at least 95% sequence identity, optionally at least 99% sequence identity, optionally 100% sequence identity with one or more sequences of SEQ ID NO: 1-16, all values and subranges therebetween inclusive.

[0074] In some embodiments, the one or more bonds as described herein is one or more of a peptide bond, an ionic bond, a disulfide bond, a hydrogen bond, or a hydrophobic/hydrophilic interaction. In some embodiments, the one or more bonds is a covalent bond. As will be understood by those of skill in the art, a peptide bond is an amide type of covalent chemical bond linking two consecutive alpha-amino acids from Cl (carbon number one) of one alpha- amino acid and N2 (nitrogen number two) of another, along a peptide or protein chain. An ionic bond is a type of chemical bonding that involves the electrostatic attraction between oppositely charged ions, or between two atoms with different electronegativities, and is the primary interaction occurring in ionic compounds. A disulfide bond is derived by the coupling of two thiol groups in two cysteine residues. A hydrogen bond is an electrostatic force of attraction between a hydrogen atom which is covalently bound to a more electronegative atom or group, and another electronegative atom bearing a lone pair of electrons. A hydrophobic/hydrophilic interaction are forces of interactions that serve to keep chemical groups positioned close to one another. A covalent bond is a chemical bond that involves the sharing of electron pairs between atoms.

[0075] Also provided herein is a kit for amplifying one or more nucleic acids. In some embodiments, the kit may comprise a first tube having a heterologous protein comprising a polymerase domain joined by one or more bonds of a chassis linker domain to a DNA clamp protein, wherein the DNA clamp protein comprises one or more clamp domains, in accordance with any one of the embodiments described herein. The kit may further comprise a second tube having deoxyribose nucleoside triphosphate (dNTPs). In some embodiments, the kit may also include a reaction buffer suitable for polymerase chain reaction as would be understood by one of skill in the art. In some embodiments, the kit may comprise a third tube having a secondary protein comprising a one or more clamp domains, the one or more clamp domains joined by one or more bonds of an interdomain connector loop or a protomer connector sequence, wherein this second heterologous protein complexes with the heterologous protein thus forming a complete DNA clamp protein.

[0076] Also contemplated herein is a method of manufacturing a protein complex in accordance with any of the embodiments described herein. [0077] While it is contemplated that the use of the present invention is directed towards polymerase chain reaction (PCR), it is contemplated herein that the utility of the present invention may extend to other nucleic acid amplification techniques and/or sequencing technologies.

[0078] Further provided herein is a protein complex as described herein, wherein the DNA clamp protein improves performance, activity, processivity, extension rate, or stutter-resistant capabilities of the DNA polymerase allowing for more complete or longer amplicon products while reducing the DNA polymerase error rate.

[0079] PCR stutter is an artifact that arises when amplifying repeat sequences such as short tandem repeats (STRs) (Genomics Proteomics Bioinformatics, 2007 Feb 5(1): 7-14). If a DNA polymerase dissociates while amplifying an STR, the last repeat unit of the newly generated strand may slide up (stream) or down (stream) along the template strand (Frontiers in Microbiology, 06 Aug 2014, 5: 403). This DNA slippage event results in the addition or subtraction of one or more repeat units to the final product or amplicon. This creates a mixed population of amplicons with varying lengths that are observed as multiple signals or peaks. With respect to microsatellite instability (MSI) analysis, DNA slippage results in multiple peaks per target locus arranged as a distribution centered around the ‘true peak’ flanked by the false peaks bearing or missing repeat units. The intensity of the true peak will decrease, and the distribution of peaks will flatten as DNA slippage and PCR stutter worsen. PCR stutter can be a function of DNA slippage across nucleotide repeats, which may occur in the absence of polymerase following stalling and dissociation. As such, reducing PCR stutter may be possible by decreasing DNA slippage. Decreasing DNA slippage may occur by preventing polymerase dissociation from the growing amplicon, and/or, in the event that the polymerase does dissociate from a double- stranded DNA amplicon, DNA slippage can be reduced by preventing dissociation or melting of the growing amplicon from the template.

[0080] Processivity of an enzyme is the ability of the enzyme to perform consecutive or successive reactions between the association and/or dissociation of its substrate. With respect to template-based synthesis by a nucleic acid modifying enzyme or polymerase ( e.g ., a DNA polymerase), processivity is the average number of nucleotides added to a growing amplicon (opposite the template strand) per associate event.

[0081] In some embodiments, the DNA clamp protein is configured to increase processivity and/or polymerase performance by reducing k _0ff (e.g., polymerase dissociation) or increasing k _on (polymerase association). In some embodiments, the DNA clamp protein is configured to encircle and/or slide along double-stranded DNA. In some embodiments, a DNA polymerase is linked to the DNA clamp protein, and, when the DNA polymerase is bound to double- stranded DNA, the DNA clamp protein functions to enhance amplification by increasing processivity and preventing DNA polymerase dissociation.

[0082] In the event of DNA polymerase dissociation, the DNA clamp protein may function to maintain the DNA polymerase in close proximity to the double-stranded DNA until reassociation of the DNA polymerase to the double-stranded DNA occurs. In some embodiments, the DNA clamp protein further prevents the dissociation or melting of dsDNA from the growing end of the amplicon whilst the DNA polymerase remains dissociated.

[0083] In some embodiments, the DNA clamp protein reduces the DNA polymerase dissociate rate (k _0ff ) or increases the DNA polymerase association rate (k _on ).

Example 1

[0084] The foundational theory for designing the Impala polymerase system is a simple observation. PCR stutter is less about the polymerase itself and more a function of DNA slippage across repeats, which occurs in the absence of polymerase following stalling and dissociation. Hence, reducing PCR stutter is possible by addressing (decreasing or eliminating) DNA slippage. First, prevent polymerase dissociation from the growing amplicon in the first place. Second, in the event that the polymerase does dissociate from a double-stranded DNA amplicon, DNA slippage can be blocked by preventing dissociation or melting of the growing amplicon from the template.

[0085] Towards this end, we developed the Impala polymerase system. Impala is a multi subunit protein complex or molecular chassis that grants a polymerase the functionality of a PCNA or DNA clamp in a PCR-compatible manner without the need for a clamp loader and other accessory proteins.

[0086] The Impala polymerase system has been designed such that the DNA clamp increases processivity and polymerase performance by reducing k _0ff (polymerase dissociation) and increasing k _on (polymerase association). However, and unlike the disclosure of U.S. Patent No. 6,627,424, the one or more clamp domains physically encircle and have the capacity to slide along double-stranded DNA. Once assembled, the DNA clamp protein of the Impala protein complex effectively connects its covalently-bound polymerase domain to the surrounded double-stranded DNA amplicon. When polymerase is bound to the double-stranded DNA amplicon, the DNA clamp protein of the Impala protein complex functions to enhance amplification by increasing processivity and preventing polymerase dissociation. In the event of polymerase dissociation during elongation, the DNA clamp protein of the Impala protein complex functions to keep its polymerase domain in close proximity to the double-stranded DNA amplicon until the two can reassociate.

[0087] As depicted in FIG. 1, a non-limiting embodiment of the Impala system is a two- component system wherein the protein complex is composed of two distinct polypeptide chains that assemble to form an irregular two-piece, oligomeric DNA clamp, which derives from a DNA clamp. More specifically, the three protomers of a trimeric DNA clamp would be divided between the two distinct proteins with the first protein termed the ‘heterologous protein’ comprising one or more protomers fused to a polymerase and the second protein comprising the remaining protomer(s) present as a single polypeptide termed the ‘secondary protein’. When heterologous protein and secondary protein complex with one another, they reconstitute a complete DNA clamp that functions as the DNA clamp protein of the Impala protein complex. [0088] In FIG. 1, the proteins of this Impala two-component system are represented by the Connolly surface (e.g., solvent-accessible surface area). The heterologous protein (mPCNA- pol) comprises a DNA polymerase (e.g., Taq DNA polymerase; PDB: 1TAU) in dark green and a single protomer of a trimeric PCNA clamp (mPCNA, PDB: 6T8H) colored light green, which would be connected by a chassis linker domain. The two remaining protomers of the PCNA clamp (blue and purple) can be linked together by a protomer connector sequence to form a dimer of two PCNA protomers that is the secondary protein (dPCNA) of this two- component Impala polymerase system. In this embodiment (i.e., mPCNA-pokdPCNA), the Impala protein complex forms when mPCNA-pol and dPCNA associate with one another thus forming the complete, six-domain DNA clamp protein. Given a homotrimeric DNA clamp, it is further possible that a complete, six-domain DNA clamp protein could assemble from three individual mPCNA-pol proteins. If a heterotrimeric DNA clamp is employed, then it is impossible for three mPCNA-pol proteins to assemble into a complete, six-domain DNA clamp.

[0089] Another non-limiting embodiment of the two-component Impala system is the protein complex with the heterologous protein formed by joining a DNA polymerase via chassis linker domain to one PCNA protomer that is then joined to another PCNA protomer by a protomer connector sequence. The secondary protein in this embodiment of the two- component Impala system is the remaining protomer or single monomer of the DNA clamp employed (mPCNA). In this embodiment (i.e., dPCNA-pokmPCNA), the Impala protein complex forms when dPCNA-pol and mPCNA associate with one another thus forming the complete, six-domain DNA clamp protein. Given a homotrimeric DNA clamp, it is further possible that a complete, six-domain DNA clamp protein could assemble from three individual mPCNA proteins. If a heterotrimeric DNA clamp is employed, assembly of three individual PCNA proteins is not possible.

[0090] As illustrated in FIG. 2, for an Impala protein complex with a homotrimeric DNA clamp, three mPCNA-pol proteins are capable of associating to form a complete trimeric DNA clamp. A protein complex in accordance with some embodiments forms when one heterologous protein (i.e., mPCNA-pol) protein associates with one secondary protein (i.e., dPCNA). For the mPCNA-pol :dPCN A embodiment, adding an excess of secondary protein (dPCNA) relative to heterologous protein (mPCNA-pol) can drive the formation of the Impala protein complex and reduces the formation of the alternative complex formed from three mPCNA-pol proteins.

Example 2

[0091] DNA clamps, also known as sliding clamps, PCNA clamps, or b-clamps, are multimeric toroidal proteins whose assembled structure is composed of six clamp domains that encircles dsDNA and function as a processivity factors for replicative DNA polymerases (Acta Cryst (2008) D64, 941-949). As illustrated in FIGS. 3A-3C, each clamp domain of the DNA clamp adopts an a+b fold where two four-stranded b-sheets meet at an outer point while sandwiching two internal a-helices that contact the encircled dsDNA. These six clamp domains are further assembled into multi-domain subunits termed protomers, where each subunit or protomer constitutes a single peptide chain and the association of two or more protomers functions to assemble the complete structure of the DNA clamp toroid. FIGS. 3A-3C represent a trimeric PCNA clamp (colored pink, blue, and green), each protomer having two clamp domains ( e.g ., an N-terminal clamp domain (shaded darker) and a C-terminal clamp domain (shaded lighter) which are joined by an interdomain connector loop (grey).

[0092] Typically, DNA clamps assemble as dimers or trimers. More specifically, bacterial DNA clamps are dimeric whereas eukaryotes, archaea, and the viruses that use DNA clamps all employ a trimeric structure. The DNA clamps of eukaryotes and archaea are generally referred to as PCNA (from “proliferating cell nuclear antigen”) clamps. For trimeric PCNA clamps, the individual subunits are composed of an N-terminal domain (Domain A) that is linked to the C-terminal domain (Domain B) by a linear linker, the interdomain connector loop (IDCL). It is also thought that the IDCL functions as the binding site for protein-protein interactions between the DNA clamp and interacting partners (e.g., polymerase) (Acta Cryst. (2009). D65, 560-566.

[0093] For the mPCNA-pokdPCNA embodiment of the Impala system (FIGS. 1, 2, 7, 8), the first protein component or heterologous protein (mPCNA-pol) may be the fusion of a polymerase to one protomer of a trimeric DNA clamp through a chassis linker domain. As the secondary protein of that Impala protein complex, dPCNA is a fusion of two protomers from a trimeric DNA clamp that is complementary or cognate to the protomer from mPCNA-pol. The individual DNA clamp protomers of the dPCNA protein are fused by a protomer connector sequence. Finally, as with the polymerases employed in PCR, the individual protomers of the DNA clamp selected for use in the Impala polymerase system would also be thermostable and have the ability to survive repeated thermal challenges at the elevated temperatures encountered during in vitro amplification such as PCR.

[0094] FIG. 4 represents a b-clamp that is a dimer, each protomer having 3 clamp domains which are colored blue, green, and yellow. The N-terminal domain (blue), internal domain (green), and the C-terminal domain (yellow) are each joined by an interdomain connector loop (grey). In certain embodiments of the Impala system, it is possible to generate mPCNA- pokdPCNA and dPCNA-pokmPCNA components from a dimeric b-clamp by removing the clamp domain of protomer and correctly fusing it to its cognate protomer. This transfer of one clamp domain results in a two new peptides, a two clamp domain peptide that is complemented or cognate to a now four clamp domains peptide. In this manner, transfer of one clamp domain can convert the dimeric b-clamp into the familiar mPCNA:dPCNA format of the two- component Impala system.

Example 3

[0095] The assembly of mPCNA-pol and dPCNA (or dPCNA-pol and mPCNA) is a protein complex referred to as the “Impala complex” or “Impala polymerase complex”. For the model shown in FIG. 1, an explicit polymerase is named, so the protein assembly would be referred to as the “Impala-Taq complex” or “Impala-Taq”. Although the polymerase selected may affect design and final sequence, the secondary protein (mPCNA or dPCNA) of this two-component Impala complex functions to complete the DNA clamp and is designed to be polymerase agnostic. Here, the Impala polymerase system allows for reaction customization with functional variants of the secondary protein.

[0096] With the two-component Impala system, association of the heterologous protein and secondary protein forms a complete DNA clamp. When these two proteins further associate with template-primer double-stranded DNA, the result is a strong or favorable initiation complex with the formed DNA clamp protein completely encircling template-primer duplex. This Impala design is further defined as “Convertible” to denote its two-protein, two- component nature and highlight its compatibility with thermocycling-based amplification. [0097] At the beginning of each PCR cycle, the reaction is incubated at an elevated temperature in order to melt template DNA, as well as any DNA product generated from a previous cycle. That same melt step at the start of each PCR cycle functions to melt or dissociate the heterologous and secondary proteins of the Impala polymerase complex. Similar to template and oligonucleotide primer annealing, heterologous and secondary components would reassociate and bind to template-primer duplex, as the reaction temperature dropped from the elevated melting temperature to that of the lower annealing temperature.

[0098] With respect to PCR stutter, the Impala complex addresses both methods for reducing or eliminating DNA slippage. The first method is to prevent polymerase dissociation in the first place, which is addressed through DNA clamp -based processivity gains. If polymerase is bound and amplifying, then DNA slippage is not possible. However, in the event of polymerase dissociation from amplicon, the Impala system prevents diffusion of polymerase and amplicon away from one another through the chassis linker domain. This permits faster reassociation than possible from diffusion and binding of a new polymerase complex. Furthermore, the DNA clamp protein of the Impala protein complex passively reduces DNA slippage by encircling the growing end of the dsDNA amplicon, which increases stability of that duplex while sterically blocking movement of the growing strand along the template strand.

[0099] For the mPCNA-pokdPCNA embodiment of Impala polymerase system, a complete homotrimeric DNA clamp can assemble from either three heterologous proteins (mPCNA-pol) or one heterologous protein (mPCNA-pol) bound to cognate secondary protein (dPCNA). Given this definition of the Impala polymerase system, it is impossible for any number of dPCNA proteins to assemble into a trimeric DNA clamp. Instead, dPCNA is effectively inert in the absence of its cognate protomer represented by mPCNA-pol. The easiest strategy is to employ Le Chatelier’s principle to drive equilibrium toward the formation of the Impala complex by adding excess dPCNA relative to mPCNA-pol (FIG. 2). Although this may lead to Impala complex formation in the absence of template and primer, it is contemplated that those Impala complexes assembled will be transient assemblies constantly associating and dissociating. Finally, the use of heterotrimeric PCNA clamps would eliminate the potential for formation of a three mPCNA-pol complex.

[00100] It is further contemplated that the interactions between the DNA duplex (annealed primer and template) and the Impala complex will favor the formation of that greater complex leading to initiation and, through gains to processivity, the rapid formation of product/amplicon. Finally, augmenting a polymerase with a PCR-compatible DNA clamp permits variation of the extension temperature used. It is possible to lower the extension temperature down to 60°C for Taq DNAP (standard extension temperature is 72°C). However, with the added stability of a DNA clamp, amplification at higher temperatures becomes possible. An optimal temperature could be used to tune the best performance for a two- component Impala system by balancing DNA clamp protein melting with polymerase performance as well as the flexibility and shape of chassis linker domain and protomer connector sequences.

[00101] There are several caveats to consider for design of the two-component Impala system. During amplification, the heterologous protein and secondary protein must complex and remain associated at the extension temperature and throughout that step. Selection of a thermostable DNA clamp should address this concern. If not, increasing the ratio of dPCNA:mPCNA-pol is one method for driving the formation of a complete DNA clamp. [00102] However, it remains possible to over-engineer a system. With respect to Impala, the association of heterologous protein and secondary protein ( e.g ., mPCNA-pol and dPCNA) could be so favorable that the DNA clamp would assemble without binding and encircling dsDNA. The likelihood and severity of this second scenario is expected to increase at higher ratios of dPCNA:mPCNA-pol. As previously discussed, adjustments to the chassis linker domain and more importantly the protomer connector sequence can function to tune the stability and formation of the DNA clamp. Similarly, the overall stability of a DNA clamp could be further tuned by replacing naturally-occurring interdomain connector loops (IDCLs) with protomer connector sequences. Another means for addressing this concern is to weaken the interactions between the two clamp protomers of dPCNA. In this manner, the dPCNA can more freely transition between the closed form of associated clamp protomers and an unassociated open form. Residue Fill from the PCNA homotrimer of Pyrococcus abyssi was identified as an ideal candidate for gentle perturbations to the subunit interface. Mutation of this residue to tyrosine, histidine, or valine is expected to provide the desired effect. Finally, we have further identified residue F241 from the P. abyssi PCNA homotrimer as a site for similar mutagenesis as Fill (i.e. mutation to tyrosine, histidine, or valine) in order to gently perturb the interface between clamp domains of a single clamp protomer. Similar strategies can be applied to tune the performances of different DNA clamps.

Example 4

[00103] FIG. 6 illustrates the structure of the DNA-bound PolD-PCNA processive complex from P. abyssi (PDB: 6T8H) (Nat Commun 11, 1591 (2020). However, in nature, native polymerase and all DNA clamp protomers are discrete proteins whose assembly and function is guided by important protein-protein interactions and the workings of the larger replication fork complex with no appreciable space between a polymerase and its assembled DNA clamp. For the Impala polymerase system, it is possible to vary the distance or “chassis gap” between polymerase and DNA clamp protein. The chassis linker domain - between and connecting the polymerase and the DNA clamp protein - determines the chassis gap distance and its occupancy as well as the flexibility or rigidity of the Impala complex.

[00104] When considering the structures of some trimeric DNA clamps (e.g., 2HIK, 1RWZ, 3LX2), the N- and C-termini of the clamp protomers point toward one side or “face” of the toroidal DNA clamp, which is referred to as the “termini-face”. While the N-terminus is angled steeper and enters the clamp protomer above the IDCL, the C-terminus exits the clamp protomer below the IDCL and so points out perpendicular to the plane formed by the toroidal DNA clamp. The other side of the DNA clamp is referred to as the “smooth-face.” For the DNA-bound PolD-PCNA processive complex from P. abyssi (FIG. 6), the termini-face of the assembled DNA clamp is oriented toward the polymerase.

[00105] For the Impala system, it is possible to have either the termini-face or the smooth- face of the DNA clamp protein oriented toward the polymerase, which is determined by the chassis linker domain of Impala protein complex. Design of the Impala complex protein is much simpler with the termini-face of the DNA clamp oriented toward polymerase. However, this design could impede polymerase activity as the various linker domains (chassis linker domain and protomer connection sequence(s)) of the Impala complex increase the occupancy of and so potential clutter the chassis gap. By contrast, orienting the smooth-face of the DNA clamp toward polymerase keeps the chassis gap free of unnecessary clutter while affording the greatest degree of design flexibility. The chassis linker domain between polymerase and DNA clamp must balance between flexible license that allows optimal polymerization and rigid structure that holds the complex in place during and throughout amplification and thermocycling.

[00106] FIGS. 7A-7D illustrate various views of a non-limiting embodiment of the Compact Impala-Taq polymerase. FIG. 7A is a right lateral view. FIG. 7B is a left lateral view. FIG. 7C is a dorsal (top) view. FIG. 7D is a ventral (bottom) view. In FIGS. 7A-7D, structural elements are color coded: clamp protomer and Taq DNA polymerase domains (green), chassis linker domain (yellow), and the generic dPCNA (blue). Example 5

[00107] A consideration for design of an Impala polymerase system is DNA clamp orientation. Again, orienting the termini-face toward polymerase reduces the complexity of the chassis linker domain used to connect DNA clamp and polymerase with the caveat of potential steric crowding in the chassis gap. This design configuration of the Impala system where the termini-face of a DNA clamp is oriented toward polymerase is termed a “minimalist configuration” or a “minimalist design configuration”. Accordingly, the design configuration where the smooth-face of a DNA clamp is oriented toward the polymerase is termed an “elaborate configuration” or an “elaborate design configuration”.

[00108] With respect to the elaborate configuration of the Impala system, three principal models of the heterologous protein (mPCNA-pol) for the mPCNA-pokdPCNA embodiment of the two-component Impala polymerase system were designed: Compact (FIG. 6A), Coupe (FIG. 6B), and Sedan (FIG. 6C). We have further designed three variant of the secondary protein (dPCNA) for the mPCNA-pokdPCNA embodiment of the two-component Impala polymerase system: Elastic, Turbo-C, and Turbo-S. As its design does not crowd the chassis gap, the elaborate configuration of the Impala polymerase system affords the ability to include further DNA-binding protein functionality through fusion into a posterior-facing protomer connection sequence. These secondary proteins with expanded DNA-binding functionality are colloquially called Turbo to continue our car metaphors. The Turbo-C dPCNA variant includes a Cren7 DNA-binding protein in its posterior-facing protomer connector sequence whereas the Turbo-S and Turbo-G dPCNA variants include a Sul7 DNA-binding protein and GR mut (Sul7d-Cren7 chimera) in that same sequence respectively. The below elaborate configuration models of the Impala polymerase system utilize the homotrimeric PCNA from Pyrococcus abyssi (SEQ ID NO: 17).

[00109] An amino acid sequence for mPCNA-pol for Compact Impala-Taq may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 17] [SEQ ID NO: 154] [SEQ ID NO: 2].

[00110] An amino acid sequence for mPCNA-pol for Coupe Impala-Taq may be: SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 151] [SEQ ID NO: 134] [SEQ ID NO: 17] [SEQ ID NO: 154] [SEQ ID NO: 2]

[00111] An amino acid sequence for mPCNA-pol for Sedan Impala-Taq may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 17] [SEQ ID NO: 155] [SEQ ID NO: 2] [00112] An amino acid sequence for Elastic dPCNA may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 17] [SEQ ID NO: 159] [SEQ ID NO: 17] [SEQ ID NO: 131] [SEQ ID NO: 148] [SEQ ID NO: 131] [SEQ ID NO: 132] [00113] An amino acid sequence for Turbo-C dPCNA may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 17] [SEQ ID NO: 160] [SEQ ID NO: 17] [SEQ ID NO: 131] [SEQ ID NO: 148] [SEQ ID NO: 131] [SEQ ID NO: 132] [00114] An amino acid sequence for Turbo-G dPCNA may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 17] [SEQ ID NO: 161] [SEQ ID NO: 17] [SEQ ID NO: 131] [SEQ ID NO: 148] [SEQ ID NO: 131] [SEQ ID NO: 132] [00115] An amino acid sequence for Turbo-S dPCNA may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 17] [SEQ ID NO: 162] [SEQ ID NO: 17] [SEQ ID NO: 131] [SEQ ID NO: 148] [SEQ ID NO: 131] [SEQ ID NO: 132] [00116] Additionally, the adaptation of the Impala polymerase systems to polymerases with N- and C-terminal fusion requirements should be considered when designing the heterologous protein. Taq polymerase is an example of a DNA polymerase that can successfully tolerate N- terminal fusions, but not fusion to its C-terminus. By contrast, KOD polymerase (and others) tolerate protein fusion to its C-terminus. The below models represent the same elaborate configuration Impala designs utilizing the same homotrimeric PCNA from Pyrococcus abyssi (SEQ ID NO: 17), which will remain cognate and compatible with the three above-mentioned dPCNA designs.

[00117] An amino acid sequence for mPCNA-pol for Compact Impala-KOD may be: [SEQ ID NO: 13] [SEQ ID NO: 156] [SEQ ID NO: 17] [SEQ ID NO: 133] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00118] An amino acid sequence for mPCNA-pol for Coupe Impala-KOD may be: [SEQ ID NO: 13] [SEQ ID NO: 156] [SEQ ID NO: 17] [SEQ ID NO: 133] [SEQ ID NO: 151] [SEQ ID NO: 134] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00119] An amino acid sequence for mPCNA-pol for Sedan Impala-KOD may be: [SEQ ID NO: 13] [SEQ ID NO: 157] [SEQ ID NO: 17] [SEQ ID NO: 133] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00120] With this convertible, two-component Impala system, it is possible to prepare twelve design configurations from the seven individual proteins (three heterologous proteins and four secondary proteins) (Table 1). While some configurations may lead to poorer performance than others, it is also possible that different configurations may be better suited for specific tasks or applications. As mentioned previously, these dPCNA proteins should be universally compatible with any subsequent mPCNA-pol regardless of the polymerase used so long as cognate DNA clamp protomers are used.

Table 1. Potential combinations of the mPCNA-pol models with different dPCNA for the two- component Impala polymerase system.

[00121] FIGS. 7A-7D illustrate various views of a non-limiting embodiment (mPCNA- pohdPCNA) for the Compact Impala-Taq polymerase employing an elaborate design configuration. FIG. 7A is a right lateral view. FIG. 7B is a left lateral view. FIG. 7C is a dorsal (top) view. FIG. 7D is a ventral (bottom) view. In FIGS. 7A-7D, structural elements are color coded: mPCNA and Taq DNA polymerase of the heterologous protein (green), chassis linker domain (yellow), and a generic dPCNA representing the secondary protein (blue).

[00122] FIGS. 8 A and 8B illustrate various views of a non-limiting embodiment of the Compact Impala-Taq polymerase. In FIGS. 8 A and 8B, the dorsal side is generally depicted at 802, the posterior side is generally depicted at 804, the anterior side is generally depicted at 806, and the ventral side is generally depicted at 808. Arrows 810 in FIGS. 8A and 8B illustrate the direction of DNA synthesis.

[00123] With respect to the minimalist configuration of the Impala system, we have designed two principal models for the heterologous protein (dPCNA-pol) for the dPCNA- pokmPCNA embodiment of the Impala polymerase system. Whether with a dPCNA- pokmPCNA or mPCNA-pokdPCNA embodiment, steric crowding of the chassis gap should be considered for the minimalist configuration of the Impala polymerase. Crowding of the chassis gap would result from the chassis linker domain as well as protomer connector sequences and any termini fusion sequences such as solubility fusion proteins or affinity purification tags. The fast-folding protein known as the “tryptophan cage” (SEQ ID NO: 138) as well as its variants presents the ideal elemental peptide for design of a protomer connector sequence (SEQ ID NO: 158) that avoids steric crowding of the chassis gap. The melting temperature of a tryptophan cage can be tuned through minor mutation (Protein Engineering, Design and Selection, Volume 21, Issue 3, March 2008, Pages 171-185,), which would afford considerable tuning of the transition state for a protomer connector sequence with minimal cost to steric bulk. In this manner, a tryptophan cage is an ideal elemental peptide for minimalist configuration design as it avoids crowing the chassis gap while serving as a kind of ‘thermal hinge’. Similarly, a tryptophan cage is included in the design of two version of the chassis linker domain for a minimalist configuration (SEQ ID NO: 152, 153). In order to accommodate the dPCNA-pokmPCNA embodiment, these models utilize the heterotrimeric PCNA from Saccharolobus solfataricus P2 {PCNA1_SACS2 (SEQ ID NO: 35), PCNA2_SACS2 (SEQ ID NO: 38), PCNA3_SACS2 (SEQ ID NO: 41)} with PCNA3_SACS2 serving as the secondary protein (mPCNA) of this embodiment.

[00124] An amino acid sequence for dPCNA-pol for minimalistic-1 Impala-Taq may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 35] [SEQ ID NO: 158] [SEQ ID NO: 38] [SEQ ID NO: 152] [SEQ ID NO: 2]

[00125] An amino acid sequence for dPCNA-pol for minimalistic-2 Impala-Taq may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 35] [SEQ ID NO: 158] [SEQ ID NO: 38] [SEQ ID NO: 153] [SEQ ID NO: 2]

[00126] An amino acid sequence for dPCNA-pol for minimalistic-1 Impala-KOD may be: [SEQ ID NO: 13] [SEQ ID NO: 152] [SEQ ID NO: 35] [SEQ ID NO: 158] [SEQ ID NO: 38] [SEQ ID NO: 133] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00127] An amino acid sequence for dPCNA-pol for minimalistic-2 Impala-KOD may be: [SEQ ID NO: 13] [SEQ ID NO: 153] [SEQ ID NO: 35] [SEQ ID NO: 158] [SEQ ID NO: 38] [SEQ ID NO: 133] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00128] An amino acid sequence for minimalistic mPCNA may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 41] [SEQ ID NO: 131] [SEQ ID NO: 132]

Example 6

[00129] As a final contemplation, the one-component or single polypeptide Impala polymerase system was considered. With respect to interactions with a dsDNA (e.g., template:primer), a one-component Impala system reduce the reaction order from third to second. It is further contemplated that this system would be rather rigid and may not encircle and capture dsDNA as effectively as a two-component system with skewed molar ratios. It is further contemplated that these issues - observed as performance loss - could be corrected by increasing flexibility across the DNA clamp protein. This could be accomplished by increasing the length and flexibility of the protomer connector sequence, that of interdomain connector loops, and/or through the replacement of interdomain connector loops with protomer connector sequences. Below are several embodiments for a one component Impala polymerase systems varying between the polymerase used (Taq or KOD) and so the fusion terminus (N- or C-) as well as the DNA clamp configuration (minimalist or elaborate) and finally the DNA clamp utilized (homotrimeric or heterotrimeric). More specifically, the heterotrimeric PCNA from Saccharolobus solfataricus P2 {PCNA1_SACS2 (SEQ ID NO: 35), PCNA2_SACS2 (SEQ ID NO: 38), PCNA3_SACS2 (SEQ ID NO: 41)} and the homotrimeric PCNA from Pyrococcus abyssi (SEQ ID NO: 17) may be utilized.

[00130] An amino acid sequence for one-component, minimalistic configuration of Impala- Taq utilizing a homotrimeric DNA clamp may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 17] [SEQ ID NO: 158] [SEQ ID NO: 17] [SEQ ID NO: 158] [SEQ ID NO: 17] [SEQ ID NO: 153] [SEQ ID NO: 2]

[00131] An amino acid sequence for one-component, elaborate configuration of Impala-Taq utilizing a homotrimeric DNA clamp may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 17] [SEQ ID NO: 159] [SEQ ID NO: 17] [SEQ ID NO: 159] [SEQ ID NO: 17] [SEQ ID NO: 154] [SEQ ID NO: 2]

[00132] An amino acid sequence for one-component, minimalistic configuration of Impala- KOD utilizing a homotrimeric DNA clamp may be: [SEQ ID NO: 13] [SEQ ID NO: 153] [SEQ ID NO: 17] [SEQ ID NO: 158] [SEQ ID NO: 17] [SEQ ID NO: 158] [SEQ ID NO: 17] [SEQ ID NO: 133] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00133] An amino acid sequence for one-component, elaborate configuration of Impala- KOD utilizing a homotrimeric DNA clamp may be: [SEQ ID NO: 13] [SEQ ID NO: 156] [SEQ ID NO: 17] [SEQ ID NO: 159] [SEQ ID NO: 17] [SEQ ID NO: 159] [SEQ ID NO: 17] [SEQ ID NO: 133] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00134] An amino acid sequence for one-component, minimalistic configuration of Impala- Taq utilizing a heterotrimeric DNA clamp may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 35] [SEQ ID NO: 158] [SEQ ID NO: 38] [SEQ ID NO: 158] [SEQ ID NO: 41] [SEQ ID NO: 153] [SEQ ID NO: 2] [00135] An amino acid sequence for one-component, elaborate configuration of Impala-Taq utilizing a heterotrimeric DNA clamp may be: [SEQ ID NO: 132] [SEQ ID NO: 131] [SEQ ID NO: 141] [SEQ ID NO: 133] [SEQ ID NO: 35] [SEQ ID NO: 159] [SEQ ID NO: 38] [SEQ ID NO: 159] [SEQ ID NO: 41] [SEQ ID NO: 154] [SEQ ID NO: 2].

[00136] An amino acid sequence for one-component, minimalistic configuration of Impala- KOD utilizing a heterotrimeric DNA clamp may be: [SEQ ID NO: 13] [SEQ ID NO: 153] [SEQ ID NO: 35] [SEQ ID NO: 158] [SEQ ID NO: 38] [SEQ ID NO: 158] [SEQ ID NO: 41] [SEQ ID NO: 133] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00137] An amino acid sequence for one-component, elaborate configuration of Impala- KOD utilizing a heterotrimeric DNA clamp may be: [SEQ ID NO: 13] [SEQ ID NO: 156] [SEQ ID NO: 35] [SEQ ID NO: 159] [SEQ ID NO: 38] [SEQ ID NO: 159] [SEQ ID NO: 41] [SEQ ID NO: 133] [SEQ ID NO: 141] [SEQ ID NO: 131] [SEQ ID NO: 132]

[00138] Although the invention has been described in detail for the purpose of illustration based on what is currently considered to be the most practical and preferred embodiments, it is to be understood that such detail is solely for that purpose and that the invention is not limited to the disclosed embodiments, but on the contrary, is intended to cover modifications and equivalent arrangements that are within the spirit and scope of the appended claims. For example, it is to be understood that the present invention contemplates that, to the extent possible, one or more features of any embodiment can be combined with one or more features of any other embodiment.