# Patent

## Title
Fusion polymerases

## Abstract

Fusion polypeptides having a heterologous 5′-3′ exonuclease domain linked to a polymerase that does not naturally have 5′-3′ exonuclease activity, as well as methods of their use are provided. Other aspects are also disclosed.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
The present application claims benefit of priority to U.S. Provisional Patent Application No. 61/912,981, filed Dec. 6, 2013 and U.S. Provisional Patent Application No. 62/006,409, filed Jun. 2, 2014, each of which are incorporated by reference for all purposes.
REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The Sequence Listing written in file -1009-2.TXT, created on Jan. 7, 2015, 110,592 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.


BACKGROUND OF THE INVENTION
Nucleic acid amplification reactions, such as polymerase chain reaction (PCR), are generally template-dependent reactions in which a desired nucleic acid sequence is amplified by treating separate complementary strands of a target nucleic acid with an excess of two oligonucleotide primers. The primers are extended to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. In such processes, the nucleic acid sequence between the primers on the respective DNA strands is selectively amplified.
A variety of thermostable polymerases have been discovered that can be used in PCR. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. There is little or no structural or sequence similarity among the various families. Most family A polymerases are single chain proteins that can contain multiple enzymatic functions including polymerase, 3′ to 5′ exonuclease activity and 5′ to 3′ exonuclease activity. Family B polymerases typically have a single catalytic domain with polymerase and 3′ to 5′ exonuclease activity, as well as accessory factors. Family C polymerases are typically multi-subunit proteins with polymerization and 3′ to 5′ exonuclease activity.
Taq polymerase has inherent polymerase and 5′-3′ exonuclease activity, but does not have 3′-5′ exonuclease (“proofreading”) activity. Utilizing the inherent 5′ to 3′ exonuclease activity of Taq, it is possible to achieve PCR amplification and signal release from a target-specific fluorogenic probe (e.g., a “Taqman” probe). The 5′ to 3′ exonuclease activity of Taq cleaves the 5′ terminus of a hybridized oligo probe to release both mono- and oligonucleotides. The probe is hydrolyzed during strand replication so that the accumulating fluorescent signal correlates with amplification.
Pfu DNA polymerase and other family B polymerases has superior thermostability, inhibitor tolerance, and proofreading properties compared to Taq DNA polymerase. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3′ to 5′ exonuclease proofreading activity, meaning that it works its way along the DNA from the 5′ end to the 3′ end and corrects nucleotide-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. However, Pfu and other family B polymerases lack 5′-3′ exonuclease activity and thus do not work in probe-based quantitative PCR methods such as those involving Taqman probes.
BRIEF SUMMARY OF THE INVENTION
Provided herein are polypeptides having at least polymerase activity and 5′-3′ exonuclease activity, wherein the polypeptides (“fusion polypeptide”) comprise a 5′-3′ exonuclease domain linked to a heterologous polymerase that does not naturally have 5′-3′ exonuclease activity. In some embodiments, the polymerase activity and 5′-3′ exonuclease activity are thermostable.
In some embodiments, the heterologous polymerase is a family B polymerase. In some embodiments, the heterologous polymerase is derived from two parental polymerases.
In some embodiments, the 5′-3′ exonuclease domain is a flap endonuclease 5′-3′ exonuclease domain. In some embodiments, the 5′-3′ exonuclease domain is a 5′-3′ exonuclease domain from a polymerase.
In some embodiments, the polymerase comprises a uracil-sensing domain (USD). In some embodiments, the USD comprises one or more point mutation substantially eliminating uracil-sensing activity.
In some embodiments, the polymerase lacks at least 10 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 125) amino acids of a native uracil-sensing domain (USD). In some embodiments, the uracil-sensing domain (USD) is removed or otherwise absent.
In some embodiments, the fusion polypeptide further comprises a heterologous sequence non-specific double-stranded or single-stranded DNA binding domain. In some embodiments, the heterologous sequence non-specific double stranded DNA binding domain comprises a Sso7 DNA binding domain or a Sso7-like DNA binding domain. In some embodiments, the heterologous sequence non-specific double stranded DNA binding domain substantially (e.g., at least 60%) identical to any of SEQ ID NOs: 27, 28, 29, 30, or 31.
In some embodiments, the 5′-3′ exonuclease domain and the heterologous family B polymerase are linked by a linker. In some embodiments, the linker is an amino acid linker.
In some embodiments, the carboxyl terminus of the 5′-3′ exonuclease domain is linked via a linker to the amino terminus of the family B polymerase. In some embodiments, the amino terminus of the 5′-3′ exonuclease domain is linked via a linker to the carboxyl terminus of the family B polymerase.
In some embodiments, the polypeptide has 3′-5′ exonuclease activity.
In some embodiments, the polypeptide substantially lacks 3′-5′ exonuclease activity. In some embodiments, the polymerase comprises at least one point mutation that substantially eliminates 3′-5′ exonuclease activity. In some embodiments, the polymerase comprises a deletion that substantially eliminates 3′-5′ exonuclease activity.
In some embodiments, the polypeptide is bound to a reagent that prevents the polymerase activity until the polypeptide is heated.
In some embodiments, the reagent is one or more antibody or aptamer bound to the polymerase.
In some embodiments, the reagent is a reversible covalent chemical modification.
Also provided are kits comprising the fusion polypeptide and other components as described above or elsewhere herein. In some embodiments, the kit further comprises a reverse transcriptase.
Also provided are reaction mixtures, e.g., comprising the fusion polypeptide and other components as described above or elsewhere herein. In some embodiments, the reaction mixture further comprises a polynucleotide primer. In some embodiments, the reaction mixture comprises a sample nucleic acid. In some embodiments, the reaction mixture does not comprise a sample nucleic acid.
In some embodiments, the reaction mixture further comprises a reverse transcriptase.
In some embodiments, the reaction mixture further comprises dUTP and/or a nucleic acid template comprising incorporated uracil.
In some embodiments, the reaction mixture comprises at least one polynucleotide primer and at least one probe with a fluorophore and quencher that is hybridized to a target polynucleotide sequence, wherein during amplification of the target polynucleotide sequence the 5′-3′ exonuclease activity releases the fluorophore from the probe, thereby generating fluorescent signal.
Also provided are nucleic acids comprising a polynucleotide encoding the fusion polypeptide as described above or elsewhere herein.
Also provided are methods of performing polymerase chain reaction (PCR) or other type (e.g., isothermal) of amplification. In some embodiments, the method comprises: contacting in an amplification reaction mixture the fusion polypeptide as described herein to a sample comprising nucleic acids under conditions to allow for amplification of a target sequence in the nucleic acids, if present; and detecting the presence or absence of amplified target sequence.
In some embodiments, the amplification reaction comprises at least one polynucleotide primer and at least one probe with a fluorophore and quencher that is hybridized to a target polynucleotide sequence, wherein during amplification of the target polynucleotide sequence the 5′-3′ exonuclease activity releases the fluorophore from the probe, thereby generating fluorescent signal.
In some embodiments, the sample comprises one or more inhibitor of PCR. In some embodiments, the sample is crude sample that has not undergone nucleic acid purification. In some embodiments, the sample is blood or serum.
In some embodiments, the amplification reaction comprises dUTP and/or a nucleic acid template comprising incorporated uracil.
In some embodiments, the sample comprises a RNA target nucleic acid and the reaction mixture comprises a reverse transcriptase, and wherein the method further comprises: reverse transcribing the RNA target nucleic acid with the reverse transcriptase to generate a cDNA; and amplifying the cDNA with the polypeptide.
Also provided is a method of making the fusion polypeptide. In some embodiments, the method comprises incubating cells comprising a polynucleotide encoding the polypeptide under conditions to cause expression of the polypeptide in the cells; and purifying the expressed polypeptide.
Also provided are polypeptides having polymerase activity (e.g., thermostable polymerase activity), the polypeptide comprising a family B polymerase but lacking at least 10 amino acids of a native uracil-sensing domain (USD). In some embodiments, the uracil-sensing domain (USD) is absent.
In some embodiments, the polypeptide further comprises a heterologous sequence non-specific double stranded DNA binding domain. In some embodiments, the heterologous sequence non-specific double stranded DNA binding domain comprises a Sso7 DNA binding domain or a Sso7-like DNA binding domain.
Also provided is a kit comprising the polypeptide having polymerase activity (e.g., thermostable polymerase activity), the polypeptide comprising a family B polymerase but lacking at least 10 amino acids of a native uracil-sensing domain (USD).
Also provided are reaction mixtures comprising the polypeptide having polymerase activity (e.g., thermostable polymerase activity), the polypeptide comprising a family B polymerase but lacking at least 10 amino acids of a native uracil-sensing domain (USD).
In some embodiments, the reaction mixture further comprises a polynucleotide primer. In some embodiments, the reaction mixture comprises a sample nucleic acid. In some embodiments, the reaction mixture does not comprise a sample nucleic acid.
In some embodiments, the reaction mixture further comprises a reverse transcriptase.
In some embodiments, the reaction mixture further comprises dUTP and/or a nucleic acid template comprising incorporated uracil.
Also provided are nucleic acids comprising a polynucleotide encoding the polypeptide having polymerase activity (e.g., thermostable polymerase activity), the polypeptide comprising a family B polymerase but lacking at least 10 amino acids of a native uracil-sensing domain (USD).
Also provided are methods of performing polymerase chain reaction (PCR). In some embodiments, the method comprises: contacting in an amplification reaction mixture polypeptide having polymerase activity (e.g., thermostable polymerase activity) to a sample comprising nucleic acids under conditions to allow for amplification of a target sequence in the nucleic acids, if present, wherein the polypeptide comprises a family B polymerase but lacks at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 125 amino acids of a native uracil-sensing domain (USD); and detecting the presence or absence of amplified target sequence.
In some embodiments, the amplification reaction comprises dUTP and/or a nucleic acid template comprising incorporated uracil.
In some embodiments, the sample comprises a RNA target nucleic acid and the reaction mixture comprises a reverse transcriptase, and wherein the method further comprises: reverse transcribing the RNA target nucleic acid with the reverse transcriptase to generate a cDNA; and amplifying the cDNA with the polypeptide.
Also provided are methods of making the polypeptide. In some embodiments, the method comprises incubating cells comprising a polynucleotide encoding the polypeptide under conditions to cause expression of the polypeptide in the cells; and purifying the expressed polypeptide.
Other aspects of the invention will be evident as described below.
Definitions
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthetic described below are those well-known and commonly employed in the art.
The term “Sso7” or “Sso7 DNA binding domain” or “Sso7-like DNA binding domain” or “Sso7 domain” refers to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity to SEQ ID NO:27, 28, 29, 30, or 31. The term includes both full-length Sso7d polypeptides and fragments of the polypeptides that have sequence non-specific double-stranded binding activity. Sso7-like proteins include, but are not limited to, Sso7d, Sac7d and Sac7e.
“Domain” refers to a unit of a protein or protein complex, comprising a polypeptide subsequence, a complete polypeptide sequence, or a plurality of polypeptide sequences where that unit has a defined function.
“Heterologous”, when used with reference to portions of a protein, indicates that the protein comprises two or more domains that are not found in the same relationship (e.g., do not occur in the same polypeptide) to each other in nature. Such a protein, e.g., a fusion protein, contains two or more domains from unrelated proteins arranged to make a new functional protein.
“Thermally stable polymerase activity” or “thermostable polymerase activity” of a polypeptide as used herein refers to enzyme activity that catalyzes polynucleotide synthesis by addition of nucleotide units to a nucleotide chain using DNA or RNA as a template and has an optimal activity at a temperature above 45° C., e.g., above 60° C.
The term “amplification reaction mixture” refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid. These include enzymes, aqueous buffers, salts, amplification primers, target nucleic acid, and nucleoside triphosphates. As discussed further herein, amplification reaction mixtures may also further include stabilizers and other additives to optimize efficiency and specificity. Depending upon the context, the mixture can be either a complete or incomplete amplification reaction mixture
“Polymerase chain reaction” or “PCR” refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990. Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization/elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.
A “primer” refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety of lengths and are often less than 50 nucleotides in length, for example 12-30 nucleotides, in length. The length and sequences of primers for use in PCR can be designed based on principles known to those of skill in the art, see, e.g., Innis et al., supra.
A “template” refers to a polynucleotide sequence that comprises the polynucleotide to be amplified, flanked by primer hybridization sites. Thus, a “target template” comprises the target polynucleotide sequence flanked by hybridization sites for a 5′ primer and a 3′ primer.
As used herein, “nucleic acid” means DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, points of attachment and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like. Nucleic acids can also include non-natural bases, such as, for example, nitroindole. Modifications can also include 3′ and 5′ modifications such as capping with a fluorophore (e.g., quantum dot) or another moiety.
The terms “oligonucleotide” or “polynucleotide” or “nucleic acid” interchangeably refer to a polymer of monomers that can be corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleic acid (DNA) polymer, or analog thereof. This includes polymers of nucleotides such as RNA and DNA, as well as modified forms thereof, peptide nucleic acids (PNAs), locked nucleic acids (LNATM), and the like. In certain applications, the nucleic acid can be a polymer that includes multiple monomer types, e.g., both RNA and DNA subunits.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the 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, e.g., hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon atom that is bound to a hydrogen atom, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The term “encoding” refers to a polynucleotide sequence encoding one or more amino acids. The term does not require a start or stop codon. An amino acid sequence can be encoded in any one of six different reading frames provided by a polynucleotide sequence.
The term “promoter” refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
A “vector” refers to a polynucleotide, which when independent of the host chromosome, is capable replication in a host organism. Preferred vectors include plasmids and typically have an origin of replication. Vectors can comprise, e.g., transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid. Any of the polynucleotides described herein can be included in a vector.
A “DNA polymerase” or a “polymerase,” as used herein, refers to an enzyme that performs template-directed synthesis of DNA. The term encompasses both the full length polypeptide and a domain that has polymerase activity. DNA polymerases are well-known to those skilled in the art, including but not limited to DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, Bacillus stearothermophilus, and Thermotoga maritime, or modified versions thereof. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. There is little or no sequence similarity among the various families. Most family A polymerases are single chain proteins that can contain multiple enzymatic functions including polymerase, 3′ to 5′ exonuclease activity and 5′ to 3′ exonuclease activity. Family B polymerases typically have a single catalytic domain with polymerase and 3′ to 5′ exonuclease activity, as well as accessory factors. Family C polymerases are typically multi-subunit proteins with polymerizing and 3′ to 5′ exonuclease activity. In E. coli, three types of DNA polymerases have been found, DNA polymerases I (family A), II (family B), and III (family C). In eukaryotic cells, three different family B polymerases, DNA polymerases α, δ, and ε, are implicated in nuclear replication, and a family A polymerase, polymerase γ, is used for mitochondrial DNA replication. Other types of DNA polymerases include phage polymerases.
Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Accelrys), or by manual alignment and visual inspection.
Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates results of quantitative detection of probe-based qPCR reactions using two different kinds of control polymerases.
FIG. 1, upper portion: Probe based real-time PCR amplification curves of 100 ng to 100 pg cDNA input using qPCR reagent containing Taq DNA polymerase, which has intrinsic 5′-3′ exonuclease activity. Cross labelled traces are no template control (NTC). A standard curve of Cq (signal take-off cycle) against input concentration is shown on the right. PCR efficiency in percentage is shown as E vlaue.
FIG. 1, lower portion: Probe based real-time PCR amplification curves of 100 ng to 100 pg cDNA input using qPCR reagent containing Pfu-based DNA polymerase (a fusion polymerase of Sso7d and pfu/deepVent hybrid DNA polymerase), which lacks intrinsic 5′-3′ exonuclease activity. Cross labelled traces are no template control (NTC). A standard curve of Cq (signal take-off cycle) against input concentration is shown on the right. PCR efficiency in percentage is shown as E vlaue.
FIG. 2 illustrates results of quantitative detection of probe-based qPCR reactions using Pfu FEN1 5′-3′ exonuclease domain fused to DNA polymerase (upper), and Pfu FEN1 5′-3′ exonuclease domain fused to DNA polymerase lack of uracil-sensing domain (lower).
FIG. 2, upper portion: Probe based real-time PCR amplification curves of 100 ng to 100 pg cDNA input using qPCR reagent containing a fusion DNA polymerase of Pfu flap endonuclease 1 (Pfu FEN1) and Pfu-based DNA polymerase. Cross labelled traces are no template control (NTC). A standard curve of Cq (signal take-off cycle) against input concentration is shown on the right. PCR efficiency in percentage is shown as E vlaue.
FIG. 2, lower portion: Probe based real-time PCR amplification curves of 100 ng to 100 pg cDNA input using qPCR reagent containing a fusion DNA polymerase of Pfu flap endonuclease 1 (Pfu FEN1) and uracil-sensing domain minus Pfu-based DNA polymerase. Cross labelled traces are no template control (NTC). A standard curve of Cq (signal take-off cycle) against input concentration is shown on the right. PCR efficiency in percentage is shown as E vlaue.
FIG. 3 illustrates results of quantitative detection of probe-based qPCR reactions using Da FEN1 5′-3′ exonuclease domain fused to DNA polymerase (upper), and Da FEN1 5′-3′ exonuclease domain fused to DNA polymerase lack of uracil-sensing domain (lower).
FIG. 3, upper portion: Probe based real-time PCR amplification curves of 100 ng to 100 pg cDNA input using qPCR reagent containing a fusion DNA polymerase of Da flap endonuclease 1 (Da FEN1) and Pfu-based DNA polymerase. Cross labelled traces are no template control (NTC). A standard curve of Cq (signal take-off cycle) against input concentration is shown on the right. PCR efficiency in percentage is shown as E vlaue.
FIG. 3, lower portion: Probe based real-time PCR amplification curves of 100 ng to 100 pg cDNA input using qPCR reagent containing a fusion DNA polymerase of Da flap endonuclease 1 (Da FEN1) and uracil-sensing domain minus Pfu-based DNA polymerase. Cross labelled traces are no template control (NTC). A standard curve of Cq (signal take-off cycle) against input concentration is shown on the right. PCR efficiency in percentage is shown as E vlaue.
FIG. 4 illustrates results of quantitative detection of probe-based qPCR reactions using a PFU/DEEPVENT polymerase alone (upper) or a Taq 5′-3′ exonuclease domain fused to the PFU/DEEPVENT polymerase (lower) as explained in Example 3.



DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
It has been surprisingly discovered that polymerases that do not naturally have a 5′-3′ exonuclease activity can be fused with a heterologous 5′-3′ exonuclease domain to generate a fusion protein that retains both polymerase and 5′-3′ exonuclease activity. This discovery thus allows, for example, for use of family B polymerases and other polymerases that do not naturally have a 5′-3′ exonuclease activity in probe-based quantitative PCR (qPCR) applications that rely on a polymerase having 5′-3′ exonuclease activity. It is also expected that the fusion protein will retain the improved tolerance of inhibitors of family B polymerases (compared, for example, to the lesser inhibitor tolerance of family A polymerases such as Taq polymerase).
As demonstrated in the examples, 5′-3′ exonuclease domains have been fused to the amino terminus of a family B polymerase via a linker and have been shown to have activity in probe-based qPCR methods. Also demonstrated in the example is the generation of a family B polymerase lacking the uracil-sensing domain (USD) and fused to a heterologous 5′-3′ exonuclease domain. This demonstrates, for the first time to the inventor's knowledge, that a family B polymerase can retain polymerase activity without the presence (i.e., the structure) of the USD.
II. 5′-3′ Exonuclease Domains
It is believed that any domain having 5′-3′ exonuclease activity can be fused to a polymerase that does not naturally have a 5′-3′ exonuclease activity. Generally, the 5′-3′ exonuclease will be fused to the amino terminus of the polymerase, either directly or via a linker. Linkage of the 5′-3′ exonuclease and the polymerase can be achieved by any method. A convenient way to link the 5′-3′ exonuclease to the polymerase is via recombinant DNA techniques to generate a coding polynucleotide sequence encoding the fused protein and then expressing the protein from the polynucleotide in a cell or via in vitro translation.
A variety of domains having 5′-3′ exonuclease activity are known and can be used in the fusion proteins as described herein. In some embodiments, the 5′-3′ exonuclease domain is a flap endonuclease (FEN1) or a fragment thereof retaining 5′-3′ exonuclease activity. FEN1 proteins are generally from Eukarya and Archea and possess 5′-3′ exonuclease activity. A variety of FEN1 proteins (as well as active fragments or variants thereof) are known (see, e.g., Williams, et al., J. Mol. Biol. 371(1):34-38 (2007)) and can be used as the 5′-3′ exonuclease domain as described herein. In some embodiments the FEN1 protein has thermostable 5′-3′ exonuclease activity. Thermostable FEN1 proteins include, but are not limited to, the Methanococcus jannaschii FEN1 protein (see, e.g., Rao, et al., J. Bacteriol. 180(20):5406-5412 (1998)), the Pyrococcus furiosus FEN1 protein (see, e.g., Hosfield, et al., Cell 95:135-146 (1998)) or the Desulfurococcus amylolyticus FEN1 protein (see, e.g., Mase et al., Acta Crystallographica Section F F67:209-213 (2011), as well as active variants (e.g., substantially identical versions thereof) or fragments thereof. An exemplary active FEN1 protein fragment is a FEN1 protein that lacks a PCNA-interacting protein motif (PIP) box. PIP boxes are described in, e.g., Querol-Audi, et al., Proc. Natl. Acad. Sci USA 109(22):8528-8533 (2012). Exemplary thermostable FEN1 protein sequences include those substantially identical to SEQ ID NOs: 10 or 24.
In some embodiments, the 5′-3′ exonuclease domain is from a heterologous polymerase. For example family A polymerases have 5′-3′ activity and thus fragments of a family A polymerase can be used as the 5′-3′ exonuclease domain. Conserved sites within the 5′-3′ exonuclease domain of the E. coli polymerase (Pol I) has been described. See, e.g., Gutman et al., Nucleic Acids Res. 21(18):4406-7 (1993). The 5′-3′ exonuclease domain of various thermostable polymerases have also been identified and separately expressed with retained activity. See, e.g., Choi et al., Biotechnol. Letts. 23:1647-52 (2001) and Kaiser et al., J. Chem. Biol. 274(30):21387-21394 (1999). An exemplary listing of sources of 5′-3′ exonuclease domains useful in the protein fusions described herein include, but are not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Bacillus stearothermophilus DNA polymerase, and Thermotoga maritima (Tma) DNA polymerase, and mutants, and variants (e.g., substantially identical versions thereof) and derivatives thereof. An exemplary Taq 5′-3′ exonuclease domain is SEQ ID NO:35, or a substantially identical amino acid sequence thereof.
In some embodiments, the coding sequences of each polypeptide in a resulting fusion protein (e.g., the 5′-3′ exonuclease domain and the polymerase and optionally the sequence non-specific DNA binding protein discussed further below) are directly joined at their amino- or carboxy-terminus via a peptide bond. Alternatively, an amino acid linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such an amino acid linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Typical peptide linker sequences contain Gly, Ser, Val and Thr residues. Other near neutral amino acids, such as Ala can also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al. (1985) Gene 40:39-46; Murphy et al. (1986) Proc. Natl. Acad. Sci. USA 83:8258-8262; U.S. Pat. Nos. 4,935,233 and 4,751,180, each of which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to linkers. The linker sequence may generally be from 1 to about 50 amino acids in length, e.g., 3, 4, 6, or 10 amino acids in length, but can be 100 or 200 amino acids in length. Linker sequences may not be required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. In some embodiments, linker sequences of use in the present invention comprise an amino acid sequence according to SEQ ID NO: 12 or 21.
Other chemical linkers include carbohydrate linkers, lipid linkers, fatty acid linkers, polyether linkers, e.g., PEG, etc. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterobifunctional linkages.
Other methods of joining a DNA binding domain and polymerase domain include ionic binding by expressing negative and positive tails and indirect binding through antibodies and streptavidin-biotin interactions. See, e.g., Bioconjugate Techniques, Hermanson, Ed., Academic Press (1996).
As previously described, nucleic acids encoding the polymerase or DNA binding domains can be obtained using routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994-1999). Such nucleic acids may also be obtained through in vitro amplification methods such as those described herein and in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117, each of which is incorporated by reference in its entirety for all purposes and in particular for all teachings related to amplification methods.
Modifications can additionally be made to the 5′-3′ exonuclease domain (or the polymerase) without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of a domain into a fusion protein. Such modifications can include, for example, the addition of codons at either terminus of the polynucleotide that encodes the binding domain to provide, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
The fusion polypeptides described herein can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeasts, filamentous fungi, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. Techniques for gene expression in microorganisms are described in, for example, Smith, Gene Expression in Recombinant Microorganisms (Bioprocess Technology, Vol. 22), Marcel Dekker, 1994.
There are many expression systems for producing the fusion polypeptides described herein that are known to those of ordinary skill in the art. See, e.g., Gene Expression Systems, Fernandex and Hoeffler, Eds. Academic Press, 1999; Sambrook and Russell, supra; and Ausubel et al, supra.) Typically, the polynucleotide that encodes the fusion polypeptide is placed under the control of a promoter that is functional in the desired host cell. Many different promoters are available and known to one of skill in the art, and can be used in the expression vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included. Constructs that include one or more of these control sequences are termed “expression cassettes.” Accordingly, the nucleic acids that encode the joined polypeptides are incorporated for high level expression in a desired host cell.
Expression control sequences that are suitable for use in a particular host cell are often obtained by cloning a gene that is expressed in that cell. Commonly used prokaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. (1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25); and the lambda-derived PL promoter and N-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128). The particular promoter system is not critical, any available promoter that functions in prokaryotes can be used. Standard bacterial expression vectors include plasmids such as pBR322-based plasmids, e.g., pBLUESCRIPTTM, pSKF, pET23D, lambda-phage derived vectors, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc, HA-tag, 6-His (SEQ ID NO:39) tag, maltose binding protein, VSV-G tag, anti-DYKDDDDK (SEQ ID NO:40) tag, or any such tag, a large number of which are well known to those of skill in the art.
III. Polymerases
As noted above, it is believed that any polymerase not naturally having 5′-3′ exonuclease activity can be used as described herein as a fusion partner with a heterologous 5′-3′ exonuclease domain. Exemplary polymerases not naturally having 5′-3′ exonuclease activity include family B polymerases. In some embodiments, the family B polymerase is an archeal family B polymerase.
A number of DNA polymerases have been grouped under the designation of DNA polymerase family B. Six regions of similarity (numbered from I to VI) are found in all or a subset of the B family polymerases. The most conserved region (I) includes a conserved tetrapeptide with two aspartate residues. Its function is not yet known. However, it has been suggested that it may be involved in binding a magnesium ion. All naturally-occurring polymerase sequences in the B family contain a characteristic DTDS (SEQ ID NO:41) motif, and possess many functional domains, including a 5′-3′ elongation domain, a 3′-5′ exonuclease domain, a DNA binding domain, and binding domains for both dNTP's and pyrophosphate (see, e.g., Zhou M, et al., Acta Crystallographica. Section D, Biological Crystallography 54(Pt 5):994-995 (1998)). Conserved amino acid residues of family B polymerases are described, for example, Hopfner, K.-P., et al., Proc. Natl. Acad. Sci USA 96: 36003605 (1999) in general and in FIG. 3 in particular.
Exemplary polymerases useful in the fusions described herein include, but are not limited to, Pyrococcus horikoshii (e.g., accession number O59610), P. abyssi (e.g., accession number P77916), P. glycovorans (e.g., accession number CAC12849), Pyrococcus sp. GE23 (e.g., accession number CAA90887), Pyrococcus sp. GB-D (e.g., accession number Q51334), P. furiosus (e.g., accession number P61875), P. woesei (e.g., accession number P61876), Thermococcus kodakaraensis (e.g., accession number P77933), T. gorgonarius (e.g., accession number P56689), T. fumicolans (e.g., accession number P74918), T. sp. 9oN-7 (e.g., accession number Q56366), T. onnurineus NA1 (e.g., accession number ABC11972), T. litoralis (e.g., accession number P30317), and T. aggregans (e.g., accession number O33845), as well as fragments and variants (e.g., substantially identical versions thereof) thereof that retain polymerase activity. In some embodiments, the polymerase is derived from two parental polymerases, e.g., Pfu and DeepVent. Such polymerases are described for example in U.S. Application Publication Nos. 20040219558; 20040214194; 20040191825; 20030162173, each of which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to hybrid polymerases.
In some aspects, the fusion polypeptide has 3′-5′ exonuclease activity and an active uracil sensing activity, as well as polymerase activity. In other aspects, the polymerase lacks one or more 3′-5′ exonuclease activity and an active uracil sensing activity. As described in more detail below, in some aspects, the polymerase lacks or substantially lacks the uracil sensing domain (USD).
In one aspect, the fusion polypeptide lacks 3′-5′ exonuclease activity. In one embodiment, such fusion polypeptides comprise a double point mutation in the polymerase domain that provides this exonuclease deficiency. A variety of mutations can be introduced into a native or mutant polymerase domain to reduce or eliminate 3′-5′ exonuclease activity. For example, U.S. Pat. Nos. 6,015,668; 5,939,301 and 5,948,614 describe mutations of a metal-binding aspartate to an alanine residue in the 3′-5′ exonuclease domain of the Tma and Tne DNA polymerases. These mutations reduce the 3′-5′ exonuclease activities of these enzymes to below detectable levels. Similarly, U.S. Pat. No. 5,882,904 describes an analogous aspartate-to-alanine mutation in Thermococcus barossi, and U.S. Pat. No. 5,489,523 teaches the double-mutant D141A E143A of the Pyrococcus wosei DNA polymerases. Both of these mutant polymerases have virtually no detectable 3′-5′ exonuclease activity. Methods of assaying 3′-5′ exonuclease activity are well-known in the art. See, e.g., Freemont et al., Proteins 1:66 (1986); Derbyshire et al., EMBO J. 16:17 (1991) and Derbyshire et al., Methods in Enzymology 262:363 85 (1995). It will be understood that while the above-described mutations were originally identified in one polymerase, one can generally introduce such mutations into other polymerases to reduce or eliminate exonuclease activity. In a specific embodiment, a polymerase comprises the double point mutation corresponding to D141A/E143A in the polymerase domain. Sequence comparisons can be performed using any BLAST including BLAST 2.2 algorithm with default parameters, described in Altschul et al., Nuc. Acids Res. 25:3389 3402 (1977) and Altschul et al., J. Mol. Biol. 215:403 410 (1990), respectively, to determine the “corresponding” amino acid in a different polymerase.
In one aspect, the polymerase in the fusion polypeptide lacks a uracil sensing domain (USD). The USD is generally described in Kim et al., J. Microbiol. Biotechnol. 18(8):1377-1385 (2008), which also describes assays for measuring uracil sensing. FIG. 3 of Kim et al, supra, provides an alignment of various USDs. USDs are also described in, e.g., European Patent Application Publication No. EP1463809B1. As described in the Examples below, it has been surprisingly discovered the entire USD can be removed from a family B polymerase without significantly affecting polymerase activity. Accordingly, in some embodiments, the fusion polypeptides as described herein lack at least a portion (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 125 contiguous amino acids), a majority of, or all of the native USD. The USD of an exemplary Pfu/DeepVent hybrid DNA polymerase (SEQ ID NO: 20) is ILDADYITEEGKPVIRLFKKENGEFKIEHDRTFRPYIYALLKDDSKIEEVKKITAERHGKIV RIVDAEKVEKKFLGRPITVWRLYFEHPQDVPTIREKIREHSAVVDIFEYDIPFAKRY (SEQ ID NO:25) and the USD of Pfu is ILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIV RIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRY (SEQ ID NO:38), though it will be appreciated that USDs of other polymerases may vary at least somewhat in sequence from SEQ ID NO:25 (e.g., a USD can be substantially identical to SEQ ID NO:25). As shown in the Examples, the inventors have found that additional amino acids (e.g., corresponding to SEQ ID NO:26) following the USD can also be conveniently removed from the polymerase without significantly affecting polymerase activity. Removal or inactivation of the USD can be useful to enable the fusion polypeptide to amplify templates comprising incorporated uracils, deaminated bases (e.g., inosine), and/or bisulfite-converted bases, for example.
IV. Sequence Non-Specific DNA Binding Domains
In some embodiments, fusion polypeptides described herein comprise a heterologous DNA binding domain. A DNA binding domain is a protein, or a defined region of a protein, that binds to nucleic acid in a sequence-independent matter, e.g., binding does not exhibit a gross preference for a particular sequence. DNA binding domains may bind single or double stranded nucleic acids.
The DNA binding proteins of use are generally thermostable. Examples of such proteins include, but are not limited to, the Archaeal small basic DNA binding proteins Sso7d and Sso7d-like proteins (see, e.g., Choli et al., Biochimica et Biophysica Acta 950:193-203, 1988; Baumann et al., Structural Biol. 1:808-819, 1994; and Gao et al, Nature Struc. Biol. 5:782-786, 1998), Archaeal HMf-like proteins (see, e.g., Starich et al., J. Molec. Biol. 255:187-203, 1996; Sandman et al., Gene 150:207-208, 1994), and PCNA homologs (see, e.g., Cann et al., J. Bacteriology 181:6591-6599, 1999; Shamoo and Steitz, Cell:99, 155-166, 1999; De Felice et al., J. Molec. Biol. 291, 47-57, 1999; and Zhang et al., Biochemistry 34:10703-10712, 1995).
Sso7d and Sso7d-like proteins, Sac7d and Sac7d-like proteins, e.g., Sac7a, Sac7b, Sac7d, and Sac7e are small (about 7,000 kd MW), basic chromosomal proteins from the hyperthermophilic archaebacteria Sulfolobus solfataricus and S. acidocaldarius, respectively. These proteins are lysine-rich and have high thermal, acid and chemical stability. They bind DNA in a sequence-independent manner and when bound, increase the Tm of DNA by up to 40° C. under some conditions (McAfee, Biochemistry 34:10063-10077, 1995; Gao et al., Nat. Struct. Biol. 5(9):782-786, 1998). These proteins and their homologs are typically believed to be involved in stabilizing genomic DNA at elevated temperatures. Suitable Sso7d-like DNA binding domains for use in the invention can be modified based on their sequence homology to Sso7d. Typically, DNA binding domains that are identical to or substantially identical to a known DNA binding protein over a comparison window of about 25 amino acids, optionally about 50-100 amino acids, or the length of the entire protein, can be used in the invention. The sequence can be compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the described comparison algorithms or by manual alignment and visual inspection. A variety of mutations in the Sso7 binding domain have been described in, e.g., US Patent Application Nos. 2005/0048530; 2007/0141591; and WO 2012/138417.
The HMf-like proteins are archaeal histones that share homology both in amino acid sequences and in structure with eukaryotic H4 histones, which are thought to interact directly with DNA. The HMf family of proteins form stable dimers in solution, and several HMf homologs have been identified from thermostable species (e.g., Methanothermus fervidus and Pyrococcus strain GB-3a).
Certain helix-hairpin-helix motifs have been shown to bind DNA nonspecifically and enhance the processivity of a DNA polymerase to which it is fused (Pavlov et al., Proc Natl Acad Sci USA. 99:13510-5, 2002). Single-stranded DNA binding proteins have also been described.
Additional DNA binding domains suitable for use can be identified by homology with known DNA binding proteins and/or by antibody cross reactivity, or may be found by means of a biochemical assay. DNA binding domains may be synthesized or isolated using the techniques described herein and known in the art.
Sequence non-specific single-stranded or doubled-stranded nucleic acid binding domains for use can also be identified by cross-reactivity using antibodies, including but not limited to, polyclonal antibodies that bind to known nucleic acid binding domains. Polyclonal antibodies are generated using methods well known to those of ordinary skill in the art (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988)). Those proteins that are immunologically cross-reactive binding proteins can then be detected by a variety of assay methods. For descriptions of various formats and conditions that can be used, see, e.g., Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993), Coligan, supra, and Harlow & Lane, supra.
Specificity for binding to double-stranded nucleic acids can be tested using a variety of assays known to those of ordinary skill in the art. These include such assays as filter binding assays or gel-shift assays. For example, in a filter-binding assay the polypeptide to be assessed for binding activity to double-stranded DNA is pre-mixed with radio-labeled DNA, either double-stranded or single-stranded, in the appropriate buffer. The mixture is filtered through a membrane (e.g., nitrocellulose) which retains the protein and the protein-DNA complex. The amount of DNA that is retained on the filter is indicative of the quantity that bound to the protein. Binding can be quantified by a competition analysis in which binding of labeled DNA is competed by the addition of increasing amounts of unlabeled DNA. A polypeptide that binds double-stranded DNA at a 10-fold or greater affinity than single-stranded DNA is defined herein as a double-stranded DNA binding protein. Alternatively, binding activity can be assessed by a gel shift assay in which radiolabeled DNA is incubated with the test polypeptide. The protein-DNA complex will migrate slower through the gel than unbound DNA, resulting in a shifted band. The amount of binding is assessed by incubating samples with increasing amounts of double-stranded or single-stranded unlabeled DNA, and quantifying the amount of radioactivity in the shifted band.
A binding domain binds to double-stranded nucleic acids in a sequence-independent fashion, i.e., a binding domain of the invention binds double-stranded nucleic acids with a significant affinity, but, there is no known double-stranded nucleic acid that binds to the domain with more than 100-fold more affinity than another double stranded nucleic acid with the same nucleotide composition, but a different nucleic acid sequence. Non-specific binding can be assayed using methodology similar to that described for determining double-stranded vs. single-stranded nucleic acid binding. Filter binding assays or gel mobility shift assays can be performed as above using competitor DNAs of the same nucleotide composition, but different nucleic acid sequences to determine specificity of binding.
Sequence non-specific single-stranded or double-stranded nucleic acid binding domains can also be assessed, for example, by assaying the ability of the single-stranded or double-stranded binding domain to increase processivity or efficiency of a modifying enzyme or, in the case of double-stranded nucleic acid binding domains, to increase the stability of a nucleic acid duplex by at least 1° C. can be determined.
A binding domain of the invention can also be identified by direct assessment of the ability of such a domain to stabilize a double-stranded nucleic acid conformation. For example, a melting curve of a primer-template construct can be obtained in the presence or absence of protein by monitoring the UV absorbance of the DNA at 260 nm. The Tm of the double-stranded substrate can be determined from the midpoint of the melting curve. The effect of the sequence-non-specific double-stranded nucleic-acid-binding protein on the Tm can then be determined by comparing the Tm obtained in the presence of the modified enzyme with that in the presence of the unmodified enzyme. (The protein does not significantly contribute to the UV absorbance because it has a much lower extinction coefficient at 260 nm than DNA). A domain that increases the Tm by 1° C., often by 5° C., 10° C. or more, can then be selected for use in the invention.
Novel sequence non-specific double-stranded nucleic acid binding proteins of the invention can also be isolated by taking advantage of their DNA binding activity, for instance by purification on DNA-cellulose columns. The isolated proteins can then be further purified by conventional means, sequenced, and the genes cloned by conventional means via PCR. Proteins overexpressed from these clones can then be tested by any of the means described above.
In some embodiments, the fusion polypeptides described herein comprise an Sso7 polypeptide sequence that is substantially identical to SEQ ID NOs: 27, 28, 29, 30, or 31. In some embodiments, the Sso7 polypeptide sequence has amino acid substitutions compared to the native (wildtype) Sso7d sequence. In some embodiments, the amino acid substitutions include amino acid changes from the native amino acid at the positions corresponding to K28 and/or R43 of SEQ ID NO:27. It should be understood that such position designations do not indicate the number of amino acids in the claimed molecule per se, but indicate where in the claimed molecule the residue occurs when the claimed molecule sequence is maximally aligned with SEQ ID NO:27.
Any Sso7 DNA binding protein domain can be substituted at the K28 and/or R43 position corresponding to SEQ ID NO:27. Thus, for example, in some embodiments, the variant Sso7 polypeptide sequence is substantially (e.g., at least 60, 70, 80, 85, 90, or 95%) identical to any of, e.g., SEQ ID NOS:27, 28, 29, 30, or 31, and comprises an amino acid other than K at the amino acid position corresponding to K28. In some embodiments, the amino acid position corresponding to K28 is serine (S), threonine (T), cysteine (C), proline (P), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), alanine (A), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), arginine (R), valine (V), tryptophan (W), or tyrosine (Y).
In some embodiments, the variant Sso7 polypeptide sequence is substantially (e.g., at least 60, 70, 80, 85, 90, or 95%) identical to any of, e.g., SEQ ID NOS: 27, 28, 29, 30, or 31, and comprises an amino acid other than R at the amino acid position corresponding to R43. In some embodiments, the amino acid position corresponding to R43 is alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y), or proline (P).
In some embodiments, the variant Sso7 polypeptide sequence is substantially (e.g., at least 60, 70, 80, 85, 90, or 95%) identical to any of, e.g., SEQ ID NOS: 27, 28, 29, 30, or 31, and comprises an amino acid other than K at the amino acid position corresponding to K28 and an amino acid other than R at the amino acid position corresponding to R43. For example, in some embodiments, the amino acid at position K28 is selected from: serine (S), threonine (T), cysteine (C), proline (P), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), alanine (A), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), valine (V), tryptophan (W), or tyrosine (Y) and the amino acid at position R43 is selected from: alanine (A), cytosine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine (Y), or proline (P).
V. Methods
In some embodiments, the fusion polypeptides described herein are used in nucleic acid amplification reactions. Such amplification reactions can include without limitation polymerase chain reaction (PCR), DNA ligase chain reaction (LCR), QBeta RNA replicase, and RNA transcription-based (such as TAS and 3SR) amplification reactions as well as others known to those of skill in the art. Polymerase chain reactions that can be conducted using the compositions described herein include without limitation reverse-transcription PCR (rt-PCR) and quantitative PCR (qPCR).
In some embodiments, the PCR is quantitative PCR in which the accumulation of amplicon is monitored in “real time” (i.e., continuously, e.g., once per cycle—rather than only following the completion of amplification). Quantitative amplification methods (e.g., quantitative PCR or quantitative linear amplification) can involve amplification of an nucleic acid template, directly or indirectly (e.g., determining a Ct value) determining the amount of amplified DNA, and then calculating the amount of initial template based on the number of cycles of the amplification. Amplification of a DNA locus using reactions is well known (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Innis et al., eds, 1990)). Typically, PCR is used to amplify DNA templates. However, alternative methods of amplification have been described and can also be employed, as long as the alternative methods amplify intact DNA to a greater extent than the methods amplify cleaved DNA. Methods of quantitative amplification are disclosed in, e.g., U.S. Pat. Nos. 6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., Gibson et al., Genome Research 6:995-1001 (1996); DeGraves, et al., Biotechniques 34(1):106-10, 112-5 (2003); Deiman B, et al., Mol Biotechnol. 20(2):163-79 (2002).
In some embodiments, quantitative amplification is based on the monitoring of the signal (e.g., fluorescence of a probe) representing copies of the template in cycles of an amplification (e.g., PCR) reaction. In the initial cycles of the PCR, a very low signal is observed because the quantity of the amplicon formed does not support a measurable signal output from the assay. After the initial cycles, as the amount of formed amplicon increases, the signal intensity increases to a measurable level and reaches a plateau in later cycles when the PCR enters into a non-logarithmic phase. Through a plot of the signal intensity versus the cycle number, the specific cycle at which a measurable signal is obtained from the PCR reaction can be deduced and used to back-calculate the quantity of the target before the start of the PCR. The number of the specific cycles that is determined by this method is typically referred to as the cycle threshold (Ct). Exemplary methods are described in, e.g., Heid et al. Genome Methods 6:986-94 (1996) with reference to hydrolysis probes.
One method for detection of amplification products is the 5′-3′ exonuclease “hydrolysis” PCR assay (also sometimes referred to as the TaqManTM assay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al., PNAS USA 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res. 21: 3761-3766 (1993)). This assay detects the accumulation of a specific PCR product by hybridization and cleavage of a doubly labeled fluorogenic probe (e.g., the “TaqManTM probe) during the amplification reaction. The fluorogenic probe consists of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye. During PCR, this probe is cleaved by the 5′-3′ exonuclease activity of DNA polymerase if, and only if, it hybridizes to the segment being amplified. Cleavage of the probe generates an increase in the fluorescence intensity of the reporter dye.
Another method of detecting amplification products that relies on the use of energy transfer is the “beacon probe” method described by Tyagi and Kramer, Nature Biotech. 14:303-309 (1996), which is also the subject of U.S. Pat. Nos. 5,119,801 and 5,312,728. This method employs oligonucleotide hybridization probes that can form hairpin structures. On one end of the hybridization probe (either the 5′ or 3′ end), there is a donor fluorophore, and on the other end, an acceptor moiety. In the case of the Tyagi and Kramer method, this acceptor moiety is a quencher, that is, the acceptor absorbs energy released by the donor, but then does not itself fluoresce. Thus, when the beacon is in the open conformation, the fluorescence of the donor fluorophore is detectable, whereas when the beacon is in hairpin (closed) conformation, the fluorescence of the donor fluorophore is quenched. When employed in PCR, the molecular beacon probe, which hybridizes to one of the strands of the PCR product, is in the open conformation and fluorescence is detected, while those that remain unhybridized will not fluoresce (Tyagi and Kramer, Nature Biotechnol. 14: 303-306 (1996)). As a result, the amount of fluorescence will increase as the amount of PCR product increases, and thus may be used as a measure of the progress of the PCR. Those of skill in the art will recognize that other methods of quantitative amplification are also available.
Various other techniques for performing quantitative amplification of a nucleic acids are also known. For example, some methodologies employ one or more probe oligonucleotides that are structured such that a change in fluorescence is generated when the oligonucleotide(s) is hybridized to a target nucleic acid. For example, one such method involves is a dual fluorophore approach that exploits fluorescence resonance energy transfer (FRET), e.g., LightCyclerTM hybridization probes, where two oligo probes anneal to the amplicon. The oligonucleotides are designed to hybridize in a head-to-tail orientation with the fluorophores separated at a distance that is compatible with efficient energy transfer. Other examples of labeled oligonucleotides that are structured to emit a signal when bound to a nucleic acid or incorporated into an extension product include: ScorpionsTM probes (e.g., Whitcombe et al., Nature Biotechnology 17:804-807, 1999, and U.S. Pat. No. 6,326,145), SunriseTM (or AmplifluorTM) probes (e.g., Nazarenko et al., Nuc. Acids Res. 25:2516-2521, 1997, and U.S. Pat. No. 6,117,635), and probes that form a secondary structure that results in reduced signal without a quencher and that emits increased signal when hybridized to a target (e.g., Lux ProbesTM).
In some embodiments, the PCR reaction mixture does not include a labeled probe oligonucleotide. For example, the reaction mixture lacks a Taqman or other labeled oligonucleotide probe for monitoring real-time or endpoint accumulation of the amplicon. In some of these embodiments, an intercalating fluorescent dye is included. In some embodiments, the intercalating dye changes signal (increases or decreases) when bound to double stranded nucleic acids compared to single stranded nucleic acids. Exemplary agents include SYBR GREENTM, SYBR GOLDTM, and EVAGREENTM. Since these agents are not template-specific, it is assumed that the signal is generated based on template-specific amplification. This can be confirmed by monitoring signal as a function of temperature because melting point of template sequences will generally be much higher than or different from, for example, primer-dimers, non-specifically amplified sequences, etc.
A number of components of a PCR reaction are well known and can be determined readily by a skilled artisan. In certain aspects, it may be desirable to include an additional compound as an additive to improve efficiency in amplification reactions, such as qPCR. For example, there may be situations in which a polymerase of the invention that lacks exonuclease activity exhibits low efficiency for certain targets when used in a formulation that includes certain binding dyes (such as, in one non-limiting example, an EvaGreen DNA binding dye) or in the presence of certain amplification inhibitors. Such low efficiency may in some embodiments be a result of delay in Ct values associated with low input DNA concentrations. Methods for measuring efficiency of a particular reaction are known in the art.
In some embodiments, an osmolyte may be included in an amplification reaction of the invention to improve efficiency. See, e.g., WO2010/080910, incorporated by reference. Members of the osmolyte family have been shown to improve the thermal stability of proteins (Santoro, Biochemistry, 1992) as well as decrease DNA double helix stability (Chadalavada, FEBS Letters, 1997). Osmolytes of use in the present invention may include without limitation sarcosine, trimethylamine N-oxide (TMAO), dimethylsulfoniopropionate, and trimethylglycine. Sarcosine is chemically similar to betaine, a chemical which has been shown to improve conventional PCR (Henke, Nucleic Acids Research, 1997).
In conventional uses of osmolytes, the stabilizing effects of such compounds are generally observed at relatively high concentrations (>1M). However, in methods of the present invention, millimolar concentrations of osmolytes have been found to be effective for improving the reaction efficiency of amplification reactions such as qPCR. See, e.g., WO2010/080910, incorporated by reference. Without being bound by a mechanism of action, it is possible that the improvement in efficiency is the result of improvement of the Ct values for the reactions that contain low DNA template concentration. In some embodiments, concentrations of about 100 to about 1000 mM of osmolytes are used in methods and kits of the present invention. In still further embodiments, concentrations of about 50 to about 700, about 100 to about 600, about 150 to about 500, about 200 to about 400 mM, or about 300 to about 350 mM osmolytes are used in methods and kits of the invention. In some embodiments, the osmolyte used in methods and kits of the invention is sarcosine. Indeed, it has been found that addition of sarcosine improved the efficiency of the amplification reaction as compared to control comprising water.
In some embodiments, particularly in the amplification of low-copy target nucleic acids or in the presence of amplification inhibitors, efficiency decreases due to the binding of polymerase to non-primed double-stranded nucleic acid targets. Binding of the polymerase to the double-stranded targets will prevent those targets from denaturation, hybridizing to primers, and undergoing an amplification reaction. To improve the specificity of the polymerase for primed templates, in some embodiments methods and kits of the invention utilize heparin. See, e.g., WO2010/080910, incorporated by reference. Heparin molecules, which are negatively charged, can be included in the reaction mixture to mimic the electrostatic property of double stranded nucleic acids. The addition of heparin can, without being limited to a mechanism of action, prevent excess polymerase from binding to the double-stranded template until a single-stranded primed-template becomes available. In some exemplary embodiments, heparin is used in methods and kits of the invention at concentrations of about 50 to about 750 pg/μl. In further exemplary embodiments, heparin is used in methods and kits of the invention at concentrations of about 75 to about 700, about 100 to about 600, about 125 to about 500, about 150 to about 400, about 175 to about 300, or about 200 to about 250 pg/μl.
Non-specific amplification can be reduced by reducing the formation of extension products from primers bound to non-target sequences prior to the start of the reaction. In one method, referred to as a “hot-start” protocol, one or more critical reagents are withheld from the reaction mixture until the temperature is raised sufficiently to provide the necessary hybridization specificity. In this manner, the reaction mixture cannot support primer extension during the time that the reaction conditions do not insure specific primer hybridization. In some embodiments, the polypeptides as described herein can be reversibly inactivated by a reagent bound to the polymerase. The inhibitory reagent can be removed by heat (e.g., above 50 or at 95° C.). Thus, in some embodiments, the amplification reaction comprises a hot start reagent.
In some embodiments, the reagent is a “hot start” antibody. Hot-start antibodies increase the specificity of amplification reactions, because they render the polymerase inactive at room temperature, thus avoiding extension of nonspecifically annealed primers or primer dimers. See, e.g., U.S. Pat. No. 5,338,671. The functional activity of the polymerase is restored by disassociating the antibody from the polymerase, generally through incubation at a higher temperature. In some embodiment, such a “higher temperature” is from about 90° to about 99° C. for about 2 to about 10 minutes. It will be appreciated that the temperature and length of time for incubation to disassociate the antibody and activate the polymerase can be varied according to known parameters to provide the most effective method of activating the polymerase in these hot-start methods. In other embodiments, the reagent is an aptamer that inhibits polymerase activity until the polymerase is heated to disassociate the aptamer. Exemplary aptamers include, but are not limited to, slow off-rate modified aptamers (e.g., SOMAmersTM).
Alternatively, a polymerase can be substantially inactivated by covalently linking a chemical reagent to the polymerase. For example a dicarboxylic acid anhydride can be linked to one or more lysine residue of the polymerase, thereby substantially inactivating the polymerase activity. See, e.g., U.S. Pat. Nos. 5,773,258 and 5,677,152. The reagents are thermally labile and thus can be removed upon heating.
In some embodiments, the fusion polypeptide comprising a FEN1 protein or active fragment thereof can be used to generate a 5′ cleaved primer flap that subsequently is used to prime a second amplification reaction to generate a detectable signal. An example of such a method includes the TOCETM assay (Seegene, KR). Such assays detect a target nucleic acid sequence in which the PTO (Probing and Tagging Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved to release a fragment and the fragment is hybridized with the CTO (Capturing and Templating Oligonucleotide) to form an extended duplex, followed by detecting the presence of the extended duplex. The extended duplex provides signals (generation, increase, extinguishment or decrease of signals) from labels indicating the presence of the extended duplex and has adjustable Tm value, which are well adoptable for detection of the presence of the target nucleic acid sequence. See, e.g., US Patent Publication No. 2013/0109588.
In other embodiments, the fusion polypeptide can be used to cause recombination between DNA strands, thereby replacing one strand of a DNA duplex with a homologous third strand. For example, the fusion polypeptide comprising 5′-3′ exonuclease activity can be used to facilitate “somatic recombination” type of cross-linking
VI. Reaction Mixtures
The present invention also provides for reaction mixtures comprising one or more of the fusion polypeptides as described herein. Other reagents as described herein can also be included in the reaction mixture. For example, in some embodiments, the reaction mixtures comprise a fluorogenic probe comprises an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye that generates signal in a 5′-3′ exonuclease “hydrolysis” PCR assay (also referred to as a TaqManTM assay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al., PNAS USA 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res. 21: 3761-3766 (1993)).
In some embodiments, the fusion polypeptides described herein have increased tolerance for common PCR inhibitors, e.g., inhibitors of Taq polymerase. Exemplary PCR inhibitors include, but are not limited to, heparin, bile salts, polysaccharides, collagen, heme, humic acid, melanin and eumelanin, urea, hemoglobin, lactoferrin, immunoglobulin G (IgG), and indigo dye. Thus in some embodiments the reaction mixture comprises a sample having inhibitors that would significantly inhibit activity of Taq polymerase (e.g., degrading Ct values by at least 1 compared to the Ct of a polymerase as described herein).
In some embodiments, the sample is a crude sample, i.e., a sample in which minimal or no purification of nucleic acids has occurred. For example, the crude sample can be a blood or serum sample, cell lysate, a plant or animal tissue sample, etc.
In some embodiments, the amplification reaction comprises dUTP and/or a nucleic acid template comprising incorporated uracil. In some embodiments, dUTP is included in an amplification reaction mixture so that amplification products can be prevented from contaminating future amplification reactions. This is achieved by an additional incubation step in the presence of the enzyme, UNG, followed by inactivation of UNG, prior to the amplification reactions. UNG renders uracil-containing templates unable to be amplified by polymerases.
In some embodiments, the target template to be amplified contains incorporated uracil. Polymerases having an active uracil-sensing domain (e.g., most or all native B family polymerases) typically stall at an incorporated uracil in the template. In contrast, polypeptides lacking an active USD as described herein will not stall at incorporated uracils.
In some embodiments, the reaction mixture is formulated as a ready-to-use formulation, meaning the mixture contains all components needed for a polymerase reaction except for a sample or except for a sample and oligonucleotide primers.
In some embodiments, the reaction mixture further comprises a reverse transcriptase and optionally reagents necessary for reverse transcription. Thus in some embodiments, the reaction mixture can be used to generate a cDNA from RNA in a sample and then the fusion polypeptide can subsequently amplify the cDNA in the same reaction mixture. Alternatively, the cDNA can be generated in a previous reverse transcription reaction and the resulting cDNA can be added to a reaction mixture in a two-step reaction (a first step for the RT reaction, and a second for the cDNA amplification).
VII. Polynucleotides
Also provided are polynucleotides encoding (1) a fusion polypeptide comprising a heterologous 5′-3′ exonuclease domain and a polymerase (e.g., family B polymerase) as described herein or (2) a family B polymerase lacking the uracil sensing domain (USD). In some embodiments, the polynucleotides are isolated, i.e., are separated from the cell in which the polypeptide was translated and optionally purified. In some embodiments, expression cassettes (i.e., a heterologous promoter operably linked to the coding sequence) or vectors comprising the above-described polynucleotide are provided, as well as host cells (including but not limited to bacterial, fungal, yeast, insect, or mammalian cells) comprising such expression cassettes or vectors. Such host cells can be incubated under conditions to result in expression of the encoded polypeptide, which can subsequently be purified as desired.
VIII. Kits
In one aspect, kits comprising a fusion polypeptide as described herein is provided. Kits can be adapted, for example, for conducting nucleic acid amplification reactions. In some embodiments, such kits include dNTPs, and at least one buffer. Such kits may also include one or more primers as well as instructions for conducting nucleic acid amplification reactions using the components of the kits.
In still further embodiments, kits can include optimized buffer (e.g., Tris-HCl), KCl, (NH4)2SO4, stabilizer, detergent, dNTPs, MgCl2, and/or DMSO.
In still further embodiments, kits can include double stranded DNA binding dyes. Such double stranded DNA binding dyes can include without limitation: EvaGreen and SYBR Green, as well as any other double stranded DNA binding dyes known in the art.
Alternatively, or in addition, the kit can comprise one of more nucleic acid probe for use in a 5′-3′ exonuclease “hydrolysis” PCR assay (also referred to as a TaqManTM assay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al., PNAS USA 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res. 21: 3761-3766 (1993)). This assay detects the accumulation of a specific PCR product by hybridization and cleavage of a doubly labeled fluorogenic probe (the “TaqManTM probe) during the amplification reaction. The fluorogenic probe comprises an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye. During PCR, this probe is cleaved by the 5′-3′ exonuclease activity of DNA polymerase if, and only if, it hybridizes to the segment being amplified. Cleavage of the probe generates an increase in the fluorescence intensity of the reporter dye.
It will be appreciated that kits can also encompass any combination of the above-described components.
The following examples are offered to illustrate, but not to limit the claimed invention.
EXAMPLES
Example 1
Proof-reading DNA polymerases, such as pfu DNA polymerase, oftentimes are hyper-thermalphilic (or highly thermal stable) and have ability to perform well in the presence of common DNA polymerase inhibitors, such as salt and solvent. These proof-reading DNA polymerases usually have a 3′-5′ exonuclease activity that enhances fidelity; however they lack of 5′-3′ exonuclease activity that is essential for signal generation in probe-based qPCR applications.
We constructed fusion DNA polymerases comprising a proof-reading DNA polymerase fused to two different flap endonucleases (Archaeon Pyrococcus furiosus flap endonuclease (pfu FEN1 SEQ ID NO:24)) and Archaeon Desulfurococcus amylolyticus flap endonuclease (Da FEN1 SEQ ID NO:10)). A flexible linker (SEQ ID NO:8) was used to link the carboxyl terminus of the flap endonuclease domain to the amino terminus of the polymerase. The polymerase used was a Pfu/Vent-hybrid DNA polymerase (SEQ ID NO: 20) and further included a carboxyl terminal Sso7d domain with a K28 mutation (SEQ ID NO: 22) and then a poly-His tag. The full length amino acid and coding sequence of the pfu FEN1-polymerase-Sso7d fusion (referred to as “fusion protein #1”) as tested is SEQ ID NO:1 and 5, respectively. The full length amino acid and coding sequence of the Da FEN1-polymerase-Sso7d fusion (referred to as “fusion protein #2”) as tested is SEQ ID NO:2 and 6, respectively.
We also constructed similar fusions, however without the uracil sensing domain (USD) of polymerase. In fact, in addition to the removal of the entire USD, a small number of additional amino acids were removed from the polymerase based on the predicted structure of the polymerase. The full length amino acid and coding sequence of the pfu FEN1-polymerase (USD minus)-Sso7d fusion (referred to as “fusion protein #3”) as tested is SEQ ID NO:3 and 7, respectively. The full length amino acid and coding sequence of the Da FEN1-polymerase (USD minus)-Sso7d fusion (referred to as “fusion protein #4”) as tested is SEQ ID NO:4 and 8, respectively.
The fusions were tested in a probe qPCR assay that required 5′-3′ exonuclease activity to generate signal. As shown in the top part of FIG. 1, the positive control (Taq polymerase) generated signal in a concentration dependent manner. In contrast, the negative control (a pfu/DeepVent hybrid polymerase lacking 5′-3′ exonuclease activity) did not generate significant signal. However, each of the above-described fusion proteins (fusions #1-4) had activity at least comparable to Taq.
Example 2
Taq polymerase has 5′-3′ exonuclease activity but is generally considered to have a relatively low tolerance for the presence of inhibitors in the reaction mixture. In contrast, pfu and other family B polymerases lack 5′-3′ exonuclease activity, but have a higher inhibitor tolerance. We tested one of the fusions described herein (Pfu FEN1 fused to a Pfu/DeepVent hybrid DNA polymerase (SEQ ID NO:20) and an Sso7d domain) to determine whether the protein fusions retain the higher inhibitor tolerance of the B family polymerases with the fusion of the 5′-3′ exonuclease domain. iSTaq DNA polymerase (a fusion Sso7d DNA-binding protein and Taq DNA polymerase) was used as a control polymerase in these experiments. The effect of the following inhibitors was tested: heparin with ammonium and sodium salt, hematin, and humic acid. Inhibitor tolerance is reported in term of Cq value.










Heparin Ammonium Salt










iSTaq Pol
fusion protein


ng per 20 ul
(Control)
#1 (Test)


reaction
Cq value
Cq value












0
16.3
16.0


0.4
16.3
16.1


1.6
17.1
16.1


6.3
n.d.
16.0


25
n.d.
18.5



















Heparin Sodium Salt










iSTaq Pol
fusion protein


ng per 20 ul
(Control)
#1 (Test)


reaction
Cq value
Cq value












0
16.2
16.3


0.4
16.0
16.2


1.6
17.3
15.9


6.3
n.d.
15.8


25
n.d.
21.7



















Hematin










iSTaq Pol
fusion protein



(Control)
#1 (Test)


nM
Cq value
Cq value












0
16.2
16.1


150
15.8
15.8


187.5
16.8
15.7


225
29.6
16.0


262.5
38.2
16.1


300
n.d.
29.4



















Humic Acid










iSTaq Pol
fusion protein


ng per 20 ul
(Control)
#1 (Test)


reaction
Cq value
Cq value












0
16.0
16.2


0.8
16.0
16.1


3.1
18.7
16.0


12.5
n.d.
27.2


50
n.d.
n.d.









As shown in the data above, the 5′-3′ exonuclease-Pfu/DeepVent hybrid DNA polymerase-Sso7d fusion had a higher tolerance for a variety of inhibitors compared to the Taq polymerase-based Sso7d fusion, demonstrating that the fusions described herein retain the higher inhibitor tolerance of the family B polymerases even when fused to the 5′-3′ exonuclease domain.
Example 3
A fusion DNA polymerases comprising a proof-reading DNA polymerase (SEQ ID NO: 20) was fused to 5′-3′ exonuclease domain of Taq polymerase 5′-3′ exonuclease domain (SEQ ID NO:35). The resulting DNA and amino acid sequence of the fusion was SEQ ID NO: 32 and 33, respectively. A flexible linker (SEQ ID NO:37) was used to link the carboxyl terminus of the 5′-3′ exonuclease domain to the amino terminus of the polymerase. The fusion was further fused with a carboxyl terminal Sso7d domain with a K28 mutation (SEQ ID NO: 22) and then a poly-His tag.
FIG. 4 (lower portion) demonstrates that, in a probe-based qPCR assay, a fusion polymerase with the Taq polymerase 5′-3′ exonuclease domain can amplify DNA targets and generate detection signal through probe hydrolysis. In contrast, a polymerase lack of Taq polymerase 5′-3′ exonuclease domain cannot generate detectable signal (FIG. 4).
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

## Claims

1. A polypeptide having polymerase activity and 5′-3′ exonuclease activity, the polypeptide comprising a 5′-3′ exonuclease domain linked to a heterologous polymerase that does not naturally have 5′-3′ exonuclease activity, wherein the heterologous polymerase comprises a family B polymerase catalytic domain having polymerase activity and the 5′-3′ exonuclease domain is a flap endonuclease, wherein the polypeptide substantially lacks 3′-5′ exonuclease activity.

2. The polypeptide of claim 1, wherein the polymerase activity and 5′-3′ exonuclease activity are thermostable.

3. The polypeptide of claim 1, further comprising a heterologous sequence non-specific double-stranded DNA binding domain or sequence non-specific single-stranded DNA binding domain.

4. The polypeptide of claim 3, wherein the heterologous sequence non-specific double stranded DNA binding domain comprises a Sso7 DNA binding domain or a Sso7-like DNA binding domain.

5. The polypeptide of claim 4, wherein the heterologous sequence non-specific double stranded DNA binding domain is at least 60% identical to any of SEQ ID NOs: 27, 28, 29, 30, or 31.

6. The polypeptide of claim 1, wherein the 5′-3′ exonuclease domain and the family B polymerase catalytic domain are linked by a linker.

7. The polypeptide of claim 6, wherein the linker is an amino acid linker.

8. The polypeptide of claim 1, wherein the carboxyl terminus of the 5′-3′ exonuclease domain is linked via a linker to the amino terminus of the family B polymerase catalytic domain.

9. The polypeptide of claim 1, wherein the polymerase comprises a deletion that substantially eliminates 3′-5′ exonuclease activity.

10. A reaction mixture comprising the polypeptide of claim 1.

11. The reaction mixture of claim 10, further comprising a polynucleotide primer.

12. The reaction mixture of claim 10, wherein the reaction mixture comprises a sample nucleic acid.

13. A method of performing polymerase chain reaction (PCR), the method comprising:
contacting in an amplification reaction mixture the polypeptide of claim 1 to a sample comprising nucleic acids under conditions to allow for amplification of a target sequence in the nucleic acids, if present; and
detecting the presence or absence of amplified target sequence.

14. The polypeptide of claim 5, wherein the heterologous sequence non-specific double stranded DNA binding domain is at least 95% identical to any of SEQ ID NOs: 27, 28, 29, 30, or 31.

15. The polypeptide of claim 7, wherein the amino acid linker is between 1-50 amino acids in length.