Patent Application
20250215413
Enzymes are provided for isothermal amplification of nucleic acid sequences, without the need for heating and cooling cycles associated with traditional polymerase chain reaction (PCR).
DNA and RNA amplification is an essential method involved in rapid molecular diagnostics, gene manipulations, and genetic analyses including the detection of bacteria, viruses, and diagnosis of genetic disorders. The most widely used method for DNA amplification is polymerase chain reaction (PCR), implemented through thermal cycling. Promising next generation methods include enzymatic isothermal amplification; however, existing isothermal methods are limited to producing only short amplicons, or complex heterogeneous mixed and branched products, and often involve using multiple sets of complicated pairs of primers (Y. Zhao, F. Chen, Q. Li, L. Wang and C. Fan, “Isothermal Amplification of Nucleic Acids,” Chemical Reviews, vol. 115, p. 12491-12545, 11 2015). As a result, no existing isothermal method can approach the versatility of thermal cycler-based PCR.
Provided herein is a polymerase chain reaction (PCR) which eliminates thermal cycling while retaining many of the desired traditional PCR features, termed herein as SHARP (SSB-Helicase Assisted Rapid PCR). Compositions include a novel helicase and an SSB (Single-Stranded Binding protein) for effective strand separation at constant temperature, thus avoiding the need for periodic heating. SHARP uses the same starting set of primers and template DNA as PCR does, carries out the reaction at a constant temperature, and outputs the same amplicon as PCR with lengths of up to 6000 base pairs, a feature no existing isothermal amplification method can match.
Accordingly, in certain embodiments, a bacterial derived helicase comprises a PcrA helicase having at least two or more mutations. In certain embodiments, the PcrA helicase comprises at least a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises SEQ ID NO: 1. In certain embodiments, the PcrA helicase is derived from Geobacillus. In certain embodiments, the Geobacillus is Geobacillus stearothermophilus.
In certain embodiments, a vector encodes a PcrA helicase, wherein the PcrA helicase comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises SEQ ID NO: 1. In certain embodiments, the PcrA helicase is derived from Geobacillus stearothermophilus.
In certain embodiments, a vector comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, a vector comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, a vector comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, a vector comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, a vector comprises SEQ ID NO: 2.
In certain embodiments, a nucleic acid sequence comprises at least a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3. In certain embodiments, the nucleic acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In certain embodiments, the nucleic acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In certain embodiments, a nucleic acid sequence comprises SEQ ID NO: 3. In certain embodiments, SEQ ID NO: 3 encodes an amino acid comprising amino acid substitutions at positions H93A, C96A, N187C, C247A, L384V and L409C.
In certain embodiments, an engineered helicase comprises a PcrA helicase wherein said PcrA helicase comprises one or more amino acid mutations. In certain embodiments, the PcrA helicase is derived from Geobacillus stearothermophilus. In certain embodiments, the PcrA helicase comprises mutations at amino positions 93, 96, 187, 247, 384 and 409 of wild type PcrA helicase.
In certain embodiments, a method of amplifying nucleic acids comprises: mixing a first composition with one or more primers, a target nucleic acid sequence, and a second composition. In certain embodiments, the mixed compositions are incubated for about 20-120 minutes preferably at a substantially constant temperature and preferably in a temperature range of less than 100° C. such as between 37° C. to 60° C., 70° C., 80° C. or 90°° C. In certain embodiments, the mixed composition is incubated up to about 10, 20, 30 or 40 minutes at a substantially constant temperature (substantially contant temperature being e.g. within 1° C., 2° C., 3° C., 4° C. or 5° C., 6° C., 7° C. 8° C., 9° C. or 10° C. of a specified temperature), such as, for example, of up to about 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C. or 90° C. In certain embodiments, the mixed composition is incubated at a constant temperature or substantially constant temperature, such as, for example, of about 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C.
In certain embodiments, the first composition comprises a nucleic acid dye, deoxyribonucleotide triphosphates (dNTPs), adenosine triphosphate (ATP) or combinations thereof. In certain embodiments, the second composition comprises: an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase) buffer or combinations thereof. In certain embodiments, the engineered PcrA helicase comprises SEQ ID NO: 1.
In certain embodiments, the first and second compositions further comprise: water, buffers or the combination thereof. In certain embodiments, the polymerase comprises: DNA polymerase (DNAP) I, DNAP II, DNAP III, DNAP IV, DNAP V, Klenow fragment, reverse transcriptases, Exo-polymerase, high-fidelity polymerases, Taq polymerases, bacteriophage polymerases, Pyrococcus furiosus Vc1 (Pfu) polymerase, Hot Start DNA Polymerase, engineered polymerases, engineered reverse transcriptases or eukaryotic polymerases. In certain embodiments, the polymerase is a Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment, Exo-polymerase, Bst, Bst 2.0 or Bst 3.0. In certain embodiments, the Geobacillus stearothermophilus DNA polymerase I is Bst polymerase or Bst-large fragment polymerase (Bst-LF). In certain embodiments, the reverse transcriptase comprises murine leukemia virus (MMLV) reverse transcriptase or Reverse Transcription Xenopolymerase (RTX).
In certain embodiments, a kit comprises an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase). In certain embodiments, the engineered PcrA helicase comprises SEQ ID NO: 1. In certain embodiments, the polymerase comprises: DNA polymerase (DNAP) I, DNAP II, DNAP III, DNAP IV, DNAP V, Klenow fragment, reverse transcriptases, Exo-polymerase, high-fidelity polymerases, Taq polymerases, bacteriophage polymerases, Pyrococcus furiosus Vc1 (Pfu) polymerase, Hot Start DNA Polymerase engineered polymerases, engineered reverse transcriptases or eukaryotic polymerases. In certain embodiments, the polymerase is a Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment, Exo-polymerase, Bst, Bst 2.0 or Bst 3.0. In certain embodiments, the Geobacillus stearothermophilus DNA polymerase I is Bst polymerase or Bst-large fragment polymerase (Bst-LF). In certain embodiments, the reverse transcriptase comprises murine leukemia virus (MMLV) reverse transcriptase or Reverse Transcription Xenopolymerase (RTX).
In certain embodiments, the kit further comprises buffers, deoxyribonucleotide triphosphates (dNTPs), nucleoside triphosphates (NTPs), a detectable nucleic acid label, water, dithiothreitol (DTT) or combinations thereof. In certain embodiments, the detectable nucleic acid label comprises a fluorescent dye.
In certain embodiments, a kit comprises a first composition and a second composition. In certain embodiments, the first composition comprises: a nucleic acid dye, deoxyribonucleotide triphosphates (dNTPs), adenosine triphosphate (ATP) or combinations thereof. In certain embodiments, the second composition comprises: an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase) buffer or combinations thereof. In certain embodiments, the first and second compositions further comprise; water, buffers or the combination thereof. In certain embodiments, the polymerase comprises: DNA polymerase (DNAP) I, DNAP II, DNAP III, DNAP IV, DNAP V, Klenow fragment, reverse transcriptases, Exo-polymerase, high-fidelity polymerases, Taq polymerases, bacteriophage polymerases, Pyrococcus furiosus Vc1 (Pfu) polymerase, Hot Start DNA Polymerase, engineered polymerases, engineered reverse transcriptases or eukaryotic polymerases. In certain embodiments, the polymerase is a Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment, Exo-polymerase, Bst, Bst 2.0 or Bst 3.0. In certain embodiments, the Geobacillus stearothermophilus DNA polymerase I is Bst polymerase or Bst-large fragment polymerase (Bst-LF). In certain embodiments, the reverse transcriptase comprises murine leukemia virus (MMLV) reverse transcriptase or Reverse Transcription Xenopolymerase (RTX). In certain embodiments, the first composition is contained in a first vial and the second composition is contained within a second vial.
In certain embodiments, a method of amplifying nucleic acids comprises incubating a composition comprising a target sequence with primers and an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase) buffer or combinations thereof, at a substantially constant temperature and preferably in a temperature range of less than 100° C. such as between 37° C. to 90° C.
In certain embodiments, the composition is incubated at a temperature (preferably isothermal temperature) of about 45° C. or less, or 40° C. or less, or 38° C. or less such as about 30° C., 31° C., 32° C., 35° C., 36° C., 37° C., 38° C., 39° C. or 40° C.
In certain embodiments, the composition is incubated for about 5 to 120 minutes, or more typically 10 or 20 minutes to about 60 minutes, including up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 or 180 minutes, at isothermal conditions.
In certain embodiments, the mixed composition is incubated at a constant temperature, such as, for example, of up to or about 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., or 90° C.
In certain embodiments, the engineered PcrA helicase comprises at least a 95% sequence identity to SEQ ID NO: 1. In certain embodiments, the engineered PcrA helicase comprises SEQ ID NO: 1. In certain embodiments, the target nucleic acid sequence is an RNA, DNA or the combination thereof.
In one preferred aspect, a method of amplifying nucleic acids comprising: incubating at a substantially isothermal temperature i) a composition comprising a target sequence, ii) primers and iii) an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, and/or a thermostable pyrophosphatase (PPase) buffer, or combinations thereof.
In a further provided aspect, a method of amplifying nucleic acids is provided comprising: incubating in the absence of thermal cycling i) a composition comprising a target sequence, iii) primers and iii) an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, and/or a thermostable pyrophosphatase (PPase) buffer, or combinations thereof.
In such methods, the composition may be suitably incubated at about 40° C. to about 80° C., including at about 45° C. or less, or about 40° C. or less. The composition is suitably incubated for a sufficient time, such as 5 or 10 minutes to 60, 120 or 180 minutes or more.
In certain preferred aspects of such methods, suitably the engineered PcrA helicase comprises at least an 80%, 85% or 90% sequence identity to SEQ ID NO: 1. In certain aspects, the engineered PcrA helicase comprises at least a 95% sequence identity to SEQ ID NO: 1. In certain aspects, the engineered PcrA helicase comprises SEQ ID NO: 1.
In preferred methods, assays and systems, the amplification is sensitive, specific and/or can generate greater (e.g. 1.05, 1.1, 1.2, 1.4, 1.6, 1.8, 2, 3, 4 or 5 times greater) than or equal to kilo-base pair amplified product length as compared to PCR methods or reference PCR assay. Such sensitivity can be determined by one or more assays, e.g. wherein the one or more assays comprise quantitative or real-time PCR (qPCR) or by detecting a cycle threshold value, suitably wherein the cycle threshold value is the number of cycles after which the fluorescence of a PCR product can be detected above a background. See M. Gavrilov et al., Nature Communications (2022) 13:6312, incorporated herein by reference in its entirety.
In certain embodiments, a method of screening for cancer comprises the method or kits embodied herein. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastatses. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
In certain embodiments, a method of detecting infectious agents or sepsis comprising the method or kits embodied herein. The infectious agents comprise, for example, viruses, bacteria, fungi, prions, or parasites.
In certain embodiments, a method of diagnosing a disease or disorder in a subject comprising the method or kits embodied herein. The diseases or disorders comprise, for example: autoimmune diseases, cancer, inflammatory diseases, neurological diseases or disorders, neuroinflammatory diseases or disorders, cardiovascular disease, diabetes.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”
As used herein, the term “about” in the context of a numerical value or range means±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.
In the descriptions above and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible
The term “amino acid” as used herein refers to naturally occurring and synthetic α, β, γ, and δ amino acids, and includes but is not limited to, amino acids found in proteins, i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleucinyl, β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl. When the term amino acid is used, it is considered to be a specific and independent disclosure of each of the esters of α, β, γ, and δ glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine in the D and L-configurations.
As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements—or, as appropriate, equivalents thereof—and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.
As used herein, the term “dNTP” refers to deoxyribonucleoside triphosphates. Non-limiting examples of such dNTPs are dATP, dGTP, dCTP, dTTP, dUTP, which may also be present in the form of labelled derivatives, for instance comprising a fluorescence label, a radioactive label, a biotin label. dNTPs with modified nucleotide bases are also encompassed, wherein the nucleotide bases are for example hypoxanthine, xanthine, 7-methylguanine, inosine, xanthinosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, pseudouridine, dihydrouridine, 5-methylcytidine. Furthermore, ddNTPS of the above-described molecules are encompassed in the present invention.
The term “helicase” refers here to any enzyme capable of unwinding a double stranded nucleic acid enzymatically. For example, helicases are enzymes that are found in all organisms and in all processes that involve nucleic acid such as replication, recombination, repair, transcription, translation and RNA splicing. (Kornberg and Baker, DNA Replication, W. H. Freeman and Company (2nd ed. (1992)), especially chapter 11).
“Isothermal amplification” refers to amplification which occurs at a single or constant temperature. This may not include the single brief time period (less than 15 minutes) at the initiation of amplification which may be conducted at the same temperature as the amplification procedure or at a higher temperature. Depending on the source of enzymes that are used for HDA, the reaction can be performed at low temperatures (<50° C.) e.g., at least or about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 degrees Celsius; or at high temperatures (>50° C.), e.g., at least or about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 or more degrees Celsius.
As used herein, a “natural amino acid” refers to the twenty genetically encoded alpha-amino acids. See, e.g., Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York for structures of the twenty natural amino acids.
“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In embodiments, the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms also 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. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed or chemically synthesized as a single moiety.
“Polypeptide fragment” refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, in which the remaining amino acid sequence is usually identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20 amino acids long, at least 50amino acids long, or at least 70 amino acids long.
As used herein, an “unnatural amino acid,” “non-natural”, “modified amino acid” or “chemically modified amino acid” refers to any amino acid, modified amino acid, or amino acid analogue other than the twenty genetically encoded alpha-amino acids. Unnatural amino acids have side chain groups that distinguish them from the natural amino acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha-amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural amino acids may have an extended backbone such as beta-amino acids.
Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl) alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid containing amino acid; an α,α disubstituted amino acid; a β-amino acid; and a cyclic amino acid other than proline. In an embodiment of the helicases described herein, one or more amino acids of the helicase are substituted with one or more unnatural amino acids and/or one or more natural amino acids.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
The terms “subject”, “patient” or “individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the disclosure find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates. Patients in need of therapy comprise those at risk of developing a certain condition, disease or disorder (e.g. due to genetic, environmental or physical attributes, such as for example, obesity). Patients in need of therapy also include those afflicted with a condition, disease or disorder. The diseases or disorders comprise, for example: autoimmune diseases, cancer, inflammatory diseases, neurological diseases or disorders, neuroinflammatory diseases or disorders, cardiovascular disease, obesity, diseases or disorders caused by infectious agents such as, for example, viruses, bacteria, fungi, prions, or parasites.
Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
As used herein, “variant” of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
A novel method named herein as SHARP (SSB-Helicase Assisted Rapid Polymerase chain reaction) uses helicase and SSB (Single-Stranded Binding protein) to initiate, and DNAP (DNA polymerase) to carry out the isothermal amplification reaction.
Amplification methods overview: PCR is a fast and sensitive technique to amplify or copy segments of DNA starting from the initially small amount of DNA (3, 4) as the first step in any downstream detection or application. PCR alternates the temperature between high (≈98° C.) to melt the DNA duplex and low (45 to 72° C.) to replicate the missing strand by a DNAP (Taq, Pfu, Q5 etc.) (
PCR and isothermal methods really on a DNAP to carry out the in vitro replication through primer extension (1, 2), but the methods differ in their approach to initiate each subsequent reaction cycle and allow primers to bind the template. PCR heat denatures the DNA duplex and subsequently cools it down to initiate each replication cycle (
Isothermal amplification methods based on gp32, a single stranded DNA binding protein, uses a set of two primers, just like PCR, and can produce over 1 kbp amplicons (2); however, the template is amplified only a few times, far from the exponential growth in PCR, and the product also contains high-molecular weight multimers and other undesired bands. RPA (recombinase polymerase amplification) uses two primers and a recombinase (7, 8). RPA primers are sometimes longer than PCR primers, 30 to 38 bases, which is necessary for recombinase binding. The recombinase-primer complex then searches for homologous template sequence resulting in the strand invasion and primer-template pairing, followed by the polymerase extension. RPA provides an exponential amplification and competitive detection limit, but it can produce only short amplicons, 100 to 200 bp in length. Longer primers used by RPA are more prone to forming secondary structure and nonspecific product, requiring more careful primer design, or using unnatural bases (9). SIBA (strand invasion-based amplification) also uses a recombinase, UvsX, but it reduces the possibility of the undesired product formation by using an invasion oligonucleotide in addition to primers (10, 11) but still generates only short amplicons. SDA (strand displacement amplification) (12) and NEAR (nicking enzyme amplification reaction) (13) are similar methods that use a nicking enzyme to assist the amplification initiation. Both methods provide exponential amplification, but the product is also only several hundred base pairs long. Both methods use a set of 4 primers, with additional sequence requirements for the nicking enzyme. HDA or helicase-dependant amplification (14) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNAP. HDA is also commercially available, and it uses similar primers to PCR. Combining UvrD helicase, MutL accessory protein, T4 gene 32 protein (SSB), and Klenow Exo-DNAP, Vincent et al. achieved an exponential amplification of 100 bp product (14); however, for longer amplicons reaction yield dropped significantly, limiting possible applications of the method. Due to the limitations summarized above, existing isothermal methods are used for specific applications, and cannot be used as broadly as PCR (1).
SHARP (SSB-Helicase Assisted Rapid Polymerase chain reaction): SHARP removes thermal cycling from PCR, keeps all other desired PCR features in place, and adds new features. SHARP performs equally well or better than PCR according to 8 criteria we benchmarked against: (1) up to 6000 bp amplicon length, (2) 5 to 30 minutes amplification time, (3) primer design principles and convenience, (4) detection limit, (5) real-time detection, (6) amplification-result interpretation, (7) downstream product applications, and (8) no initial heat-denaturing step. Numerous benefits of removing a thermal cycler from PCR for faster diagnostics are often discussed in the literature (Y. Zhao, et al., “Isothermal Amplification of Nucleic Acids,” Chemical Reviews, vol. 115, p. 12491-12545, 11 2015; Y. Zhang and N. A. Tanner, “Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein,” Scientific Reports, vol. 7, 8 2017), and here the suitability of isothermal reaction was tested for other common tasks in the wet lab as described in the examples section which follows. Briefly, it was demonstrated that E. coli cells transformed with 3.2 kbp SHARP-made plasmid can replicate the plasmid and display antibiotic resistance; hence, SHARP is suitable for cloning, sequencing and gene manipulation. It was also shown that DNA sequences with potential to form non-canonical structures, such as sequence containing (CAG)47 repeats, do not inhibit SHARP. Because PCR heats the sample up to 98° C., it narrows down possible enzymes one may use to improve the reaction speed, yield, product fidelity, detection specificity, and ability to simultaneously perform other reactions. SHARP works optimally at 65° C., but can be carried out in the range between 37-65° C., leaving many possibilities for future improvements and combination with other techniques.
Accordingly, in certain embodiments, a method of amplifying nucleic acids comprises: mixing a first composition with one or more primers, a target nucleic acid sequence, and a second composition. In certain embodiments, the mixed compositions are incubated for about 20-60 minutes at a constant temperature in a temperature range of between 37° C. to 90° C. In certain embodiments, the mixed composition is incubated for about 30 minutes at a constant temperature of about 65° C. In certain embodiments, the first composition comprises a nucleic acid dye, deoxyribonucleotide triphosphates (dNTPs), adenosine triphosphate (ATP) or combinations thereof. In certain embodiments, the second composition comprises: an engineered PcrA helicase, a single stranded binding protein (SSB), a Bst polymerase, a thermostable pyrophosphatase (PPase) buffer or combinations thereof. In certain embodiments, the first and second compositions further comprise: water, buffers or the combination thereof.
Accordingly, in certain embodiments, a bacterial derived helicase comprises a PcrA helicase having at least two or more mutations. In certain embodiments, the PcrA helicase comprises at least a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises SEQ ID NO: 1. In certain embodiments, the PcrA helicase is derived from Geobacillus. In certain embodiments, the Geobacillus is Geobacillus stearothermophilus.
In certain embodiments, a vector encodes a PcrA helicase, wherein the PcrA helicase comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises SEQ ID NO: 1. In certain embodiments, the PcrA helicase is derived from Geobacillus stearothermophilus.
In certain embodiments, a vector comprises a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, a vector comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, a vector comprises at least an 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, a vector comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 2. In certain embodiments, a vector comprises SEQ ID NO: 2.
In certain embodiments, a nucleic acid sequence comprises at least a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 3. In certain embodiments, the nucleic acid sequence comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In certain embodiments, the nucleic acid sequence comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In certain embodiments, a nucleic acid sequence comprises SEQ ID NO: 3. In certain embodiments, SEQ ID NO: 3 encodes an amino acid comprising amino acid substitutions at positions H93A, C96A, N187C, C247A, L384V and L409C.
In certain embodiments, an engineered helicase comprises a PcrA helicase wherein said PcrA helicase comprises one or more amino acid mutations. In certain embodiments, the PcrA helicase is derived from Geobacillus stearothermophilus. In certain embodiments, the PcrA helicase comprises mutations at amino positions 93, 96, 187, 247, 384 and 409 of wild type PcrA helicase.
SHARP enables new generation of portable, point-of-care, point-of-need, and home medical molecular diagnostics tests. Traditional PCR requires a thermocycler, a substantial and high-power-consuming device, and it heats biomolecules above 95° C., making PCR hardly available outside specialized labs. SHARP uses bioengineered enzymes to eliminate the need for complex hardware. SHARP can be carried out with a simple low power heater; hence, molecular diagnostics tests can be performed where needed. SHARP can be used in applications for detecting, for example: infectious diseases, infectious disease agents, disease agents, cancer screening, sepsis, veterinary medicine, agriculture and food processing, forestry cannabis research, and as a general biotechnology tool instead of PCR.
A traditional definition of a helicase is an enzyme that catalyzes the reaction of separating/unzipping/unwinding the helical structure of nucleic acid duplexes (DNA, RNA or hybrids) into single-stranded components, using nucleoside triphosphate (NTP) hydrolysis as the energy source (such as ATP). However, it should be noted that not all helicases fit this definition anymore. A more general definition is that they are motor proteins that move along the single-stranded or double stranded nucleic acids (usually in a certain direction, 3′ to 5′ or 5 to 3, or both), i.e., translocases, that can or cannot unwind the duplexed nucleic acid encountered. In addition, some helicases simply bind and “melt” the duplexed nucleic acid structure without an apparent translocase activity.
Helicases exist in all living organisms and function in all aspects of nucleic acid metabolism. Helicases are classified based on the amino acid sequences, directionality, oligomerization state and nucleic-acid type and structure preferences. The most common classification method was developed based on the presence of certain amino acid sequences, called motifs. According to this classification helicases are divided into 6 super families: SF1, SF2, SF3, SF4, SF5 and SF6. SF1 and SF2 helicases do not form a ring structure around the nucleic acid, whereas SF3 to SF6 do. Superfamily classification is not dependent on the classical taxonomy.
DNA helicases are responsible for catalyzing the unwinding of double-stranded DNA (dsDNA) molecules to their respective single-stranded nucleic acid (ssDNA) forms. Although structural and biochemical studies have shown how various helicases can translocate on ssDNA directionally, consuming one ATP per nucleotide, the mechanism of nucleic acid unwinding and how the unwinding activity is regulated remains unclear and controversial (T. M. Lohman, E. J. Tomko, C. G. Wu, “Non-hexameric DNA helicases and translocases: mechanisms and regulation,” Nat Rev Mol Cell Biol 9:391-401 (2008)). Since helicases can potentially unwind all nucleic acids encountered, understanding how their unwinding activities are regulated can lead to harnessing helicase functions for biotechnology applications.
The term “HDA” refers to Helicase Dependent Amplification, which is an in vitro method for amplifying nucleic acids by using a helicase preparation for unwinding a double stranded nucleic acid to generate templates for primer hybridization and subsequent primer-extension. This process utilizes two oligonucleotide primers, each hybridizing to the 3′-end of either the sense strand containing the target sequence or the anti-sense strand containing the reverse-complementary target sequence. The HDA reaction is a general method for helicase-dependent nucleic acid amplification.
The SHARP method utilizes a novel PcrA M6 helicase enzyme which was engineered to eliminate a thermal cycling machine from Polymerase Chain Reaction (PCR), while retaining all PCR characteristics.
Accordingly, in certain embodiments, a bacterial derived helicase comprising a PcrA helicase having at least two or more mutations. In certain embodiments, the PcrA helicase comprises at least a 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises SEQ ID NO: 1.
In certain embodiments, the PcrA helicase is derived from Geobacillus. In certain embodiments, the Geobacillus is Geobacillus stearothermophilus. In certain embodiments, a nucleic acid sequence, SEQ ID NO: 3, encodes an amino acid comprising amino acid substitutions at positions H93A, C96A, N187C, C247A, L384V and L409C.
In certain embodiments, an engineered helicase comprising a PcrA helicase wherein said PcrA helicase comprises one or more amino acid mutations. In certain embodiments, the PcrA helicase is derived from Geobacillus stearothermophilus. In certain embodiments, the PcrA helicase comprises mutations at amino positions 93, 96, 187, 247, 384 and 409 of wild type PcrA helicase.
In certain embodiments, the mutations at positions 93, 96, 187, 247, 384 and 409 of wild type PcrA helicase comprise both naturally-occurring amino acids and non-naturally-occurring amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, D-amino acids (i.e. an amino acid of an opposite chirality to the naturally-occurring form), N-α-methyl amino acids, C-α-methyl amino acids, β-methyl amino acids and D- or L-β-amino acids. Other non-naturally occurring amino acids include, for example, β-alanine (β-Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (orn), sarcosine, α-amino isobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3-diaP), D- or L-phenylglycine, D-(trifluoromethyl)-phenylalanine, and D-p-fluorophenylalanine.
In certain embodiments, the PcrA helicase comprises one or more modified peptide bonds. As used herein, “peptide bond” can be a naturally-occurring peptide bond or a non-naturally occurring (i.e. modified) peptide bond. Examples of suitable modified peptide bonds are well known in the art and include, but are not limited to, —CH2NH—, —CH2S——CH2CH2—, —CH═CH— (cis or trans), —COCH2—, —CH(OH)CH2—, —CH2SO—, —CS—NH— and —NH—CO— (i.e. a reversed peptide bond) (see, for example, Spatola, Vega Data Vol. 1, Issue 3, (1983); Spatola, in Chemistry and Biochemistry of Amino Acids Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267 (1983); Morley, J. S., Trends Pharm. Sci. pp. 463-468 (1980); Hudson et al., Int. J. Pept. Prot. Res. 14:177-185 (1979); Spatola et al., Life Sci. 38:1243-1249 (1986); Hann, J. Chem. Soc. Perkin Trans. I 307-314 (1982); Almquist et al., J. Med. Chem. 23:1392-1398 (1980); Jennings-White et al., Tetrahedron Lett. 23:2533 (1982); Szelke et al., EP 45665 (1982); Holladay et al., Tetrahedron Lett. 24:4401-4404 (1983); and Hruby, Life Sci. 31:189-199 (1982)).
Vectors: In certain embodiments, a vector encodes for a PcrA helicase comprising one or more mutations. In certain embodiments, a vector encodes a PcrA helicase comprising a 60% sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 75% sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 90% sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises at least a 95% sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises SEQ ID NO: 1. In certain embodiments, the PcrA helicase is derived from Geobacillus stearothermophilus.
In certain embodiments a vector comprises the sequence, SEQ ID NO: 2.
The nucleic acids encoding the PcrA polypeptides can be adapted to suitable expression systems for producing the helicase polypeptides for PcrA helicase production. For DNAs encoding PcrA helicase genes, the representative genes can be operably-linked to suitable expression vectors for expressing the proteins in bacterial, fungal, insect or other suitable expression host. For RNAs encoding PcrA helicase polypeptides, the representative RNAs can be engineered for enabling efficient expression in vitro of the polypeptides in extract lysates produced from bacterial, fungal, insect or other suitable expression host sources. Such systems are well known in the art. Following expression, the PcrA helicase polypeptides can be purified by methods known in the art, including affinity-tag chromatography, SDS-PAGE, and size-exclusion chromatography, among others.
In certain embodiments, vectors such as, for example, expression vectors, containing a nucleic acid encoding one or more PcrA helicase polypeptides described herein are provided. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably. However, this disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
In certain embodiments, the recombinant expression vectors comprise a nucleic acid sequence (e.g., a nucleic acid sequence encoding one or more PcrA helicase polypeptides described herein) in a form suitable for expression of the nucleic acid sequence in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence encoding one or more PcrA helicase polypeptides is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors described herein can be introduced into host cells to thereby produce proteins or portions thereof, including fusion proteins or portions thereof, encoded by nucleic acids as described herein (e.g., one or more PcrA helicase polypeptides).
Recombinant expression vectors can be designed for expression of one or more encoding one or more PcrA helicase polypeptides in prokaryotic or eukaryotic cells. For example, one or more vectors encoding one or more PcrA helicase polypeptides can be expressed in bacterial cells such as E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40); pMAL (New England Biolabs, Beverly, Mass.); and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
In another embodiment, the expression vector encoding one or more PcrA helicase polypeptides is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et. al., (1987) EMBO J. 6:229-234); pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943); pJRY88 (Schultz et al., (1987) Gene 54:113-123); pYES2 (Invitrogen Corporation, San Diego, Calif.); and picZ (Invitrogen Corporation).
Alternatively, one or more PcrA helicase polypeptides can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In certain embodiments, a nucleic acid described herein is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see Green M., and Sambrook; J. Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012.
In certain embodiments, host cells into which a recombinant expression vector of the invention has been introduced are provided. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, one or more PcrA helicase polypeptides can be expressed in bacterial cells such as E. coli, viral cells such as retroviral cells, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Delivery of nucleic acids described herein (e.g., vector DNA) can be by any suitable method in the art. For example, delivery may be by injection, gene gun, by application of the nucleic acid in a gel, oil, or cream, by electroporation, using lipid-based transfection reagents, or by any other suitable transfection method.
As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as, for example, LIPOFECTIN™ (Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE™ (Invitrogen), FUGENE™ (Roche Applied Science, Basel, Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, N.Y.), EFFECTENE™ (Qiagen, Valencia, Calif.), DREAMFECT™ (OZ Biosciences, France) and the like), or electroporation (e.g., in vivo electroporation). Suitable methods for transforming or transfecting host cells can be found in Green and Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012), and other laboratory manuals.
In certain embodiments, a kit comprises an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase). In certain embodiments, the engineered PcrA helicase comprises SEQ ID NO: 1. In certain embodiments, the polymerase comprises: DNA polymerase (DNAP) I, DNAP II, DNAP III, DNAP IV, DNAP V, Klenow fragment, reverse transcriptases, Exo-polymerase, high-fidelity polymerases, Taq polymerases, bacteriophage polymerases, Pyrococcus furiosus Vc1 (Pfu) polymerase, Hot Start DNA Polymerase, or eukaryotic polymerases. In certain embodiments, the polymerase is a Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment or Exo-polymerase. In certain embodiments, the Geobacillus stearothermophilus DNA polymerase I is Bst polymerase or Bst-large fragment polymerase (Bst-LF). In certain embodiments, the reverse transcriptase is murine leukemia virus (MMLV) reverse transcriptase.
In certain embodiments, the kit further comprises buffers, deoxyribonucleotide triphosphates (dNTPs), nucleoside triphosphates (NTPs), a detectable nucleic acid label, water, dithiothreitol (DTT) or combinations thereof. In certain embodiments, the detectable nucleic acid label comprises a fluorescent dye.
In certain embodiments, a kit comprises a first composition and a second composition. In certain embodiments, the first composition comprises: a nucleic acid dye, deoxyribonucleotide triphosphates (dNTPs), adenosine triphosphate (ATP) or combinations thereof. In certain embodiments, the second composition comprises: an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase) buffer or combinations thereof. In certain embodiments, the polymerase comprises: DNA polymerase (DNAP) I, DNAP II, DNAP III, DNAP IV, DNAP V, Klenow fragment, reverse transcriptases, Exo-polymerase, high-fidelity polymerases, Taq polymerases, bacteriophage polymerases, Pyrococcus furiosus Vc1 (Pfu) polymerase, Hot Start DNA Polymerase, or eukaryotic polymerases. In certain embodiments, the polymerase is a Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment or Exo-polymerase. In certain embodiments, the Geobacillus stearothermophilus DNA polymerase I is Bst polymerase or Bst-large fragment polymerase (Bst-LF). In certain embodiments, the reverse transcriptase is murine leukemia virus (MMLV) reverse transcriptase.
In certain embodiments, the kit further comprises buffers, deoxyribonucleotide triphosphates (dNTPs), nucleoside triphosphates (NTPs), a detectable nucleic acid label, water, dithiothreitol (DTT) or combinations thereof. In certain embodiments, the detectable nucleic acid label comprises a fluorescent dye.
In certain embodiments, the first and second compositions further comprise; water, buffers or the combination thereof. In certain embodiments, first composition is contained in a first vial and the second composition is contained within a second vial.
In certain embodiments, the kit may further comprise a helicase, a primer pair, a polymerase, and optionally, a detection system for detecting amplification of the target nucleic acid, as described herein. In certain embodiments, the primer pair may comprise a first and second primer, with the first primer comprising a portion that is complementary to the first strand of the target nucleic acid and the second primer comprising a portion that is complementary to the second strand of the target nucleic acid. The kit may also include a set of instructions for use. In certain embodiments, the kit may include reagents for purifying the double-stranded nucleic acid in the sample. In some embodiments, the kit may be a kit for amplifying and/or detecting a target single-stranded nucleic acid in a sample and may include reagents for purifying the single-stranded nucleic acid in the sample. The kit may also include a set of instructions for use.
A new robust isothermal amplification method is described herein, which is analogous to PCR in that it uses the same set of primers and template as PCR at the input. Further, the method outputs the same amplicon as PCR, with the length of up to 6000 base pairs (bp) but is performed at a constant temperature, thus obviating the need for thermal cycling. This new method is named SHARP (SSB-Helicase Assisted Rapid Polymerase chain reaction) herein, because it uses helicase and SSB (Single-Stranded Binding protein) to initiate, and DNAP (DNA polymerase) to carry out the isothermal amplification reaction.
SHARP reaction mix: Three principal enzymes, Bst-LF DNAP, E.coli SSB, and PcrA M6 helicase, were overexpressed and purified. The following enzymes were used for SHARP with stock concentrations: SSB (9 mg/mL), PcrA helicase (0.2 mg/mL), Bst-LF DNAP (1.5 mg/mL), PPase (2000 units/mL, NEB catalog #M0296S). Other stock components are dNTP's (10 mM each), ATP (100 mM), Evagreen dye (20× from Biotium #31000), DTT (Dithiothreitol, 100 mM in water). The 10× reaction buffer contains 500 mM Potassium acetate, 200 mM Tris-acetate, 100 mM Magnesium acetate, 1 mg/mL BSA, pH 7.9. Primer stock concentrations are at 10 or 20 μM, while the template concentration is variable.
For each SHARP reaction, 40 μL total volume was prepared and the volume was split to 2 separate wells on a 96-well plate to have 2 independent fluorescence readouts of the same reaction. SYBR (EvaGreen) intensity was monitored in real time every 10 s for each well on BioRad CFX96 machine. Table 1 contains raw component volumes.
indicates data missing or illegible when filed
Component 1 and Component 2 were separately prepared and were mixed together on the plate, before the temperature was increased to 65 degrees and the recording of fluorescence starts. After incubation, the product with was purified with Qiagen PCR cleanup kit and test the product on the gel.
Primers and template: Primers were ordered from Integrated DNA technologies (IDT). DNA template vector containing 2019 coronavirus nCOV-2 N protein sequence (2019-nCOV_N_Positive Control, Catalog #10006625) was also ordered from IDT. For PCR and SHARP in
DNA unwinding assays: The FRET unwinding assay uses the following buffer: 10 mM TRIS pH8.0, 10 mM MgCl2, 50 mM NaCl, 1 mM ATP, 1% BSA. DNA concentration is at 5 nM and the helicase concentration varies. The reaction is performed in 0.2 mL cuvette. The spectrofluorometer excites the sample at 550 nm and records the fluorescence at 570 nm (green, Cy3) and 667 nm (red, Cy5). Unwinding of longer DNA substrates in the presence of EvaGreen intercalating dye is carried out in 20 μL volume on qPCR machine in the same buffer as the SHARP reaction (50 mM Potassium acetate, 20 mM Tris-acetate, 10 mM Magnesium acetate, 0.1 mg/mL BSA, pH 7.9). The reaction mix is prepared on ice, and the qPCR machine is precooled to 4° C. Upon placing the same on qPCR machine, the machine starts recording and then rapidly increases the temperature to 37° C.
Protein overexpression and purification: PcrA M6 helicase with 6xHis tag: PcrA M6 helicase, PcrA and all mutants are purified as described previously (Arslan, R. et al. “Engineering of a superhelicase through conformational control,” Science, vol. 348, p. 344-347, 4 2015; J. Park, et al., “PcrA Helicase Dismantles RecA Filaments by Reeling in DNA in Uniform Steps,” Cell, vol. 142, p. 544-555, 8 2010) using a standard Ni-NTA purification column, followed by single-stranded DNA cellulose column. Briefly, the pET-11b vector containing PcrA M6 sequence between NdeI and BamHI sites was used. The vector including 6x-His tag on N terminus has been synthetized by GenScript and GenScript synthetized all point mutations. The vector was transformed to E. coli. BL21 (DE3) pLysS. Cells were grown at 37° C. in the presence of ampicillin and chloramphenicol, moved to 18° C. when OD600 reached 0.3 and induced at OD600=0.5 with 0.5 mM IPTG and harvested after an overnight incubation at 18° C. Cell pellets, previously stored at −80° C. were resuspended in the lysis buffer (50 mM Tris, 5 mM Imidazole, 200 mM NaCl, 20% Sucrose, 15% Glycerol, 0.5 mg/mL Lysozyme, pH 7.6) and sonicated followed by centrifugation at 35,000 g. The Ni-NTA agarose resin is preequilibrated with the wash buffer (50 mM Tris, 5 mM Imidazole, 150 mM NaCl, 25% (v/v) Glycerol, pH 7.6). 40 mL of cell lysate supernatant is added to 2 mL of equilibrated Ni-NTA resin and incubated for 1 hour at 4° C. with constant stir mixing by inverting the 50 ml tube. After 1 hour, the resin is gently centrifuged at 1000 g for 2 minutes and supernatant is discarded carefully, and the tube with the resin refilled with the wash buffer. The batch wash is repeated 3 times, protein-loaded resin is then poured into a disposable gravity flow column, washed with 20 mL of wash buffer, and eluted with elution buffer made by dissolving 200 mM imidazole in wash buffer. The protein is then loaded to single-stranded DNA cellulose column, washed with buffer (100 mM NaCl, 50 mM Tris, 1 mM EDTA, 20% (v/v) Glycerol), and eluted with (1 M NaCl, 50 mM Tris, 1 mM EDTA, 20% (v/v) Glycerol). The presence of the 6xHis does not affect any downstream application. The protein concentration was always kept below 4 mg/ml (≈50 mM) to avoid aggregation, and the final PcrA protein was stored at −80° C. or −20° C. in the storage buffer containing 600 mM NaC1, 50 mM TRIS pH 7.6, and 50% glycerol. This protocol leads to a diluted PcrA to between 0.2 and 1 mg/mL, if necessary one can concentrate protein with a membrane filtering.
PcrA helicase without 6xHis tag: Plasmid expressing wild-type PcrA helicase without any tag is a gift from Tim Lohman lab. Obtaining the cell lysate is done in the same way as in the previous protocol. The cell lysate is also spun down, but supernatant is mixed with 0.7 volume of saturated ammonium sulfate solution. Ammonium sulfate precipitates PcrA, and PcrA is collected by spinning at 5000 g. PcrA was suspended in the wash buffer (50 mM Tris, 5 mM Imidazole, 150 mM NaCl, 25% (v/v) Glycerol, pH 7.6), spun down, and the supernatant loaded to ssDNA-cellulose column. The rest of the protocol is the same as for PcrA with the 6xHis tag.
Single Stranded binding protein (SSB): E. coli SSB is purified without tag through polymin-P and ammonium sulfate precipitation, followed by the heparin Sepharose column, similar to the protocol by Lohman et al. (“Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under λ PL control,” Biochemistry, vol. 25, p. 21-25, 1 1986). SSB sequence is cloned into pET21a vector using the NdeI and BamHI sites. The vector was used to transform BL21 (DE3) cells, the colonies were picked, grown at 37° C., overexpression was induced with 0.5 mM IPTG at OD600=0.5 and grown for an additional 5 hours and the pellet collected. Overexpression levels were usually very high, often SSB made over 60% of the total cell protein. The cell pellet was resuspended in the lysis buffer (50 mM Tris, 200 mM NaCl, 20% Sucrose, 15% Glycerol, 0.5 mg/mL Lysozyme, pH 7.6) and sonicated, followed by the centrifugation at 35,000 g for 30 minutes. The supernatant containing soluble SSB is collected, and Polymin-P of the final concentration 0.2% was added to the supernatant to precipitate SSB. Precipitated SSB was collected by spinning at 4000 g and then resuspended in buffer containing 50 mM TRIS pH 8.3, 20% glycerol, 1 mM EDTA, 400 mM NaCl, and stir mixed at 4° C. for 30 minutes. To remove undissolved protein, the mixture was centrifuged at 10,000 g for 20 minutes and the supernatant containing soluble SSB was collected. Finally, solid ammonium sulfate was added to give the final concentration of 150 g/L, precipitate SSB, and collect the pellet containing SSB after centrifugation at 12,000 for 30 minutes. The pellet was resuspended in 50 mM TRIS pH 8.3, 20% glycerol, 1 mM EDTA, 200 mM NaCl, stir mixed at 4° C. for 30 minutes, centrifuged at 18,000 g for 20 minutes, and filtered through 200 μm filter. We equilibrate heparin-Sepharose column with a wash buffer (50 mM TRIS pH 8.3, 20% glycerol, 1 mM EDTA), dilute the SSB solution in the wash buffer 5 times and load on the column. Next, the bound SSB was washed with 50 to 100 mL wash buffer and eluted with NaCl gradient from 100 mM to 1 M. The final SSB solution was dialyzed against the storage buffer (20 mM Tris, pH 8.1, 50% Glycerol, 0.5M NaCl, 1 mM EDTA, 1 mM BME). SSB concentrated between 6 and 9 mg/mL was obtained. The SHARP reaction uses highly concentrated SSB; hence, it is important to use SSB stock concentrated above 5 mg/mL.
Bst-LF DNA polymerase: Bst-LF purification protocol uses Ni-NTA resin. Bst-LF DNAP expressing vector available through addgene.org/145799/was used and a modified version of the protocol described in (S. Bhadra, et al. “High-surety isothermal amplification and detection of SARS-COV-2, including with crude enzymes,” 4 2020) was followed for the overexpression and purification. Bst-LF expressing plasmid containing 6xHis tag on N terminus was transformed to expressing cells, select individual colonies, grow medium until OD600 reaches 0.5 and overexpression was induced with 200 ng/ml anhydrotetracycline (aTC) or 100 ng/ml of tetracycline. The total culture volume of 500 mL was grown at 25 degrees for 12 hour and spun down to form the pellet kept at −80 degrees. The pellet is resuspended in the Lysis buffer (20 mM Tris pH 7.4, 300 mM NaCl, 0.1% Tween-20, 10 mM imidazole, 1×EDTA-free protease inhibitor tablet, 0.5 mg/mL Lysozyme) in the cold room and sonicated to lyse the cells. Lysed cells were centrifuged at 35,000 g for 30 min at 4° C. and the supernatant was collected. The lysate was heat treated at 65° C. for 10 min, cooled down on ice for 10 min at 4° C., and filtered using 60 mL syringe with 200 μm filter. The Ni-NTA agarose resin was pre-equilibrated with the wash buffer (20 mM Tris, pH 7.4, 300 mM NaCl, 0.1% Tween-20, 40 mM imidazole). 40 mL of filtered lysate is added to 2 mL of equilibrated Ni-NTA resin and incubated for 1 hour at 4° C. with constant stir mixing by inverting the 50 ml tube. After 1 hour, the resin was gently centrifuged at 1000 g for 2 minutes, supernatant was discarded carefully, and the tube with the resin refilled with the wash buffer. The batch wash was repeated 3 times, protein-loaded resin is then poured into a disposable gravity flow column, washed with 10 mL of wash buffer and eluted with a buffer containing 20 mM Tris pH 7.4, 300 mM NaCl, 0.1% Tween-20, 250 mM imidazole in 500 μL fractions. Collected fractions were tested on the gel, and dialyzed against the storage buffer (50% Glycerol, 10 mM Tris pH 7.4, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% Tween-20, 0.5% Triton-X100) overnight. Small aliquots of Bst-LF DNAP concentrated at 1.5 mg/mL are kept at −80° C. for long term storage or at −20° C. for daily use. Optimal Bst-LF concentration in the SHARP reaction is between 0.0015 and 0.0075 mg/mL.
Reference PCR assay: To introduce SHARP a linearized 4012-bp DNA template vector containing 2019 coronavirus nCOV-2 N protein sequence between M13 primers (
SHARP amplification reaction: SHARP was used to carry out the amplification reaction at 65° C., with the same template copy numbers and the same primer set as the reference PCR. SHARP produces the same 1463 bp amplicon as PCR (
SHARP kinetics (
Quantitative or real-time PCR (qPCR) uses and defines the cycle threshold (CT) value as the number of cycles when the fluorescence of a PCR product can be detected above the background. Because the number of amplification cycles in SHARP could not be directly determined, the time it takes to detect the products in qSHARP were instead determined, termed the detection time. The detection threshold was chosen so that the signal for non-template control remains below the threshold for 25 minutes.
SHARP was also demonstrated with other template-primers sets, including ≈3 kbp amplicon (
Next, it was tested how SHARP behaves with the respect to some common applications in the wet lab. It was first tested whether SHARP makes amplicons that can be propagated in living cells. Starting from a 4012 bp template vector (
It was further tested whether SHARP could amplify sequences prone to secondary structure formation. A 385-bp region was selected, containing 47 trinucleotide CAG repeats known to undergo multivalent intermolecular interactions in the single stranded form (A. Jain and R. D. Vale, “RNA phase transitions in repeat expansion disorders,” Nature, vol. 546, p. 243-247, 5 2017). Trinucleotide CAG repeats are known to have low PCR yields (L. Aeschbach and V. Dion, “Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications,” Scientific Reports, vol. 7, 12 2017; V. Dion, “Tissue specificity in DNA repair: lessons from trinucleotide repeat instability,” Trends in Genetics, vol. 30, p. 220-229, 6 2014). In the example in
Finally, the temperature dependence of the SHARP reaction was tested. With nCOV-2 sequence template and primers targeting 155 bp region, the SHARP reactionwas run in the temperature range between 45° C. and 65° C., and the kinetics curves were recorded (
Overall, it was demonstrated with the examples in
Engineering PcrA M6 helicase for SHARP: The DNA amplification reaction requires separation of duplex strands and the original patent for PCR (U.S. Pat. No. 4,800,159) proposed separating DNA either through heating or enzymatically with helicases or RecA1. Apart from HAD (M. Vincent, et al., “Helicase-dependent isothermal DNA amplification,” EMBO reports, vol. 5, p. 795-800, 8 2004), which is limited in performance and applications, the strand separation by helicases has not been widely utilized for DNA amplification which it is attributed to the lack of helicases suitable for this application. It was found that PcrA M6 helicase, which was engineered from the Geobacillus stearothermophilus PcrA, serves the purpose well after testing various other candidate helicases.
A bulk FRET unwinding assay was used to test the unwinding activity of different helicase candidates for SHARP (S. Arslan, et al., “Engineering of a superhelicase through conformational control,” Science, vol. 348, p. 344-347, 4 2015). The DNA construct (
Wild-type Geobacillus stearothermophilus PcrA helicase showed low unwinding activity at elevated concentration of 600 nM (
A new isothermal amplification method named SHARP was produced with the engineered PcrA M6 helicase. SHARP eliminates thermal cycling from PCR, while keeping all desirable PCR features in place. SHARP can generate simple, linear, and several kilo base pair long amplicons from a template and a set of two primers in under 30 minutes. Apart from replacing thermal cycling from PCR in simple molecular diagnostic, sequencing and gene manipulation reactions such as cloning, SHARP can also detect and amplify sequences prone to forming secondary structures. SHARP has also a potential of being a universal biotechnology tool for many new point-of-care detections methods in medicine, it simplifies many procedures in research labs, and outside labs it enables new applications such as environmental screening, farming pest detection, and many other future consumer-focused diagnostic home tests. The method simply eliminates a bulky and expensive thermal cycler machine from the amplification reaction, thus increasing its versatility.
We further show that engineered PcrA M6 is 3 to 4 times faster than prior PcrA M5.
We further analyzed data in
We also carried out the nanopore assay, where we directly observe the speed at which a helicase unwinds DNA duplex with Single Molecule Picometer Nanopore Tweezers (SPRNT) in
The higher activity and speed of PcrA M6 demonstrated in both bulk and single-molecule assay facilitates the SHARP amplification.
In the provisional application, we stated that SHARP can amplify RNA targets. We also listed Moloney Murine Leukemia Virus (MMLV) reverse transcriptase; however, we did not include any data to support this claim. To demonstrate SHARP can amplify RNA targets, we used MMLV reverse transcriptase. MMLV first generate cDNA from RNA template, and further acts as a DNA polymerase of moderate efficiency to carry our the amplification. We used nCOV-2 RNA sequence and CDC N2 primers: forward 5′-TTACAAACATTGGCCGCAAA and reverse 5′-GCGCGACATTCCGAAGAA. To generate a 67 bp amplicon at different temperatures. The highest product yield was obtained at 39.4° C. The overall efficiency of RNA amplification is lower than DNA amplification.
SHARP can be used for portable, point-of-care, point-of-need, and home medical molecular diagnostics tests. SHARP product can be easily detected using a lateral flow device. We carried out SHARP amplification where the forward primer is labeled with biotin and we included DIG-labeled dNTP mix,
In
SHARP can amplify regions in genomic DNA from human cells. We purified gDNA from HEK293T cells and amplified a 474 bp DNA region near the DMNT3B gene and another 410 bp region near FANCF gene. We used following primers 5′-AGTTCGCTAATCCCGGAACT (FANCF_F), 5′-AGTTGCCCAGAGTCAAGGAA (FANCF_R), 5′-CCAGTGGTTCAATGGTCATCC (DNMT3B_F), and 5′-GGCCAGTGAAATCACCCTG (DNMT3B_R).
From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
This application claims the benefit of priority of U.S. Provisional Application No. 63/304,189 filed on Jan. 28, 2022, which is incorporated herein by reference in its entirety and for all purposes.
This invention was made with government support under grant GM122569 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US23/61592 | 1/30/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63304189 | Jan 2022 | US |
