Bioengineered enzymes and uses thereof

Abstract

Enzymes engineered for the amplification of nucleic acid sequences function at a constant temperature, thereby eliminating the heating and cooling cycles associated with conventional polymerase chain reaction (PCR).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 304,189, filed January 28, 2022, the entirety of which is incorporated by reference herein for all purposes.

[0002] government support This invention was made with Government support under Grant GM122569 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Technical Field Enzymes are provided for the isothermal amplification of nucleic acid sequences that do not require the heating and cooling cycles associated with conventional polymerase chain reaction (PCR). [Background technology]

[0004] DNA and RNA amplification are essential methods involved in rapid molecular diagnostics, genetic manipulation, and genetic analysis, including detection of bacteria, viruses, and diagnosis of genetic disorders. The most widely used method for DNA amplification is the polymerase chain reaction (PCR) performed via thermal cycling. Promising next-generation methods include enzymatic isothermal amplification, but existing isothermal methods are limited to generating only short amplicons, or complex heterogeneous and branched products, and often involve the use of multiple sets of complex primer pairs (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, existing isothermal methods cannot approach the versatility of thermal cycler-based PCR. Summary of the Invention [Means for solving the problem]

[0005] Provided herein is a polymerase chain reaction (PCR) that retains many of the desirable features of conventional PCR while eliminating thermal cycling, referred to herein as SHARP (SSB-helicase-assisted rapid PCR). The composition contains a novel helicase and SSB (single-stranded binding protein) for effective strand separation at a constant temperature, thus avoiding the need for cyclic heating. SHARP uses the same starting set of primers and template DNA as PCR, performs the reaction at a constant temperature, and outputs the same amplicons as PCR with lengths up to 6,000 base pairs, a feature that existing isothermal amplification methods cannot match.

[0006] Thus, in certain embodiments, the bacterial helicase comprises a PcrA helicase having at least two or more mutations. In certain embodiments, the PcrA helicase comprises at least 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 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 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 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.

[0007] In certain embodiments, the vector encodes a PcrA helicase, wherein the PcrA helicase comprises 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 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 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 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.

[0008] In certain embodiments, the vector comprises 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, the vector comprises at least 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, the vector comprises at least 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, the vector comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In certain embodiments, the vector comprises SEQ ID NO: 2.

[0009] In certain embodiments, the nucleic acid sequence comprises at least 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 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 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 SEQ ID NO: 3. In certain embodiments, SEQ ID NO: 3 encodes amino acids comprising amino acid substitutions at positions H93A, C96A, N187C, C247A, L384V, and L409C.

[0010] In certain embodiments, the engineered helicase comprises a PcrA helicase, wherein the 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.

[0011] In certain embodiments, a method for amplifying a nucleic acid includes mixing a first composition with one or more primers, a target nucleic acid sequence, and a second composition. In certain embodiments, the mixed composition is incubated for about 20 to 120 minutes at a substantially constant temperature, preferably at a temperature range of less than 100°C, such as 37°C to 60°C, 70°C, 80°C, or 90°C. In certain embodiments, the mixed composition is incubated for about 10, 20, 30, or 40 minutes at a substantially constant temperature, such as up to about 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, or 90°C (wherein the substantially constant temperature is, for example, within 1°C, 2°C, 3°C, 4°C, or 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C of the specified temperature). In certain embodiments, the mixed composition may be, for example, at 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, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 102°C, 104°C, 105°C, 106°C, 107°C, 108 The culture may be incubated at a constant or substantially constant temperature, such as 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.

[0012] In certain embodiments, the first composition comprises a nucleic acid dye, a deoxyribonucleotide triphosphate (dNTP), an adenosine triphosphate (ATP), or a combination 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 a combination thereof. In certain embodiments, the engineered PcrA helicase comprises SEQ ID NO:1.

[0013] In certain embodiments, the first and second compositions further comprise water, a buffer, or a combination thereof. In certain embodiments, the polymerase comprises DNA polymerase (DNAP) I, DNAP II, DNAP III, DNAP IV, DNAP V, Klenow fragment, reverse transcriptase, exo-polymerase, high-fidelity polymerase, Taq polymerase, bacteriophage polymerase, Pyrococcus furiosus Vc1 (Pfu) polymerase, hot-start DNA polymerase, engineered polymerase, engineered reverse transcriptase, or eukaryotic polymerase. In certain embodiments, the polymerase is Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment, exopolymerase, Bst, Bst2.0, or Bst3.0. In certain embodiments, the Geobacillus stearothermophilus DNA polymerase I is Bst polymerase or Bst-large fragment polymerase (Bst-LF). In certain embodiments, the reverse transcription comprises murine leukemia virus (MMLV) reverse transcription or reverse transcription xenopolymerase (RTX).

[0014] In certain embodiments, the kit comprises an engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, and 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 transcriptase, exo-polymerase, high fidelity polymerase, Taq polymerase, bacteriophage polymerase, Pyrococcus furiosus Vc1 (Pfu) polymerase, hot start DNA polymerase engineered polymerase, engineered reverse transcriptase, or eukaryotic polymerase. In certain embodiments, the polymerase is Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment, exopolymerase, Bst, Bst2.0, or Bst3.0. In certain embodiments, the Geobacillus stearothermophilus DNA polymerase I is Bst polymerase or Bst-large fragment polymerase (Bst-LF). In certain embodiments, the reverse transcription comprises murine leukemia virus (MMLV) reverse transcription or reverse transcription xenopolymerase (RTX).

[0015] In certain embodiments, the kit further comprises a buffer, deoxyribonucleotide triphosphates (dNTPs), nucleoside triphosphates (NTPs), a detectable nucleic acid label, water, dithiothreitol (DTT), or a combination thereof. In certain embodiments, the detectable nucleic acid label comprises a fluorescent dye.

[0016] In certain embodiments, the kit comprises a first composition and a second composition. In certain embodiments, the first composition comprises a nucleic acid dye, a deoxyribonucleotide triphosphate (dNTP), an adenosine triphosphate (ATP), or a combination thereof. In certain embodiments, the second composition comprises an engineered PcrA helicase, a single-stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase), a buffer, or a combination thereof. In certain embodiments, the first and second compositions further comprise water, a buffer, or a combination thereof. In certain embodiments, the polymerase comprises DNA polymerase (DNAP) I, DNAP II, DNAP III, DNAP IV, DNAP V, Klenow fragment, reverse transcriptase, exo-polymerase, high fidelity polymerase, Taq polymerase, bacteriophage polymerase, Pyrococcus furiosus Vc1 (Pfu) polymerase, hot-start DNA polymerase, engineered polymerase, engineered reverse transcriptase or eukaryotic polymerase. In certain embodiments, the polymerase is Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment, exopolymerase, Bst, Bst2.0 or Bst3.0. In certain embodiments, the Geobacillus stearothermophilus DNA polymerase I is Bst polymerase or Bst-large fragment polymerase (Bst-LF). In certain embodiments, the reverse transcription comprises murine leukemia virus (MMLV) reverse transcription or reverse transcription xenopolymerase (RTX). In certain embodiments, the first composition is contained in a first vial and the second composition is contained in a second vial.

[0017] In certain embodiments, a method for amplifying a nucleic acid comprises incubating a composition comprising a target sequence with a primer and an engineered PcrA helicase, a single-stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase) buffer or a combination thereof at a substantially constant temperature, preferably below 100°C, for example in the range of 37°C to 90°C.

[0018] In certain embodiments, the composition is incubated at a temperature of about 45° C. or less, or 40° C. or less, or 38° C. or less, e.g., about 30° C., 31° C., 32° C., 35° C., 36° C., 37° C., 38° C., 39° C. or 40° C. (preferably an isothermal temperature).

[0019] In certain embodiments, the composition is incubated under isothermal conditions for about 5 to 120 minutes, or more typically for 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).

[0020] In certain embodiments, the mixed composition may be, for example, at or above 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, Incubated at a constant temperature such as 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.

[0021] In certain embodiments, the engineered PcrA helicase comprises at least 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 RNA, DNA, or a combination thereof.

[0022] In one preferred embodiment, a method for amplifying a nucleic acid comprises incubating i) a composition comprising a target sequence, ii) a primer, and iii) an engineered PcrA helicase, single-stranded binding protein (SSB), polymerase, and / or a thermostable pyrophosphatase (PPase) buffer, or combinations thereof, at a substantially isothermal temperature.

[0023] In further provided aspects, a method of amplifying a nucleic acid is provided, comprising incubating in the absence of thermal cycling: i) a composition comprising a target sequence; iii) a primer; and iii) an engineered PcrA helicase, single-stranded binding protein (SSB), polymerase, and / or a thermostable pyrophosphatase (PPase) buffer, or combinations thereof.

[0024] In such methods, the composition may be suitably incubated at about 40° C. to about 80° C., including about 45° C. or less, or about 40° C. or less. The composition is suitably incubated for a sufficient period of time, such as from 5 or 10 minutes to 60, 120 or 180 minutes or more.

[0025] In certain preferred aspects of such methods, suitably the engineered PcrA helicase comprises at least 80%, 85% or 90% sequence identity to SEQ ID NO: 1. In certain aspects, the engineered PcrA helicase comprises at least 95% sequence identity to SEQ ID NO: 1. In certain aspects, the engineered PcrA helicase comprises SEQ ID NO: 1.

[0026] In preferred methods, assays and systems, the amplification is sensitive, specific and / or can generate a length greater than or equal to a kilobase pair amplification product length (e.g., 1.05, 1.1, 1.2, 1.4, 1.6, 1.8, 2, 3, 4 or 5 times greater) compared to a PCR method or a reference PCR assay. Such sensitivity can be determined by one or more assays, for example, when one or more assays include quantitative or real-time PCR (qPCR), or by detecting a cycle threshold, suitably the cycle threshold being the number of cycles after which the fluorescence of the PCR product can be detected above background. See M. Gavrilov et al., Nature Communications (2022) 13: 6312 (incorporated herein by reference in its entirety).

[0027] In certain embodiments, the method for screening cancer comprises the method or kit embodied herein.Cancer includes benign and malignant cancer, as well as dormant tumor or micrometastasis.Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric 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 cancer, salivary gland cancer, renal or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphomas (low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NH L; 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 blood vessel growth, edema (such as that associated with brain tumors) associated with phakomatosis, and Meigs' syndrome.

[0028] In certain embodiments, the method of detecting an infectious agent or sepsis comprises a method or kit embodied herein. Infectious agents include, for example, viruses, bacteria, fungi, prions, or parasites.

[0029] In certain embodiments, the method of diagnosing a disease or disorder in a subject comprises a method or kit embodied herein, including, for example, an autoimmune disease, cancer, an inflammatory disease, a neurological disease or disorder, a neuroinflammatory disease or disorder, a cardiovascular disease, or diabetes.

[0030] definition

[0031] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, and biochemistry).

[0032] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used anywhere in the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0033] As used herein, the term "about" in the context of a numerical value or range means ±10% of the recited or claimed numerical value or range, unless the context requires a more limited range.

[0034] In the above description and in the claims, phrases such as "at least one" or "one or more" may appear following a connected list of elements or features. The term "and / or" may also appear in a list of two or more elements or features. Unless implicitly or explicitly contradicted by the context in which it is used, such phrases are intended to mean any of the listed elements or features individually, or any of the listed elements or features in combination with any of the other listed 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 intended to mean "A only, B only, or A and B together," respectively. A similar interpretation is intended for lists containing more than two 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 intended to mean "A only, B only, C only, A and B together, A and C together, B and C together, or A, B and C together," respectively. Additionally, use of the term "based on" above and in the claims is intended to mean "based at least in part on," and thus allows for unrecited features or elements.

[0035] The term "amino acid" as used herein refers to naturally occurring and synthetic alpha, beta, gamma, and delta amino acids, including, but not limited to, those amino acids found in proteins, i.e., glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, 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, aspartate, 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-aspartate, 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 D- and L-configuration esters of alpha, beta, gamma, and delta glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0036] As used herein, the terms "comprising," "comprise," or "comprised," and variations thereof, with respect to defined or described elements of an item, composition, apparatus, method, process, system, etc., are meant to be inclusive or open-ended, allowing for additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specific elements--or equivalents thereof, where appropriate--and that other elements may be included and still be included within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0037] As used herein, the term "dNTP" refers to deoxyribonucleoside triphosphate. Non-limiting examples of such dNTPs are dATP, dGTP, dCTP, dTTP, dUTP, which may also be present in the form of labeled derivatives, including, for example, fluorescent labels, radioactive labels, biotin labels. Also included are dNTPs with modified nucleotide bases, where the nucleotide bases are, for example, hypoxanthine, xanthine, 7-methylguanine, inosine, xanthinosine, 7-methylguanosine, 5,6-dihydrouracil, 5-methylcytosine, pseudouridine, dihydrouridine, 5-methylcytidine. Furthermore, the ddNTPs of the above molecules are included in the present invention.

[0038] The term "helicase" as used herein refers to any enzyme that can enzymatically unwind double-stranded nucleic acid. For example, helicases are enzymes found in all organisms and in all processes involving nucleic acids, such as replication, recombination, repair, transcription, translation and RNA splicing. (Kornberg and Baker, DNA Replication, WH Freeman and Company (2006) nd ed.(1992), especially chapter 11).

[0039] "Isothermal amplification" refers to amplification that occurs at a single or constant temperature. It may not include a single short period (less than 15 minutes) at the beginning of the amplification, which may be carried out at the same temperature as the amplification procedure or at a higher temperature. Depending on the source of enzyme used in HDA, the reaction may be carried out 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°C; 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, 116, 117, 118, 119, 120, 121, 122, 6, 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°C or higher.

[0040] As used herein, "natural amino acid" refers to the 20 genetically encoded α-amino acids. For the structures of the 20 natural amino acids, see L. Stryer, 3 rd ed. 1988, Freeman and Company, New York.

[0041] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. In an embodiment, the percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0042] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which in embodiments may be conjugated to a moiety not composed of amino acids. The term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring 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.

[0043] "Polypeptide fragment" refers to a polypeptide having an amino- and / or carboxy-terminal deletion, where the remaining amino acid sequence is usually identical to the corresponding positions in the naturally occurring sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, at least 14 amino acids in length, at least 20 amino acids in length, at least 50 amino acids in length, or at least 70 amino acids in length.

[0044] As used herein, "unnatural amino acid", "non-natural", "modified amino acid" or "chemically modified amino acid" refers to any amino acid, modified amino acid or amino acid analog other than the 20 genetically encoded α-amino acids. Unnatural amino acids have side chain groups that distinguish them from the natural amino acids, but unnatural amino acids can be naturally occurring compounds other than the 20 proteinogenic α-amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural amino acids can have elongated backbones, such as beta-amino acids.

[0045] Non-limiting examples of unnatural amino acids include selenocysteine, pyrrolysine, homocysteine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphatase, and the like. phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, non-natural analogues of the amino acid tyrosine; non-natural analogues of the amino acid glutamine; non-natural analogues of the amino acid phenylalanine; non-natural analogues of the amino acid serine; non-natural analogues of the amino acid threonine; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl The amino acids may be substituted or unsubstituted amino acids, such as aryl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, or any combination thereof; amino acids having a photoactivatable crosslinker; spin-labeled amino acids; fluorescent amino acids; amino acids having novel functional groups; amino acids that interact covalently or non-covalently with another molecule; metal-binding amino acids; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; biotin or biotin analog-containing amino acids; glycosylated or carbohydrate-modified amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyethers; heavy atom substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar-substituted amino acids, such as sugar-substituted serine; carbon-linked sugar-containing amino acids; redox-active amino acids; alpha-hydroxy-containing acids; aminothioacid-containing amino acids; alpha,alpha disubstituted amino acids; beta-amino acids; and cyclic amino acids other than proline.In one embodiment of the helicase 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.

[0046] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0047] As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0048] The terms "subject", "patient" or "individual" are used interchangeably herein and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the present disclosure are used in laboratory animals, veterinary applications, and in the development of animal models of disease, including, but not limited to, rodents, including mice, rats, and hamsters; and primates. Patients in need of treatment include those at risk of developing a particular condition, disease, or disorder (e.g., due to genetic, environmental, or physical attributes, such as obesity). Patients in need of treatment also include those suffering from a condition, disease, or disorder. Diseases or disorders include, for example, autoimmune diseases, cancer, inflammatory diseases, neurological diseases or disorders, neuroinflammatory diseases or disorders, cardiovascular diseases, obesity, diseases or disorders caused by infectious agents, such as viruses, bacteria, fungi, prions, or parasites.

[0049] Ranges: Throughout this disclosure, various aspects of the disclosure may 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. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and 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.

[0050] As used herein, a "variant" of a polypeptide refers to an amino acid sequence that is altered by one or more amino acid residues. A variant may have "conservative" changes, where the substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "non-conservative" changes (e.g., replacement of glycine with tryptophan). Similar 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 losing biological activity may be found using computer programs well known in the art (e.g., LASERGENE software (DNASTAR)).

[0051] Genbank and NCBI submissions indicated by accession numbers cited herein are incorporated herein by reference.All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference.In case of conflict, the present specification, including definitions, will control.In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

[0052] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein. [Brief description of the drawings]

[0053] [Figure 1] Figures 1A and 1B are schematics showing a comparison between PCR and SHARP. Figure 1A: PCR alternates temperatures between a high temperature (approximately 98 °C) to melt the DNA duplex and a low temperature (45-65 °C) to replicate the complementary strand by DNAP. Figure 1B: SHARP uses PcrA M6 helicase and SSB to open the duplex to allow primer binding, and then uses Bst-LF DNAP to replicate the strand.

[0054] [Diagram 2] Figures 2A-2F are a series of plots, blots and schematic diagrams showing a comparison of PCR and SHARP amplification products. Figure 2A: 2019 coronavirus nCoV-2 N protein sequence between M13 primers. Figure 2B: PCR products and detection limits for the template-primer set of Figure 2A. Figure 2C: Kinetics of the SHARP reaction for the template-primer set of Figure 2A. Figure 2D: SHARP products and detection limits allow direct comparison with PCR. Figure 2E: SHARP doubling time estimates. Detection time is the intercept of the intensity curve with threshold in Figure 2C. Figure 2F: SHARP essential components. In the absence of ATP, SSB, or PcrA M6, the reaction fails.

[0055] [Diagram 3]Figures 3A-3F are a series of blots and graphs showing other SHARP products. Figure 3A: 3 kbp SHARP amplicon. Figure 3B: 6 kbp SHARP amplicon. Figure 3C: 200 bp SHARP amplicon using lambda DNA as template. Figure 3D: SHARP doubling time estimates for the 200 bp product. Figure 3E: Limit of detection vs. amplicon length. For short amplicons, SHARP can detect one molecule in a test tube. Figure 3F: Doubling times show that short amplicons replicate at the fastest rate.

[0056] [Figure 4] Figures 4A-4E are a series of graphs, blots and schematics showing the transformation and properties of SHARP plasmids. Figure 4A: Transformation of SHARP-made plasmid into E. coli. Figure 4B: SHARP amplifies sequences that tend to form secondary structures, CAG repeats. Figure 4C: SHARP kinetic curves at different temperatures, inset shows detection time. Figure 4D: SHARP products at different temperatures. Figure 4E: SHARP at 37°C with Bsu and Klenow Exo-DNAp at varying SSB concentrations.

[0057] [Diagram 5]Figures 5A-5I are a series of graphs and schematics demonstrating the bulk unwinding activity of different DNA helicases at 37 °C. Each helicase is at a different concentration while all other conditions are fixed (see Methods). Figure 5A: Unwinding reaction of FRET versus labeled DNA constructs. Figure 5B: Rep-X superhelicase shows the highest unwinding activity even at a low concentration of 10 nM. C) E. coli UvrD helicase at 34 nM shows a relatively high initial unwinding activity, but the reaction is rapidly terminated, leading to DNA reannealing. Figure 5D: Thermostable Tte UvrD helicase at 40 nM shows no unwinding activity at 37 °C. Figure 5E: Wild-type PcrA expressed with a 6xHis tag shows low unwinding activity at 600 nM. Figure 5F: Wild-type PcrA without the 6xHis tag shows low unwinding activity at 600 nM. Figure 5G: PcrA M5 mutant at 400 nM shows high unwinding activity. Figure 5H: PcrA M5 mutant at 40 nM. Figure 5I: PcrA M6 shows higher unwinding activity than M5 even at 40 nM concentration. SHARP uses about 40 nM PcrA M6.

[0058] [Figure 6] Figures 6A-6D are a series of schematics and graphs showing the structure and characterization of PcrA M6 mutants for unwinding long DNA. Figure 6A: Structure of wild-type PcrA and location of point mutations H93A, C96A, N187C, C247A, L384V, L409C. Figure 6B: Unwinding of λ DNA occurred in the presence of SSB and ATP. Figure 6C: Unwinding of different DNA substrates. Figure 6D: Time required for unwinding of different DNA substrates by PcrA M6.

[0059] [Figure 7]Figures 7A-7L show the bulk unwinding activity of different DNA helicases at 37 °C. Each helicase is at a different concentration, while all other conditions are fixed. Figure 7A: Unwinding reaction of FRET versus labeled DNA constructs. Figure 7B: Rep-X superhelicase shows the highest unwinding activity even at a low concentration of 10 nM. Figure 7C: E. coli UvrD helicase at 34 nM shows high initial unwinding activity, but the reaction terminates rapidly, likely resulting in DNA reannealing due to ATP depletion. Figure 7D: Thermostable Tte UvrD helicase at 40 nM shows very low unwinding activity at 37 °C. Figure 7E: Wild-type PcrA expressed with a 6xHis tag shows low unwinding activity at 600 nM. Figure 7F: Wild-type PcrA without the 6xHis tag shows low unwinding activity at 600 nM. Figure 7G: PcrA M5 mutant at 400 nM shows high unwinding activity. Figure 7H: PcrA M5 mutant at 40 nM. Figure 7I: PcrA M6 shows higher unwinding activity than M5 even at 40 nM concentration. SHARP uses PcrA M6 at almost 40 nM. Figure 7J: Unwinding rates obtained from fits to FRET efficiencies in Figures 7B-7I. Figure 7K: Unwinding activity for Rep-X is obtained by dividing the unwinding rates in Figure 7J by the concentration and normalizing. Inset shows relative activity on the log10 axis. Data in Figures 7J and 7K are shown as mean values ​​± standard error obtained from the fits. Source data and fitting parameters are provided as source data files.

[0060] [Figure 8]Figures 8A-8C show single molecule nanopore assays measuring the unwinding rates of PcrA M5 and PcrA M6 helicases. Figure 8A: The single molecule picometer resolution nanopore tweezers (SPRNT) setup consists of two separate chambers connected to a nanopore. The helicase unwinds a periodic DNA sequence, driving it through the pore and a current signal is recorded. By measuring the period of the signal repeats, where each period corresponds to a 103 base pair sequence, the helicase rate can be inferred. Figure 8B: PcrA M5 shows about 3 repeats in 8 s, therefore we estimate the rate to be 41 bp / s. Figure 8C: PcrA M5 shows more than 10 repeats in just 8 s, therefore its rate is estimated to be 134 bp / s.

[0061] [Figure 9] Figure 9: SHARP can amplify RNA targets. Using MMLV reverse transcriptase and CDC N2 primers, we amplified a 67 bp region in the nCoV-2 RNA sequence at different temperatures. The highest yield was obtained at 39.4 °C.

[0062] [Figure 10] Figures 10A-10C show that the SHARP product can be easily detected using a lateral flow device for use in portable, point-of-care, point-of-demand, and home molecular diagnostics. Figure 10A: SHARP with biotin-labeled primer and DIG-labeled dNTP. Figure 10B: SHARP is detected with a lateral flow device. Figure 10C: When no labeled dNTP is used, the product does not show lateral flow devoice.

[0063] [Figure 11] Figure 11 shows SHARP generating amplicons of 6000 and 7923 bp. The 6000 bp product has a high yield. The generation of the 7929 amplicon is possible with SHARP, with the amount of product being much less than the 6000 bp amplicon. The 7929 product was amplified only a few times, whereas the 6000 bp product was amplified exponentially.

[0064] [Figure 12] Figures 12A-12C: SHARP amplification of genomic DNA (gDNA) from human cells. Figure 12A: The amount of amplified product is monitored in real time. SHARP detects the correct sequence within 30 minutes. Non-specific amplification in a control experiment (-gDnA) occurred after 45 minutes, giving a clear temporal separation between specific and non-specific products. Figure 12B: A gel-based assay compares SHARP and PCR products for both genes. Figure 10C: Both SHARP and PCR products can be sequenced in the same manner to confirm amplification of the correct gene.

[0065] [Figure 13] Figure 13: Amplification of 4 repeats of the 601 nucleosome positioning sequence with repeats, linkers (65 or 80 bp), showed cleavage products and smears in PCR but not in SHARP (unpublished data), presumably because in SHARP the DNA is not melted globally, which would otherwise cause pairing with internal segments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The novel method, named herein as SHARP (SSB-helicase-assisted rapid polymerase chain reaction), uses a helicase and SSB (single-stranded binding protein) to initiate an isothermal amplification reaction, and DNAP (DNA polymerase) to carry out the isothermal amplification reaction.

[0067] Overview of amplification methods: PCR is a rapid and sensitive technique to amplify or copy a segment of DNA, starting from an initial small amount of DNA (3, 4), as the first step in any downstream detection or application. PCR alternates temperatures between a high temperature (approximately 98 °C) to melt the DNA double strand and a low temperature (45-72 °C) to replicate the missing strand by DNAPs (Taq, Pfu, Q5, etc.) (Figure 1A). Given a template and a set of two template-specific primers, each PCR cycle doubles the template copy number, resulting in an exponential increase, thereby resulting in a product detectable by the naked eye after approximately 30 cycles. Currently, there are more than 10 different approaches to isothermal amplification (1, 2); however, existing isothermal methods are not as universal and versatile as PCR.

[0068] Although PCR and isothermal methods do indeed perform in vitro replication via primer extension on DNAP (1, 2), these methods differ in their approach to initiate each subsequent reaction cycle and allow the primer to bind to the template. Whereas PCR heat denatures the DNA duplex and subsequently cools it to initiate each replication cycle (Figure 1A), isothermal methods use either specially designed primers or other auxiliary proteins to allow the primer to bind to the template. LAMP (loop-mediated isothermal amplification) uses 4–6 loop-forming primers to generate 3' ends that can be extended by Bst DNAP (5). LAMP is a rapid, commercially available (6), and highly specific exponential amplification method that results in a product that is detectable by the naked eye; however, LAMP products are highly complex and difficult to interpret, compare, and use in many applications, including cloning, sequencing, and genetic manipulation. LAMP, unlike PCR, does not amplify a specific region of interest. Also, dedicated software is required to design LAMP primers, and primers optimized for PCR cannot be used as LAMP primers.

[0069] Isothermal amplification methods based on gp32, a single-stranded DNA binding protein, use a set of two primers similar to PCR and can generate amplicons over 1 kbp (2); however, the template is only amplified a few-fold, far from the exponential growth in PCR, and the product also contains high molecular weight multimers and other undesirable bands. RPA (recombinase polymerase amplification) uses two primers and a recombinase (7, 8). RPA primers can be longer than PCR primers, 30-38 bases required for recombinase binding. The recombinase-primer complex then searches for homologous template sequences, resulting in strand invasion and primer-template pairing, followed by polymerase extension. RPA provides exponential amplification and a competitive detection limit, but can only generate short amplicons of 100-200 bp in length. The longer primers used by RPA are more prone to form secondary structures and nonspecific products, require more careful primer design, or use unnatural bases (9). SIBA (strand invasion-based amplification) also uses the recombinase UvsX, but reduces the chance of unwanted product formation by using an invading oligonucleotide in addition to the primer (10, 11), but still produces 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 in amplification initiation. Both methods provide exponential amplification, but the products are also only a few hundred base pairs long. Both methods use a set of four primers with additional sequence requirements for the nicking enzyme. HDA, or helicase-dependent amplification (14), utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by DNAP. HDA is also commercially available and uses primers similar to PCR.By combining UvrD helicase, MutL accessory protein, T4 gene 32 protein (SSB), and Klenow Exo-DNAP, Vincent et al. achieved exponential amplification of a 100-bp product (14); however, for longer amplicons the reaction yield decreased significantly, limiting the possible applications of this method. Due to the limitations summarized above, existing isothermal methods are used for specific applications and cannot be used as widely as PCR (1).

[0070] SHARP (SSB-Helicase-Assisted Rapid Polymerase Chain Reaction): SHARP removes thermal cycling from PCR, keeping all other desired PCR features in place and adding new features. SHARP performs as well as or better than PCR according to eight criteria that we benchmarked: (1) amplicon length up to 6000 bp, (2) amplification time from 5 to 30 minutes, (3) primer design principle and convenience, (4) detection limit, (5) real-time detection, (6) interpretation of amplification results, (7) application of downstream products, and (8) no initial heat denaturation step. The numerous advantages of removing the thermal cycler from PCR for more rapid diagnosis have been frequently 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 NA Tanner, "Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein," Scientific Reports, vol. 7, 8 2017), where the suitability of isothermal reactions was tested for other common tasks in wet labs, as described in the Examples section below. Briefly, it was demonstrated that E. coli cells transformed with a 3.2 kbp SHARP-made plasmid can replicate the plasmid and exhibit antibiotic resistance; therefore, SHARP is suitable for cloning, sequencing and genetic manipulation. It was also shown that DNA sequences with the potential to form non-canonical structures, such as sequences containing (CAG)47 repeats, do not inhibit SHARP. Because PCR involves heating samples to 98°C, it narrows down the possible enzymes that could be used to improve reaction speed, yield, product fidelity, detection specificity, and the ability to perform other reactions simultaneously. SHARP works optimally at 65°C, but can be performed in the range of 37°C to 65°C, leaving many possibilities for future improvements and combination with other techniques.

[0071] Thus, in certain embodiments, a method for amplifying a nucleic acid includes mixing a first composition with one or more primers, a target nucleic acid sequence, and a second composition. In certain embodiments, the mixed composition is incubated at a constant temperature in the temperature range of 37°C to 90°C for about 20 to 60 minutes. In certain embodiments, the mixed composition is incubated at a constant temperature of about 65°C for about 30 minutes. In certain embodiments, the first composition includes a nucleic acid dye, a deoxyribonucleotide triphosphate (dNTP), an adenosine triphosphate (ATP), or a combination thereof. In certain embodiments, the second composition includes an engineered PcrA helicase, a single-stranded binding protein (SSB), a Bst polymerase, a thermostable pyrophosphatase (PPase), a buffer, or a combination thereof. In certain embodiments, the first and second compositions further include water, a buffer, or a combination thereof.

[0072] Thus, in certain embodiments, the bacterial helicase comprises a PcrA helicase having at least two or more mutations. In certain embodiments, the PcrA helicase comprises at least 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 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 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 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.

[0073] In certain embodiments, the vector encodes a PcrA helicase, wherein the PcrA helicase comprises 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 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 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 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.

[0074] In certain embodiments, the vector comprises 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, the vector comprises at least 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, the vector comprises at least 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, the vector comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In certain embodiments, the vector comprises SEQ ID NO: 2.

[0075] In certain embodiments, the nucleic acid sequence comprises at least 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 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 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 SEQ ID NO: 3. In certain embodiments, SEQ ID NO: 3 encodes amino acids comprising amino acid substitutions at positions H93A, C96A, N187C, C247A, L384V, and L409C.

[0076] In certain embodiments, the engineered helicase comprises a PcrA helicase, wherein the 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.

[0077] SHARP enables a new generation of portable, point-of-care, point-of-demand, and home medical molecular diagnostic tests. Conventional PCR requires a substantial, high-power consuming device, a thermocycler, which heats biomolecules to over 95°C, making PCR rarely available outside specialized laboratories. SHARP uses bioengineered enzymes to eliminate the need for complex hardware. SHARP can be performed with a simple low-power heater, and therefore molecular diagnostic tests can be performed wherever necessary. SHARP can be used in applications, for example, to detect 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 in place of PCR.

[0078] Helicase Enzyme

[0079] The traditional definition of a helicase is an enzyme that catalyzes the reaction of separating / dissociating / unwinding the helical structure of a nucleic acid duplex (DNA, RNA or hybrid) into single-stranded components using nucleoside triphosphate (NTP) hydrolysis as an energy source (such as ATP). However, it should be noted that not all helicases fit this definition any more. A more common definition is that they are motor proteins that move (usually in a certain direction, 3' to 5' or 5 to 3 or both) along single-stranded or double-stranded nucleic acids, i.e., translocases that can or cannot unwind duplex nucleic acids that they encounter. Furthermore, some helicases simply bind and "melt" to duplex nucleic acid structures without any apparent translocase activity.

[0080] Helicases exist in all organisms and function in all aspects of nucleic acid metabolism. Helicases are classified based on their amino acid sequence, directionality, oligomerization state, and selection of nucleic acid types and structures. The most common classification method was developed based on the presence of specific amino acid sequences called motifs. According to this classification, helicases are divided into six superfamilies: SF1, SF2, SF3, SF4, SF5 and SF6. SF1 and SF2 helicases do not form ring structures around nucleic acids, while SF3-SF6 do. Superfamily classification does not depend on classical taxonomy.

[0081] DNA helicases are responsible for catalyzing the unwinding of double-stranded DNA (dsDNA) molecules into their respective single-stranded nucleic acid (ssDNA) forms. Although structural and biochemical studies have shown how various helicases can translocate directionally on ssDNA and consume one ATP per nucleotide, the mechanism of nucleic acid unwinding and how the unwinding activity is regulated are unclear and controversial (TM Lohman, EJ Tomko, CG 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 they encounter, understanding how their unwinding activity is regulated may lead to the exploitation of helicase function for biotechnological applications.

[0082] The term "HDA" refers to helicase-dependent amplification, which is an in vitro method for amplifying nucleic acids by using a helicase preparation to unwind double-stranded nucleic acids to generate templates for primer hybridization and subsequent primer extension. The method utilizes two oligonucleotide primers, each of which hybridizes to the 3' end of either the sense strand containing the target sequence or the antisense strand containing the reverse-complementary target sequence. The HDA reaction is a common method for helicase-dependent nucleic acid amplification.

[0083] The SHARP method utilizes a novel PcrA M6 helicase enzyme that has been engineered to eliminate the thermal cycling mechanism from the polymerase chain reaction (PCR), yet retain all the properties of PCR.

[0084] The amino acid sequence of PcrA M6 contains the following mutations: H93A, C96A, N187C, C247A, L384V, L409C (SEQ ID NO:1). JPEG2025504929000002.jpg42169 JPEG2025504929000003.jpg29169

[0085] Plasmid sequence of PcrA M6 H93A, C96A, N187C, C247A, L384V, L409C (vector name: pET-11b, cloning sites: NdeI / BamHI, mutant sequence contains N-terminal 6xHis tag and C-terminal FLAG tag). SEQ ID NO: 2: JPEG2025504929000004.jpg168169 JPEG2025504929000005.jpg39169

[0086] The amino acid sequence of wild-type PcrA (NCBI Reference Sequence WP_033016687.1) is listed below and is available at ncbi.nlm.nih.gov / protein / WP_033016687.1 (SEQ ID NO: 3). JPEG2025504929000006.jpg66169

[0087] Thus, in certain embodiments, the bacterial helicase comprises a PcrA helicase having at least two or more mutations. In certain embodiments, the PcrA helicase comprises at least 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 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 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 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. In certain embodiments, the PcrA helicase comprises SEQ ID NO: 1.

[0088] In certain embodiments, the PcrA helicase is derived from Geobacillus. In certain embodiments, the Geobacillus is Geobacillus stearothermophilus. In certain embodiments, the nucleic acid sequence, SEQ ID NO:3, encodes amino acids containing amino acid substitutions at positions H93A, C96A, N187C, C247A, L384V, and L409C.

[0089] In certain embodiments, the engineered helicase comprises a PcrA helicase, wherein the 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.

[0090] In certain embodiments, the mutations at positions 93, 96, 187, 247, 384, and 409 of wild-type PcrA helicase include both naturally occurring and non-naturally occurring amino acids. Examples of non-natural amino acids include, but are not limited to, D-amino acids (i.e., amino acids of opposite chirality to the natural 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 (β-Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic acid (β-Ahx), ornithine (orn), sarcosine, α-aminoisobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3-diaP), D- or L-phenylglycine, D-(trifluoromethyl)-phenylalanine, and Dp-fluorophenylalanine.

[0091] In certain embodiments, the PcrA helicase comprises one or more modified peptide bonds. As used herein, a "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., reverse peptide bond). (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, JS, 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); See 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).

[0092] Vector: In certain embodiments, the vector encodes a PcrA helicase that comprises one or more mutations. In certain embodiments, the vector encodes a PcrA helicase that comprises 60% sequence identity to SEQ ID NO:1. In certain embodiments, the PcrA helicase comprises at least 75% sequence identity to SEQ ID NO:1. In certain embodiments, the PcrA helicase comprises at least 90% sequence identity to SEQ ID NO:1. In certain embodiments, the PcrA helicase comprises at least 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.

[0093] In one embodiment, the vector comprises the sequence of SEQ ID NO:2.

[0094] The nucleic acid encoding the PcrA polypeptide can be adapted to a suitable expression system to produce a helicase polypeptide for PcrA helicase production. For DNA encoding the PcrA helicase gene, the representative gene can be operably linked to a suitable expression vector for expressing the protein in bacteria, fungi, insects or other suitable expression hosts. For RNA encoding the PcrA helicase polypeptide, the representative RNA can be engineered to allow efficient expression of the polypeptide in vitro in the extract lysate produced from bacteria, fungi, insects or other suitable expression host sources. Such systems are well known in the art. After expression, the PcrA helicase polypeptide can be purified by methods known in the art, including affinity tag chromatography, SDS-PAGE, and size exclusion chromatography, among others.

[0095] In certain embodiments, vectors are provided, such as, for example, expression vectors, that contain a nucleic acid encoding one or more PcrA helicase polypeptides described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be linked. Another type of vector is a viral vector, in which additional DNA segments can be linked to the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0096] In certain embodiments, a recombinant expression vector comprises 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, meaning that the recombinant expression vector comprises one or more regulatory sequences selected based on the host cell to be used for expression, operably 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 a regulatory sequence in a manner that allows 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 that direct the constitutive expression of a nucleotide sequence in many types of host cells, and those that direct the expression of a nucleotide sequence only in a specific host cell (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of an expression vector may depend on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. The expression vectors described herein can be introduced into a host cell to thereby produce the protein or a portion thereof (including a fusion protein or a portion thereof) (e.g., one or more PcrA helicase polypeptides) encoded by the nucleic acid described herein.

[0097] Recombinant expression vectors can be designed for the expression of one or more encoding 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 further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif (1990). Alternatively, recombinant expression vectors can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

[0098] Expression of proteins in prokaryotes is most frequently carried out in E. coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add several amino acids to the protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase the expression of the 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 allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, DB and Johnson, KS (1988) Gene 67:31-40); pMAL (New England Biolabs, Beverly, Mass); and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0099] 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).

[0100] Alternatively, one or more PcrA helicase polypeptides can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expressing 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).

[0101] In certain embodiments, the 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 control function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Green M., and Sambrook; J. Molecular Cloning: A Laboratory Manual.4 th, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012.

[0102] In one embodiment, a host cell is provided into which the recombinant expression vector of the present invention is introduced.The terms "host cell" and "recombinant host cell" are used interchangeably herein.It is understood that such terms refer not only to a particular target cell, but also to the progeny or potential progeny of such a cell.Since certain modifications may occur in later generations, either due to mutation or environmental influence, such progeny may not actually be identical to the parent cell, but still fall within the scope of the terms used herein.

[0103] The 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.

[0104] The delivery of nucleic acid (e.g., vector DNA) described herein can be by any suitable method in the art.For example, delivery can be by injection, gene gun, application of nucleic acid in gel, oil or cream, electroporation, use of lipid-based transfection reagent, or any other suitable transfection method.

[0105] As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques, including calcium phosphate or calcium chloride co-precipitation, DEAF-dextran mediated transfection, lipofection (e.g., using commercially available reagents such as LIPOFECTIN™ (Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE™ (Invitrogen), FUGENE™ (Roche Applied Science, Basel, Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, NY), EFFECTENE™ (Qiagen, Valencia, Calif.), DREAMFECT™ (OZ Biosciences, France)), or electroporation (e.g., in vivo electroporation). For suitable methods for transforming or transfecting host cells, see Green and Sambrook et al. (Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012), and other laboratory manuals.

[0106] kit

[0107] In certain embodiments, the kit comprises an engineered PcrA helicase, a single-stranded binding protein (SSB), a polymerase, and 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 transcriptase, exopolymerase, high fidelity polymerase, Taq polymerase, bacteriophage polymerase, Pyrococcus furiosus Vc1 (Pfu) polymerase, hot start DNA polymerase, or eukaryotic polymerase. In certain embodiments, the polymerase is Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment, or exopolymerase. 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.

[0108] In certain embodiments, the kit further comprises a buffer, deoxyribonucleotide triphosphates (dNTPs), nucleoside triphosphates (NTPs), a detectable nucleic acid label, water, dithiothreitol (DTT), or a combination thereof. In certain embodiments, the detectable nucleic acid label comprises a fluorescent dye.

[0109] In certain embodiments, the kit comprises a first composition and a second composition. In certain embodiments, the first composition comprises a nucleic acid dye, a deoxyribonucleotide triphosphate (dNTP), an adenosine triphosphate (ATP), or a combination 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 a combination thereof. In certain embodiments, the polymerase comprises DNA polymerase (DNAP) I, DNAP II, DNAP III, DNAP IV, DNAP V, Klenow fragment, reverse transcriptase, exopolymerase, high fidelity polymerase, Taq polymerase, bacteriophage polymerase, Pyrococcus furiosus Vc1 (Pfu) polymerase, a hot-start DNA polymerase, or a eukaryotic polymerase. In certain embodiments, the polymerase is Geobacillus stearothermophilus DNA polymerase I, Bacillus subtilis DNA polymerase I, Klenow fragment, or exopolymerase. 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.

[0110] In certain embodiments, the kit further comprises a buffer, deoxyribonucleotide triphosphates (dNTPs), nucleoside triphosphates (NTPs), a detectable nucleic acid label, water, dithiothreitol (DTT), or a combination thereof. In certain embodiments, the detectable nucleic acid label comprises a fluorescent dye.

[0111] In certain embodiments, the first and second compositions further comprise water, a buffer, or a combination thereof. In certain embodiments, the first composition is contained in a first vial and the second composition is contained in a second vial.

[0112] 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 a second primer, the first primer comprising a portion complementary to a first strand of the target nucleic acid, and the second primer comprising a portion complementary to a second strand of the target nucleic acid. The kit may also comprise a set of instructions for use. In certain embodiments, the kit may comprise a reagent for purifying double-stranded nucleic acid in a 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 comprise a reagent for purifying single-stranded nucleic acid in a sample. The kit may also comprise a set of instructions for use. EXAMPLES

[0113] Example 1: Rapid isothermal amplification of multi-kilobase DNA

[0114] A novel robust isothermal amplification method is described herein, which is similar to PCR in that it uses the same set of primers and templates as PCR in input. Furthermore, this method outputs the same amplicons as PCR, with lengths up to 6000 base pairs (bp), but is performed at a constant temperature, thus eliminating the need for thermal cycling. This new method is named SHARP (SSB-helicase-assisted rapid polymerase chain reaction) herein, since it uses helicase and SSB (single-stranded binding protein) to initiate and DNAP (DNA polymerase) to carry out the isothermal amplification reaction.

[0115] material and method

[0116] SHARP reaction mixture: Three key enzymes, Bst-LF DNAP, E. coli SSB, and PcrA M6 helicase, were overexpressed and purified. The following enzymes were used for SHARP at 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 number M0296S). Other stock components are dNTPs (10 mM each), ATP (100 mM), Evagreen dye (20X from Biotium #31000), DTT (dithiothreitol, 100 mM in water). 10X 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 10 or 20 μM, while template concentrations are variable.

[0117] For each SHARP reaction, a total volume of 40 μL was prepared and the volume was divided into two separate wells on a 96-well plate to obtain two independent fluorescence readings of the same reaction. SYBR (EvaGreen) intensity was monitored in real time for each well every 10 seconds on a BioRad CFX96 instrument. Table 1 contains the volumes of the raw components.

[0118] [Table 1]

[0119] Component 1 and component 2 were prepared separately and mixed together on a plate, after which the temperature was raised to 65 degrees and fluorescence recording began. After incubation, the products were purified with a Qiagen PCR cleanup kit and the products were tested on a gel.

[0120] Primers and templates: Primers were ordered from Integrated DNA technologies (IDT). A DNA template vector containing the 2019 coronavirus nCoV-2 N protein sequence (2019-nCoV_N_Positive Control, catalog number 10006625) was also ordered from IDT. M13 primers were used for PCR and SHARP in Figures 2A-2D and 3C.

[0121] 5'-CCCAGTCACGACGTTGTAAAACG (forward; SEQ ID NO: 4) and

[0122] 5'-AGCGGATAACAATTTCACACAGG (reverse; SEQ ID NO: 5) and the nCoV-2 N template. In Figures 2E and 3A, the following primers were used:

[0123] 5'-AATTTTGGGGACCAGGAAC (forward; SEQ ID NO: 6) and

[0124] The primers themselves contain a vector backbone containing ampicillin resistance: 5'-TCTGGTTACTGCCAGTTGAATCTG (reverse; SEQ ID NO: 7) and the nCoV-2 N template. The t-DNA amplicon in Figure 2G uses t-DNA from NEB (catalog number N3011S) as a template with the following primers to generate a 200 bp amplicon:

[0125] 5'-CGGCTTCTGACTCTCTTTCC (forward; SEQ ID NO: 7) and

[0126] 5'-TTCCTTCAAGCTTTGCCACA (reverse; SEQ ID NO: 8). To test SHARP with the CAG repeat sequence in Figure 4B, the pBluescript-CTG-47 sequence from A. Jain and RD Vale ("RNA phase transitions in repeat expansion disorders," Nature, vol. 546, p. 243-247, 5 2017) deposited at Addgene #99150 and the M13 primers above were used. To test the temperature dependency of SHARP in Figure 4C, the nCoV-2 N template and the following primers were used:

[0127] 5'-AATTTTGGGGACCAGGAAC (forward; SEQ ID NO: 9) and

[0128] 5'-GCACCTGTGTAGGTCAAC (reverse; SEQ ID NO: 10) generates an amplicon of 155 bp.

[0129] DNA unwinding assay: The FRET unwinding assay uses the following buffer: 10 mM TRIS pH 8.0, 10 mM MgCl2, 50 mM NaCl, 1 mM ATP, 1% BSA. The DNA concentration is 5 nM and the helicase concentration is varied. The reaction is carried out in a 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 a volume of 20 μL in a qPCR instrument 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 mixture is prepared on ice and the qPCR instrument is pre-cooled to 4 °C. This is placed into the qPCR machine, which begins recording and then rapidly increases the temperature to 37°C.

[0130] Protein overexpression and purification: PcrA M6 helicase with 6xHis tag: PcrA M6 helicase, PcrA and all mutants are purified using a standard Ni-NTA purification column followed by a single-stranded DNA cellulose column as previously described (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). Briefly, the pET-11b vector containing the PcrA M6 sequence between the NdeI and BamHI sites was used. The vector containing an N-terminal 6x-His tag was synthesized by GenScript, which synthesized all point mutations. The vector was transformed into E. coli BL21(DE3)pLysS. Cells were grown at 37°C in the presence of ampicillin and chloramphenicol and grown to an OD 600 When the OD reached 0.3, the culture was transferred to 18°C ​​and incubated with 0.5 mM IPTG. 600The cells were induced at pH = 0.5 and harvested after overnight incubation at 18 °C. The cell pellet, previously stored at -80 °C, was resuspended in lysis buffer (50 mM Tris, 5 mM imidazole, 200 mM NaCl, 20% sucrose, 15% glycerol, 0.5 mg / mL lysozyme, pH 7.6), sonicated, and subsequently centrifuged at 35,000 g. Ni-NTA agarose resin is pre-equilibrated with wash buffer (50 M 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 h at 4 °C with constant agitation mixing by inverting the 50 mL tube. After 1 h, the resin is gently centrifuged for 2 min at 1000 g, the supernatant is carefully discarded, and the tube containing the resin is refilled with wash buffer. The batch wash is repeated three times, and then the protein-loaded resin is 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 the wash buffer. The protein is then loaded onto a 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 6xHis does not affect downstream applications. Protein concentration was always kept below 4 mg / ml (approximately 50 mM) to avoid aggregation, and the final PcrA protein was stored at -80 °C or -20 °C in a storage buffer containing 600 mM NaCl, 50 mM TRIS pH 7.6, and 50% glycerol. This protocol results in PcrA diluted to between 0.2-1 mg / mL and, if necessary, the protein can be concentrated using membrane filtration.

[0131] PcrA helicase without 6xHis tag: A plasmid expressing wild-type PcrA helicase without any tag was kindly provided by Tim Lohman lab. Obtaining cell lysate is performed in the same way as in the previous protocol. Cell lysate is also centrifuged, but the supernatant is mixed with 0.7 volumes of saturated ammonium sulfate solution. Ammonium sulfate precipitates PcrA, which is recovered by spinning at 5000g. PcrA is suspended in wash buffer (50 mM Tris, 5 mM imidazole, 150 mM NaCl, 25% (v / v) glycerol, pH 7.6), spun down, and the supernatant was loaded onto a ssDNA-cellulose column. The rest of the protocol is the same as for PcrA with 6xHis tag.

[0132] Single-stranded binding protein (SSB): E. coli SSB is purified without tags by polymin-P and ammonium sulfate precipitation followed by a heparin sepharose column, similar to the protocol by Lohman et al. ("Large-scale overproduction and rapid purification of the E. coli ssb gene product. Expression of the ssb gene under λPL control", Biochemistry, vol. 25, p. 21-25, 1986). The SSB sequence is cloned into the pET21a vector using the NdeI and BamHI sites. The vector is used to transform BL21(DE3) cells, colonies are picked, grown at 37°C, and OD 600Overexpression was induced with 0.5 mM IPTG at pH = 0.5, grown for an additional 5 hours, and pellets were harvested. Overexpression levels were usually very high, with SSB often producing more than 60% of total cellular protein. Cell pellets were resuspended in lysis buffer (50 mM Tris, 200 mM NaCl, 20% sucrose, 15% glycerol, 0.5 mg / mL lysozyme, pH 7.6), sonicated, and then centrifuged at 35,000 g for 30 minutes. The supernatant containing soluble SSB was collected, and Polymin-P was added to the supernatant to a final concentration of 0.2% to precipitate SSB. The precipitated SSB was collected by spinning at 4000 g, then resuspended in a buffer containing 50 mM TRIS pH 8.3, 20% glycerol, 1 mM EDTA, 400 mM NaCl, and mixed by vortexing for 30 minutes at 4 °C. To remove undissolved proteins, the mixture was centrifuged at 10,000g for 20 min and the supernatant containing soluble SSB was collected. Finally, solid ammonium sulfate was added to obtain a final concentration of 150 g / L to precipitate the SSB, and the pellet containing SSB was collected after centrifugation at 12,000 for 30 min. The pellet was resuspended in 50 mM TRIS pH 8.3, 20% glycerol, 1 mM EDTA, 200 mM NaCl, agitated and mixed for 30 min at 4 °C, centrifuged at 18,000g for 20 min, and filtered through a 200 μm filter. The heparin-Sepharose column was equilibrated with wash buffer (50 mM TRIS pH 8.3, 20% glycerol, 1 mM EDTA), and the SSB solution was diluted 5-fold with wash buffer and loaded onto the column. The bound SSB was then washed with 50–100 mL of wash buffer and eluted with a NaCl gradient from 100 mM to 1 M. The final SSB solution was dialyzed against storage buffer (20 mM Tris, pH 8.1, 50% glycerol, 0.5 M NaCl, 1 mM EDTA, 1 mM BME). A concentrated SSB between 6-9 mg / mL was obtained. The SHARP reaction uses a high concentration of SSB; therefore, it is important to use an SSB stock concentrated above 5 mg / mL.

[0133] Bst-LF DNA polymerase: The Bst-LF purification protocol uses Ni-NTA resin. Overexpression and purification were performed using the Bst-LF DNAP expression vector available through addgene.org / 145799 / , following 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). The Bst-LF expression plasmid containing an N-terminal 6xHis tag was transformed into expressing cells, individual colonies were selected, and OD 600The cells were grown in culture medium until the β-actin concentration reached 0.5, and overexpression was induced with 200 ng / ml anhydrotetracycline (aTC) or 100 ng / ml tetracycline. A total culture volume of 500 mL was grown for 12 hours at 25°C, spun down to form a pellet, and kept at -80°C. The pellet was resuspended in lysis buffer (20 mM Tris pH 7.4, 300 mM NaCl, 0.1% Tween-20, 10 mM imidazole, 1x EDTA-free protease inhibitor tablet, 0.5 mg / mL lysozyme) in a cooling room and sonicated to lyse the cells. Lysed cells were centrifuged at 35,000g for 30 minutes at 4°C, and the supernatant was collected. The lysate was heat-treated at 65°C for 10 minutes, cooled on ice at 4°C for 10 minutes, and filtered using a 60 mL syringe with a 200 μm filter. Ni-NTA agarose resin was pre-equilibrated with 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 h at 4 °C with constant agitation mixing by inverting the 50 mL tube. After 1 h, the resin is gently centrifuged at 1000 g for 2 min, the supernatant was carefully discarded, and the tube containing the resin was refilled with wash buffer. The batch wash is repeated three times, and then the protein-loaded resin is poured into a disposable gravity flow column, washed with 10 mL of wash buffer, and eluted in 500 μL fractions with a buffer containing 20 mM Tris pH 7.4, 300 mM NaCl, 0.1% Tween-20, 250 mM imidazole. The collected fractions were run on a gel and dialyzed overnight against 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). Small aliquots of Bst-LF DNAP concentrated to 1.5 mg / mL are kept at -80 °C for long-term storage or at -20 °C for daily use. The optimal Bst-LF concentration in the SHARP reaction is 0.0015-0.0075 mg / mL.

[0134] result

[0135] Reference PCR assay: To introduce SHARP, we used a linearized 4012 bp DNA template vector (Figure 2A) containing the 2019 coronavirus nCoV-2 N protein sequence between the M13 primers. Figure 1A summarizes the conventional thermal cycler-based PCR reaction that serves as a control and reference for introducing the new SHARP method. The nCoV-2 N template and 250 nM of two M13 primers were mixed with the commercially available NEB (New England Biolabs, #M0531S) Phusion master mix containing the PCR components. The thermal cycler alternates temperatures between 98 °C to melt the duplex, 65 °C to bind the primers, and 72 °C to extend the primers bound to the template. After 30 cycles, each template molecule theoretically produces 2 30 ≒10 9 The amount of single-band PCR amplicon in a 2% agarose gel is 6 × 10 per microliter. 2 ~6×10 7 From initial template copy numbers ranging from 1 × 10 to 1 × 10 (Figure 2B), the reference PCR reaction yielded at least 6 × 10 copies for gel-based detection of the 1463 bp product. 3 It was decided that a template for copying was required.

[0136] SHARP amplification reaction: Using SHARP, amplification reactions were performed at 65 °C with the same template copy number and the same primer set as the reference PCR. SHARP produces the same 1463 bp amplicon as PCR (Figure 2D). DNA amplification was monitored in real time using an intercalating dye (Figure 2C). The main components of SHARP are one template, two primers at 250 nM each, dNTPs, Bst-LF DNAP, E. coli SSB, engineered PcrA M6 helicase (for SHARP, see the following section entitled Engineering PcrA helicase), ATP, and thermostable inorganic pyrophosphatase (see Table 1). If SSB is excluded, PcrA M6 or ATP prevents amplification (Figure 2F). During the development phase, other polymerases were tested (Bsu large fragment, Klenow Exo-, phi29, and reverse transcriptases M-MLV and AMV). Although they all showed some amplification, none of these other polymerases met all eight criteria. Figure 2C shows SHARP kinetics as a function of initial template copy number. 7 For 1,463 copies, the SHARP product was detectable after 5 minutes, whereas for 6 × 103, it takes approximately 22 minutes. The 1,463 SHARP product imaged on a 2% agarose gel in Figure 2D reproduces the same band observed for the reference PCR product (Figure 2B). The detection limit for SHARP is 6 × 103 per microliter. 2 The detection limit of PCR in Figure 2B exceeds 6 x 10 copies. 3 It was more sensitive than 6×10 6 Comparing SHARP (Figure 2D) and PCR (Figure 2B) products at higher template amounts over copies, it was noted that PCR was more prone to forming nonspecific products resulting in smears, whereas SHARP produced clean, well-defined amplicons.

[0137] The SHARP kinetics (Figure 2C) shows an exponential increase in the amount of product. The gel image (Figure 2D) shows one major band of 1463 bp corresponding to the desired product. 4For template copy numbers below 100 and NTC (no template control), a small amount of low molecular weight products due to primer dimers was observed. As judged by the NTC SHARP kinetic curve, primer dimers start to accumulate between 20 and 25 min (Figure 2C). Primer dimer bands were also observed in PCR, but were not prominent at 30 cycles. It was speculated that ATP hydrolysis by PcrA helicase M6 stimulated by single-stranded DNA may deplete ATP in 20 to 25 min at 65 °C, shifting the reaction balance toward primer dimer formation when the initial template amount is below the detection limit.

[0138] Quantitative or real-time PCR (qPCR) uses a cycle threshold (CT) value, defined as the cycle number at which the fluorescence of the PCR product can be detected above background. Since the number of amplification cycles in SHARP could not be determined directly, instead, the time it takes to detect the product in qSHARP was determined and termed the detection time. The detection threshold was chosen such that the signal of the non-template control remained below the threshold for 25 min. Figure 2E shows the initial template copy number versus the measured detection time. Assuming an exponential model, from the fit, a doubling time of t1 / 2 ≈ 1.5 min was determined for the 1464 bp amplicon. For the PCR reaction using the Pfusion mix and the 1.5 kbp amplicon, each cycle or doubling time is longer than in SHARP. It typically takes 2-3 min for the thermal cycle to melt the duplex, bind the primers, replicate, and change temperature.

[0139] SHARP was also demonstrated with other template-primer sets, including an amplicon of approximately 3 kbp (Figure 3A), and an amplicon of approximately 6 kbp (Figure 3B). Figure 3C shows the kinetics of the short 200 bp amplicon and amplification reaction using λ-DNA as template. For the 200 bp amplicon, gel bands were observed even at single-digit template copy numbers per microliter. For each kinetic curve of the 200 bp amplicon (Figure 3C), the detection time was determined (Figure 3D), and the t1 / 2 = 0.83 minutes or 50 seconds doubling time.

[0140] Figure 3E summarizes the SHARP detection limit for different amplicon lengths (200 bp, 1.4 kbp, 3 kbp, and 6 kbp). For short amplicons less than 200 bp, SHARP is extremely sensitive and can detect and amplify only a few template molecules per microliter. As the DNA template length increases, the detection limit of SHARP increases. This is also expected for thermal cycler-based PCR. Other isothermal amplification methods do not achieve amplification of long templates and do not report their detection limits (Y. Zhao, et al. "Isothermal Amplification of Nucleic Acids," Chemical Reviews, vol. 115, p. 12491-12545, 11 2015). Figure 3F shows the doubling time (t 1 / 2 ) are shown. Short amplicons have a minimum doubling time of less than 1 min, but for longer amplicons, the time increases. For the 6 kbp amplicon, the low detection limit (Figures 3B and 3E) resulted in an insufficient number of kinetic curves to determine the doubling time.

[0141] application

[0142] We next tested how SHARP behaves for some common applications in the wet lab. We first tested whether SHARP creates an amplicon that can be propagated in living cells. Starting with a 4012 bp template vector (Figure 2A), primers were selected to contain a vector backbone containing ampicillin resistance (see Methods), a 3245 bp region was amplified with SHARP (Figure 3A), and the blunt ends were ligated. E. coli DH5α cells were transformed with this plasmid and then plated. Three randomly picked colonies were picked, the plasmid extracted, linearized, and the correct 3245 bp product was confirmed in all three (Figure 4A). Thus, E. coli can take up the SHARP-made plasmid, acquire ampicillin resistance, survive, and replicate.

[0143] We further tested whether SHARP can amplify sequences prone to secondary structure formation. We selected a 385 bp region containing 47 trinucleotide CAG repeats that are known to undergo multivalent intermolecular interactions in single-stranded form (A. Jain and RD 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). The example in Figure 4B shows that sequences containing (CAG)47 do not inhibit the SHARP reaction over a range of SSB concentrations.

[0144] Finally, the temperature dependence of the SHARP reaction was tested. Using the nCoV-2 sequence template and primers targeting the 155 bp region, SHARP reactions were performed in the temperature range of 45 °C to 65 °C, and kinetic curves were recorded (Figure 4C). After 30 min, the reactions were stopped and the products were imaged on a 2% agarose gel (Figure 4D). In reactions ranging from 48.9 °C to 65 °C, a 155 bp amplification product was observed, but at lower temperatures, no amplification occurred within 30 min. At 65 °C, it takes about 5 min to detect the product, and at 48.9 °C, the amplification time increases to 25 min (Figure 4C, inset). Thus, SHARP with Bst-LF DNAP works optimally at 65 °C, but can also occur at lower temperatures. If SHARP must be performed at 37 °C, Bsu and Klenow Exo- are better choices of DNAP. Figure 4E shows a 155 bp product amplified at 37 °C with Bsu and Klenow Exo-DNAP over a range of SSB concentrations. The reaction was quenched after 1 h without generating primer dimers, presumably because PcrA M6 ATPase activity is also lower at 37 °C than at 65 °C. It was observed that Klenow Exo- requires a higher SSB concentration than Bsu for successful application. SHARP at 37 °C has two limitations, the amplicon length is less than 1 kbp and the amplification time can take more than 1 h, but the lower temperature makes SHARP more versatile when it comes to combining it with other techniques and running it in parallel with other temperature-sensitive DNA modifying enzymes.

[0145] Overall, using the examples in Figures 2A-2F and 3A-3F, SHARP was demonstrated to perform as well as or better than PCR according to eight criteria. (1) SHARP can generate large amounts of amplicons up to 6000 bp, unlike existing isothermal amplification methods that only generate a few hundred base pair products. (2) SHARP amplification time is template and primer dependent, but typically occurs within 5-30 min. (3) Primers optimized for PCR can be used with SHARP, unlike many other isothermal methods that require more complex primer design. (4) SHARP detection limits are comparable to PCR based gel assays. (5) SHARP is also suitable for real-time product monitoring in the presence of intercalating dyes, as an isothermal alternative to qPCR. (6) Correct SHARP and PCR products appear as a single band on the gel; therefore, it is easy to determine the presence, length, and amount of the product. Unlike SHARP and PCR, LAMP generates multiband products, often containing more than 10 bands, making the results difficult to interpret and quantify. (7) The SHARP product can be used for downstream applications in the same way as a PCR product. For example, it can be gel purified and used for cloning, as shown in Figures 4A-4E. (8) Even when the template is much longer than the amplicon (see the example of a lambda DNA template), SHARP works without the need for an initial heat denaturation step.

[0146] Engineering PcrA M6 helicase for SHARP: DNA amplification reactions require separation of the two strands, and the original patent for PCR (US Pat. No. 4,800,159) proposed separating the DNA via heating or enzymatically using helicase or RecA1. Strand separation by helicase, HAD (M. Vincent et al., "Helicase-dependent isothermal DNA amplification", EMBO reports, Vol. 5, pp. 795-800, 8 2004), has not been widely utilized for DNA amplification, due to the lack of a suitable helicase for this application. PcrA M6 helicase engineered from Geobacillus stearothermophilus PcrA was found to serve the purpose well after testing a variety of other candidate helicases.

[0147] The bulk FRET unwinding assay was used to test the unwinding activity of different helicase candidates in SHARP (S. Arslan, et al., "Engineering of a superhelicase through conformational control," Science, vol. 348, p. 344-347, 4 2015). The DNA construct (Figure 5A) consisted of an 18 bp duplex with a 3' overhang labeled with a FRET pair of Cy3 and Cy5 in such a manner that FRET decreased upon DNA unwinding. A reference reaction using the highly processively engineered Rep-X superhelicase gave a rapid FRET decrease upon addition of ATP (Figure 5B). Rep-X showed the highest unwinding activity at 37°C, but its activity decreased at higher temperatures and is not suitable for SHARP, as it cannot unwind blunt-ended DNA. Rep-X showed no amplification with SHARP. Since UvrD was used with HDA to amplify 100-200 bp fragments, we also tested E. coli UvrD helicase for SHARP (M. Vincent et al., "Helicase-dependent isothermal DNA amplification," EMBO reports, vol. 5, p. 795-800, 8 2004). Although 34 nM UvrD was roughly equivalent to 10 nM Rep-X when it came to unwinding the DNA construct (Figure 5C), the DNA underwent subsequent reannealing shown as FRET recovery, presumably due to ATP depletion. UvrD did not support SHARP. Thermostable Thermoanaerobacter tengcongensis Tte UvrD showed no unwinding activity at 37°C (Figure 5D) and no SHARP activity at 65°C.

[0148] Wild-type Geobacillus stearothermophilus PcrA helicase showed low unwinding activity at a high concentration of 600 nM (Figures 5E and 5F) and did not support SHARP. The PcrA M5 mutant has two natural cysteines mutated (C96A and C247A) (Figure 6A) and two new cysteines introduced (N187C and L409C), as well as one additional mutation L384V. Engineered PcrA M5 at 400 nM showed unwinding activity (Figure 5G), which was significantly reduced at 40 nM (Figure 5H). PcrA M5 could also be crosslinked into a closed conformation to create a superhelicase, but such crosslinking was not required for SHARP. An additional H93A mutation was introduced into M5 to create PcrA M6. PcrA M6 shows high unwinding activity even at 40 nM (Figure 5I).

[0149] Figure 6A shows the locations of the six point mutations in PcrA M6 mapped onto the PcrA structure (SS Velankar et al., "Crystal Structure of PcrA DNA Helicase Complexed with DNA Substrates," Inchworm Mechanism, vol. 97, pp. 75-84, 4 1999). The unwinding activity of PcrA M6 was further characterized on various DNA substrates using an intercalating dye at 37°C (Figure 6B). Unwinding was detected as a decrease in fluorescence intensity over time. PcrA M6 at 40 nM with ATP did not unwind the 48 kbp λ DNA, whereas in the presence of 0.2 mg / mL SSB, nearly complete unwinding occurred (Figure 6B). This ability to unwind long DNAs is likely important for DNA amplification without an initial heat denaturation step. PcrA M6 at a concentration of 40 nM was able to unwind 1.5 kbp linear DNA, long human genomic DNA (gDNA), λ DNA, and 4 kbp circular plasmid in the presence of 0.2 mg / mL SSB and 2 mM ATP (Figure 6C). For the same mass concentration of DNA substrate used (20 ng / µL), PcrA M6 showed the fastest unwinding for 1.5 kbp linear DNA and also unwinding activity for circular plasmids, albeit at the slowest rate among those tested. PcrA plays an important role in plasmid replication and can unwind circular plasmids, suggesting that PcrA can unwind DNA that does not have ends. Although some of the unwinding properties of PcrA M6 and M5 are important for their ability to function in SHARP, more mechanistic studies are needed to show exactly why these engineered PcrA helicases support SHARP while wild-type PcrA and other related helicases do not.

[0150] conclusion

[0151] A new isothermal amplification method named SHARP was created using engineered PcrA M6 helicase. SHARP eliminates thermal cycling from PCR while keeping all the desirable PCR characteristics in place. SHARP can generate simple, linear, multi-kilobase pair long amplicons from a template and a set of two primers in less than 30 minutes. Apart from replacing thermal cycling from PCR in genetic engineering reactions such as simple molecular diagnostics, sequencing and cloning, SHARP can also detect and amplify sequences that tend to form secondary structures. SHARP also has the potential to be a universal biotechnology tool for many new point-of-care detection methods in medicine, simplifying many procedures in the laboratory and enabling new applications outside the laboratory such as environmental screening, agricultural pest detection, and many other future consumer-focused diagnostic home tests. This method simply eliminates the bulky and expensive thermal cycler equipment from the amplification reaction, thus increasing its versatility.

[0152] References [1] 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. [2] Y. Zhang and NA Tanner, "Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein," Scientific Reports, vol. 7, 8 2017. [3] K. Mullis, F. Faloona, S. Scharf, R. Saiki, G. Horn and H. Erlich, "Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction," Cold Spring Harbor Symposia on Quantitative Biology, vol. 51, p. 263-273, 1 1986. [4] K. B. Mullis, H. A. Erlich, N. Arnheim, G. T. Horn, R. K. Saiki and S. J. Scharf, "Process for amplifying, detecting, and / or cloning nucleic acid sequences". United States of America Patent US4800159A, 24 1 1989. [5] T. Notomi, "Loop-mediated isothermal amplification of DNA," Nucleic Acids Research, vol. 28, p. 63e-63, 6 2000. [6] A. Ganguli, A. Mostafa, J. Berger, M. Y. Aydin, F. Sun, S. A. S. de Ramirez, E. Valera, B. T. Cunningham, W. P. King and R. Bashir, "Rapid isothermal amplification and portable detection system for SARS-CoV-2," Proceedings of the National Academy of Sciences, vol. 117, p. 22727-22735, 8 2020. [7] O. Piepenburg, C. H. Williams, D. L. Stemple and N. A. Armes, "DNA Detection Using Recombination Proteins," PLoS Biology, vol. 4, p. e204, 6 2006. [8] I. Magrina Lobato and C. O'Sullivan, "Recombinase polymerase amplification: Basics, applications and recent advances," TrAC Trends in Analytical Chemistry, vol. 98, p. 19-35, 1 2018. [9] N. Sharma, S. Hoshika, D. Hutter, K. M. Bradley and S. A. Benner, "Recombinase-Based Isothermal Amplification of Nucleic Acids with Self-Avoiding Molecular Recognition Systems (SAMRS)," ChemBioChem, vol. 15, p. 2268-2274, 9 2014.

[10] M. J. Hoser, H. K. Mansukoski, S. W. Morrical and K. E. Eboigbodin, "Strand Invasion Based Amplification (SIBA(registered trademark)): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte," PLoS ONE, vol. 9, p. e112656, 11 2014.

[11] K. E. Eboigbodin and M. J. Hoser, "Multiplex Strand Invasion Based Amplification (mSIBA) assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae," Scientific Reports, vol. 6, 2 2016.

[12] M. C. Little, J. Andrews, R. Moore, S. Bustos, L. Jones, C. Embres, G. Durmowicz, J. Harris, D. Berger, K. Yanson, C. Rostkowski, D. Yursis, J. Price, T. Fort, A. Walters, M. Collis, O. Llorin, J. Wood, F. Failing, C. O'Keefe, B. Scrivens, B. Pope, T. Hansen, K. Marino, K. Williams and M. Boenisch, "Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, BDProbeTecET," Clinical Chemistry, vol. 45, p. 777-784, 6 1999.

[13] J. V. Ness, L. K. V. Ness and D. J. Galas, "Isothermal reactions for the amplification of oligonucleotides," Proceedings of the National Academy of Sciences, vol. 100, p. 4504-4509, 4 2003.

[14] M. Vincent, Y. Xu and H. Kong, "Helicase-dependent isothermal DNA amplification," EMBO reports, vol. 5, p. 795-800, 8 2004.

[15] A. Jain and R. D. Vale, "RNA phase transitions in repeat expansion disorders," Nature, vol. 546, p. 243-247, 5 2017.

[16] L. Aeschbach and V. Dion, "Minimizing carry-over PCR contamination in expanded CAG / CTG repeat instability applications," Scientific Reports, vol. 7, 12 2017.

[17] V. Dion, "Tissue specificity in DNA repair: lessons from trinucleotide repeat instability," Trends in Genetics, vol. 30, p. 220-229, 6 2014.

[18] S. Arslan, R. Khafizov, C. D. Thomas, Y. R. Chemla and T. Ha, "Engineering of a superhelicase through conformational control," Science, vol. 348, p. 344-347, 4 2015.

[19] S. S. Velankar, P. Soultanas, M. S. Dillingham, H. S. Subramanya and D. B. Wigley, "Crystal Structures of Complexes of PcrA DNA Helicase with a DNA Substrate Indicate an Inchworm Mechanism," vol. 97, p. 75-84, 4 1999.

[20] J. Park, S. Myong, A. Niedziela-Majka, K. S. Lee, J. Yu, T. M. Lohman and T. Ha, "PcrA Helicase Dismantles RecA Filaments by Reeling in DNA in Uniform Steps," Cell, vol. 142, p. 544-555, 8 2010.

[21] T. M. Lohman, J. M. Green and R. S. Beyer, "Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under .lambda. PL control," Biochemistry, vol. 25, p. 21-25, 1 1986.

[22] S. Bhadra, T. E. Riedel, S. Lakhotia, N. D. Tran and A. D. Ellington, "High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes," 4 2020.

[23] L. An, W. Tang, TA Ranalli, H.-J. Kim, J. Wytiaz and H. Kong, "Characterization of a Thermostable UvrD Helicase and Its Participation in Helicase-dependent Amplification," Journal of Biological Chemistry, vol. 280, p. 28952-28958, 8 2005.

[24] AF Slatter, CD Thomas and MR Webb, "PcrA Helicase Tightly Couples ATP Hydrolysis to Unwinding Double-Stranded DNA, Modulated by the Initiator Protein for Plasmid Replication, RepD," Biochemistry, vol. 48, p. 6326-6334, 6 2009.

[25] LT Chisty, CP Toseland, N. Fili, GI Mashanov, MS Dillingham, JE Molloy and MR Webb, "Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD," vol. 41, p. 5010-5023, 3 2013.

[0153] Example 2

[0154] We further show that the engineered PcrA M6 is 3-4 times faster than the previous PcrA M5. Figures 5H and 5I of the provisional application qualitatively compare the unwinding activity of PcrA M5 and M6, respectively. Figure 5I shows that PcrA M6 is more active than M5 in Figure 5H.

[0155] We further analyzed the data in Figure 5 to obtain the unwinding rate of each helicase. Figures 7B-7I show the extracted FRET efficiencies. From the fits, we obtained the unwinding rates, summarized in Figure 7J. Furthermore, we divided the rates by the helicase concentrations to obtain the activities (Figures 7K-L). From the obtained normalized activities, we conclude that PcrA M6 is 3 times more active than PcrA M5.

[0156] We also performed nanopore assays to directly observe the rate at which the helicase unwinds the DNA duplex using a Single Molecule Picometer Nanopore Tweezer (SPRNT) in Figure 8. Briefly, the SPRNT setup consists of two separate wells connected with a nanopore. At room temperature (22 °C), the well near the positive electrode contains signal buffer (500 mM KCl and 50 mM HEPES pH 8.0), while the well near the negative electrode contains reaction buffer (200 mM KCl, 50 mM HEPES pH 8.0, 5 mM MgCl2, and 2 mM ATP) and the helicase-DNA construct (Figure 8A). The DNA construct has 64 repeats of the spinach DNA sequence, with each repeat being 103 bp long. A potential difference of 180 mV is applied between the electrodes and picoamp (pA) current is measured using an Axon Axopatch 200B instrument controlled by code written in LabVIEW 2018. As the helicase unwinds the DNA and drives one strand into the nanopore, the current passing through the nanopore generates a repeat signal (Figure 8B-C). By counting the number of 103 bp long repeats over time, the helicase unwinding rate can be determined. For PcrA M5 in Figure 8B, 3.2 repeats are observed over 8 seconds. For PcrA M6, we identified 10.4 repeats over 8 seconds. From the nanopore data, we extract unwinding rates of 41 bp / s for PcrA M5 and 134 bp / s for PcrA M6. We conclude that PcrA M6 is more than three times faster than PcrA M5.

[0157] The higher activity and rate of PcrA M6 demonstrated in both bulk and single molecule assays facilitates SHARP amplification.

[0158] In this provisional application, the inventors stated that SHARP can amplify RNA targets. The inventors also listed Moloney Murine Leukemia Virus (MMLV) reverse transcriptase; however, the inventors did not include any data to support this claim. To demonstrate that SHARP can amplify RNA targets, the inventors used MMLV reverse transcriptase. MMLV first generates cDNA from an RNA template and then acts as a moderately efficient DNA polymerase to carry out the amplification. The nCoV-2 RNA sequence and CDC N2 primers: forward 5'-TTACAAACATTGGCCGCAAA and reverse 5'-GCGCGACATTCCGAAGAA were used. 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 that of DNA amplification.

[0159] SHARP can be used for portable, point-of-care, point-of-need, and home medical molecular diagnostic testing. SHARP products can be easily detected using lateral flow devices. We performed SHARP amplification in which the forward primer was labeled with biotin and contained a DIG-labeled dNTP mixture (Figure 10A). With this modification of the SHARP mixture, we can easily pull out and detect the SHARP product using a lateral flow device (Figure 10B). If no labeled dNTPs are included, no product is shown in the lateral flow device (Figure 10C).

[0160] In FIG. 3B, it is shown that SHARP can generate amplicons up to 6000 bp, and a 6000 bp amplicon was shown. This is not a limitation. SHARP can generate amplicons longer than 6000 base pairs (e.g., up to or at least 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800, or 8000 base pairs or more). Yields and detection limits may vary, as shown in FIG. 3E. FIG. 11 shows the previously disclosed 6000 base pair amplicon, as well as the 7929 amplicons obtained using a lambda DNA template and forward: 5'-GGCAAAGCGGTGTCCTATAA and reverse 5'-CGGTGTACCTCTCTCGTTTG primers. The correct 7929 product is amplified, but in less amount than the 6000 bp product. Generating amplicons much longer than >8000 base pairs would require extremely high template concentrations and would be impractical.

[0161] 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 the FANCF gene. We used the following primers 5'-AGTTCGCTAATCCCGGAACT (FANCF_F), 5'-AGTTGCCCAGAGTCAAGGAA (FANCF_R), 5'-CCAGTGGTTCAATGGTCATCC (DNMT3B_F), and 5'-GGCCAGTGAAATCACCCTG (DNMT3B_R). Figure 12 shows the amplification of the products in real time (Figure 12A), compared to the PCR products in a gel-based assay (Figure 12B), and confirmed the correct products using Sanger sequencing (Figure 12C).

[0162] Figure 4B shows that SHARP can amplify the repeat sequence (CAG)n. We also demonstrated that SHARP can amplify four repeats of the 601 nucleosome positioning DNA sequence, and compared SHARP with PCR in Figure 13. The 601 nucleosome positioning sequence is 147 base pairs in length, while the linkers between the repeats are 65 and 80 base pairs in length.

[0163] Other embodiments

[0164] From the above description, it is apparent that variations and modifications may be made to the disclosure described herein to adapt it to various usages and conditions. Such embodiments also fall within the scope of the following claims.

[0165] All citations to sequences, patents, and publications in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A bacterial helicase comprising a PcrA helicase having at least two or more mutations.

2. The bacterial helicase of claim 1 , wherein the PcrA helicase has at least 90% sequence identity with SEQ ID NO:

1.

3. The bacterial helicase of claim 1 , wherein the PcrA helicase comprises SEQ ID NO:

1.

4. The bacterial helicase according to claim 1 , wherein the PcrA helicase is derived from Geobacillus.

5. A vector comprising a nucleic acid encoding a PcrA helicase that contains 60% sequence identity to SEQ ID NO:

1.

6. An isolated nucleic acid comprising a nucleic acid sequence that comprises at least 60% sequence identity to SEQ ID NO:

3.

7. An engineered helicase comprising a PcrA helicase, wherein said PcrA helicase comprises one or more amino acid mutations.

8. 8. The engineered helicase of claim 7, wherein the PcrA helicase comprises mutations at amino positions 93, 96, 187, 247, 384, and 409 of wild-type PcrA helicase.

9. A kit containing engineered PcrA helicase, single-stranded binding protein (SSB), Bst polymerase, and thermostable pyrophosphatase (PPase).

10. 1. A method of amplifying a nucleic acid, comprising mixing a first composition with one or more primers, a target nucleic acid sequence, and a second composition. The method, wherein the second composition comprises an engineered PcrA helicase, a single-stranded binding protein (SSB), a polymerase, a thermostable pyrophosphatase (PPase) buffer, or a combination thereof.

11. An isolated nucleic acid comprising the sequence of SEQ ID NO:

2.

12. A method for amplifying nucleic acids, comprising incubating at a substantially isothermal temperature: i) a composition comprising a target sequence; ii) a primer; and iii) an engineered PcrA helicase, single-stranded binding protein (SSB), a polymerase, and / or a thermostable pyrophosphatase (PPase) buffer, or a combination thereof.

13. A method for amplifying nucleic acids, comprising incubating, in the absence of thermal cycling, i) a composition comprising a target sequence, iii) a primer, and iii) an engineered PcrA helicase, single-stranded binding protein (SSB), polymerase, and / or a thermostable pyrophosphatase (PPase) buffer, or a combination thereof.

14. A method for screening for cancer, comprising the method or kit according to any one of claims 9 to 10 and 12 to 13.

15. A method for detecting an infectious agent or sepsis, comprising the method or kit according to any one of claims 9 to 10 and 12 to 13.

16. A composition for use in a method for diagnosing a disease or disorder in a subject, comprising the method or kit according to any one of claims 9 to 10 and 12 to 13.

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