Methods and compositions for primer-independent cell-free DNA amplification

Abstract

A method for amplifying a DNA template, comprising: a) providing a composition comprising a DNA template, an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide, wherein said composition does not comprise a ribonucleotide; and b) incubating said composition in a suitable condition to allow amplification of said DNA template. Also provided is a method for priming DNA amplification from a DNA template, comprising: incubating a DNA template, an RNA polymerase and a deoxyribonucleotide in a suitable condition to allow the initiation of DNA amplification; wherein said method does not comprise adding a ribonucleotide.

Description

METHODS AND COMPOSITIONS FOR PRIMER-INDEPENDENT CELL-FREE DNA AMPLIFICATIONFIELD

[0001] The present disclosure relates generally to methods and compositions for DNA amplification in a cell-free system.BACKGROUND

[0002] Nucleic acid amplification techniques such as polymerase chain reaction (PCR) which requires a thermocycling process and various isothermal amplification techniques have become an integral part of nucleic acid-based therapeutics and diagnostics.

[0003] Examples of isothermal reactions include rolling circle amplification (RCA), multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and nicking enzyme amplification reaction (NEAR).

[0004] Replication of a DNA molecule requires a free hydroxyl group that serves as a starting point for the addition of deoxyribonucleotides by a DNA polymerase. Typically, short, single stranded oligonucleotides or primers complementary to the template DNA molecule are exogenously added in order to prime replication of a DNA molecule by a DNA polymerase.

[0005] The use of primers generally requires denatured DNA for efficient priming, and thus is associated with some shortcomings including damage to the initial template DNA during template denaturation by heat or chemical methods.

[0006] Rolling circle amplification (RCA) is an efficient isothermal method for DNA amplification using Phi29 DNA polymerase, which may require primers to initiate the strand displacement process. While RCA is valuable in diagnostics for detecting low quantities of DNA, the conventional method involves a DNA denaturation step that can cause strand breaks and other damage to the template DNA, potentially affecting downstream analyses.

[0007] DNA replication is a complicated process in vivo. It requires numerous components working together to synthesize new DNA strands. Since no known replicative DNA polymerase can initiate DNA synthesis de novo, primers that are synthesized by a primase are required to provide a free 3’-hydroxyl group for DNA polymerase.

[0008] A few primer-independent DNA amplification methods have been developed (e.g., US2022 / 0213538A1 ; EP3697930B1 ), for example, by using a DNA primase, a T7 RNA polymerase, or a DNA polymerase with priming activity. These methods each has its own limitations, such as, some primases recognize a specific sequence for primer synthesis, which might lead to amplification bias. Thus, there is still a need in the art for an alternative and improved method for primer-independent cell-free isothermal DNA amplification.BRIEF SUMMARY

[0009] In an aspect, the present disclosure relates to a method for amplifying a DNA template, comprising: a) providing a composition comprising a DNA template, an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide, wherein said composition do not comprise a ribonucleotide; and b) incubating said composition in a suitable condition to allow amplification of said DNA template.

[0010] In an embodiment, the RNA polymerase is a wide-type or mutant RNA polymerase. The RNA polymerase can be selected from the group consisting of T7 RNA polymerase or its mutants, T3 RNA polymerase or its mutants, SP6 RNA polymerase or its mutants, K11 RNA polymerase or its mutants, or a combination thereof. In a preferred embodiment, the RNA polymerase is wide-type T7 RNA polymerase or its mutants. In another preferred embodiment, the T7 RNA polymerase mutant can use deoxyribonucleotide as a substrate. In another preferred embodiment, the T7 RNA polymerase mutant is the Y639F mutant.

[0011] In an embodiment, the DNA template is a single strand or double strand circularor linear DNA.

[0012] In another embodiment, the DNA polymerase has strand displacement activity. In a preferred embodiment, the DNA polymerase is Phi29 polymerase.

[0013] In an embodiment, incubating said composition in a suitable condition comprises incubating at 20°C to 37°C for 3-36 hours.

[0014] In an embodiment, said composition further comprise suitable buffer. In a preferred embodiment, said buffer comprises potassium acetate, Tris-acetate, magnesium acetate, albumin, and DTT.

[0015] In an embodiment, said composition comprise a DNA template at a concentration of 0.1 to 10 ng / pl , an RNA polymerase at a concentration of 0.1 to 10 U / p I , a DNA polymerase at a concentration of 0.1 to 10 U / pl , and a deoxyribonucleotide at a concentration of 0.1 to 30 mM.

[0016] In an embodiment, said composition in step a) further comprises at least one oligonucleotide complementary to the DNA template, a mix of random oligonucleotides, or a combination thereof.

[0017] In another aspect, the present disclosure relates to a method for priming DNA amplification from a DNA template, comprising: incubating a DNA template, an RNA polymerase and a deoxyribonucleotide in a suitable condition to allow the initiation of DNA amplification; wherein said method does not comprise adding a ribonucleotide.

[0018] In an embodiment, said method further comprises, after the incubation, adding a DNA polymerase to elongate the DNA amplification.

[0019] In another aspect, the DNA template may not contain a promoter.

[0020] In another aspect, the composition may not contain an exogenously added oligonucleotide primer.

[0021] In another aspect, the incubating may be performed in the presence dNTP at a concentration from 0.1 mM to 10 mM, from 0.5 mM to 9 mM, from 1 mM to 8 mM, from 1 mM to 7 mM, from 1 mM to 6 mM, from 1 mM to 5 mM, from 1 mM to 4 mM, from 1 mM to3 mM, from 1 mM to 2.5 mM, from 1 mM to 2 mM, 0.1 mM, 0.5 mM, 1 mM, 1 .5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, or 10 mM.

[0022] In another aspect, methods of the present disclosure may further contain performing an in vitro transcription (IVT) of the amplified DNA template.

[0023] In another aspect, the amplified DNA template may be purified prior to IVT.

[0024] In another aspect, the amplified DNA template may be not purified prior to IVT.

[0025] In another aspect, the incubating may be performed in the presence of Inorganic pyrophosphatase (IPP) at a concentration of from 0.1 U / ml to 10 U / ml, from 0.1 U / ml to 5 U / ml, from 0.1 U / ml to 4 U / ml, from 0.1 U / ml to 3 U / ml, from 0.1 U / ml to 2 U / ml, from 0.1 U / ml to 1 U / ml, from 0.1 U / ml to 0.9 U / ml, from 0.1 U / ml to 0.8 U / ml, from 0.1 U / ml to 0.7 U / ml, from 0.1 U / ml to 0.6 U / ml, from 0.1 U / ml to 0.5 U / ml, from 0.1 U / ml to 0.4 U / ml, from 0.1 U / ml to 0.3 U / ml, from 0.1 U / ml to 0.2 U / ml, 0.1 U / ml, 0.2 U / ml, 0.3 U / ml, 0.4 U / ml, 0.5 U / ml, 0.6 U / ml, 0.7 U / ml, 0.8 U / ml, 0.9 U / ml, or 1 U / ml.

[0026] In another aspect, the present disclosure relates to a kit comprising: a template DNA molecule, an RNA polymerase, a DNA polymerase, a deoxyribonucleotide, and instructions for amplifying a DNA template in the presence of an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide but without a ribonucleotide.

[0027] In another aspect, the RNA polymerase may not contain a primase activity.

[0028] In another aspect, the RNA polymerase may be a promoter-specific RNA polymerase.

[0029] In another aspect, the RNA polymerase may be a single-unit RNA polymerase.

[0030] In another aspect, the DNA template may be a circular DNA.

[0031] In another aspect, the DNA template may be a plasmid DNA.

[0032] In another aspect, the DNA template may be a linear DNA.

[0033] In an aspect, the present disclosure may relate to a method for in vitrotranscription (IVT) of a DNA template into RNA, including a) providing a composition containing DNA template, RNA polymerase, DNA polymerase, deoxyribonucleotide, and restriction enzyme, in which composition may not contain ribonucleotide; b) incubating composition in a suitable condition to allow amplification of DNA template, in which restriction enzyme may be capable of linearizing amplified DNA template, and c) performing IVT by incubating linearized amplified DNA template in the presence of ribonucleotides at from about 15°C to about 35°C for from about 1 hour to about 12 hours, thereby producing the RNA.

[0034] In another aspect, performing IVT may be further in the presence of cap analogues.

[0035] In another aspect, linearized amplified DNA template may be not purified prior to performing IVT at step c).BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a gel image that shows the results of the amplification reactions under different conditions.

[0037] FIG. 2 is a gel image that shows the comparison of DNA amplifications using WT vs. a mutant T7 polymerase in the presence of dNTPs.

[0038] FIG. 3 shows the yields of DNA amplifications under different conditions.

[0039] FIG. 4 is a gel image that shows the digestion of the DNA amplification products obtained using WT in the presence of both dNTPs and rNTPs vs. using a mutant T7 polymerase in the presence of dNTPs alone.

[0040] FIG. 5A shows yields of RCA products prepared by methods in accordance with some embodiments of the present disclosure.

[0041] FIG. 5B shows yields of RCA products prepared by methods in accordance with some embodiments of the present disclosure.

[0042] FIG. 6 shows RCA products prepared by methods in accordance with some embodiments of the present disclosure.

[0043] FIG. 7 shows gene expression levels of DNA templates obtained from RCA products prepared by methods in accordance with some embodiments of the present disclosure.

[0044] FIG. 8 shows RCA products prepared by methods in accordance with some embodiments of the present disclosure.

[0045] FIG. 9 shows RCA products prepared by methods in accordance with some embodiments of the present disclosure.

[0046] FIG. 10 shows RCA products prepared by methods in accordance with some embodiments of the present disclosure.

[0047] FIG. 11 shows RCA products prepared by methods in accordance with some embodiments of the present disclosure.

[0048] FIG. 12 shows RCA products prepared by methods in accordance with some embodiments of the present disclosure.DETAILED DESCRIPTION

[0049] Definitions

[0050] The term “template DNA”,” or “DNA template”, as is used herein, refers to a DNA molecule that is to be replicated. Said DNA template can be any DNA molecule, including a single or double stranded DNA molecule, a linear or circular DNA molecule, and a small or a large DNA molecule, ranging from linear or circular plasmid molecules of less than 10 kilo bases to large DNA molecules such as genomic DNA molecules or BAC plasmids. A template DNA can be, for example, a target DNA sequence contained in a sample. A circular DNA template can be a naturally circular DNA such as a plasmid, or can be a DNA molecular that is circularized from a linear format.

[0051] DNA Primase

[0052] In most replication systems, synthesis of the lagging strand may be initiated from short oligoribonucleotide primers that are synthesized at specific sites by primases. Specific interactions between a primase and the DNA polymerase allow the DNA polymerase to initiate DNA synthesis from the oligoribonucleotide resulting in the synthesis of the laggingstrand. A unique primase mechanism may be found in the terminal protein exemplified by adenovirus and bacteriophage 029 (Salas, Ann. Rev. Biochem. 60:39, 1991 ). In these organisms, duplex linear DNA may be replicated by the covalent binding of the terminal protein to each 5’ end, followed by initiation of DNA synthesis from a mononucleotide bound to the terminal protein by a DNA polymerase.

[0053] The term “RNA polymerase”, as is used herein, refers to an DNA-dependent RNA polymerase enzyme that produces a DNA templated RNA transcript. Said RNA polymerase preferably is a member of the family of single-subunit RNA polymerases that includes many phage RNA polymerases (T7, T3, K11 , SP6, N4, and others) as well as the mitochondrial RNA polymerases (McAllister and Raskin, 1993. Mol Microbiol. 10: 1-6). A preferred RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, K11 RNA polymerase, or mutants thereof. Such mutants include for instance mutant T7 RNA polymerases capable of utilizing both canonical and non-canonical ribonucleotides and deoxynucleotides as substrates (Kostyuk et al., 1995. FEBS Lett. 369: 165-168; Sousa et al., 1995. EMBO J. 14(18): 4609-4621 ;Gudima et al., 1998. FEBS Lett. 439: 302-306; Padilla et al., 2002. Nucl. Acids Res. 30(24): e138), RNA polymerase variants displaying higher thermostability (Hi-T7TMRNAPolymerase from New England Biolabs; Boulain et al., 2013. Protein Eng Des Sei. 26(11 ): 725-734), or a mutant RNA polymerase with decreased promoter specificity (Ikeda et al., 1993. Biochemistry 32(35):9115-9124).

[0054] Another category of a preferred mutant RNA polymerase (e.g. a T7 RNA polymerase mutant) is a mutant that uses deoxyribonucleotides as substrates, either naturally or engineered to. It can be a mutant RNA polymerase that exclusively uses deoxyribonucleotides but does not use ribonucleotides. Alternatively, it can be a mutant RNA polymerase that uses both deoxyribonucleotides and ribonucleotides, and such mutant may or may not preferentially uses deoxyribonucleotides over ribonucleotides. An example of a mutant T7 RNA polymerase that can be used in the present disclosure is a Y639F mutant, also called DuraScribe T7 RNA polymerase, that efficiently incorporates 2'- Fluorine-CTP (2'-F-dCTP), 2'-Fluorine-UTP (2'-F-dUTP), ATP and GTP into RNase- resistant RNA transcripts (shop.biosearchtech.com / molecular-biology-enzymes / in-vitro- transcription-kits-and-reagents / nuclease-resistant-in-vitro-transcription-kits / durascribe-t7-transcription-kits / p / MBTOOL-007).

[0055] In some embodiments of the present disclosure, T7 RNA polymerase can use RNA templates as well as DNA templates and may be capable of both primer extension and de novo initiation. In some embodiments of the present disclosure, T7 RNA polymerase (Y639F) mutant may retain the ability to use RNA or DNA templates. This Y639F mutant can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase, or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction. (Sousa et al.. “A mutant T7 RNA polymerase as a DNA polymerase.” EMBO J. 1995 Sep 15;14(18):4609-21).

[0056] T7 RNA polymerase can incorporate 2'-deoxynucleotides into oligoribonucleotides with greater than 50% efficiency per deoxynucleotide incorporated as compared to the yield of the fully ribonucleotide product. (Wyatt et al., “Deoxynucleotide- containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H.” Nucleic Acids Res. 1989 Oct 11 ;17(19)7833-42). The efficiency of incorporation may be dependent on the template sequence and the dNMP incorporated; compared to the corresponding rNMP, 70% for dA, 85% for dT or dll, 50% for dC, and 25% for dA plus dT. During the incorporation of dNMP , rNMP misincorporations may occur at a frequency of less than 5%. Wild type T7 RNA polymerase can incorporate dNTP with Mn. For example, substitution of MnCl2 for MgCl2 may result in a relaxation of the specificity of T7 RNA polymerase, allowing incorporation of deoxynucleotides to 90-100% efficiency. Overall yield, however, may be lowered by a factor of 30 when compared with that obtained with ribonucleotides only. Transcription buffer containing 2.0-2.5 mM Mn2+may support T7 RNA polymerase-mediated insertion of 8-N3AMP into RNA, a mixture of 2.5 mM Mn2+and 2.5 mM Mg2+may further improve the yield of 8-NsAMP-containing transcript. (Gopalakrishna, et al., “Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase.” RNA. 2004 Nov; 10(11 ): 1820-30). Manganese ions may be critical in the method for the incorporation of dNTPs into the transcription products. Preferably, Mn2+is present in a concentration of between 10 pM to 20 mM. In an especially preferred embodiment, the concentration is 10 mM.

[0057] T7 RNA polymerase (Y639F) mutant may possess DNA primase and DNA polymerase activities. T7 RNA polymerase (Y639F) mutant may exhibit reduced discrimination between dNTP and rNTP and, as if a DNA Polymerase, may incorporate dNMP as efficiently as rNMP. T7 RNA polymerase (Y639F) mutant may perform DNA amplification in the presence of dNTPs, in the absence of rNTPs, and in the absence of any specific or random primers.

[0058] In certain embodiments, T7 RNA polymerase may be competent to incorporate dNTPs into a template nucleic acid. For example, T7 RNA polymerases with a Y639F mutation, or a S641A mutation, or Y639F / S641A mutations can be used. In certain embodiments, T7 RNA polymerase may contain at least two mutations. In certain embodiments, manganese ions may be used for the incorporation of dNTPs into transcription products. Preferably, the Mn2+may be present in a concentration of between 10 pM to 20 mM, 100 pM to 15 mM, 500 pM to 15 mM, 1 mM to 14 mM,. 2 mM to 13 mM, 5 mM to 12 mM, 6 mM to 11 mM, 7 mM to 11 mM, 8 mM to 11 mM, 9 mM to 11 mM, 9 mM to 10 mM, 10 mM to 11 mM, 10 mM to 12 mM, 10 mM to 13 mM, 10 mM to 14 mM, 10 mM to 15 mM, 15 mM to 16 mM, 15 mM to 17 mM, 15 mM to 18 mM, 15 mM to 19 mM, or 10 mM to 20 mM. In a preferred embodiment, the concentration may be 10 mM.

[0059] A wide-type (WT) RNA polymerase (e.g., T7 RNA polymerase: Genomic Sequence: NC_001604.1 , ncbi.nlm.nih.gov / gene / 1261050) or mutants hereof that can be used in the present disclosure may or may not exhibit a conventional primase activity, i.e. , the ability to generate a shorter primer (especially an RNA primer) on a DNA template. Without being bound to any theories, an RNA polymerase can be used in the present disclosure as long as it can initiate DNA amplification in the presence of the other required materials and conditions for the present disclosure.

[0060] DNA Polymerase

[0061] In general, a polymerase may refer to one that may carry out extensive DNA synthesis on both strands of a DNA template, with the synthesized DNA in turn being capable of being used as a template for new DNA synthesis. This may result in an exponential increase in the amount of DNA synthesized with time. Many replicative DNA polymerases have accessory proteins such as processivity factors, helicases, primasesand DNA binding proteins that are specific for each DNA polymerase (for review, see Kornberg and Baker, DNA Replication, Freeman and Co., New York, 1992). Many such combinations may result in efficient DNA synthesis.

[0062] The term “DNA polymerase”, as is used herein, refers to an DNA dependent DNA polymerase enzyme that produces a DNA templated DNA molecule. Said DNA polymerase adds deoxyribonucleotides to the 3’ end of a DNA strand.

[0063] The term “strand-displacing DNA polymerase” or “DNA polymerase with strand displacing activity”, as is used herein, refers to an DNA polymerase which is able to displace a downstream DNA strand. Hence, said enzyme is able to unwind a double stranded DNA molecule during replication of said molecule. Said polymerase preferably lacks a 5’ —3’ exonuclease activity. A suitable strand-displacing DNA-dependent DNA polymerase includes the Klenow fragment of DNA polymerase I, a Klenow fragment of a Bacillus stearothermophilus DNA polymerase termed Bst polymerase (New England Biolabs, Ipswich, Mass.), Bsm DNA Polymerase, BcaBEST DNA polymerase (Takara Bio, Kusatsu, Japan), Thermoanaerobacter thermohydrosulfuricus (Tts) DNA polymerase (U.S. Pat. No. 5,744,312), a DNA polymerases from Thermus aquaticus, Thermus flavus or Thermus thermophiles, a variant of T7 DNA-dependent DNA polymerase termed SEQUENASE (Thermo Fisher Scientific, Waltham, Mass.), a T5 DNA dependent DNA polymerase (Fujimura and Roop, 1976. J Biol Chem 251 : 2168-2174), a T4 DNA polymerase holoenzyme (Kaboord and Benkovic, 1995. Curr Biol 5:149-157), a DNA dependent DNA polymerase from Thermococcus litoralis, termed VENT polymerase (New England Biolabs, Ipswich, Mass.), phage M2 DNA polymerase (Matsumoto et al., 1989. Gene 84: 247), phage PRD1 DNA polymerase (Jung et al., 1987. Proc Natl Aced Sci USA 84: 8287; Zhu and Ito, 1994. Biochim Biophys Acta 1219: 267-276), Phi29 DNA polymerase, and any strand-displacing DNA-dependent DNA polymerase mutant thereof including, for example, a fusion of a polymerase with a DNA binding protein (de Vega et al., 2010. PNAS 107: 16506-16511 ).

[0064] A preferred example of the DNA polymerase that can be used in the present disclosure may be Phi29 DNA polymerase. Phi29 DNA polymerase combines high processivity, strand displacement activity, and 3’ to 5’ proofreading exonuclease activityresulting in synthesis of DNA fragments >70 kb with very low error rates (Blanco et al., 1989. J Biol Chem 264: 8935-8940; Garmendia et al., 1992. J Biol Chem 267: 2594-2599; Esteban et al., 1993. J Biol Chem 268: 2719-26).

[0065] The term “ribonucleotide”, as is used herein, refers a molecule comprising a ribose sugar group, a nucleobase and at least one phosphate group. Said nucleobase includes adenine, guanine, cytosine, uracil, and any modifications thereof. The nucleobases adenine and guanine are collectively termed purines, while cytosine and uracil are collectively termed pyrimidines. The term ribonucleotide includes reference to an analogue of a ribonucleotide such as a fluorescent molecule, for example 1 ,3-diaza-2- oxophenothiazine-ribose-5’ -triphosphate (tCTP), and / or other analogues such as inosine, xanthosine, N4-hydroxycytosine, N4-methoxycytosine and 6H, 8H-3,4-dihydropyrimido[4,5- c][1 ,2]oxazin-7-one (Suzuki et al., 2005. Nucleic Acids Symp Series 49: 97-98). Ribonucleotides are the monomeric units of the biopolymer, ribonucleic acid (RNA). The term “rNTP” or “NTP”, as is used herein, refers to ribonucleotide triphosphate. rNTP is a ribonucleotide in the context of the present disclosure.

[0066] The term “deoxyribonucleotide”, as is used herein, refers a molecule comprising a deoxyribose sugar group, a nucleobase and at least one phosphate group. Said nucleobase includes the naturally occurring adenine, guanine, cytosine, thymidine, and any modifications thereof. The nucleobases adenine and guanine are collectively termed purines, while cytosine and thymidine are collectively termed pyrimidines. The term deoxyribonucleotide includes reference to an analogue of a deoxyribonucleotide such as a deoxyuridine, deoxyinosine, and / or deoxyxanthosine. Deoxyribonucleotides are the monomeric units of the biopolymer, deoxyribonucleic acid (DNA). The term “dNTP”, as is used herein, refers to deoxyribonucleotide triphosphate. dNTP is a deoxyribonucleotide in the context of the present disclosure. Suitable dNTPs may include dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate), dllTP (deoxyuridine triphosphate), dCTP (deoxycytidine triphosphate), diTP (deoxyinosine triphosphate), dXTP (deoxyxanthosine triphosphate), and derivatives and modified versions thereof. It is preferred that the dNTPs comprise one or more of dATP, dGTP, dTTP or dCTP, or modified versions or derivatives thereof. It is preferred to use a mixture of dATP, dGTP, dTTP and dCTP or modified version thereof.

[0067] In some embodiments of the present disclosure, the concentration of dNTP may be from 0.1 mM to 10 mM, from 0.5 mM to 9 mM, from 1 mM to 8 mM, from 1 mM to 7 mM, from 1 mM to 6 mM, from 1 mM to 5 mM, from 1 mM to 4 mM, from 1 mM to 3 mM, from 1 mM to 2.5 mM, from 1 mM to 2 mM, 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, or 10 mM.

[0068] The term “nucleotide”, as is used herein, refers to a ribonucleotide, a deoxyribonucleotide, or any variant or modification thereof. It will be appreciated that a great variety of nucleotide modifications have been made and described that serve many useful purposes known to those skilled in the art. The term nucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of nucleotides.

[0069] The nucleotides may comprise modified nucleotides, and these modified nucleotides may be in a biologically inactive form. The modified nucleotide may thus be a biologically inactive nucleotide. A biologically inactivated nucleotide may have a removable moiety protecting a group of the nucleotide, such as the 3' carbon oxygen or the terminal phosphate oxygen. A biologically inactive nucleotide may be a caged nucleotide or a blocked nucleotide. Suitable moieties include but are not limited to photoactivatable (caging) group, including a-carboxy-2-nitrobenzyl (CNB), 1 -(2-nitrophenyl)ethyl (NPE), 4,5- dimethoxy-2-nitrobenzyl (DMNB), 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) and 5- carboxymethoxy-2-nitrobenzyl (CMNB) (Molecular Probes). These groups are generally attached via terminal phosphate oxygen. The moiety may be removed by any suitable means. For example if the moiety is photolabile, flash photolysis of the moiety by ultraviolet light leads to a rapid and highly localized release of the biologically active nucleotide at the site of illumination. The moiety may be heat labile, and thus the biologically active nucleotide may be released by heat. If heat is used to biologically activate the nucleotide, the temperature requirements of the polymerase must be considered, and this is within the remit of the skilled person. For example, the terminal phosphate may be esterified with a caging or blocking moiety.

[0070] The term “rolling circle amplification” (RCA) may refer to the ability of RCA-typepolymerases to continuously progress around a circular DNA template strand whilst extending a hybridized primer. This leads to formation of linear single stranded products with multiple repeats of amplified DNA. The sequence of the circular template (a single unit) may be multiply repeated within a linear product. For a circular template, the initial product of strand displacement amplification may be a single stranded concatamer, which may be either sense or antisense, depending on the polarity of the template. These linear single stranded products may serve as the basis for multiple hybridisation, primer extension, and strand displacement events, resulting in formation of concatameric double stranded DNA products, again comprising multiple repeats of amplified DNA. There are thus multiple copies of each amplified “single unit” DNA in the concatameric double stranded DNA products. RCA polymerases may be preferred for use in the process of the present disclosure. The products of RCA-type strand displacement replication processes may require processing to release single unit DNAs. This may be desirable if single units of DNA are required.

[0071] In order to allow for amplification, the DNA template may be also contacted with one or more primers. The primers may be non-specific (i.e. random in sequence) or may be specific for one or more sequences comprised within the DNA template. If the primers are of random sequence they allow for non-specific initiation at any site on the DNA template. This may allow for high efficiency of amplification through multiple initiation reactions from each template strand. Examples of random primers may be hexamers, heptamers, octamers, nonamers, decamers or sequences greater in length, for example of 12, 15, 18, 20 or 30 nucleotides in length. A random primer may be of 6 to 30, 8 to 30 or 12 to 30 nucleotides in length. Random primers may be provided as a mix of oligonucleotides, which may be representative of all potential combinations of e.g. hexamers, heptamers, octamers or nonamers in the DNA template.

[0072] In some embodiments of the present disclosure, RCA may be performed in the absence of exogenously added oligonucleotide primers.

[0073] The terms "amplification" and "replication" and their various forms are used herein interchangeably to refer to the synthesis of complementary DNA from a template DNA and thus increasing the number of copies of a template DNA.

[0074] The term “primer”, as is used herein, refers to an oligonucleotide, i.e. , short DNA or RNA nucleotides, which is effective in annealing to a template nucleic acid and act as a starting point for the addition of nucleotides in the copying process of a strand complementary to the sequence of said template nucleic acid.

[0075] The term “priming” or “prime”, as is used herein, refers to the initiation of DNA amplification, and it does not necessarily require formation of a primer (e.g. a DNA or RNA primer) . After the priming (i.e., the initiation) of the DNA amplification, a DNA polymerase can then take the work and extend the DNA product.

[0076] The term “suitable buffer”, as is used herein, refers to an aqueous buffered solution of which the pH is at a nearly constant value. A suitable buffer for replication reactions comprises a divalent metal salts, preferably a magnesium salt, such as magnesium chloride, magnesium sulphate and / or magnesium acetate.

[0077] The term “suitable condition”, as is used herein in the context of allowing amplification of a DNA template, refers to or comprises suitable concentrations of the reaction materials, and temperature and time duration of the incubation, optionally a suitable buffer comprised in the reaction materials, as discussed in more details elsewhere in the present disclosure. In light of the teachings in the present disclosure, those skilled in the art should have no difficulty in determining the suitable condition to allow amplification of a desired DNA template.

[0078] The article “a” or “an”, as is used herein, means one or more, unless specifically defined otherwise.

[0079] Provided herein are methods for amplifying a DNA template, comprising:

[0080] a) providing a composition comprising a DNA template, an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide, wherein said composition does not comprise a ribonucleotide;

[0081] b) incubating said composition in a suitable condition to allow amplification of said DNA template.

[0082] The inventors of the present disclosure have surprisingly found that DNA templated amplification can be carried out in the presence of only a RNA polymerase, aDNA polymerase, and a deoxyribonucleotide (such as in the form of one or more dNTPs, for example, one or more of dATP (adenine), dCTP (cytosine), dGTP (guanine), and dTTP (thymine), without any externally added ribonucleotide. This finding is particularly surprising in view of the fact that an RNA polymerase was used to initiate the DNA replication process and an RNA polymerase, by its definition, will naturally use rNTPs as substrates. Without being bound by any theories, the priming process in the present disclosure may or may not involve the formation of a primer.

[0083] RNA polymerases may be used in the methods of the present disclosure for, among other things, transcribing substrates in order to provide transcripts that are part of amplification cycle. Typically, RNA polymerases may utilize ribonucleotides and cannot utilize deoxyribonucleotides. In the methods of the present disclosure, e.g., in the presence of Mn++and dNTPs, RNA polymerases can incorporate deoxyribonucleotides into transcripts. RNA polymerases can be obtained from many sources, including from prokaryotes, phage, bacteriophage, eukaryotes, fungi, plants, archaebacteria, etc. RNA polymerases should be stable and active under the conditions of amplification methods.

[0084] In an embodiment, the RNA polymerase may be a wide-type or mutant RNA polymerase. The RNA polymerase can be selected from the group consisting of T7 RNA polymerase or its mutants, T3 RNA polymerase or its mutants, SP6 RNA polymerase or its mutants, K11 RNA polymerase or its mutants, or a combination thereof. In a preferred embodiment, the RNA polymerase may be T7 RNA polymerase or its mutants. In another preferred embodiment, the T7 RNA polymerase mutant can use deoxyribonucleotide as a substrate. In another preferred embodiment, the T7 RNA polymerase mutant may be DuraScribe T7 RNA polymerase. In certain embodiments, T7 RNA polymerase mutant may be competent to incorporate dNTPs into a template nucleic acid. For example, T7 RNA polymerase with a Y639F mutation (e.g., DuraScribe T7 RNA polymerase), or a S641A mutation, or Y639F / S641A mutations can be used. In certain embodiments, T7 RNA polymerase mutant may contain at least two mutations, e.g., Y639F / S641A mutations.

[0085] Inorganic Pyrophosphatase (IPP)

[0086] During DNA synthesis reaction, inorganic pyrophosphate may accumulate as a product of the reaction. If the concentration becomes too high, it can reduce the amount ofDNA synthesis due to product inhibition. IPP of the present disclosure may catalyze the enzymatic conversion of PPi into 2Pi. Thus, IPP may used in methods for producing nucleic acid molecules, such as RCA of the present disclosure and RNA in vitro transcription reactions but also in DNA sequencing reactions and cDNA transcription reactions because addition of IPP may increase transcription yields and may minimize the effect of variation of magnesium concentration (see for example Cunningham P. R. and Ofengand J. (1990) Biotechniques 9(6): 713-714). Addition of IPP to a reaction mixture (e.g., to RNA in vitro transcription reactions using DNA dependent RNA polymerases or to cDNA in vitro transcription using RNA dependent DNA polymerases) may catalyze the hydrolysis of inorganic pyrophosphate and thus may prevent its direct inhibitory action of the transcription enzyme.

[0087] IPP may be used in some embodiments of the present disclosure at a concentration of from 0.1 U / ml to 10 U / ml, from 0.1 U / ml to 5 U / ml, from 0.1 U / ml to 4 U / ml, from 0.1 U / ml to 3 U / ml, from 0.1 U / ml to 2 U / ml, from 0.1 U / ml to 1 U / ml, from 0.1 U / ml to 0.9 U / ml, from 0.1 U / ml to 0.8 U / ml, from 0.1 U / ml to 0.7 U / ml, from 0.1 U / ml to 0.6 U / ml, from 0.1 U / ml to 0.5 U / ml, from 0.1 U / ml to 0.4 U / ml, from 0.1 U / ml to 0.3 U / ml, from 0.1 U / ml to 0.2 U / ml, 0.1 U / ml, 0.2 U / ml, 0.3 U / ml, 0.4 U / ml, 0.5 U / ml, 0.6 U / ml, 0.7 U / ml, 0.8 U / ml, 0.9 U / ml, or 1 U / ml.

[0088] Manganese Ion Concentration

[0089] The presence of manganese ions may be required for methods of the present disclosure, which may involve transcription of substrates with RNA polymerase in order to incorporate dNTPs. Manganese ions may be provided in the form of a salt, e.g., manganese chloride. In some embodiments, the Mn2+may be present in a concentration of between 1 pM to 30 mM. In more preferred embodiments, Mn2+may be present in a concentration of between 10 pM to 100 pM, 10 pM to 500 pM, 10 pM to 1 mM, 10 pM to 2 mM, 10 pM to 3 mM, 10 pM to 4 mM, 10 pM to 5 mM, 10 pM to 6 mM, 10 pM to 7 mM, 10 pM to 8 mM, 10 pM to 9 mM, 10 pM to 10 mM, 10 pM to 11 mM, 10 pM to 12 mM, 10 pM to 13 mM, 10 pM to 14 mM, 10 pM to 15 mM, 10 pM to 16 mM, 10 pM to 17 mM, 10 pM to 18 mM, 10 pM to 19 mM, 10 pM to 20 mM, 1 mM to 20 mM, 2 mM to 19 mM, 3 mM to 18 mM, 4 mM to 17 mM, 5 mM to 16 mM, 6 mM to 15 mM, 7 mM to 14 mM, 8 mM to 13 mM, 9 mMto 12 mM, 9 mM to 11 mM, 9 mM to 10 mM, 10 mM to 11 mM. In a preferred embodiment, the concentration may be 10 mM.

[0090] In an embodiment, the DNA template is a single strand or double strand circular or linear DNA. The method in the present disclosure does not require specific sequences of DNA templates, thus allowing its usage on a wide range of DNA template sequences.

[0091] In another embodiment, the DNA polymerase has strand displacement activity. In a preferred embodiment, the DNA polymerase is Phi29 polymerase.

[0092] In an embodiment, incubating said composition in a suitable condition comprises incubating at 20°C to 37°C for 3-36 hours. For example, 20°C , 25°C, 30°C, 35°C, or 37°C, for 3, 6, 9, 12, 15, 18, 21 , 24, 30, or 36 hr.

[0093] In an embodiment, said composition further comprise suitable buffer. In a preferred embodiment, said buffer comprises rCutSmart™ Buffer (1X buffer components include 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, and 100 pg / ml Recombinant Albumin), and DTT. The composition of 1X rCutSmart™ Buffer is as follows: 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 pg / ml Recombinant Albumin, pH 7.9 at 25°C.

[0094] In another embodiment, a suitable buffer can comprise Potassium salt (e.g, Potassium Acetate), Tris-acetate, Magnesium salt (e.g., Magnesium Acetate), Albumin (e.g. Recombinant Albumin), each at a suitable concentration to allow DNA amplification (including DNA amplification initiation and / or the ensuing elongation), pH 6.5-8.5 (e.g., pH 7-8, such as 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9) at a temperature range of 20°C- 37 °C (e.g., 25°C-35°C, 25°C-30°C, such as 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C).

[0095] In another embodiment, a suitable buffer can comprise 25-75 mM (e.g. 30, 40, 50, 60, or 70 mM) Potassium ion, 10-40 mM (e.g. 10, 20, 30, 40 mM) Tris-acetate, 10-40 mM (e.g. 10, 20, 30, 40 mM) Magnesium ion, 50-200 pg / ml (e.g. 50, 100, 150, 200 pg / ml) Albumin, pH 6.5-8.5 (e.g., pH 7-8, such as 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9) at a temperature range of 20°C-37°C (e.g., 25°C-35°C, 25°C-30°C, such as 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C).

[0096] In another embodiment, a suitable buffer can comprise 25-75 mM (e.g. 30, 40, 50, 60, or 70 mM) Potassium Acetate, 10-40 mM (e.g. 10, 20, 30, 40 mM) Tris-acetate, IQ- 40 mM (e.g. 10, 20, 30, 40 mM) Magnesium Acetate, 50-200 pg / ml (e.g. 50, 100, 150, 200 pg / ml) Recombinant Albumin, pH 6.5-8.5 (e.g., pH 7-8, such as 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9) at a temperature range of 20°C-37°C (e.g., 25°C-35°C, 25°C-30°C, such as 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C).

[0097] In an embodiment, said composition may comprise a DNA template at a concentration of 0.1 to 10 ng / pl (e.g. 0.1 -0.5 ng / pl , 0.1 -1 ng / pl , 0.2-1 ng / pl , 0.1 -2.5 ng / pl , or 0.2-2 ng / pl , such as 0.1 , 0.25, 0.5, 0.75, 1 , 2, 3, ,4, 5, 6, 7, 8, 9 and 10 ng / pl ), an RNA polymerase at a concentration of 0.1 to 10 U / pl (e.g., 0.25-1.5 ll / pl , 0.5-1 ll / pl , 0.5-2.5 U / pl , or 0.5-5 U / pl , such as 0.1 , 0.25, 0.5, 0.75, 1 , 2, 2.5, 3, ,4, 5, 6, 7, 8, 9 and 10 U / pl ), a DNA polymerase at a concentration of 0.1 to 10 U / pl (e.g., 0.25-1.5 U / pl , 0.25-1 U / pl , 0.25-2.5 U / pl , or 0.25-5 U / pl 0.1 , such as 0.25, 0.5, 0.75, 1 , 2, 2.5, 3, ,4, 5, 6, 7, 8, 9 and 10 U / pl ), and a deoxyribonucleotide at a concentration of 0.1 -30 mM (e.g., 0.5-20 mM, 0.5- 10 mM, or 1 -10 mM, or 1 -4 mM, such as, 0.1 , 0.25, 0.5, 0.75, 1 , 2, 3, ,4, 5, 6, 7, 8, 9, 10, 20, and 30mM) .

[0098] In an embodiment, said composition in step a) further comprises at least one primer complementary to the DNA template, a mix of random primers, or a combination thereof.

[0099] In another aspect, the present disclosure relates to a method for priming DNA amplification from a DNA template, comprising:

[0100] incubating a DNA template, an RNA polymerase and a deoxyribonucleotide in a suitable condition to allow the initiation of DNA amplification;

[0101] wherein said method does not comprise adding a ribonucleotide.

[0102] In an embodiment, said method further comprises, after the incubation, adding a DNA polymerase to elongate the DNA amplification.

[0103] In another aspect, the present disclosure relates to a kit comprising:

[0104] a template DNA molecule, an RNA polymerase, a DNA polymerase, a deoxyribonucleotide, and instructions for amplifying a DNA template in the presence of anRNA polymerase, a DNA polymerase, and a deoxyribonucleotide but without a ribonucleotide.

[0105] Provided herein is also a method for amplifying a DNA template, comprising a) providing a DNA template, an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide; b) incubating the composition from step a) in a suitable condition to allow amplification of said DNA template; wherein said method does not comprise providing ribonucleotide.

[0106] The present method can generate DNA amplification product of various lengths, such as, for example, between a few bp to 200 kb, for example, 1 kb, 10 kb, 50bp, 10Obp, and etc.

[0107] The present disclosure also contemplates utilizing PrimPol in the system of the disclosure. PrimPol is a class of enzymes, termed primase-polymerases, which comprises both DNA polymerase and DNA primase activities and may be use both for primer synthesis and replication of a DNA molecule. PrimPols, unlike conventional primases, generate DNA primers (Lipps et al., 2003. EMBO J 22: 2516-2525). A PrimPol isolated from Thermus thermophiles was recently found to provide excellent results in the amplification of whole-genomes from single cells, when compared to random priming (Picher et al., 2016. Nature Comm 7: 13296).

[0108] Advantages of the present disclosure may include, for example, no need of using cells e.g. E.coli for cloning (i.e. cell-free), no need of using specific or random primers, isothermal so no need of thermocycling, no need for denaturation of templates, no requirement of specific DNA template sequences, and ease and simplicity to carry out.

[0109] For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned in these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.EXAMPLES

[0110] EXAMPLE 1

[0111] Objective: To test if the mutant T7 polymerase can prime DNA amplification in the presence of dNTPs (and in the absence of rNTPs) and if the strand displacement amplification of the template can begin with Phi29 polymerase, DNA amplification was performed under the following conditions (Table 1 ):Table 1

[0112] Condition 1 : Template EGFP plasmid DNA (5 ng) was incubated with rCutsmart™ Buffer (NEW ENGLAND BIOLABS; 1X was used in all the Examples), 1 mM DTT, 1 mM ATP and 1 mM GTP, 2 mM dNTP (a mixture of dATP, dGTP, dCTP and dTTP was used in all the experiments that used dNTP), 10 U Phi29 polymerase, and 15 U WT T7 RNA polymerase at 31 °C for 18 hours in a 20 pl reaction volume.

[0113] Condition 2: Template EGFP plasmid DNA (5 ng) was incubated with rCutsmart™ Buffer, 2 mM dNTP, 10 U Phi29 polymerase, and 1 U Reverse transcriptase at 31 °C for 18 hours in a 20 pl reaction volume.

[0114] Condition 3: Template EGFP plasmid DNA (5 ng) was incubated with rCutsmart™ Buffer, 1 mM DTT, 2 mM dNTP, 10 U Phi29 polymerase, and 200 U mutant T7 RNA polymerase [DuraScribe (Y639F)] at 31 °C for 18 hours in a 20 pl reaction volume.

[0115] Condition 4: Template EGFP plasmid DNA (5 ng) was incubated with rCutsmart™ Buffer, 1 mM DTT, 2 mM dNTP, and 10 U Phi29 polymerase at 31 °C for 18 hours in a 20 pl reaction volume.

[0116] After 18 hours of incubation, 5 pl of each reaction was loaded on a 2% EX gel (ThermoFisher) gel to observe amplification. The results from the amplification reaction are depicted in the gel image shown in FIG. 1 .

[0117] The samples in FIG. 1 are as follows:Lanes Samples1 E gel sizing ladder2 Sample from condition 13 Sample from condition 24 Sample from condition 35 Sample from condition 4

[0118] Results: The results, in particular, Lane 4, clearly show that the mutant T7 RNA polymerase initiated the DNA amplification when only dNTPs but no rNTPs were present, and the Phi29 polymerase successfully amplified the DNA template.

[0119] EXAMPLE 2

[0120] Comparison of Priming Activity of WT and Mutant T7 RNA Polymerase in the Presence of dNTPs to Enable Rolling Circle Amplification (RCA) of the Template

[0121] In this experiment, Table 2 shows that four different conditions were tested to compare the priming effect of WT T7 RNA polymerase with that of the mutant (Y639F) T7 RNA polymerase.Table 2

[0122] Condition 5: Template EGFP plasmid DNA (5 ng) was incubated with rCutsmart™ Buffer, 1 mM DTT, 1 mM ATP and 1 mM GTP, 2 mM dNTP, 10 U Phi29 polymerase, and 15 U WT T7 RNA polymerase at 31 °C for 18 hours in a 20 pl reaction volume.

[0123] Condition 6: Template EGFP plasmid DNA (5 ng) was incubated with rCutsmart™ Buffer, 2 mM dNTP, 10 U Phi29 polymerase, and 50 U WT T7 RNA polymerase at 31 °C for 18 hours in a 20 pl reaction volume.

[0124] Condition 7: Template EGFP plasmid DNA (5 ng) was incubated with rCutsmart™ Buffer, 1 mM DTT, 2 mM dNTP, 10 U Phi29 polymerase, and 200 U mutant (Y639F) T7 RNA polymerase [DuraScribe] at 31 °C for 18 hours in a 20 pl reaction volume.

[0125] Condition 8: Template EGFP plasmid DNA (5 ng) was incubated with rCutsmart™ Buffer, 1 mM DTT, 2 mM dNTP, 1 mM ATP, 1 mM GTP, and 10 U Phi29 polymerase at 31 °C for 18 hours in a 20 pl reaction volume.

[0126] After the RCA, 5 pl of each sample was loaded onto a 2% agarose EX gel to analyse the results, which is shown in FIG. 2.

[0127] The samples in FIG. 2 are as follows:Lanes Samples1 E gel sizing ladder2 Sample from condition 53 Sample from condition 64 Sample from condition 75 Sample from condition 8

[0128] Purification: Samples were purified using Yeasen size exclusion magnetic beads.A 0.4X bead-to-sample ratio was used. The beads were added to the sample and incubated for 5 minutes at room temperature (RT). The samples were then placed on amagnetic stand, and the supernatant was removed. The beads were washed twice with 80% ethanol and then resuspended in 50 pl of Tris-EDTA buffer (pH 7). After a 5-minute incubation, the supernatant was eluted. DNA from the supernatant was quantified using a Nanodrop spectrophotometer and the yields from the RCA were calculated

[0129] Results: Figures 2 and 3 demonstrate that priming activity in the presence of dNTPs occurs primarily with the mutant (Y639F) T7 RNA polymerase (condition 7 in Lane 4, FIG. 2). FIG. 3 shows that the RCA yields using only dNTPs with Phi29 polymerase and mutant (Y639F) T7 RNA polymerase (condition 7) were comparable to those obtained with rNTP, dNTP, WT T7 RNA polymerase, and Phi29 (condition 5 in Lane 2, FIG. 2). WT T7 polymerase also produced DNA amplification products as can be seen in Lane 3 (condition 6) (FIG. 2), but the yield was scarce compared with the mutant (Y639F) T7 RNA polymerase (FIG. 3).

[0130] Sanger Sequencing: Sanger sequencing confirmed the findings described above. RCA samples from conditions 5 and 7 were Sanger sequenced using EGFP-specific primers. The sequences obtained from the control condition (WT T7 RNA polymerase + rNTP + dNTP) aligned with those from the RCA conducted with mutant (Y639F) T7 RNA polymerase + dNTP (in the absence of rNTP). These results confirm the priming activity of T7 RNA polymerase (especially mutant (Y639F) T7 polymerase) in the presence of dNTPs in the absence of rNTPs.

[0131] EXAMPLE S

[0132] Digestion of RCA Products with BspQI Restriction Enzyme to Compare Digestion Patterns of RCA Samples from WT and Mutant T7 RNA Polymerases

[0133] 10 pg of purified RCA samples from conditions 5 and 7 were digested using 1X NEB3.1 buffer and 0.5 U / pl of BspQI enzyme in a 20 pl reaction. The reaction was incubated for 2 hours. After incubation, 5 pl of each sample was loaded onto a 2% agarose gel alongside a E gel sizing ladder for analysis. The results are shown in FIG. 4.

[0134] The samples in FIG. 4 are as follows:Lanes Samples1 E gel sizing ladder2 Condition 5- Bspql digested3 Condition7 - Bspql digested

[0135] Results: The digestion pattern for the RCA product from WT T7 RNA polymerase with rNTP and dNTP was identical to that from the mutant (Y639F) T7 RNA polymerase RCA reaction with dNTP alone. This suggests that the RCA products generated by condition 7 were identical to that generated by condition 5. EXAMPLE 4

[0136] dNTP TitrationTable 3

[0137] Table 3 shows RCA conditions with increasing concentration of dNTP, e.g., 2 mM (Condition #1), 3 mM (Condition #2), 4 mM (Condition #3), and 5 mM (Condition #4). The positive control contains 1 mM dNTP.

[0138] FIG. 5A shows that highest yield of RCA product was obtained from Condition # 1 with 2 mM final dNTP input (221 pg), e.g., Condition #1 (2 mM dNTP) yielded 1.4-fold more RCA product than the positive control (1 mM dNTP). Whereas yields (pg) of RCA products obtained from Condition #2 (3 mM dNTP), Condition #3 (4 mM dNTP), and Condition #4 (5 mM dNTP) were too low to be determined. RCA product yields were further tested in the presence of between 1 mM and 2.5 mM dNTP. FIG. 5B shows RCA products are detectable between 1 mM and 2.5 mM dNTP. The highest RCA product yield was obtained in the presence of 2 mM dNTP.

[0139] FIG. 6 shows that linearized RCA products obtained from Condition #1 and positive control exhibit same size, indicating that these DNA templates appear identical.

[0140] Table 4 shows that the yield increase observed during RCA with elevated dNTP concentrations correlates with higher mRNA integrity.Table 4

[0141] RCA products obtained from Condition #1 and positive control were used as DNA templates for in vitro transcription (IVT). Table 3 shows that the quality of RNA generated by using DNA templates obtained from Condition #1 (2 mM dNTP) is higher, e.g., 79.9%, than that obtained from positive control (1 mM dNTP), e.g., 68.8%.

[0142] FIG. 7 shows Luciferase expression levels of DNA templates obtained from Condition #1 (2 mM dNTP) used in IVT is greater than that obtained from positive control (1 mM dNTP), e.g., about 1.07-fold increase.

[0143] These results suggest that Conditions #1 (2 mM dNTP) may be used to generate higher yield of RCA products as DNA templates for generating RNA with higher quality in IVT and with higher functionality, e.g., higher luciferase expression levels, than positive control (1 mM dNTP), Condition #2 (3 mM dNTP), Condition #3 (4 mM dNTP), and Condition #4 (5 mM dNTP).

[0144] EXAMPLE 5

[0145] Inorganic pyrophosphatase (IPP) Titration: An experiment was conducted to evaluate the impact of inorganic pyrophosphatase (IPP) titration during the rolling circle amplification (RCA) process. In certain embodiments, IPP was added to the RCA reaction mixture to catalyze the hydrolysis of inorganic pyrophosphate (PPi), a byproduct formed upon the incorporation of each nucleotide, into two molecules of inorganic phosphate (Pi). The purpose of this enzymatic cleavage was to prevent the accumulation of pyrophosphate, which can chelate magnesium ions and lead to the precipitation of magnesium pyrophosphate, thereby potentially inhibiting the RCA reaction. By including IPP, the reaction aimed to maintain free magnesium in solution, supporting sustained polymerase activity and improving overall yield and reaction efficiency. Various concentrations of IPP were tested to determine the optimal conditions for maximizing RCA output while preserving reaction component stability.

[0146] In some embodiments, inorganic pyrophosphatase (IPP) may be included in the RCA to cleave the inorganic pyrophosphate produced after each nucleotide has beenincorporated into 2 units of the inorganic phosphate. This may ensure that magnesium remains in solution and may not precipitate as magnesium pyrophosphate.

[0147] Table 5

[0148] Table 5 shows RCA reactions in the presence of IPP at 0 ll / rnl (positive control), 1 U / ml (Condition A), 0.5 U / ml (Condition B), and 0.2 U / ml (Condition C). FIG. 8 shows that linearized RCA products and linearized plasmids obtained from Conditions A-C and Positive control exhibit same size, indicating that these DNA templates appear identical. These results suggest that IPP may have little effect on the quality of RCA products under these conditions.

[0149] FIG. 9 shows the initial yields* (pg) of RCA products obtained in the presence of IPP concentrations at 0 U / m I, 1 ll / ml, 0.5 ll / ml, and 0.2 U / ml.Table 6* Initial yields refer to yields obtained right after RCA.** Final yields refer to yields obtained after purification.

[0150] Table 6 shows the final yields (pg) of RCA products obtained in the presence of IPP concentrations of 0 U / ml, 1 U / ml, 0.5 U / ml, or 0.2 U / ml. The results show that Conditions B (0.5 U / ml IPP) increased in yield compared to Condition C (0.2 U / ml IPP) or Condition A (1 U / ml IPP).

[0151] EXAMPLE S

[0152] DNA Polymerase Comparison

[0153] Phi29 polymerase mutant (EquiPhi29) (ThermoFisher) may exhibit improved protein thermostability, reaction speed, product yield, and amplification bias. For example, EquiPhi29 may have optimal activity at about 42°C. In contrast, Phi29 may have optimal activity at about 31 °C; and T7 RNA polymerase may have optimal activity at about 37°C.

[0154] To determine effects of temperatures on RCA products generated by Phi29 or EquiPhi29, the reaction conditions at various temperatures were tested as shown in Table 7 below:Table 7Table 8ND = Not determined (concentration falls outside assay range, too low, i.e. , no amplification.)

[0155] Table 8 and FIG. 10 show that highest yield of RCA product and fold increase were obtained by using EquiPhi29 at about 37°C as compared with that using Phi29 at about 31 °C, about 37°C, and about 42°C and compared with that using EquiPhi29 at about 31 °C and about 42°C. Low or no RCA products by using Phi29 at about 37°C and about 42°C suggest that Phi29 activity may be inhibited under these conditions. In the presence of EquiPhi29, low T7 polymerase activity at 42°C, however, may contribute to lower RCA product yield as compared with that at 31 °C and 37°C. These results suggest that Equ iPh i29 yielded highest amount of RCA products at 37°C and that T7 RNA polymerase and EquiPhi29 activity may be optimized at 37°C.

[0156] EXAMPLE ?

[0157] Effect of promoter on primer free RCA

[0158] Plasmid DNA containing T7 promoter or single stranded circular DNA (ssDNA), which is promoter-less, were incubated in RCA reaction containing dNTP (2 mM), Phi29 polymerase (0.5 U), T7 RNA polymerase mutant (Y639F / S641A) (1 U), and DTT (2 mM) at 31 °C for 18 hours. RCA products was analyzed by gel electrophoresis. FIG. 11 shows that RCA products can be generated using plasmid DNA containing T7 promoter (+) and ssDNA (-), suggesting that RCA can be performed using DNA template with or without promoter. M denotes size markers.

[0159] EXAMPLE S

[0160] One-pot primer free RCA

[0161] To test whether RCA and IVT can be performed in the same test tube, experiment was conducted to compare a conventional two-step rolling circle amplification(RCA) workflow with a simplified one-pot RCA approach for downstream in vitro transcription (IVT). In the two-step method, RCA was performed on 100 ng of plasmid DNA encoding firefly luciferase (Flue) and allowed to proceed overnight (~18 hours). The RCA products were then purified and subsequently linearized using restriction enzymes prior to IVT. In contrast, the one-pot RCA method combined RCA and enzymatic linearization within the same reaction tube, eliminating the need for intermediate purification. The RCA reactions were followed directly by the addition of restriction enzymes, allowing linearization to occur in situ. Following preparation by either method, IVT was performed using the respective linearized RCA products as templates. The RNA yield from the one-pot RCA workflow was 13.77 pg, while the two-step RCA method produced 12.51 pg of RNA.

[0162] As shown in FIG. 12, the linearized DNA products from both workflows are visible and comparable, indicating successful processing in each case. These results demonstrate that one-pot RCA, without intermediate purification, can generate starting material suitable for IVT with RNA yields comparable to the traditional two-step process, offering a streamlined and efficient alternative for RNA synthesis.

[0163] EXAMPLE S

[0164] Effect of T7 RNA polymerase on primer free RCA

[0165] To test the effect of WT T7 RNA polymerase and mutant T7 RNA polymerases on primer free RCA, T7 RNA polymerases listed in Table 9 were used in RCA reactions.Table 9

[0166] RCA reactions were performed with 0.5 ng / pl DNA template, e.g., Flue plasmid, 2 mM dNTP, 0.5 U / pl Phi29 DNA polymerase, 0.2 U / ml IPP, 2 mM DTT, and T7 RNA polymerase at 31 °C for 18 hours.Table 10

[0167] Table 10 shows that RCA with Y639F / S641 A T7 RNA polymerase mutant yielded 16.3% more RCA product in initial yield than the control RCA with Y639F T7 RNA polymerase mutant. In contrast, RCA with Y639F / H784A T7 RNA polymerase mutant or S641A T7 RNA polymerase mutant yielded less RCA product.

[0168] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0169] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

Claims

CLAIMS1 . A method for amplifying a DNA template, comprising: a) providing a composition comprising a DNA template, an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide, wherein said composition does not comprise a ribonucleotide; and b) incubating said composition in a suitable condition to allow amplification of saidDNA template.

2. The method according to claim 1 , wherein the RNA polymerase is a wide-type or mutant RNA polymerase.

3. The method according to claim 1 or 2, wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase or its mutants, T3 RNA polymerase or its mutants, SP6 RNA polymerase or its mutants, K11 RNA polymerase or its mutants, or a combination thereof.

4. The method according to any one of claims 1-3, wherein the RNA polymerase is T7 RNA polymerase or its mutants.

5. The method according to claim 4, wherein the T7 RNA polymerase mutant can use deoxyribonucleotide as a substrate.

6. The method according to claim 5, wherein the T7 RNA polymerase mutant comprises at least one substitution selected from Y639F, S641A, and H784A,.

7. The method according to any one of claims 1-6, wherein the DNA template is a single strand or double strand circular or linear DNA.

8. The method according to any one of claims 1-7, wherein the DNA polymerase has strand displacement activity.

9. The method according to any one of claims 1-8, wherein the DNA polymerase is Phi29 polymerase.

10. The method according to any one of claims 1 -9, wherein the incubating said composition in a suitable condition comprises incubating at 20° C to 37° C for 3-36 hours.11 . The method according to any one of claims 1 -10, wherein said composition further comprises a suitable buffer.

12. The method according to claim 11 , where said buffer comprises potassium acetate, Tris-acetate, magnesium acetate, albumin, and DTT.

13. The method according to any one of claims 1 -12, wherein said composition comprise a DNA template at a concentration of 0.1 to 10 ng / pl, an RNA polymerase at a concentration of 0.1 to 10 U / pl, a DNA polymerase at a concentration of 0.1 to 10 U / pl, and a deoxyribonucleotide at a concentration of 0.1 to 30 mM.

14. The method according to any one of claims 1 -13, wherein said composition in step a) further comprises at least one oligonucleotide complementary to the DNA template, a mix of random oligonucleotides, or a combination thereof.

15. A method for priming DNA amplification from a DNA template, comprising: incubating a DNA template, an RNA polymerase, and a deoxyribonucleotide in a suitable condition to allow the initiation of DNA amplification; wherein said method does not comprise adding a ribonucleotide.

16. The method according to claim 15, wherein said method further comprises, after the incubation, adding a DNA polymerase to elongate the DNA amplification.

17. A kit comprising: a DNA template, an RNA polymerase, a DNA polymerase, a deoxyribonucleotide, and instructions for amplifying a DNA template in the presence of an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide and in the absence of a ribonucleotide.

18. The method of any one of claims 1 -16, wherein the DNA template does not comprise a promoter.

19. The method of any one of claims 1 -16 and 18, wherein the composition does not comprise an exogenously added oligonucleotide primer.

20. The method of any one of claims 1 -16, 18, and 19, wherein the incubating is performed in the presence of dNTP at a concentration from 0.1 mM to 10 mM, from 0.5 mM to 9 mM, from 1 mM to 8 mM, from 1 mM to 7 mM, from 1 mM to 6 mM, from 1 mM to 5 mM, from 1 mM to 4 mM, from 1 mM to 3 mM, from 1 mM to 2.5 mM, from 1 mM to 2 mM, 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, or 10 mM.

21. The method of any one of claims 1 -16 and 18-20, further comprising performing an in vitro transcription (IVT) of the amplified DNA template.

22. The method of claim 21 , wherein the amplified DNA template is purified prior to IVT.

23. The method of claim 21 , wherein the amplified DNA template is not purified prior to IVT.

24. The method of any one of claims 1 -16 and 18-23, wherein the incubating is performed in the presence of Inorganic pyrophosphatase (IPP) at a concentration of from 0.1 U / ml to 10 U / ml, from 0.1 U / ml to 5 U / ml, from 0.1 U / ml to 4 U / ml, from 0.1 U / ml to 3 U / ml, from 0.1 U / ml to 2 U / ml, from 0.1 U / ml to 1 U / ml, from 0.1 U / ml to 0.9 U / ml, from 0.1 U / ml to 0.8 U / ml, from 0.1 U / ml to 0.7 U / ml, from 0.1 U / ml to 0.6 U / ml, from 0.1 U / ml to 0.5 U / ml, from 0.1 U / ml to 0.4 U / ml, from 0.1 U / ml to 0.3 U / ml, from 0.1 U / ml to 0.2 U / ml, 0.1 U / ml, 0.2 U / ml, 0.3 U / ml, 0.4 U / ml, 0.5 U / ml, 0.6 U / ml, 0.7 U / ml, 0.8 U / ml, 0.9 U / ml, or 1 U / ml.

25. The method of any one of claims 1 -16 and 18-24, wherein the RNA polymerase does not comprise a primase activity.

26. The method of any one of claims 1 -16 and 18-25, wherein the RNA polymerase is a promoter-specific RNA polymerase.

27. The method of any one of claims 1 -16 and 18-26, wherein the RNA polymerase is a single-unit RNA polymerase.

28. The method of any one of claims 1 -16 and 18-27, wherein the DNA template is a circular DNA.

29. The method of claim 28, wherein the circular DNA is a plasmid DNA.

30. The method of any one of claims 1 -16 and 18-27, wherein the DNA template is a linear DNA.31 . A method for in vitro transcription (IVT) of a DNA template into RNA, comprising a) providing a composition comprising a DNA template, an RNA polymerase, a DNA polymerase, a deoxyribonucleotide, and a restriction enzyme, wherein said composition does not comprise a ribonucleotide; b) incubating said composition in a suitable condition to allow amplification of saidDNA template, wherein the restriction enzyme is capable of linearizing the amplified DNA template; and c) performing IVT by incubating the linearized amplified DNA template in the presence of ribonucleotides at from about 15°C to about 35°C for from about 1 hour to about 12 hours, thereby producing the RNA.

32. The method of claim 31 , wherein the performing IVT is further in the presence of a cap analogue.

33. The method of claim 31 or 32, wherein the linearized amplified DNA template is not purified prior to the performing IVT at step c).

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