Nucleic acid extraction

Abstract

The present invention relates to nucleic acid extraction, and in particular, to methods of extracting nucleic acid molecules from a nucleic acid synthesis reaction, without the need to interrupt or terminate the reaction.

Description

[0001] Nucleic Acid Extraction

[0002] The present invention relates to nucleic acid extraction, and in particular, to methods of extracting nucleic acid molecules from a nucleic acid synthesis reaction, without the need to interrupt or terminate the reaction.

[0003] All living organisms make use of DNA to store their genetic information. Most of these genes code for specific proteins which control all cellular functions, including metabolic processes, cellular signalling to allow cells to respond to their environment, the structure of cells and ultimately the organism, and also intercellular communication through cell-cell contacts and hormones. Key to translating the genetic information from the level of DNA into proteins is a molecule called messenger RNA (mRNA). Each gene is first transcribed into an mRNA which is then translated through ribosomes into a protein. The key elements of this process are very similar across all kingdoms of life from bacteria to fungi to plants to animals. Following transcription of the mRNA, so- called posttranscriptional modifications occur that further modify the primary mRNA structure, however, these posttranscriptional processes and / or their functional implications differ between kingdoms of life.

[0004] Messenger RNA is a polymer of ribonucleic acids with a backbone formed from the pentose sugar ribose and phosphate. The nucleobases adenine, guanine, cytosine and uridine are attached to these sugars via the l'C-atom. The resulting nucleosides are coupled to each other via phosphate molecules that form esters with the hydroxy group of the 3'C-atom of one ribose and of the hydroxy group of the 5'C-atom of another ribose. The 5'- and 3'C-atoms give the mRNA molecule a directionality. During transcription, the mRNA is synthesized from 5' to 3' along the DNA template. Also, translation through the ribosome occurs from 5' to 3'. One unit of the mRNA polymer consisting of nucleoside and phosphate is called a nucleotide.

[0005] As one of the structural modifications, for all eukaryotic mRNAs, a stretch of adeninenucleotides is attached to the 3'-end of the mRNA molecule. This poly-A tail plays a key role for controlling the lifetime and expression of the mRNA as the poly-A tail is bound through proteins that protect the mRNA from degradation by RNA exonucleases. Poly-A tails also occur in prokaryotes, however, in this case rather destabilize than stabilize mRNA.

[0006] Considering that (i) mRNA can code for virtually any protein and that (ii) the presence of mRNA in the cytosol is transient, thereby not posing the risk of permanent changes of the genome, and also that (iii) mRNA, unlike DNA, does not have to enter the nucleus to show expression, thus can also be applied to nondividing, differentiated cells, synthetic mRNA introduced into cells may be considered a highly interesting modality to yield cellular protein expression. In 1990, it was first demonstrated that injection of naked mRNA yielded an immune response against the protein that the mRNA encoded for, thus demonstrating that mRNAs could be applied in vivo as a message for protein production (Wolff JA, et al. Science. 1990;247:1465-1468). Two years later, it was also demonstrated that in vivo delivery of mRNA could yield expression of a bioactive protein (Jirikowski GF, et al. Science. 1992;255:996-998).

[0007] Now, more than twenty years later, the full potential of mRNA as a highly valuable molecular modality in medicine and biotechnology is broadly recognized. Following the break-through success of the mRNA-based SARS-CoV2 vaccines, Comirnaty (marketed by Pfizer / BioNTech) and SpikeVax (marketed by Moderna), RNA (in particular, mRNA) is considered to be one of the fastest growing molecular modalities in therapy development and biotechnology with a multi-billion dollar market, the ultimate scope of which is still difficult to predict but which will, to a major extent, depend on costs- of-goods, speed of development / manufacture and safety.

[0008] Costs-of-goods of RNA therapeutics and vaccines are heavily influenced by the method of manufacturing. For short RNAs, chemical, enzymatic or enzyme-supported solid- state syntheses are frequently used. However, this is not an option for longer RNAs due to cumulative inefficiencies in nucleotide additions, thereby exponentially decreasing the yield of full-length, in-frame products. The laboratory and commercial production of RNA with lengths above 150-300nt, including the vast majority of messenger RNA (mRNA), self-amplifying and circular RNA, relies on enzymatic conversion of ribonucleotide triphosphate monomers into an RNA polymer. The sequence of the resulting RNA polymer is determined by the template strand of a suitable nucleic acid polymer, usually DNA. This process is referred to as in vitro transcription or IVT for short. IVT is typically an efficient process that can transcribe more than 1000 RNA polymers from a single DNA template under most contemporary batch reaction conditions.

[0009] Despite this efficiency, industrial applications required even higher yields from the enzymatic reaction than currently or theoretically can be achieved from batch reactions to reach desired cost-levels. Since the majority of the ingredients, such as the DNA template, the RNA polymerase, RNAse-inhibitor protein, and cap-analogue molecules are the most part non-consumed or suffer only minor losses during the reaction, one approach to seek cost-effectiveness is to feed the batch with the consumed reagents. Coincidentally, the least consumed reagents make up the majority of the cost of a synthesis reaction. Another is to terminate the reaction, break the reaction mixture up into fractions and recycle specific components. Usually, the enzymes, and optionally the DNA template in case of RIMA synthesis, are the components that are recycled.

[0010] Both methods can extend the reaction or service of its ingredients, but are for reasons of solubility or volume limits, as well as inherent losses and degradation of ingredients during recycling, incapable of the magnitude in increases in yield sought after by the industry. Furthermore, the recycling process introduces additional, potentially complex process steps and the requirement to monitor the quality of the materials that have undergone one or more recycling steps.

[0011] One increasingly popular method of recycling specific components is to couple one or more components to a solid support, such as a bead or a column matrix. Examples include the coupling of the template strand of the DNA and the RNA polymerase to the same solid support (US 2023 / 0265477 Al), or coupling a restriction enzyme to a solid support (EP 3289077 Bl). The limitations to these strategies, however, include:

[0012] (i) only a limited number of components can be coupled to solid supports as the IVT process requires several components to come in close proximity (usually within a few Angstrom), meaning that a significant portion of the reagents still have to be recycled in another way,

[0013] (ii) coupling to solid supports often requires chemical modifications, which may alter the natural activities of the enzyme or reagent and may leave traces in the nucleic acid product, which may be toxic or form toxic metabolites after processing by the human body,

[0014] (iii) not all reagents are suitable for coupling to a solid support, especially reagents that need to be incorporated in the nucleic acid product, for example, capanalogues during IVT need the physical flexibility to reach the active site of the enzyme in an unencumbered manner, and

[0015] (iv) by coupling to solid supports, the maximum concentration or amount that may be added to the reaction volume may be limited to the amount of said solid support that can be added to the reaction volume or be brought in contact with said reaction volume.

[0016] Second to cost-effectiveness, turn-around time or manufacturing throughput is a relevant parameter for single, but especially multi-product, manufacturing sites. Long manufacturing cycles keep limited and valuable equipment and personnel occupied, incapable on working on the next batch or other projects / products. Often, a shorter turn-around time can be achieved by scaling the reaction, which may not always be an option for economic, equipment or physics-related reasons. Therefore, there is an increased need for technologies that enable continuous manufacturing of nucleic acid drug substance and drug product. Typical continuous manufacturing equipment utilizes continuous flow, wherein in a continuous fashion the reaction ingredients are combined in a defined ratio, and after a specified incubation time, the reaction is terminated, and the reaction mixture disassembled to extract the nucleic acid produced. Although this type of continuous manufacturing provides flexibility towards the total volume of the manufacturing run (and thus yield), the consumption of all (unless selected ingredients are recovered via a downstream process) ingredients is ratiometric to the total manufacturing volume. This results in significant waste in at least template nucleic acid and enzymes, which contribute the most labour-intensive and costly ingredients of a nucleic acid synthesis reaction. Even if such elements can be recovered, it is doubtful if that can be done at a rate that is matched to the flow rate of the continuous flow, and thus provide a meaningful recycling within the same production run. Recycling towards other manufacturing runs (of other products) is unlikely to be compatible with GMP.

[0017] Further to practical and economic realities, contemporary nucleic acid (NA) manufacturing, especially mRNA manufacturing, typically yields mixtures of various types of nucleic acids, and specific nucleic acid contaminants may, depending on the dose-level and application, be the cause of (serious) side-effects. Prime examples include double stranded RNA (dsRNA) and plasmid DNA (pDNA) contaminants generated or contained during mRNA synthesis. This problem was initially targeted during the downstream processing (DSP) step of the NA manufacturing, with high pressure liquid chromatography (HPLC) methods being among the most favoured methods. Recently, there has been a strong increase in approaches that target the source of the contaminant formation, such as by optimizing the reaction conditions (e.g., temperature, incubation period, etc.), the reaction mixture (e.g., salts, (relative) nucleotide concentrations, co-factors, etc.) and / or the enzymes (e.g., mutant RNA polymerases with a lower dsRNA production). These methods are limited for several reasons:

[0018] (i) not all contaminants can be avoided, as some are key ingredients of the reaction mixture, such as the (p)DNA template during in vitro transcription (IVT),

[0019] (ii) certain contaminants have near-identical physicochemical properties and can therefore not be easily distinguished nor separated from the intended NA species, (iii) purification of related nucleic acid species from a mixture often requires stringent conditions, which nearly always coincides with significant losses of the intended NA fraction,

[0020] (iv) column-based purification techniques cannot be scaled indefinitely, since at a certain point, the limits of the materials and / or physics are reached (e.g., the maximum amount of pressure in an HPLC system),

[0021] (v) adjustments to the reaction conditions, mixture or enzymatic components might lessen the tendency to form spurious NA by-products, but when the intended NA accumulates during the reaction, the change of side-reactions increases (e.g., the formation of dsRNA as the consequence of an RNA-dependent RNA polymerase activity hiding under the promoter / DNA-dependent RNA polymerase activity of many single subunit bacteriophage RNA polymerases), and

[0022] (vi) adjustments to the reaction conditions, mixture or enzymatic components may overlap with lower catalytic activity, thus longer reaction incubation, which by itself increases the chances of the formation of by-products.

[0023] Therefore, there is an urgent need for methods of extracting nucleic acids to enable truly continuous nucleic acid synthesis reactions, with defined nucleic acid products and high cost-efficiency.

[0024] The inventors have devised a nucleic acid (NA) purification method capable of continuously or intermittently extracting nucleic acid from a continuing batch and / or continuous nucleic acid synthesis reaction, as illustrated in Figures 1 and 2. The method utilizes sequence-specific hybridization to bind the nucleic acid of interest (NAI) to a solid support, which is subsequently captured and / or separated from the reaction volume. The method is capable of being selective for binding the NAI, and not significantly binding the template NA, enabling the reaction to proceed.

[0025] Accordingly, in a first aspect of the invention, there is provided a method of extracting a nucleic acid molecule during an active nucleic acid synthesis reaction, the method comprising carrying out the nucleic acid synthesis reaction in the presence of at least one solid support comprising at least one sequence-specific hybridization element that binds specifically to the nucleic acid molecule, thereby resulting in the capture and extraction of the nucleic acid molecule.

[0026] Advantageously, the methods of the invention achieve surprisingly rapid and selective extraction of the intended nascent nucleic acid, keeping the concentration of the accumulating nucleic acid product of the reaction at lower levels, while the overall yield is higher than typical batch and fed-batch reactions of the current state of the art. The resultant nucleic acid molecules of the claimed method are obtained in a higher purity with less consumption of expensive reagents and a simplified purification procedure, which considerably lowers the cost of the synthesis of the nucleic acid. Furthermore, the lower levels of nucleic acid side-products causes less unwanted side effects of the nucleic acid when used in vitro or in vivo applications. For example, there is less activation of the innate immune responses by dsRNA when the nucleic acid product is mRNA and is administered to a subject, for example as a vaccine.

[0027] Advantageously, the inventors have demonstrated that the nucleic acid of interest can be extracted from an active enzymatic nucleic acid synthesis reaction, without said reaction being interrupted, terminated or otherwise disturbed (e.g. slowed down).

[0028] Accordingly, the nucleic acid molecule may be extracted from an active nucleic acid synthesis reaction, without the reaction being interrupted, terminated and / or slowed down.

[0029] It will be appreciated that an "active nucleic acid synthesis reaction" is one in which the nucleic acid molecule (e.g., a DNA or RNA molecule), is being actively synthesised. In contrast, when a reaction is interrupted or terminated, the nucleic acid molecule is no longer being synthesised.

[0030] Additionally, it will be appreciated that the nucleic acid molecule is being extracted from a reaction mixture, which is continuously produced by the nucleic acid synthesis reaction. This reaction mixture is continuously active during the different steps and phases of the method, typically by maintaining a constant permissive temperature, substrate availability, and removal of waste and reaction products. The repeated capture of nucleic acid in the ongoing reaction is continued until the desired amount of nucleic acid is obtained, or the reaction is exhausted by depletion of a critical substrate, or the reaction mixture becomes impermissible due to the accumulation of a waste or salt.

[0031] Accordingly, in some embodiments, the method comprises extracting the nucleic acid molecule from a reaction mixture. The reaction mixture may be continuously active during the nucleic acid synthesis reaction. The reaction mixture may be continuously active during the extraction of the nucleic acid molecule. In one embodiment, the nucleic acid synthesis reaction is a DNA amplification reaction, typically a continuous DNA amplification reaction. The DNA amplification reaction may be an in vitro DNA amplification reaction. The DNA amplification reaction may be polymerase chain reaction (PCR), rolling circle amplification (RCA), or strand displacement amplification (SDA). The DNA amplification reaction may be an exponential or linear amplification reaction, or combination thereof.

[0032] In another embodiment, the nucleic acid synthesis reaction is an in vitro transcription reaction, typically a continuous in vitro transcription reaction.

[0033] The nucleic acid molecule may be a DNA or RNA molecule. In embodiments in which the nucleic acid synthesis reaction is a DNA amplification reaction, the nucleic acid molecule is typically a DNA molecule. In embodiments in which the nucleic acid synthesis reaction is an in vitro transcription reaction, the nucleic acid molecule is typically an RNA molecule.

[0034] The DNA may be single-stranded or double-stranded. The DNA may be coding or noncoding. The DNA may be genomic DNA (gDNA), complementary DNA (cDNA) or plasmid DNA.

[0035] The RNA may be single-stranded or double-stranded. The RNA may be coding or noncoding. The RNA may be selected from a group of RNA molecules consisting of: messenger RNA (mRNA), micro RNA (miRNA); interference RNA (RNAi); short interfering RNA (siRNA); short hairpin RNA (shRNA); anti-sense RNA; RNA aptamers; selfamplifying RNA (saRNA); coding RNA; non-coding RNA; and circular RNA.

[0036] In some embodiments, the RNA comprises mRNA. The mRNA may comprise the basic elements of the cap, a 5' UTR, a 3' UTR, optionally an IRES, a coding sequence, and a poly(A) tail of variable length.

[0037] The nucleic acid molecule may be at least at least 50 bases in length, at least 60 bases in length, at least 75 bases in length, at least 100 bases in length, at least 200 bases in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, or at least 900 bases in length. The skilled person would appreciate that when the nucleic acid molecule is double-stranded, "bases in length" will refer to the length of base pairs. The nucleic acid molecule may be at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length, at least 10,000 bases in length, at least 11,000 bases in length or at least 12000 bases in length.

[0038] In one embodiment, the nucleic acid synthesis reaction is a batch nucleic acid synthesis reaction. In another embodiment, the nucleic acid synthesis reaction is a fed-batch nucleic acid synthesis reaction. In another embodiment, the nucleic acid synthesis reaction is a continuous nucleic acid synthesis reaction.

[0039] The term "fed-batch" will be well-known to the skilled person, and can mean a process whereby one or more substrates of the reaction are fed to the reactor during the reaction and in which the enzymatic component (i.e. the nucleic acid polymerase) remains in the reactor until the end of the run. This is in contrast to a "batch" reaction in which all of the substrates are provided in the reactor at the start of a reaction, which is then allowed to proceed to completion. Fed-batch is also different to a "continuous" reaction in which the substrates are continuously fed into the reactor, and the product is continuously removed from the reactor, normally under steadystate conditions.

[0040] In one embodiment, the nucleic acid synthesis reaction is a DNA amplification reaction, typically a continuous DNA amplification reaction.

[0041] The method may comprise contacting : (i) a reaction mixture comprising a template nucleic acid sequence, a DNA polymerase, optionally a DNA primer or a plurality of DNA primers, optionally an endonuclease, a plurality of deoxynucleotide triphosphates (dNTPs) and / or a buffer, and (ii) at least one solid support comprising at least one sequence-specific hybridization element that binds specifically to the nucleic acid molecule.

[0042] The template nucleic acid sequence is typically provided with the DNA polymerase, which synthesises the product DNA molecule. In some embodiments, therefore, the template nucleic acid sequence comprises DNA. The DNA primer(s) may be singlestranded DNA that are complementary to the template nucleic acid sequence.

[0043] In some embodiments, the method comprises the use of a DNA polymerase, which may be selected from the group consisting of: Taq DNA polymerase, Pft / DNA polymerase, DNA polymerase I, Phusion DNA polymerase, KAPA HIFI DNA polymerase, T7 DNA polymerase, Sequenase 2.0 (a thioredoxin-T7 DNA polymerase hybrid with mutations), phi29 DNA polymerase, temperature-stabilised phi29 DNA polymerase, Bst DNA polymerase, Bsll DNA polymerase, Klenow, Q5 high-fidelity DNA polymerase, or a mutated variant of any of these DNA polymerases. In some embodiments, the method comprises the use of phi29 DNA polymerase or a variant thereof.

[0044] In some embodiments, the plurality of deoxynucleotide triphosphates (dNTPs) are selected from a group consisting of dATP, dGTP, dCTP and / or dTTP, or modified variants thereof. dNTPs are the building blocks of DNA.

[0045] In one embodiment, the DNA polymerase replicates at least one strand of the template nucleic acid with the plurality of dNTPs, to thereby form the product nucleic acid molecule. The product nucleic acid molecule has at least one single stranded sequence capable of binding to the sequence-specific hybridization element on the solid support. As such, the solid support comprising the sequence-specific hybridization element specifically binds to the product nucleic acid molecule, typically in a single one-pot reaction.

[0046] The contact between (i) and (ii) may be continuous or intermittent. It will be appreciated that continuous contact is contact which continues throughout the entire nucleic acid synthesis reaction. Intermittent contact may be contact that is sufficiently long and frequent to bind the desired amount of nucleic acid molecule (DNA) in the reaction mixture to the solid support. The intermittent contact may be less than 30 minutes, or typically less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute. The duration of the intermittent contact may be dictated by the mixing process, the concentration of the nucleic acid molecule, the concentration of solid support and hybridization elements thereon, and / or extraction, or combinations thereof.

[0047] Typically, when the solid support is a bead, intermittent contact may be between 15 and 30 minutes. Alternatively, when the solid support is a column, intermittent contact may be less than 1 minute.

[0048] The first contact between (i) and (ii) may occur at the start of the DNA amplification reaction. Alternatively, the first contact between (i) and (ii) may occur at a defined interval after the start of the reaction, when a first amount of nucleic acid has accumulated in the reaction. The interval may be between 15 and 30 minutes for a typical nucleic synthesis reaction. Typically, however, the interval is shorter. The interval may be less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute. Typically, the nucleic acid molecule is not allowed to accumulate, as to avoid the nucleic acid molecule becoming a template for spurious reactions.

[0049] Advantageously, the inventors have demonstrated that the nucleic acid of interest can be extracted from an active enzymatic nucleic acid synthesis reaction, without said reaction being interrupted, terminated or otherwise disturbed. This came as a surprise to the inventors since the conditions required for the nucleic acid synthesis reaction (e.g. temperature, suboptimal salt, etc.) differ significantly from what is typically used as a binding buffer for such solid supports.

[0050] Accordingly, the contacting of (i) and (ii) may occur at reaction conditions conducive to the nucleic acid synthesis reaction (e.g. temperature, suboptimal salt, etc.). The temperature of the reaction permissible for binding of the produced nucleic acid molecule to the solid support is dictated by the length of the sequence-specific hybridization elements, the salt concentration, and the permissible temperature range of the nucleic acid polymerase.

[0051] Typically, longer sequence-specific hybridization elements provide higher affinity and permit the use of higher temperatures. For hybridization elements with a length of 10- 30 nucleotides, a temperature range of 37-75°C may be used. Typically, higher temperatures require a longer sequence-specific hybridization element. Accordingly, in some embodiments, the contacting of (i) and (ii) may occur at a temperature between 37-75°C, typically when the sequence-specific hybridization element has a length of 10-30 nucleotides.

[0052] The salt concentration may be selected from those salt concentrations that are permissible for the nucleic acid polymerase. The salt concentration may range from lOmM to IM. Accordingly, in some embodiments, the contacting of (i) and (ii) may occur at a salt concentration between lOmM and IM.

[0053] The permissible temperature range of the nucleic acid polymerase may be between 30-75°C. For example, the temperature may be between 30-37°C (e.g., phi29 DNAP, T7 DNAP, T7 RNAP, T3 RNAP, SP6 RNAP), 42°C (temperature-stabilized phi29 DNAP), 50°C (temperature stabilized T7 RNAP, e.g., HiT7 by NEB), 50-60°C (Bst DNAP), or 67- 75°C (Taq DNAP, Phusion DNAP, Pft / DNA polymerase, KAPA HiFi DNA polymerase, Q5 high-fidelity DNA polymerase).

[0054] In some embodiments, the (d)NTP concentration may be chosen so that during the majority of the reaction run, the concentration remains two times above the Km to ensure the reaction is performing at Vmax. Furthermore, in some embodiments, the concentration of the (d)NTPs multiplied by the volume is at least equal to the desired yield of produced nucleic acid.

[0055] The contacting of (i) and (ii) does not interrupt, terminate and / or slow down the reaction.

[0056] In another embodiment, the nucleic acid synthesis reaction is an in vitro transcription reaction, typically a continuous in vitro transcription reaction.

[0057] In one embodiment, the method comprises contacting : (i) a reaction mixture comprising a template nucleic acid sequence, an RNA polymerase, a plurality of nucleotide triphosphates (NTPs) and / or a buffer, and optionally a cap-analogue, and (ii) at least one solid support comprising at least one sequence-specific hybridization element that binds specifically to the nucleic acid molecule.

[0058] The template nucleic acid sequence is typically transcribed by the RNA polymerase to produce the RNA molecule. In some embodiments, therefore, the template nucleic acid sequence comprises DNA. The DNA may comprise a promoter suitable for the RNA polymerase, wherein the promoter is suitably located upstream of the nucleic acid sequence templating for the desired RNA molecule. The template nucleic acid may be made synthetically, for example by PCR, or doggybone DNA. The template nucleic acid may comprise a vector, and is typically a plasmid. The template nucleic acid may comprise a restriction site or other suitable linearization site to allow for run-off transcription by the RNA polymerase.

[0059] In some embodiments, the method comprises the use of an RNA polymerase, which may be selected from the group consisting of: T7; T3; SP6; KP34; Syn5; Vsw-3; temperature stabilised T7 RNA polymerase (e.g. HiT7 by NEB); or other DNA- dependent RNA polymerase; or a mutated variant of any of these RNA polymerases. In some embodiments, the method comprises the use of T7 RNA polymerase or a variant thereof. In an embodiment of the invention, more than one RNA polymerase is used simultaneously in the reaction (typically the IVT reaction) to transcribe multiple RNA sequences from multiple DNA templates or the same RIMA sequence as multiple RNA molecules with different properties.

[0060] In some embodiments, the plurality of nucleotide triphosphates (NTPs) is selected from a group consisting of ATP, GTP, CTP and / or UTP, or modified variants thereof, including pseudollTP, Nl-methyl-UTP, m5CTP, m6ATP, mlATP, Inosine triphosphate, hm5CTP, mlGTP, m7GTP, or m6AmTP. NTPs are the building blocks of RNA.

[0061] In one embodiment, the RNA polymerase transcribes the template nucleic acid with the plurality of NTPs and the optional cap analogue, to thereby form the (capped) RNA molecule. The RNA molecule has at least one single stranded sequence capable of binding to the sequence-specific hybridization element on the solid support. As such, the solid support comprising the sequence-specific hybridization element binds specifically to the RNA molecule, typically in a single one-pot reaction.

[0062] The contact between (i) and (ii) may be continuous or intermittent. It will be appreciated that continuous contact is contact which continues throughout the entire nucleic acid synthesis reaction. Intermittent contact may be contact that is sufficiently long and frequent to bind the desired amount of nucleic acid molecule (RNA) in the reaction mixture to the solid support. The intermittent contact may be less than 30 minutes, or typically less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute. The duration of the intermittent contact may be dictated by the mixing process, the concentration of the nucleic acid molecule, the concentration of solid support and hybridization elements thereon, and / or extraction, or combinations thereof.

[0063] Typically, when the solid support is a bead, intermittent contact may be between 15 and 30 minutes. Alternatively, when the solid support is a column, intermittent contact may be less than 1 minute.

[0064] The first contact between (i) and (ii) may occur at the start of the IVT reaction. Alternatively, the first contact between (i) and (ii) may occur at a defined interval after the start of the reaction, when a first amount of nucleic acid has accumulated in the reaction. The interval may be between 15 and 30 minutes for a typical IVT reaction. Typically, however, the interval is shorter. The interval may be less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute. Typically, the amount of RNA produced in such interval does not exceed the total binding capacity of the sequence-specific hybridization elements of the solid support added to the reaction mixture. Typically, the RNA is not allowed to accumulate as to avoid the RNA becoming a template for spurious RNA-dependent RNA polymerase activity, such as dsRNA.

[0065] Advantageously, the inventors have demonstrated that the nucleic acid of interest can be extracted from an active enzymatic nucleic acid synthesis reaction, without said reaction being interrupted, terminated or otherwise disturbed. This came as a surprise to the inventors since the conditions required for the nucleic acid synthesis reaction (e.g. temperature, suboptimal salt, etc.) differ significantly from what is typically used as a binding buffer for such solid supports.

[0066] Accordingly, the contacting of (i) and (ii) may occur at reaction conditions conducive to the nucleic acid synthesis reaction (e.g. temperature, suboptimal salt, etc.). The temperature of the reaction permissible for binding of the produced nucleic acid is dictated by the length of the sequence-specific hybridization elements, the salt concentration, and the permissible temperature range of the nucleic acid polymerase.

[0067] Typically, longer sequence-specific hybridization elements provide higher affinity and permit the use of higher temperatures. For hybridization elements with a length of 10- 30 nucleotides, a temperature range of 37-75°C may be used. Typically, higher temperatures require a longer sequence-specific hybridization element. Accordingly, in some embodiments, the contacting of (i) and (ii) may occur at a temperature between 37-75°C, typically when the sequence-specific hybridization element has a length of 10-30 nucleotides.

[0068] The salt concentration may be selected from those salt concentrations that are permissible to the RNA polymerase. The salt concentration may range from lOmM to lOOmM. Typically, the salt concentration is less than 50mM. Accordingly, in some embodiments, the contacting of (i) and (ii) may occur at a salt concentration between lOmM and lOOmM. Typically, the contacting of (i) and (ii) may occur at a salt concentration of less than 50mM.

[0069] The permissible temperature range of the RNA polymerase may be between 30-50°C. For example, the temperature may be between 30-37°C (e.g., T7 RNAP, T3 RNAP, SP6 RNAP), or 42°C-50°C (temperature stabilized T7 RNAP, e.g., HiT7 by NEB).

[0070] In some embodiments, the NTP concentration is typically chosen so that during the majority of the reaction run, the concentration remains two times above the Km to ensure the reaction performing at Vmax. Furthermore, in some embodiments, the concentration of the NTPs multiplied by the volume is at least equal to the desired yield of produce nucleic acid. For example, a typical IVT reaction contains 0.5-10mM of each NTP, 40 mM Tris-HCI at pH 7.5-8.3, typically at pH 7.9-8.0, 1-40 mM MgCI?, 1- lOmM DTT or alternative reducing agent, optionally 1-10 mM spermidine, and is performed at 37°C.

[0071] The contacting of (i) and (ii) does not interrupt, terminate and / or slow down the reaction.

[0072] The at least one solid support may be continuously or intermittently present in the nucleic acid synthesis reaction. It will be appreciated that continuously present means that the solid support is always present in the reaction mixture when the nucleic acid synthesis reaction is taking place. In contrast, intermittently present, means that the solid support is continuously fed and removed from the reaction mixture. As such, the solid support is only present in the reaction mixture for a time that is sufficiently long to bind the desired amount of nucleic acid molecule. Intermittently present may be less than 30 minutes, or typically less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute. The duration of the intermittent contact may be dictated by the mixing process, the concentration of the nucleic acid molecule, the concentration of solid support and hybridization elements thereon, and / or extraction, or combinations thereof.

[0073] In an embodiment in which the solid support is a column, the solid support may be continuously present (i.e., the reaction is performed in the column material and the reaction mixture may be flowed to unsaturated sections of the column or to another column). Alternatively, the column may be intermittently present (i.e., the reaction mixture incubates in a reservoir until sufficient nucleic acid is accumulated and then the reaction mixture is flowed through the column). The column may be situated on walls of a reaction chamber and / or microfluidic channels of an apparatus.

[0074] In an embodiment in which the solid support is a bead, the solid support may be continuously present (e.g. continuous feed and removal of beads), or intermittently present.

[0075] In some embodiments, the nucleic acid molecule is extracted at a rate substantially equal to the rate of nucleic acid synthesis. This is done by providing a suitable amount of at least one solid support comprising at least one sequence-specific hybridization element that binds specifically to the nucleic acid molecule.

[0076] In one embodiment, the nucleic acid extraction capacity present in the nucleic acid synthesis reaction is ratiometrically constant to the amount of unbound nucleic acid. It will be appreciated that unbound nucleic acid is any nucleic acid molecule which is not bound to the solid support.

[0077] Alternatively, in another embodiment, the nucleic acid extraction capacity present in the nucleic acid synthesis reaction is at any time a surplus relative to the amount of nucleic acid molecule available for binding. In one embodiment, such surplus may be ratiometric to the amount of unbound nucleic acid, for example being always twice as much as unbound nucleic acid.

[0078] A suitable amount of at least one solid support comprising at least one sequencespecific hybridization element may be a high total volume of the at least one solid support comprising at least one sequence-specific hybridization element relative to the nucleic acid synthesis reaction volume.

[0079] Accordingly, in some embodiments, a total volume of the at least one solid support comprising at least one sequence-specific hybridization element relative to the nucleic acid synthesis reaction mixture volume may be >5%, >10%, >25%, >50%, or 100% (meaning 1 :1) of the nucleic acid synthesis reaction mixture volume present at any given time. In other words, a total volume of the at least one solid support comprising at least one sequence-specific hybridization element relative to the nucleic acid synthesis reaction mixture volume may be 0.05: 1, 0.1 : 1, 0.25: 1, 0.5: 1 or 1 : 1.

[0080] In some embodiments, the nucleic acid synthesis reaction produces >lmg / ml / h, >2mg / ml / h, >5mg / ml / h, >7mg / ml / h, or >10mg / ml / h of RIMA. In some embodiments, the nucleic acid synthesis reaction comprises a total nucleic acid extraction capacity of >lmg / ml / h, >2mg / ml / h, >5mg / ml / h, >7mg / ml / h, or >10mg / ml / h.

[0081] Advantageously, the inventors have discovered that alternating the orientation of the magnetic attraction to repeatedly pull the purification beads through the reaction mixture, increases the exposure of the purification beads to the nucleic acid, and enhances mixing of the reaction mixture for the benefit of the overall reaction. The same may be achieved with a mixing element, vortex-inducing movement of the entire reaction vessel, (micro)vibration, or any other method known to those skilled in the art.

[0082] Accordingly, in one embodiment, the method may comprise mixing the reaction mixture. The reaction mixture may be mixed with a mixing element, vortex-inducing movement of the entire reaction vessel, (micro)vibration, magnetic force, or any other method known to those skilled in the art.

[0083] In an embodiment in which the solid support is a magnetic bead, the method comprises applying an external magnetic force to the reaction mixture. In one embodiment, the method comprises alternating the direction of the magnetic force on the reaction mixture.

[0084] In one embodiment, the method comprises applying a continuous magnetic gradient over at least 1 axis of a reaction chamber containing the reaction mixture, such that it causes the solid support to flow through at least a section of the reaction chamber. In some embodiments, this continuous magnetic gradient comprises a static magnet.

[0085] Advantageously, this offers a simpler method of moving magnetic or magnetized solid supports through the reaction volume than by means of a moving magnet, not requiring electric energy when performed with permanent magnets, and providing a constant gradient force on the magnetic or magnetized solid support throughout the reservoir. This constant gradient force, when combined with a constant addition of fresh or recycled solid supports, enables a constant presence of solid support in the solution and thus a constant extraction capacity.

[0086] Subsequent to the capture of the nucleic acid, the solid support is removed from the reaction mixture, or the solid support and the reaction mixture are separated. This allows the bound nucleic acid molecule to be eluted by contacting the solid support with an optional washing and subsequent release solution.

[0087] Examples of methods to separate the solid supports from the reaction mixture are shown in Figure 3. The skilled person would recognise that a large number of combinations and variants can be made of the method of separating the reaction mixture and the solid supports, and all are explicitly incorporated herein. Furthermore, Figure 3 teaches the main principles of the method of separating the reaction mixture and the solid supports, and the skilled person will recognise that various connection types can be made between reservoirs, the nature, size and shape of the reservoirs / containers may be varied, and that reservoirs may be connected by alternative (functional) elements (such as homogenizers, flow-cytometers, particle counters, QC-elements, etc.) resulting in the method of the invention.

[0088] The solid support and the reaction mixture may be separated by removing the solid support from the mixture via filtration, centrifugation, column binding, or magnetic attraction. Typically, the solid support and reaction mixture are separated by magnetic attraction.

[0089] It will be appreciated that the nucleic acid synthesis reaction, and therefore the reaction mix, is in a first container / reservoir. Accordingly, in one embodiment, the method may comprise transferring the reaction mixture and at least one solid support to a secondary container / reservoir. The solid support and the reaction mixture may be separated by removing the solid support from the mixture via filtration, centrifugation, column binding, or magnetic attraction. The solid support may be retained in the secondary container / reservoir, and the reaction mixture may be transferred back to the first container / reservoir, or a new (third) container / reservoir, to continue the nucleic acid synthesis reaction. The separated solid support may be optionally washed and / or further processed. In some embodiments, new and / or recycled solid supports may be introduced into the first container / reservoir, before the reaction mixture is returned to the first container / reservoir.

[0090] Alternatively, the reaction mixture and the solid support may be separated by moving the reaction mixture to a secondary container / reservoir. In this embodiment, the at least one solid support may be retained in the first container / reservoir in which the reaction originally took place. The retained solid support may then be processed in the first container / reservoir (e.g. via filtration, centrifugation, column binding, or magnetic attraction). Alternatively, the at least one solid support may be transferred to a new (third) container / reservoir for processing. In some embodiments, the secondary container / reservoir may already contain, or receive, new and / or recycled solid supports before the reaction mixture is received. This results in the combination of new and / or recycled solid supports with the reaction mixture.

[0091] Alternatively, the reaction mixture and the solid support may be separated by moving the reaction mixture to a secondary container / reservoir, and then to a third container / reservoir. In this embodiment, the at least one solid support may be retained in the first container / reservoir in which the reaction originally took place. The retained solid support may then be processed in the secondary container / reservoir (e.g. via filtration, centrifugation, column binding, or magnetic attraction). In some embodiments, the third container / reservoir may already contain, or receive, new and / or recycled solid supports before the reaction mixture is received. This results in the combination of new and / or recycled solid supports with the reaction mixture. In some embodiments, the third container / reservoir is connected to the first container / reservoir and receives the reaction mixture directly from the first container / reservoir (optionally combined with fresh and / or recycled solid supports).

[0092] It will be appreciated that by removing the solid support from the reaction mixture, any bound nucleic acid molecule will simultaneously be removed from the reaction mixture.

[0093] As the nucleic acid molecule produced by the nucleic acid synthesis reaction is often significantly longer than the hybridization element or a plurality of hybridization elements on the solid support, the nucleic acid molecule is not considered to be removed from participation in the reaction upon mere binding to the solid support. As such, the method typically involves repeatedly and / or frequently extracting the solid support (and therefore bound nucleic acid molecule) from the reaction mixture, to prevent or limit spurious reactions.

[0094] Accordingly, in some embodiments, the method comprises repeatedly and / or frequently extracting the solid support (and bound nucleic acid molecule) from the reaction mixture. The frequency and number of extractions is typically at least equal to the total productivity of the reaction divided by the amount of nucleic acid extracted per amount per extraction by the addition of the solid support. Typically, the interval between extractions and amount of extracted nucleic acid per extraction is matched to the amount of nucleic acid accumulating during that interval. For example, in a batch reaction, wherein in 2.5 hours the total available (d)NTP pool is depleted and converted into nucleic acid, 10 extractions, every 15 minutes is performed with an amount of solid support capable of extracting more than l / 10thof the total productivity of the reaction.

[0095] Subsequent to the removal, or separation, of the solid support from the reaction mixture, the solid support may be optionally washed and subsequently exposed to a release and / or elution solution, wherein said solution carries the released nucleic acid away from the solid support. Accordingly, in one embodiment, the method further comprises washing the solid support. Washing may comprise the use of a washing buffer. The washing buffer may comprise a low concentration of salt, wherein the salt maintains the hybridization of the nucleic acid to the sequence-specific hybridization element. Suitable salts may be selected from the group consisting of: Lithium chloride; Sodium chloride; potassium chloride; and other monovalent salts. The salt concentration may be between 0.1-1M, 0.1-0.5M, 0.1-0.25M, or 0.1-0.15M.

[0096] Examples of suitable washing buffers include: i) buffer A: 10 mM Tris, pH 7.4, 0.1 M NaCI, and optionally 1.0 mM EDTA, and / or optionally 0.1% LiDS; and ii) buffer B: lOmM Tris, pH7.4, 0.1M LiCI, and optionally l.OmM EDTA, and / or optionally 0.1% LiDS.

[0097] Alternatively, the washing buffer may be a combination of an organic solvent and water, such as 70% Ethanol or 80% Ethanol, or 70% isopropanol.

[0098] In one embodiment, the method further comprises exposing the solid support to a release solution and / or elution solution. In one embodiment, the method comprises repeatedly exposing the solid support to a release solution and / or elution solution for a second and / or third time. The release solution may be laboratory grade water, typically RNAse-free water, without any additions. Alternatively, the release solution may be 10 mM Tris-HCI, pH 7.5, or 1 mM Citrate, pH 6.0, or 1 mM Citrate, 1 mM EDTA pH 6.0, or 5 mM Tris, 1 mM EDTA pH 8.0, or any other low-salt aqueous solution.

[0099] The release solution may contain a limited concentration (e.g. less than lOOmM, less than 50mM, less than 25mM, or less than lOmM) or no salt, reducing the hybridization of the nucleic acid molecule to the solid support, thereby stimulating the release of the nucleic acid molecule.

[0100] Alternatively, the release solution may contain an endonuclease which digests the nucleic acid molecule, the sequence-specific hybridization element, or a combination thereof. Such endonucleases may be selected from the group consisting of: restriction enzymes, TALEN, Zinc-finger nucleases, and CRISPR-Cas.

[0101] Typically, the endonuclease is a restriction enzyme. The restriction enzyme may be selected from the group consisting of: Aatll, Acc65I, AccI, Acil, AcII, Acul, Afel, Aflll, AflUI, Agel-HF, AhdI, Alel-v2, Alul, Alwl, AlwNI, Apal, ApaLI, ApeKI, ApoI-HF, Asci, Asel, AsiSI, Aval, Avail, Avril, BaeGI, Bael, BamHI, BamHI-HF, BanI, Banll, BbsI, BbsI-HF, BbvCI, Bbvl, Bed, BceAI, Bcgl, BciVI, Bell, BclI-HF, BcoDI, Bfal, BfuAI, Bgll, Bglll, Blpl, BmgBI, BmrI, BmtI-HF, Bpml, BpulOI, BpuEI, BsaAI, BsaBI, BsaHI, Bsal- HF v2, BsaJI, BsaWI, BsaXI, BseRI, BseYI, Bsgl, BsiEI, BsiHKAI, BsiWI, BsiWI-HF, BslI, BsmAI, BsmBI-v2, BsmFI, BsmI, BsoBI, Bspl286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI-v2, BsrGI-HF, BsrI, BssHII, BssSI-v2, BstAPI, BstBI, BstEII-HF, BstNI, BstUI, BstXI, BstYI, BstZ17I-HF, Bsu36I, Btgl, BtgZI, BtsCI, BtsIMutl, Btsl-v2, Cac8I, Clal, CspCI, CviKI-1, CviQI, Ddel, Dpnl, DpnII, Dral, Dralll- HF, DrdI, Eael, Eagl-HF, Earl, Ecil, Eco53kl, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRI- HF, EcoRV, EcoRV-HF, Esp3I, Fatl, Faul, Fnu4HI, FokI, Fsel, FspI, Haell, Haelll, Hgal, Hhal, Hindi, Hindlll, Hindlll-HF, Hinfl, HinPlI, Hpal, Hpall, HphI, Hpyl66II, Hpyl88I, Hpyl88III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, I-Ceul, I- Scel, KasI, KpnI-HF, Mbol, MboII, Mfel-HF, MIuCI, MluI-HF, Mlyl, Mmel, MnII, Msd, Msel, MslI, MspAlI, MspI, MspJI, Mwol, Nael, Narl, Nb. BbvCI, Nb.BsmI, Nb. BsrDI, Nb.BssSI, Nb.BtsI, Neil, Ncol, NcoI-HF, Ndel, NgoMIV, Nhel-HF, Nlalll, NIaIV, NmeAIII, Notl, Notl-HF, NruI-HF, Nsil, Nsil-HF, NspI, Nt.AIwI, Nt. BbvCI, Nt. BsmAI, Nt. BspQI, Nt.BstNBI, Nt.CviPII, Pad, PaeR7I, PaqCI, Pcil, PfIFI, PfIMI, PI-PspI, PI- Scel, Piel, PluTI, Pmel, Pmll, PpuMI, PshAI, Psil-v2, PspGI, PspOMI, PspXI, PstI, Pstl- HF, PvuI-HF, PvuII, PvuII-HF, Rsal, RsrII, Sad-HF, Sadi, Sall, Sall-HF, SapI, Sau3AI, Sau96I, Sbfl-HF, Scal-HF, ScrFI, SexAI, SfaNI, Sfd, Sfil, Sfol, SgrAI, Smal, Smll, SnaBI, Spel-HF, SphI, Sphl-HF, Srfl, SspI-HF, Stul, StyD4I, Styl-HF, Swal, Taql-v2, Tfil, Tsel, Tsp45I, TspMI, TspRI, Tthllll, Xbal, Xcml, Xhol ,XmaI, XmnI, and Zral.

[0102] In some embodiments, the endonuclease is a type Ils restriction enzyme, typically a type Ils nickase digesting only the captured nucleic acid molecule of interest, releasing the hybridization element on the solid support for re-use. Examples of suitable endonucleases include: Acul, Alwl, Bael, BbsI, Bbvl, Bed, BceAI, Bcgl, BciVI, BcoDI, BfuAI, BmrI, Bpml, BpuEI, Bsal, BsaXI, BseRI, Bsgl, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, BtsIMutl, CspCI, Earl, Ecil, Esp3I, Faul, FokI, Hgal, HphI, HpyAV MboII, Mlyl, Mmel, MnII, NmeAIII, PaqCI, Piel, SapI, SfaNI, or isoschizomers, mutants or improvements thereof.

[0103] In some embodiments, the endonuclease is a nickase. Examples of suitable nickases include: Nt. BspQI, Nt.CviPII, Nt.BstNBI, Nb. BsrDI, Nb.BtsI, Nt.AIwI, Nb. BbvCI, Nt. BbvCI, Nb.BsmI, Nb.BssSI, Nt. BsmAI, preferably Nt. BspQI, Nt.BstNBI, Nt. BsmAI, or mutants / improvements thereof. Examples of suitable type Ils nickases include: Nt. BspQI, Nt.BstNBI, and Nt. BsmAI. In some embodiments, the endonuclease may be specific for the unique hybrid between the sequence-specific hybridization element and the captured nucleic acid. For example, the endonuclease may be specific for a hybrid sequence (e.g., an endonuclease sequence is completed by the hybridization), a secondary structure, or an identity of the backbone (e.g., an RNA-DNA hybrid).

[0104] The above methods may be further aided by the addition of small molecules to disrupt the hybridization (e.g., DMSO, (chaotropic) salts), and / or by heating the solution. Accordingly, the method may comprise the use of small molecules and / or heat to remove the nucleic acid from the solid support. The small molecules may be, for example, DMSO or (chaotropic) salts. Optionally, the solid support is heated to a temperature at or above the melting temperature of the nucleic acid moleculehybridization element hybrid. In one embodiment, the release of the captured nucleic acid is performed at room temperature or under isothermal conditions (e.g., a temperature similar to the temperature under which the nucleic acid synthesis method is performed).

[0105] After release of the solid support from the reaction mixture, the reaction mixture may be further incubated to produce additional nucleic acid molecules. It will be appreciated, therefore, that the nucleic acid synthesis reaction is a continuous nucleic acid synthesis reaction. Accordingly, in one embodiment, the method comprises further incubating the reaction mixture after separation from the solid support.

[0106] Furthermore, upon partial or complete release of the captured nucleic acid from the sequence-specific hybridization elements on the solid support, the solid support may be reused in the capture of new nucleic acid molecules from the reaction mixture by re-establishing contact with the reaction mixture. Any losses or diminishing binding capacity / efficiency may be compensated for by adding a compensating amount of fresh solid support, or fresh hybridization elements linked to recycled solid supports.

[0107] Accordingly, in one embodiment, the method comprises recycling the at least one solid support in the nucleic acid synthesis reaction. For example, the method may comprise combining the reaction mixture with at least one recycled solid support. In some embodiments, the method comprises combining the reaction mixture with at least one new solid support and / or at least one recycled solid support. In some embodiments, the method comprises combining the reaction mixture with at least one recycled solid support comprising at least one new sequence-specific hybridization element. In one embodiment, after removal of the solid support from the reaction mixture, the reaction mixture is immediately combined with at least one new solid support and / or at least one recycled solid support.

[0108] The solid support may be a bead, a column matrix, a container-surface modification, a hollow fibre, or a filter, membrane or sponge. Typically, the solid support is a bead. Examples of solid supports according to the invention are shown in Figure 4.

[0109] The bead may be a solid bead, a magnetic bead, a superparamagnetic bead, a solid bead comprising a superparamagnetic core, a surface-enlarged bead, or a porous bead. Typically, the bead is a superparamagnetic bead.

[0110] The bead may comprise a diameter of between 1 and 10 pm, between 1 and 5 pm, or between 1 and 2 pm. Typically, the bead comprises a diameter of 1 pm.

[0111] The at least one solid support may be at least two, three, four or five solid supports. The at least one solid support may be at least six, seven, eight, nine or ten solid supports. The at least one solid support may be at least 15, 20, 25 or 30 solid supports. The at least one solid support may be at least 100, 1000, 10,000 or 100,000 solid supports. The at least one solid support may be at least 100,000,000 or at least 100,000,000,000 solid supports.

[0112] In an embodiment in which the solid support is a column, typically the method comprises the use of one column.

[0113] Alternatively, in an embodiment in which the solid support is a bead, typically the method comprises the use of at least at least 100, 1000, 10,000, 100,000, 100,000,000 or 100,000,000,000 beads.

[0114] In some embodiments, the surface of the bead comprises polyvinylalcohol (PVA).

[0115] Advantageously, with the claimed method, the solid support binds rapidly to the nucleic acid molecule, e.g., in less than 1 second.

[0116] Accordingly, in some embodiments, the contact time of the solid support with the nucleic acid synthesis reaction is about 15 minutes, <15 minutes, <10 minutes, <5 minutes, <3 minutes, < 1 minute, <30 seconds, or <15 seconds. In some embodiments, the solid support binds the nucleic acid molecule in <5 minutes, <3 minutes, < 1 minute, <30 seconds, <15 seconds, <10 seconds, <5 seconds, or <1 second.

[0117] In some embodiments, the nucleic acid molecule is eluted from the solid support in about 5 minutes, <5 minutes, <3 minutes, <1 minute, <30 seconds, <15 seconds, <10 seconds, <5 seconds, <3 seconds, or <1 second.

[0118] In some embodiments, the nucleic acid molecule is eluted from the solid support by heat. Typically, this heat is above the melting temperature of 1 or 2 partially or fully bound oligodT strands on the solid support. In some embodiments, the nucleic acid molecule is eluted at >20°C, >30°C, >40°C, >50°C, or >60°C. In some embodiments, the nucleic acid molecule is eluted at 65°C.

[0119] In some embodiments, the method comprises repeatedly and / or frequently eluting the nucleic acid molecule from the solid support.

[0120] Advantageously, the combination of the rapid binding, rapid elution and the lack of a regeneration requirement, enables fast cycling times for the solid support and increases the efficiency of the system.

[0121] The at least one sequence-specific hybridization element may be a DNA oligonucleotide, an RIMA oligonucleotide, or comprise or consist of one or more nonnatural nucleotides, termed Xeno nucleic acids (XNA).

[0122] The at least one sequence-specific hybridization element may be selected from the group consisting of: a single stranded nucleic acid, a single stranded nucleic acid with a 3' modification, a single stranded nucleic acid with a modified sugar-phosphate backbone, a single stranded nucleic acid with a structural modification, and a hybrid single and double stranded sequence. Examples of sequence-specific hybridization elements according to the invention are shown in Figure 5.

[0123] Advantageously, the method is capable of being selective for binding the nucleic acid of interest, and not significantly binding the template nucleic acid, enabling the reaction to proceed. Accordingly, in one embodiment, the sequence-specific hybridization element is capable of selectively binding the nucleic acid molecule. Typically, the sequencespecific hybridization element does not bind to the template nucleic acid.

[0124] The at least one sequence-specific hybridization element may be at least two, three, four or five sequence-specific hybridization elements. The at least one sequencespecific hybridization element may be at least six, seven, eight, nine or ten sequencespecific hybridization elements. The at least one sequence-specific hybridization element may be at least 100, 1000, 10,000 or 100,000 sequence-specific hybridization elements. The at least one sequence-specific hybridization element may be at least 100,000,000 or at least 100,000,000,000 sequence-specific hybridization elements.

[0125] In one embodiment, the at least one sequence-specific hybridization element on the solid support is a DNA oligonucleotide (DNA oligo).

[0126] The DNA oligo may be partly or completely modified, for example with phosphothioate linkages. Advantageously, this increases resistance to cleavage, especially when the reaction mixture contains one or more endonucleases, or when the captured nucleic acid is released from the DNA oligo with an endonuclease.

[0127] In one embodiment, the DNA oligo is an oligo dA sequence. Typically, when the nucleic acid synthesis reaction is a DNA amplification reaction (producing a template for IVT wherein the IVT template contains a poly(T) sequence), the DNA oligo is an oligo dA sequence. Oligo dA sequences are oligonucleotides that contain a segment of repeating deoxyadenosine (dA). The dA anneal to the polythymine (polyT) tails of the IVT templates encoding for mRNA, including an encoded poly(A) (thus encoded poly(T) in the template). Accordingly, in one embodiment, the sequence-specific hybridization element (the oligo dA sequence) specifically binds to the polyT tail of the nucleic acid molecule.

[0128] In another embodiment, the DNA oligo is an oligo dT sequence. Typically, when the nucleic acid synthesis reaction is an in vitro transcription reaction, the DNA oligo is an oligo dT sequence. Oligo dT sequences are oligonucleotides that contain a segment of repeating deoxythymidines (dT). The dT anneal to the polyadenosine (polyA) tails of mRNA. Accordingly, in one embodiment, the sequence-specific hybridization element (the oligo dT sequence) specifically binds to the polyA tail of the nucleic acid molecule. In another embodiment, the at least one sequence-specific hybridization element on the solid support is an RIMA oligonucleotide (RNA oligo).

[0129] The RNA oligo may be partly or completely modified, for example with phosphothioate linkages. Advantageously, this increases resistance to cleavage, especially when the reaction mixture contains one or more endonucleases, or when the capture nucleic acid is released from the RNA oligo with an endonuclease. Despite the lower inherent stability of the RNA oligo, it may benefit from a higher affinity for DNA.

[0130] In another embodiment, the at least one sequence-specific hybridization element is an oligonucleotide comprising or consisting of one or more non-natural nucleotides, termed Xeno nucleic acids (XNA).

[0131] In a particular embodiment, solid supports with XNA-modified sequence-specific hybridization elements may be subjected to DNA- or RNA-specific nucleases to remove DNA or RNA retained on the solid support after an elution procedure. In such embodiment, the XNA hybridization elements will be resistant and survive the nuclease treatment. Accordingly, in some embodiments, the XNA hybridization elements may be resistant to treatment with DNA- or RNA-specific nucleases.

[0132] In another embodiment, the XNA-hybridization elements might have a higher specificity, affinity or combination thereof than similar DNA or RNA elements. Further, this enhanced specificity and / or affinity may be utilized for more specific capture of RNA in the presence of DNA, less material loss during washing, or improved elution.

[0133] XNA nucleotides have a non-(deoxy)ribose backbone and a base complementary to the natural nucleic acid, allowing them to capture, but not be accidentally incorporated or duplicated by natural DNA or RNA polymerases. Furthermore, since nature has not developed enzymes to degrade the XNA polymers, hybridization elements containing strategically placed XNA nucleotides may even be resistant to samples contaminated with exonucleases and endonucleases.

[0134] In one embodiment, therefore, the at least one sequence-specific hybridization element is gene specific or UTR-specific, i.e. it binds to the coding sequence of the nucleic acid molecule (gene specific), or to either or both of the untranslated regions (UTRs). Examples of XNAs include but are not limited to 1,5-Anhydrohexitol nucleic acid (HNA), Cyclohexene nucleic acid (CeNA), Threose nucleic acid (TNA), Glycol nucleic acid (GNA), Locked nucleic acid (LNA), Peptide nucleic acid (PNA), and Fluoroarabino nucleic acid (FANA).

[0135] In one embodiment, the at least one sequence-specific hybridization element is covalently linked to the solid support.

[0136] In one embodiment, the at least one sequence-specific hybridization element is covalently linked to the solid support by an irreversible chemical linkage. Examples of suitable chemistry / covalent linkages include: Carbodiimide Chemistry (EDC / NHS), NHS-amine (forming stable amide linkages), click chemistry including Azide-Alkyne Cycloaddition (Copper-catalyzed azide to terminal alkynes to form 1,2,3-triazoles or Strain-promoted cyclooctynes reaction with azide (e.g., azide-DBCO)), Tetrazine- Trans-Cyclooctene Ligation (forming stable dihydropyridazines), Maleimide-thiol Chemistry (producing stable thioether bonds), Aldehyde-Amine Reaction (Reductive Amination to form a stable secondary amine linkage), Thiol-ene / thiol-Yne Chemistry (thiol-vinyl), Amine-epoxy chemistry (amine-epoxy), Inverse Electron Demand Diels- Alder (Electron-deficient dienes (tetrazines) react with electron-rich dienophiles (strained alkenes)), Staudinger Ligation (Azides react with triarylphosphines to form iminophosphoranes, which hydrolyze to form amides), Sulfur(VI) Fluoride Exchange (Sulfonyl fluorides react with silyl ethers to form sulfonate esters), Copper-Free Sonogashira Coupling (Alkynes react with aryl halides in the presence of a palladium catalyst, without the need for copper), photo-crosslinking, Hydrazone chemistry (hydrazide / aldehyde or ketone), or any suitable method of covalently linking an oligonucleotide molecule to a bead surface.

[0137] In another embodiment, the at least one sequence-specific hybridization element is covalently linked to the solid support by a reversible chemical linkage. This enables the replacement of the sequence-specific hybridization element to compensate for wear and tear of such elements and re-use of the solid support. Examples of suitable reversible covalent linkages include: ester-groups (biologically degradable), disulfide bonds (reversible under reducing conditions), Imine (Schiff Base) bonds (Reaction between amines (-NH2) and aldehydes (-CHO) or ketones to form imines (C = N), can be reversed under basic or acidic conditions to the original amine and carbonyl compounds), Boronic Ester bonds, (reaction between boronic acids (R-B(OH)2) and diols (-OH) to form boronic esters (R-B(OR')2), hydrolyzed back to boronic acid and diol under acidic or basic conditions), Hydrazone bonds (Reaction between hydrazides (-CONHNH2) and aldehydes or ketones to form hydrazones (R-CH = N-NH-R'), hydrolyzed back to hydrazide and carbonyl compound under acidic conditions), Oxime ligation (amines or hydrazines react with aldehydes or ketones to form oximes or hydrazones, hydrolyzed back to hydroxylamine and carbonyl compound under acidic conditions), Diels-Alder reaction (diene reacts with a dienophile to form a cyclohexene, Retro-Diels-Alder reaction under thermal conditions), Thioester bonds (Reaction between thiols (-SH) and carboxylic acids (-COOH) or esters to form thioesters (R- CO-S-R'), Hydrolysis under acidic or basic conditions or trans-thioesterification with another thiol), Dynamic covalent Urea bonds (Reaction between amines and isocyanates to form ureas, hydrolysis under strong acidic or basic conditions), Acylhydrazone bonds (reaction between hydrazides and aldehydes or ketones to form acylhydrazones, hydrolysis under acidic conditions), Thiazolidine bonds (Reaction between 1,2-aminothiols and aldehydes or ketones to form thiazolidines, hydrolysis under acidic conditions), or any suitable method of reversible covalently linking an oligonucleotide molecule to a bead surface.

[0138] In yet another embodiment of the invention, the at least one sequence-specific hybridization element is non-covalently linked to the solid support. Examples of suitable non-covalent linkages include: biotin-streptavidin interaction, SBP- streptavidin, antibody-substrate binding (e.g., anti-FLAG antibody binding FLAG peptide (DYKDDDDK), or anti-HA antibody binding HA-tag (YPYDVPDYA), or anti-Myc antibodies binding Myc-tag (EQKLISEEDL)), aptamer-based binding, ProteinA / Protein G to capture IgG antibodies, his-tag-NiNTA (6-10 histidines binding nickel ions), his- tag - IMAC (6-10 histidines binding Cobalt ions), GST-tag - glutathione (GST protein binding immobilized glutathione), MBP-Amylose (MBP protein binding immobilized amylose), CBD-Cellulose (CBD peptide derived from cellulase enzymes binding to immobilized cellulose), or any suitable method of reversible covalently linking an oligonucleotide molecule to a bead surface.

[0139] In some embodiments, the method does not comprise treatment with a surfactant or a regeneration protocol. It will be appreciated that a regeneration protocol may comprise, for example, treatment with NaOH. Thus, in some embodiments, the solid support is recycled or re-used directly after elution of the nucleic acid molecule, without further treatment, except for resuspension in a carrier fluid.

[0140] The present disclosure also provides an apparatus for the performing the method of the claimed invention. Accordingly, in a second aspect of the invention, there is provided an apparatus configured to perform the method of the first aspect.

[0141] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0142] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:-

[0143] Figure 1 shows a method of extracting a nucleic acid molecule during a nucleic acid synthesis reaction according to the invention. A) An exemplary embodiment of the method according to the invention. Step 1. In a suitable container (Container 1), the ingredients of an enzymatic nucleic acid synthesis reaction, including a template nucleic acid, a nucleic acid (NA) polymerase (e.g., DNA polymerase or RNA polymerase), (d)NTPs, and a buffer, are combined and incubated under permissive conditions allowing the NA polymerase to synthesize the nucleic acid of interest (NAI). At the start of the incubation or at a defined period thereafter, a solid support carrying at least one sequence-specific hybridization element (the "capture element") specific for the NAI is introduced into the reaction mixture in Container 1 to capture the produced nucleic acid (the "captured NA"), but not the (template strand of the) template nucleic acid. The invention specifically provides for the combination of the capture element (solid support) to be introduced into the reaction mixture in such a manner that the NA synthesis reaction is not disrupted, not terminated and / or not slowed down. Step 2. After capturing a satisfactory amount of nucleic acid, the capture element is separated from the reaction mixture, e.g. by use of a magnet, and subjected to further processing of the captured nucleic acid (in Container 1), ultimately resulting in the elution of the captured nucleic acid from the capture element. The reaction mix is transferred to a separate container (Container 2) and combined with fresh or recycled capture element to continue the nucleic acid synthesis reaction. In a subsequent cycle, steps 1 and 2 are repeated on Container 2, for a total number of cycles m. The capture element may thereafter be optionally reused (i.e., the "recycled capture element) for a number of cycles m to capture nucleic acid in subsequent cycles of the process. The repeated capture of nucleic acid in the ongoing reaction is continued until the desired amount of nucleic acid is obtained, or the reaction is exhausted. B) A graph showing the concentration of the produced nucleic acid in the reaction mixture, illustrating that with the claimed method, repeated nucleic acid extraction prevents the accumulation of the produced nucleic acid. In contrast, the current batch reaction method used in the art allows the nucleic acid to accumulate in the reaction mix, and as such, the reaction must be terminated. C) A graph showing the total yield of the produced nucleic acid in the reaction mixture. With the claimed method, repeated nucleic acid extraction prevents the NA polymerase from being slowed down by the accumulation of the produced nucleic acid. This results in an improved yield of nucleic acid compared to the current batch reaction method used in the art.

[0144] Figure 2 shows a method of extracting a nucleic acid (NA) molecule during a nucleic acid synthesis reaction according to the invention. A) One example embodiment of the method of the invention, wherein: Step 1. (1) The reaction is assembled by combination of a template nucleic acid, a nucleic acid polymerase (e.g., DNA polymerase or RNA polymerase), (d)NTPs, a buffer, optional other ingredients and a suitable amount of capture element (solid support). Step 2. (2) The reaction mixture is incubated at permissive conditions to allow for the synthesis of the nucleic acid of interest. Meanwhile, the produced NA is captured by the capture elements in the reaction mixture. Step 3. (3) The capture elements are extracted from the reaction mix. This is followed by the processing (e.g., washing) of the capture elements containing captured nucleic acid, and elution of the captured NA. Step 4a. (4a) If required, the eluted NA is post-processed to yield the finished NA. Step 4b. (4b) Meanwhile, the eluted and optionally washed capture elements may be recycled and optionally combined with reaction mixture at (1) to capture additional produced NA. Step 4c. (4c) Meanwhile, the reaction mixture that was separated from the capture elements is further incubated at (2) to produce additional NA. B) Another example embodiment of the method of the invention, wherein the capture elements are introduced at a defined period after the start of the (further) incubation of the reaction mixture. Step 1. (1) The reaction is assembled by combination of a template nucleic acid, a nucleic acid polymerase (e.g., DNA polymerase or RNA polymerase), (d)NTPs, optional other ingredients, and a buffer. Step 2. (2) The reaction mixture is incubated at permissive conditions to allow for the synthesis of the nucleic acid of interest. Step 3. (3) The capture elements (solid support) are introduced into the reaction mixture, to capture the produced NA. Step 4. (4) The capture elements are extracted from the reaction mix. This is followed by the processing (e.g., washing) of the capture elements containing captured nucleic acid, and elution of the captured NA. Step 4b. (4b) The eluted and optionally washed capture elements may be recycled and combined with the reaction mixture at (3) to capture additional produced NA. Step 4c. (4c) Directly after extraction of the capture elements, the reaction mixture is further incubated at (2), and optionally combined with additional reaction mix or components thereof. Step 5. (5) If required, the eluted NA is post-processed to yield the finished NA. C) An example of the current state-of-the-art purification method using solid supports carrying a hybridization element for the capture of NA produced in an enzymatic mixture. Crucially, unlike with the method of the invention, such prior art methods require the reaction to be paused, terminated or disassembled to create the conditions for nucleic acid purification / extraction. Step 1. (1) The reaction is assembled by combination of a template nucleic acid, a nucleic acid polymerase (e.g., DNA polymerase or RNA polymerase), (d)NTPs, optional other ingredients, and a buffer. Step 2. (2) The reaction mixture is incubated at permissive conditions to allow for the synthesis of the nucleic acid of interest. Step 3. The reaction is terminated (3a), or suspended (3b). Step 4. The reaction mix is mixed with a binding buffer. Step

[0145] 5. The capture elements are introduced into the reaction mix to capture the NA. Step

[0146] 6. The nucleic acids are processed (e.g., washing). Step 7. The eluted NA may be post-processed to yield the finished NA.

[0147] Figure 3 shows example embodiments of methods to separate the capture elements (solid supports) from the reaction mixture. A) Step 1. The capture elements and reaction mix are present in a first container / reservoir (Rl). Step 2. The (saturated) capture elements are transferred together with / by the reaction mixture solution from the first container / reservoir (R.1) to a secondary container / reservoir (R2). Step 3. The capture elements are retained according to methods of the invention (e.g., magnetic or filter-based capture). The reaction mixture is then passed through (a series of) step(s), transferred to the same (Rl) or new container / reservoir to continue the NA synthesis reaction. Step 4. The deposited capture elements are optionally washed and / or further processed. In a variant of (A), depicted in full in (A2), fresh or recycled unsaturated capture elements are introduced in reservoir 1 (Rl) at Step 2b., before the reaction mixture is returned to reservoir 1. (B) Step 1. Alternatively, the (saturated) capture elements are retained in the container / reservoir (Rl) wherein the reaction originally took place. Step 2. The reaction mixture is transferred to another container / reservoir (R2) to continue the NA synthesis reaction. Step 3. The retained capture elements may be processed in the container / reservoir (Rl) wherein the reaction originally took place, while the reaction mix is further incubated or processed through (a series of) step(s). In a variant of (B), depicted in full in (B2), reservoir 2 (R2) already contains, or receives unsaturated capture elements during Step 1, resulting in the combination of said unsaturated capture elements with the reaction mix in Step 2. (C) Step 1. Alternatively, the (saturated) capture elements are retained in the container / reservoir (Rl) wherein the reaction originally took place. Step 2 and 3. The reaction mixture is transferred to another container / reservoir (R2), and then to a further container / reservoir (R3) to continue the NA synthesis reaction. Step 4. The retained capture elements may be transferred and processed in another container / reservoir (R.2) through (a series of) step(s). In a variant of (C), depicted in full in (C2), container / reservoir (R3) already contains or receives fresh or recycled unsaturated capture elements in Step 1, resulting in the combination of said unsaturated capture elements with the reaction mix in Step 2. In another variant of (C), depicted in full in (C3), container / reservoir (R3) is connected to container / reservoir (Rl) and receives in Step 2 the reaction mix directly from reservoir (Rl) (optionally combining with the fresh / recycled capture elements optionally introduced in container / reservoir (R3) prior or during Step 1. In Step 3, the saturated capture elements are transferred (through a series of) step(s) to container / reservoir (R2).

[0148] Figure 4 shows examples of solid supports that may, via an optional linker, carry one or a plurality of sequence-specific hybridization elements that specifically bind the nucleic acid molecule of interest. The hybridization elements are carried on the solid support such that they are readily available for the binding / capture of the nucleic acid of interest in a sequence-specific manner. A = solid bead; B = superparamagnetic bead; C = solid bead comprising a superparamagnetic core; D = surface-enlarged bead; E = porous bead; F = container-surface modification; G = hollow fibre; and H = filter, membrane or sponge.

[0149] Figure 5 shows examples of hybridizing elements that may, via an optional linker, be coupled to the solid supports of Figure 4, in a manner wherein the hybridization elements are readily available for the binding / capture of the nucleic acid of interest in a sequence-specific manner. A = regular single stranded nucleic acid; B = single stranded nucleic acid but with a 3' modification; C = single stranded nucleic acid but with a modified sugar-phosphate backbone; D = single stranded nucleic acid but with a structural modification; and E = a hybrid single and double stranded nucleic acid.

[0150] Figure 6 shows (A) the binding capacity of commercially available superparamagnetic oligo(dT) beads to capture RNA, according to two purification protocols: the manufacturer's protocol and the in-house protocol, and (B) the RNA binding capacity of oligo(dT) beads. Figure 7 shows the amount of RNA captured (pg) by oligo dT(25) magnetic beads (72pL of beads, and beads in a total in vitro transcription (IVT) reaction), compared with the control (an identical IVT reaction not containing any beads).

[0151] Figure 8 shows the amount of RNA captured (pg) when increasing amounts of oligo dT(25) beads are added to the IVT reaction.

[0152] Figure 9 shows the amount of RNA captured (pg) under differing incubation times of the oligo dT(25) beads in the IVT reaction.

[0153] Figure 10 shows the amount of RNA captured (pg) when adding oligo dT(25) beads either once or twice in an IVT reaction.

[0154] Figure 11 shows (i) the amount of RNA captured (pg) by oligo(dT) beads under differing incubation times, and (ii) the amount of unintended DNA capture by oligo(dT) beads under differing incubation times.

[0155] Figure 12 shows the RNA yield retrieved (pg) after three separate elutions for lx used beads and lOx used beads.

[0156] Figure 13 shows (A) the binding capacity of the beads (RNA concentration) following multiple rounds of use, and (B) the percentage of RNA bound to the beads after three elutions for lx used beads and lOx used beads.

[0157] Figure 14 shows the binding capacity of the polyvinylalcohol (PVA) OdT beads following multiple rounds of use.

[0158] Figure 15 shows the overall productivity and extracted RNA of batch IVT reactions supplemented with increasing amounts of PVA OdT beads.

[0159] Figure 16 shows an image of an agarose gel electrophoresis of RNA extracted after 45 and 180 min, respectively, with either polystyrene (PS) or polyvinylalcohol (PVA) OdT beads.

[0160] Figure 17 shows (A) the recovery percentage of RNA bound to OdT beads after 5-60 seconds incubation in a batch IVT reaction, (B) the recovery percentage of RNA bound to OdT beads at different elution temperatures using RNAse-free water as an eluent, and (C) the recovery percentage of RNA bound to OdT beads after 5-180 seconds incubation at 65°C in RNAse-free water.

[0161] Figure 18 shows the amount of RIMA eluted in a constant volume of RNAse-free water by reusing the RNA-containing water for repeated elutions.

[0162] Examples

[0163] Example 1 - Batch IVT reactions to determine the binding capacity of commercially available superparamagnetic olioo(dT) beads under idealized conditions.

[0164] 200ng of a PCR-generated DNA template encoding a luciferase sequence, containing a 5' T7-promoter, and 3' encoded poly-A-tail of 150nt was added to a 20 I HiScribe T7 (NEB) IVT reaction constructed according to manufacturer's specifications.

[0165] The reaction mix was subsequently incubated for 3h at 37°C until completion. The incubated samples were immediately subjected to purification by oligo(dT)25 beads (NEB #S1419, no DNAse steps was performed due to the inconsequential amount of DNA present in the solution), according to two purification protocols.

[0166] Protocol 1

[0167] Buffers

[0168] LiCI solution of IM : 2.21g 100% LiCI + 50mL RNAse free water (RFW, Invitrogen). Make DTT solution of IM : 0.77g DTT + 5mL RNAse free water (RFW).

[0169] Use HCL to set pH to 7.5.

[0170] The superparamagnetic oligo(dT)25 beads were thoroughly mixed and 72 I was preequilibrated with 200 I of lysis / binding buffer. Meanwhile, 20 I of IVT mix was mixed with 40pl of RFW and 250pl lysis / binding buffer, which was mixed until homogenous and incubated for 5 minutes. The beads were extracted from the lysis / binding buffer and mixed with the IVT-lysis mixture, and incubated for 10 minutes at RT on the agitator.

[0171] The saturated beads were extracted from the solution by permanent magnet and the supernatant was saved for further purification by silica columns.

[0172] The beads were subjected to 3 washing steps, with wash buffer I and wash buffer II and the low-salt buffer, as indicated above. Next, the RNA was 2x eluted by the addition of 50pl of elution buffer and incubation for 2 minutes at 50°C. Beads were removed from the solution by permanent magnet in each step, until the solution looked visually clear.

[0173] Protocol 2

[0174] The in-house protocol differs from the above protocol in that elution occurs with pure RNAse free water (RFW) at RT, rather than the indicated elution buffer.

[0175] The RNA not bound to the beads was thereafter purified with a RNeasy silica column according to manufacturer's protocol to quantify the total yield of the reaction by measuring the 230 / 260 / 280nm absorption on the iD3 platereader (Molecular Devices) by placing a 2pl droplet on a dedicated glass slide.

[0176] The above procedure was repeated with the same DNA template but in a Hi-T7 RNA polymerase reaction and an IVT reaction set up with proprietary T7 RNA polymerase mutant M13, with similar or identical results (data not shown).

[0177] For clarity, the bead volumes indicated are the volumes of oligo(dT) beads as provided by the manufacturer. The beads are extracted from the storage solution and added to the reaction by means of adding the reaction volume to the semi-dry beads. The results, as shown in Figure 6A, clearly demonstrate that the in-house protocol captures more RIMA than the manufacturer's protocol (nearly 18 pg vs 2 pg). The results, as shown in Figure 6B, further show that the beads have a binding capacity of around 20pg of RNA per 72pl of beads, when deployed under idealized binding, washing and elution conditions. Contrary to the method of the claimed invention, the reaction needs to be terminated for this type of extraction, as the RNA is mixed with a lysis / binding buffer.

[0178] Example 2 - Batch IVT reactions to determine the binding capacity of oligo(dT) beads and the effect on the productivity of the IVT reaction, while added directly to the IVT reaction mix.

[0179] IVT reactions were prepared as in Example 1, except that the volume of the IVT reaction was scaled (all ingredients were scaled proportionately) 3 times, to facilitate the volume of the beads (72pl worth). For clarity, the bead volumes indicated are the volumes of oligo(dT) beads as provided by the manufacturer. The beads are extracted from the storage solution and added to the reaction by means of adding the reaction volume to the semi-dry beads.

[0180] An identical IVT reaction not containing any beads was used as control, and similar to Example 1, the remaining volumes were purified with silica column to quantify the yield of an unmodified IVT reaction (i.e., under idealized conditions).

[0181] The results shown in Figure 7 clearly demonstrate that there is a minimal, if any, loss in productivity of the IVT reaction (<10%) with the introduction of a significant volume of oligo(dT) beads, capable of capturing 5% of the yield. The results also suggest that with the low binding capacity of these commercially available oligo(dT) beads, either more beads need to be packed in the reaction, or multiple exchanges of beads need to be performed during the IVT reaction to capture sufficient or all of the RNA.

[0182] 3 - Batch IVT reactions to determine the capacity of increased amounts of oliqo(dT) beads and the effect on the of the IVT reaction while added directly to the IVT reaction mix.

[0183] IVT reactions were prepared as in Example 2, except that increasing amounts of oligo(dT) superparamagnetic beads were added to the IVT reaction to study the effect on the productivity and on the total RNA capture by the beads. The bead volumes indicated are the volumes of oligo(dT) beads as provided by the manufacturer. The beads are extracted from the storage solution and added to the reaction by means of adding the reaction volume to the semi-dry beads.

[0184] The results, as displayed in Figure 8, show a dose-dependent reduction of the productivity of the IVT reaction (as measured by the amount of captured RIMA), when progressively more oligo(dT) beads are added to the reaction volume at the start of the reaction and maintained there for the duration of the 3h incubation. Interestingly, the loss in productivity is proportional to the number of beads added. Equally interesting is the fact that there seemed to be a linear relation relationship between the number of oligo(dT) beads added and the total amount of RNA extracted from the reaction. At extremely high amounts of beads added, the solution becomes difficult to handle (data not shown) and the loss of productivity is severe, leading to all the produced RNA being captured (last data point in Figure 8).

[0185] Example 4 - Batch IVT reactions to determine the time required for the beads to bind a qiven amount of RNA when mixed with the IVT solution.

[0186] In order to create a protocol for repeated addition and extraction of RNA from an IVT solution, the inventors first sought to understand the binding kinetics of the oligo(dT) beads in an IVT solution. Therefore, IVT reactions were prepared as in Example 1, except the reactions were allowed to incubate for only 1.25-1.5h before addition of the beads to the reaction, providing an average condition (~50% of RNA, ~50% of nucleotides consumed, etc.) for the binding of RNA during an IVT reaction.

[0187] Similar to Example 1, the remainder of the IVT solution was subjected to standard silica column purification to extract the remainder of the RNA from the solution to quantify the total productivity of the reaction and calculate the percentage of material bound.

[0188] Surprisingly, the results (see Figure 9) show near instant binding of the RNA, as similar amounts of RNA were extracted from the reaction regardless of whether the beads were incubated for 5, 10 or 15 minutes, or if the beads were immediately removed after mixing by contact with a permanent magnet. These results provide a clear indication of the possibility of incubating the IVT reaction multiple times with fresh or recycled beads, each time with a short incubation time. tions to verify increased capture of RIMA from the IVT re to oliqo(dT) beads.

[0189] In this experiment, the inventors sought to verify the hypothesis that repeated administration of an oligo(dT) bead would capture an increased amount of RNA than that captured by a single administration. This would provide a method of reducing the concentration of the beads (and thus the negative effect on the productivity) at any time in the reaction, while compensating for the low binding capacity of the commercially available beads.

[0190] Therefore, IVT reactions were prepared, purified and quantified as in Example 4, except the equivalent of 72p I of oligo(dT) beads was added after 60 minutes, and an equal amount after 1.5h (total incubation time). Each time, the oligo(dT) beads were incubated for 5 minutes for good measure and each time the IVT solution was completely cleared from the superparamagnetic beads by a permanent magnet. As before, the remainder of the solution was subjected to standard silica column purification as per manufacturer's protocol, and the sum of the amount of RNA purified by bead and silica column is presented as the total productivity of the IVT reaction.

[0191] Interestingly, the results shown in Figure 10 confirm the hypothesis that each dose of oligo(dT) beads captures an equal amount of RNA from the reaction, and shows that repeated administration of a given volume of oligo(dT) beads does not increase the loss in productivity of the reaction. Therefore, this result demonstrates that it is possible for a protocol to repeatedly or continuously add, and extract, small volumes of nucleic acid binding beads to a nucleic acid synthesis reaction to extract the accumulating nucleic acid.

[0192] Example 6 - Batch IVT reactions to determine the unintended loss of DNA template from the IVT reaction during RNA capture by the oliqo(dT) beads.

[0193] Repeated exposure to oligo(dT) beads could have the unintended consequence of capturing the template DNA alongside the intended RNA capture, also providing an explanation for the reduction in IVT productivity.

[0194] Therefore, batch IVT reactions were set up as in Examples 4 and 5, and the captured RNA was measured by spectrophotometer. In addition to the methods of examples 4 and 5, aliquots of the RNA samples eluted from the oligo(dT) beads were treated with RNAse to remove the RNA and subjected to qPCR to quantify the amount of co- purified DNA. Similarly, qPCR was performed directly on samples containing noneluted oligo(dT) beads (data not shown, but in agreement with Figure 11), and qPCR was performed on the remaining IVT solution (data not shown, but in agreement with Figure 11).

[0195] Briefly, 20pL of RIMA sample eluted from the oligo(dT) beads was treated with luL of 70U / mL RNAse A and incubated for lh at 37°C. Two dilutions were prepared for the qPCR. For the undiluted sample, 1 pl was taken from the RNAse-digestion and added to 7,8 pl RFW. For the lOOx dilution, 1 pl RNAse-digested sample was added to 99 pl RFW and 8,8 was mixed with the qPCR master mix.

[0196] The qPCR mix contained :

[0197] Briefly, the qpCR protocol comprises; iTaq universal SYBR Green reaction mix was thawed at 4°C (protected from the light), before being mixed and centrifuged briefly to collect the contents of the tube.

[0198] Each primer set was diluted according to the table below:

[0199] Important controls (volumes indicated for 10 pl reaction):

[0200] - Water: no sample / DNA added and 4.275 pl nuclease-free water

[0201] The qPCR mix was mixed thoroughly and dispensed in equal aliquots into a PCR plate (AmpliStar-II Semi-Skirted 96-well) and the plate was sealed with PCR tape.

[0202] Triplicates were generated.

[0203] The following protocol was performed on an ABI Prism 7000 qPCR machine:

[0204] A standard curve was generated from the same DNA template as used to set up the IVT reactions of all previous examples. Briefly, 1 pl of 100 ng / pl DNA was added to 9 pl RFW. From this first (lOx) dilution, a serial dilution was made, each sample containing lOx less DNA than the previous by mixing 18 pl RFW with 2 pl of the previous dilution (dilution line ranges from 1 to 0,00001 ng per sample in lOx dilution steps).

[0205] The results (shown in Figure 11), surprisingly, indicate that longer incubation does not necessarily increase the binding of template DNA in IVT reactions. In fact, contrarily, longer incubation of beads added after the start of the reaction resulted in a progressively lower amount of DNA being captured. Interestingly, addition of the oligo(dT) beads at the start of the IVT reaction resulted in a higher capture of DNA, presumably because of relatively high amounts of DNA over RNA being available uniquely at the start of the reaction. Performing repeated oligo(dT) bead additions resulted in more DNA being captured, the disproportional increase being attributed again to a higher DNA to RNA ratio after the first capture of RNA by the beads.

[0206] Most importantly, the amount of DNA captured by the oligo(dT) beads is extremely low at less than Ing from a reaction containing 200ng of DNA, calculated to ~0.08% of RNA being captured with each cycle of oligo(dT) bead addition.

[0207] Example 7 - Batch IVT reactions to optimize the release of the RNA from the oligo(dT) beads.

[0208] Given the plan to repeatedly add oligo(dT) beads to an IVT reaction to repeatedly extract RNA, the inventors wondered if the RNA extraction could be made more costefficient by the recycling of previously used and eluted beads, preferably within the same IVT reaction. Therefore, the completeness of the elution of the RIMA with the currently used protocol was first investigated.

[0209] A batch IVT reaction was setup similar to Example 1 and the resulting oligo(dT) beads (72pl worth) were eluted 3 times with 50pl of RFW according to the optimized protocol of Example 1. Each aliquot of eluted RNA was quantified on the spectrophotometer.

[0210] To determine the potential loss in binding capacity of the beads, repeated RNA extraction was performed per the protocol of Example 1, and the eluates of beads used lOx were determined as well.

[0211] The results shown in Figure 12 reveal that the majority of RNA captured by the oligo(dT) beads is released by the first elution, followed by a significant portion by the second elution, and minimal amount by the third. Therefore, the current double elution protocol is sufficient to recycle the beads for subsequent use in the same IVT reaction.

[0212] Surprisingly, there is a loss in binding capacity by repeated use of the beads, which is not related to the exposure time to any of the ingredients, but related to the binding of the RNA (as determined in another experiment, data not shown).

[0213] Example 8 - Batch IVT reactions to quantify the loss of binding capacity of the commercial oliqo(dT) beads.

[0214] To further quantify the loss of binding capacity of oligo(dT) beads during repeated use, and know how to compensate by mixing in fresh beads with batches of recycled beads, the experiment of Example 7 was repeated, except that each elution was quantified.

[0215] The results (see Figure 13A) clearly show a linear decrease in binding capacity of around 4-5% during each round. This can be related to the loss of beads during the manual extraction and manipulation methods used. In a closed system, the losses may be less.

[0216] Interestingly, the effect on a loss of productivity of the IVT reaction (Figure 13B) remains unchanged / trends downwards with the reuse of the beads, potentially indicating small losses of beads over the reactions. Alternative explanations include damage to the beads or its hybridization elements (oligo(dT)25 nucleic acid) or the linkage in between.

[0217] On-bead quantification of the remaining RIMA by RiboGreen assay, according to manufacturer's specifications, showed minimal (<0.4%) to no accumulation of bound RNA after repeated use.

[0218] Example 9 - Batch IVT reactions to determine the binding capacity of increased amounts of oligo(dT) beads and the effect on the productivity of the IVT reaction, while added directly to the IVT reaction mix.

[0219] IVT reactions were prepared as in Example 2, except that increasing amounts of oligo(dT) superparamagnetic beads were added to the IVT reaction to study the effect on the productivity and on the total RNA capture by the beads. The bead volumes indicated are the volumes of oligo(dT) beads as provided by the manufacturer. The beads are extracted from the storage solution and added to the reaction by means of adding the reaction volume to the semi-dry beads.

[0220] The results, as displayed in Figure 8, show a dose-dependent reduction of the productivity of the IVT reaction (as measured by the amount of captured RNA), when progressively more oligo(dT) beads are added to the reaction volume at the start of the reaction and maintained there for the duration of the 3h incubation. Interestingly, the loss in productivity is proportional to the number of beads added. Equally interesting is the fact that there seemed to be a linear relation relationship between the number of oligo(dT) beads added and the total amount of RNA extracted from the reaction. At extremely high amounts of beads added, the solution becomes difficult to handle (data not shown) and the loss of productivity is severe, leading to all the produced RNA being captured (last data point in Figure 8).

[0221] The same experimental setup was employed to determine the effect of (surface) chemistry of the beads on productivity of the IVT reaction. In a scaled-down experiment (20 pl reaction, compared to 60 pl in previous experiment), increasing amounts of M-PVA superparamagnetic beads functionalized with OdT(30) (Revvity, OdT2 M-PVA, Part # : CMG-231) were mixed into a 20 pl IVT reaction. After 45 minutes, the beads were extracted, washed and eluted. The yield was determined for both the RNA bound to the magnetic beads and the remaining IVT reaction. Surprisingly, as seen in Figure 15, the M-PVA beads did not shown the same loss in productivity as the polystyrene (PS) beads, while the dry-bead :IVT-volume ratio was increased 4x more (ratio 0.288 (PS) vs. 1.04 (M-PVA). This suggests that PVA surface chemistry is preferential over polystyrene surface chemistry for capture of nucleic acids by addition of solid supports in nucleic acid synthesis reactions.

[0222] Example 10 - Batch IVT reactions to determine the binding capacity of PVA-OdT beads underaoina repeated bindina-washina-elution cycles, with and without reaeneration

[0223] An IVT reaction was mixed with its size adjusted according to the number of cycles. The IVT reaction was made with a final DNA concentration of 5 ng / pL (Proprietary constructs - either 0.75 kb or lOkb), 7.5 mM of ATP and 5 mM of CTP, GTP, and UTP (Thermo Fisher Scientific), Inorganic pyrophosphatase (Thermo Fisher Scientific), RNase inhibitor (Thermo Fisher Scientific) and RiboPerfect™ RNA Polymerase (RIBOPRO) in IVT-buffer. For the 20-cycle experiment, a 21x 40 pL reaction was mixed and incubated for 45 minutes at 37°C. The mixture was subsequently inactivated by Turbo DNAse (Thermo Fisher Scientific) for 15 minutes to ensure equal binding conditions for every cycle.

[0224] Prior to use, M-PVA beads (Revvity, OdT2 M-PVA, Part#: CMG-231) beads were resuspended to ensure homogeneity. Unless stated otherwise, all subsequent beads- step (washing / binding / elution) followed the same procedure: the beads were thoroughly resuspended in the indicated buffer, mixed thoroughly, and recollected on the magnet before removal of the supernatant.

[0225] 150 pL of beads were first equilibrated by washing in nuclease-free water and equilibration in IVT buffer to remove storage buffer remnants. The beads were exposed to an aliquot of 40 pL of the inactivated IVT-mixture for 15 seconds. The beads were subsequently washed (2x) with 150 pL washing buffer (10 mM Tris-HCL pH 7.5, 150 mM LiCI) and eluted in 90 pL pre-heated nuclease-free water (10 seconds at 65°C). The eluent was stored under cooled conditions for further analysis. For series 1 (without regeneration); directly after elution the beads were equilibrated in 150 pL IVT buffer and continued with a new cycle, starting with exposing the beads to a fresh 40 pL aliquot of the inactivated IVT-mixture. For series 2 (with regeneration), the beads were exposed to 150 pL of 0.1N NaOH, and subsequently neutralized and equilibrated by washing 2x in IVT-buffer.

[0226] After all cycles were completed, the RNA concentration was determined by micro spectroscopy (SpectraMax iD3 microplate reader). A selection of RNA samples (start- mid-end of cycle) (750 ng / lane) were loaded onto a 1% agarose gel for electrophoreses (lOV / cm - 45 minutes).

[0227] The results (Figure 14) show that in comparison with OdT(25) PS (poly-styrene) beads from NEB, the M-PVA OdT(30) beads provide a more consistent recovery, while also not suffering from a significant drop-off of binding capacity like the PS beads. In general, the beads were handling better, with no severe clumping or plaqueformation. Under the current reaction conditions, no measurable effect of the regeneration step was observed.

[0228] Example 11 - Batch IVT reactions to demonstrate RNA quality after bead purification and to demonstrate the value of rapid binding and removal of RNA from the IVT reaction

[0229] To further investigate the purification quality of the beads, the two types of beads were exposed to two DNA constructs (a model for short and long RNA), which had been incubated for short and long incubation times. Short is a model for the repeated cycles as is employed in the manufacturing machine, and long as a model for the generally used batch-mode IVT.

[0230] For each construct (Proprietary constructs of 0.75 kb (short) or lOkb (long)), a separate 100 pL IVT reaction was made with a final DNA concentration of 5 ng / pL, 7.5 mM of ATP and 5 mM of CTP, GTP, and UTP (Thermo Fisher Scientific), Inorganic pyrophosphatase (Thermo Fisher Scientific), RNase inhibitor (Thermo Fisher Scientific) and RiboPerfect™ RNA Polymerase (RIBOPRO) in IVT-buffer. The reactions were aliquoted and either incubated for 45 minutes (short) or 3 hours (long) at 37°C. The mixture was subsequently inactivated by Turbo DNAse (Thermo Fisher Scientific) for 15 minutes.

[0231] Prior to use, M-PVA beads (Revvity, OdT2 M-PVA, Part#:CMG-231) and PS beads (NEB, catalog# : S1419S) were resuspended to ensure homogeneity. Unless stated otherwise, all subsequent beads-step (washing / binding / elution) followed the same procedure: the beads were thoroughly resuspended in the indicated buffer, mixed thoroughly, and recollected on the magnet before removal of the supernatant.

[0232] 50 pL (PS) & 150 pL (M-PVA) of beads were first equilibrated by washing in nuclease- free water and equilibration in IVT buffer to remove storage buffer remnants. The beads were exposed to an aliquot of 40 pL of the inactivated IVT-mixture for 15 seconds. The beads were subsequently washed (2x) with 50 pL (PS) or 150 pL (M- PVA) of washing buffer (10 mM Tris-HCL pH 7.5, 150 mM LiCI) and eluted in 30 pL (PS) or 60 pL (M-PVA) pre-heated nuclease-free water (10 seconds at 65°C). The eluent was stored under cooled conditions for further analysis.

[0233] The RNA concentration was determined by micro spectroscopy (SpectraMax iD3 microplate reader). RNA samples (750 ng / lane) were loaded onto a 1% agarose gel for electrophoreses (lOV / cm - 45minutes).

[0234] The results (Figure 16) show that both beads, made of polystyrene and polyvinylalcohol, produce an RNA of identical integrity. Further, the RNA extracted after 45 minutes incubation was of higher integrity than the RNA extracted after 180 minutes, which shows a secondary band. This indicates the value of repeated or continuous extraction of the nucleic acid from the synthesis reaction.

[0235] Example 12 - Batch IVT reactions to establish the binding kinetics of OdT beads added to the IVT reaction

[0236] A 385 pL IVT reaction was mixed with a final DNA concentration of 5 ng / pL (Proprietary 0.75 construct), 7.5 mM of ATP and 5 mM of CTP, GTP, and UTP (Thermo Fisher Scientific), Inorganic pyrophosphatase (Thermo Fisher Scientific), RNase inhibitor (Thermo Fisher Scientific) and RiboPerfect™ RNA Polymerase (RIBOPRO) in IVT-buffer. The reaction was incubated for 3 hours at 37°C and subsequently inactivated by the addition of EDTA.

[0237] Prior to use, PS beads (NEB, catalog# : S1419S) were resuspended to ensure homogeneity. Unless stated otherwise, all subsequent beads-step (washing / binding / elution) followed the same procedure: the beads were thoroughly resuspended in the indicated buffer, mixed thoroughly, and recollected on the magnet before removal of the supernatant.

[0238] 770 pL of beads were first equilibrated by washing in nuclease-free water and equilibration in IVT buffer to remove storage buffer remnants. The beads were separated, and the supernatant was subsequently removed . The beads were exposed to inactivated IVT-mixture for 15 seconds. The beads were subsequently washed (2x) with 770 pL washing buffer (10 mM Tris-HCL pH 7.5, 150 mM LiCI). During the second washing step, the suspension was aliquoted into 7x 100 pL aliquots and the supernatant was subsequently removed. These separate aliquots were exposed to a gradient of elution times (5 - 10 - 15 - 30 - 60 - 90 - 180 seconds) all with pre- heated nuclease free water and incubated in a dry-heat block. The eluent was stored under cooled conditions for further analysis.

[0239] The RNA concentration was determined by micro spectroscopy (SpectraMax iD3 microplate reader). RNA samples (750 ng / lane) were loaded onto a 1% agarose gel for electrophoreses (lOV / cm - 45minutes).

[0240] To determine the elution temperature, an identical experiment was performed and varying the elution temperature (25 - 37 - 40 - 45 - 50 - 55 - 60 - 65°C) instead of the elution time.

[0241] To determine the binding kinetics, an identical experiment was performed and varying the exposure time of IVT mixture to beads mixture (5 - 10 - 15 - 30 - 60 seconds) and a constant elution temperature in pre-heated nuclease-free water (90pL, 10 seconds at 65°C).

[0242] The results (Figure 17) show that under the binding conditions provided by the IVT mixture, the OdT beads bind the RNA near instantly with near 100% recovery after a mere 5 second binding time. Subsequent recovery increased from 50% at 25°C to 100% at 65°C, and was near instant with ~100% recovery after 5 seconds. This indicates that OdT beads can be rapidly cycled according to the method of the invention.

[0243] Example 13 - Batch IVT reactions to show complete elution in limited volumes In closed systems, it can be advantageous to elute the nucleic acid bound to the solid support in the same elution volume, already containing nucleic acid to reduce eluent volume or to achieve higher concentration. In this example, the inventors repeatedly eluted OdT beads with bound RNA in the same elution volume, demonstrating a linear increase in RNA concentration in the elution volume.

[0244] A 120 pL IVT reaction was mixed with a final DNA concentration of 5 ng / pL (Proprietary 0.75 construct), 7.5 mM of ATP and 5 mM of CTP, GTP, and UTP (Thermo Fisher Scientific), Inorganic pyrophosphatase (Thermo Fisher Scientific), RNase inhibitor (Thermo Fisher Scientific) and RiboPerfect™ RNA Polymerase (RIBOPRO) in IVT-buffer. The reaction was incubated for 30 minutes at 37°C and subsequently inactivated by Turbo Turbo DNAse (Thermo Fisher Scientific) for 15 minutes.

[0245] Prior to use, M-PVA beads (Revvity, OdT2 M-PVA, Part#: CMG-231) were resuspended to ensure homogeneity. Unless stated otherwise, all subsequent beads-step (washing / binding / elution) followed the same procedure: the beads were thoroughly resuspended in the indicated buffer, mixed thoroughly, and recollected on the magnet before removal of the supernatant.

[0246] 500 pL of beads were first equilibrated by washing in nuclease-free water and equilibration in IVT buffer to remove storage buffer remnants. The beads were separated, and the supernatant was subsequently removed . The beads were exposed to the inactivated IVT-mixture for 15 seconds. The beads were subsequently washed (2x) with 500 pL washing buffer (10 mM Tris-HCL pH 7.5, 150 mM LiCI). During the second washing step, the suspension was aliquoted into lOx 50 pL aliquots and the supernatant was subsequently removed. The aliquots were divided in two sets of 5x 50 pL, for which set 1 was exposed to 30 pL nuclease-free water and set 2 to 60 pL nuclease-free water.

[0247] For set 1 (30 pL), the first beads-aliquot was exposed to 30 pL pre-heated nuclease- free water. The eluent was collected, and directly afterwards, 2 pL of the sample was used to determine the RIMA concentration by micro spectroscopy (SpectraMax iD3 microplate reader). The volume used for quantitation was transferred back into the eluent. This same eluent was heated to 65°C, and this process was repeated for the second, third, fourth and fifth bead-aliquot subsequently (Figure 18).

[0248] Conclusions

[0249] The inventors have devised a nucleic acid (NA) purification method capable of continuously or intermittently extracting nucleic acid from an active nucleic acid synthesis reaction. The method utilizes sequence-specific hybridization to bind the nucleic acid of interest (NAI) to a solid support, which is subsequently captured from the reaction volume. Advantageously, the method is capable of being selective for binding the NAI, and not significantly binding the template NA, enabling the reaction to proceed continuously.

[0250] The methods of the invention achieve surprisingly rapid and selective extraction of the intended nascent nucleic acid, keeping the concentration of the accumulating nucleic acid product at lower levels, while the yield is higher than typical in batch and fed- batch reactions of the current state of the art. The resultant nucleic acid molecules of the claimed method are obtained in a higher purity with less consumption of expensive reagents and a simplified purification procedure, which considerably lowers the cost of the synthesis of the nucleic acid. Furthermore, the lower levels of nucleic acid sideproducts causes less unwanted side effects of the nucleic acid when used in vitro or in vivo applications. For example, there is less activation of the innate immune responses by dsRNA when the nucleic acid product is mRNA and is administered to a subject, for example as a vaccine. The method of the invention, through optimized solid support chemistry, the use of high relative volumes of beads, and rapid cycle times, is capable of rapidly extracting high amounts of nucleic acid, to be reused without a regeneration protocol and elution in reduced volumes, overall creating a high efficiency.

Claims

1. Claims1. A method of extracting a nucleic acid molecule during an active nucleic acid synthesis reaction, the method comprising carrying out the nucleic acid synthesis reaction in the presence of at least one solid support comprising at least one sequence-specific hybridization element that binds specifically to the nucleic acid molecule, thereby resulting in the capture and extraction of the nucleic acid molecule.

2. The method according to claim 1, wherein the nucleic acid molecule is extracted without the reaction being interrupted, terminated and / or slowed down.

3. The method according to either claim 1 or claim 2, wherein the method comprises extracting the nucleic acid molecule from a reaction mixture, optionally wherein the reaction mixture is continuously active during the extraction of the nucleic acid molecule.

4. The method according to any preceding claim, wherein the nucleic acid synthesis reaction is: (i) a DNA amplification reaction, optionally wherein the DNA amplification reaction is polymerase chain reaction (PCR), rolling circle amplification (RCA), or strand displacement amplification (SDA); or (ii) an in vitro transcription reaction.

5. The method according to any preceding claim, wherein the nucleic acid molecule is a DNA or RNA molecule.

6. The method according to any preceding claim, wherein: (i) the DNA is genomic DNA (gDNA), complementary DNA (cDNA) or plasmid DNA; and / or (ii) the RNA is selected from a group of RNA molecules consisting of: messenger RNA (mRNA), micro RNA (miRNA); interference RNA (RNAi); short interfering RNA (siRNA); short hairpin RNA (shRNA); anti-sense RNA; RNA aptamers; self-amplifying RNA (saRNA); coding RNA; non-coding RNA; and circular RNA.

7. The method according to any preceding claim, wherein the nucleic acid synthesis reaction is a DNA amplification reaction, and the method comprises contacting : (i) a reaction mixture comprising a template nucleic acid sequence, a DNA polymerase, optionally a DNA primer or a plurality of DNA primers, optionally an endonuclease, a plurality of deoxynucleotide triphosphates (dNTPs) and / or a buffer,and (ii) the at least one solid support comprising at least one sequence-specific hybridization element that binds specifically to the nucleic acid molecule.

8. The method according to claim 7, wherein the DNA polymerase is selected from the group consisting of: Taq DNA polymerase, P / uDNA polymerase, DNA polymerase I, Phusion DNA polymerase, KAPA HiFi DNA polymerase, T7 DNA polymerase, Sequenase 2.0, phi29 DNA polymerase, temperature-stabilised phi29 DNA polymerase, Bst DNA polymerase, Bsll DNA polymerase, Klenow, Q5 high-fidelity DNA polymerase, or a mutated variant of any of these DNA polymerases.

9. The method according to any one of claims 1 to 6, wherein the nucleic acid synthesis reaction is an in vitro transcription reaction, and the method comprises contacting : (i) a reaction mixture comprising a template nucleic acid sequence, an RNA polymerase, a plurality of nucleotide triphosphates (NTPs) and / or a buffer, and optionally a cap-analogue, and (ii) the at least one solid support comprising at least one sequence-specific hybridization element that binds specifically to the nucleic acid molecule.

10. The method according to claim 9, wherein the RNA polymerase is selected from the group consisting of: T7; T3; SP6; KP34; Syn5; Vsw-3; temperature stabilised T7 RNA polymerase; or other DNA-dependent RNA polymerases; or a mutated variant of any of these RNA polymerases.

11. The method according to any one of claims 7 to 10, wherein the first contact between (i) and (ii) occurs at the start of the reaction, or wherein the first contact between (i) and (ii) occurs at a defined interval after the start of the reaction, when a first amount of nucleic acid has accumulated in the reaction, optionally wherein the interval is less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute.

12. The method according to any one of claims 7 to 11, wherein the contacting of (i) and (ii) occurs at reaction conditions conducive to the nucleic acid synthesis reaction.

13. The method according to any preceding claim, wherein the at least one solid support is continuously or intermittently present in the nucleic acid synthesis reaction.

14. The method according to 13, wherein intermittently present means that the solid support is continuously fed and removed from the reaction mixture, optionally wherein the solid support is present in the reaction mixture for less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute.

15. The method according to any preceding claim, wherein the nucleic acid molecule is extracted at a rate substantially equal to the rate of nucleic acid synthesis.

16. The method according to any preceding claim, wherein the nucleic acid extraction capacity present in the nucleic acid synthesis reaction is ratiometrically constant to the amount of unbound nucleic acid.

17. The method according to any one of claims 1 to 14, wherein the nucleic acid extraction capacity present in the nucleic acid synthesis reaction is, at any time, a surplus relative to the amount of nucleic acid molecule available for binding.

18. The method according to any preceding claim, wherein a total volume of the at least one solid support comprising at least one sequence-specific hybridization element relative to the nucleic acid synthesis reaction mixture volume is >5%, >10%, >25%, >50%, or 100% (1 : 1) of the nucleic acid synthesis reaction mixture volume present at any given time.

19. The method according to any preceding claim, wherein the nucleic acid synthesis reaction produces >lmg / ml / h, >2mg / ml / h, >5mg / ml / h, >7mg / ml / h, or >10mg / ml / h of RIMA, and / or wherein the nucleic acid synthesis reaction comprises a total nucleic acid extraction capacity of >lmg / ml / h, >2mg / ml / h, >5mg / ml / h, >7mg / ml / h, or >10mg / ml / h.

20. The method according to any preceding claim, wherein the method comprises mixing the reaction mixture, optionally wherein the reaction mixture is mixed with a mixing element, vortex-inducing movement of the entire reaction vessel, (micro)vibration, or magnetic force.

21. The method according to any preceding claim, wherein the method comprises applying an external magnetic force to the reaction mixture, optionally wherein the method comprises alternating the direction of the magnetic force on the reaction mixture.

22. The method according to claim 21, wherein the method comprises applying a continuous magnetic gradient over at least 1 axis of a reaction chamber containing the reaction mixture, such that it causes the solid support to flow through at least a section of the reaction chamber.

23. The method according to any preceding claim, wherein the method comprises removing the solid support from the reaction mixture, or separating the solid support and the reaction mixture.

24. The method according to claim 23, wherein the method comprises removing the solid support from the reaction mixture via filtration, centrifugation, column binding, or magnetic attraction.

25. The method according to any preceding claim, wherein the method comprises repeatedly and / or frequently extracting the solid support from the reaction mixture.

26. The method according to any preceding claim, wherein the method further comprises washing the solid support, optionally wherein washing comprises the use of a washing buffer.

27. The method according to claim 26, wherein the washing buffer comprises a low concentration of salt, wherein the salt maintains the hybridization of the nucleic acid to the sequence-specific hybridization element, optionally wherein: (i) the salt is selected from the group consisting of: Lithium chloride; Sodium chloride; potassium chloride; and other monovalent salts; and / or (ii) the salt concentration is between 0.1-1M, 0.1-0.5M, 0.1-0.25M, or 0.1-0.15M.

28. The method according to claim 26, wherein the washing buffer is a combination of an organic solvent and water, optionally 70% Ethanol, 80% Ethanol, or 70% isopropanol.

29. The method according to any preceding claim, wherein the method further comprises exposing the solid support to a release solution and / or elution solution.

30. The method according to claim 29, wherein the release solution is selected from the group consisting of: laboratory grade water; RNAse-free water; 10 mM Tris-HCI, pH 7.5; 1 mM Citrate, pH 6.0; 1 mM Citrate; 1 mM EDTA pH 6.0; 5 mM Tris; 1 mM EDTA pH 8.0; and any low-salt aqueous solution.

31. The method according to either claim 29 or claim 30, wherein the release solution contains a limited concentration or no salt, reducing the hybridization of the nucleic acid molecule to the solid support, thereby stimulating the release of the nucleic acid molecule, optionally wherein a limited concentration of salt is less than lOOmM, less than 50mM, less than 25mM, or less than lOmM of salt.

32. The method according to any one of claims 29 to 31, wherein the release solution contains an endonuclease which digests the nucleic acid molecule, the sequence-specific hybridization element, or a combination thereof, optionally wherein the endonuclease is selected from the group consisting of: restriction enzymes, TALEN, Zinc-finger nucleases, and CRISPR-Cas.

33. The method according to any preceding claim, wherein the method comprises the use of small molecules and / or heat to remove the nucleic acid from the solid support.

34. The method according to claim 33, wherein the small molecule is DMSO or (chaotropic) salts, and / or the solid support is heated to a temperature at or above the melting temperature of the nucleic acid molecule-hybridization element hybrid.

35. The method according to any preceding claim, wherein the method comprises further incubating the reaction mixture after separation from the solid support.

36. The method according to any preceding claim, wherein the method comprises recycling the at least one solid support in the nucleic acid synthesis reaction, optionally wherein the method comprises combining the reaction mixture with at least one recycled solid support, or wherein the method comprises combining the reaction mixture with at least one new solid support and / or at least one recycled solid support.

37. The method according to any preceding claim, wherein the solid support is a bead, a column matrix, a container-surface modification, a hollow fibre, or a filter, membrane or sponge.

38. The method according to claim 37, wherein the bead is a solid bead, a magnetic bead, a superparamagnetic bead, a solid bead comprising a superparamagnetic core, a surface-enlarged bead, or a porous bead.

39. The method according to claim 38, wherein the surface of the bead comprises polyvinylalcohol (PVA).

40. The method according to any preceding claim, wherein:(i) the contact time of the solid support with the nucleic acid synthesis reaction is about 15 minutes, <15 minutes, <10 minutes, <5 minutes, <3 minutes, <1 minute, <30 seconds, or <15 seconds;(ii) the solid support binds the nucleic acid molecule in <5 minutes, <3 minutes, <1 minute, <30 seconds, <15 seconds, <10 seconds, <5 seconds, or <1 second; and / or(iii) the nucleic acid molecule is eluted from the solid support in about 5 minutes, <5 minutes, <3 minutes, <1 minute, <30 seconds, <15 seconds, <10 seconds, <5 seconds, <3 seconds, or <1 second.

41. The method according to any preceding claim, wherein the at least one sequence-specific hybridization element is a DNA oligonucleotide, an RIMA oligonucleotide, or comprises or consists of one or more non-natural nucleotides, termed Xeno nucleic acids (XNA).

42. The method according to any preceding claim, wherein the at least one sequence-specific hybridization element is selected from the group consisting of: a single stranded nucleic acid, a single stranded nucleic acid with a 3' modification, a single stranded nucleic acid with a modified sugar-phosphate backbone, a single stranded nucleic acid with a structural modification, and a hybrid single and double stranded sequence.

43. The method according to any preceding claim, wherein the at least one sequence-specific hybridization element on the solid support is a DNA oligonucleotide (DNA oligo), optionally wherein the DNA oligo is partly or completely modified, optionally with phosphothioate linkages.

44. The method according to claim 43, wherein the DNA oligo is an oligo dA sequence, optionally wherein the oligo dA sequence specifically binds to the polyT tail of the nucleic acid molecule.

45. The method according to claim 43, wherein the DNA oligo is an oligo dT sequence, optionally wherein the oligo dT sequence specifically binds to the polyA tail of the nucleic acid molecule.

46. The method according to any one of claims 1 to 42, wherein the at least one sequence-specific hybridization element on the solid support is an RIMA oligonucleotide (RNA oligo), optionally wherein the RNA oligo is partly or completely modified, optionally with phosphothioate linkages.

47. The method according to any preceding claim, wherein the at least one sequence-specific hybridization element is an oligonucleotide comprising or consisting of one or more non-natural nucleotides, termed Xeno nucleic acids (XNA), optionally wherein the XNA is selected from the group consisting of: 1,5-Anhydrohexitol nucleic acid (HNA), Cyclohexene nucleic acid (CeNA), Threose nucleic acid (TNA), Glycol nucleic acid (GNA), Locked nucleic acid (LNA), Peptide nucleic acid (PNA), and Fluoroarabino nucleic acid (FANA).

48. The method according to any preceding claim, wherein the at least one sequence-specific hybridization element binds to the coding sequence of the nucleic acid molecule, or to either or both of the untranslated regions (UTRs) of the nucleic acid molecule.

49. The method according to any preceding claim, wherein:(i) the at least one sequence-specific hybridization element is covalently linked to the solid support;(ii) the at least one sequence-specific hybridization element is covalently linked to the solid support by a reversible chemical linkage; and / or(iii) the at least one sequence-specific hybridization element is non-covalently linked to the solid support.

50. An apparatus configured to perform the method of any one of claims 1 to 49.

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