Methods of nucleic acid processing and synthesis

Abstract

Methods of filtering, concentrating, and producing nucleic acids utilize microfluidic channels separated by a permeable membrane. The permeable membrane enables the transfer of synthesis reagents, and the removal of products or byproducts.

Description

[0001] METHODS OF NUCLEIC ACID PROCESSING AND SYNTHESIS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U. S. Provisional Application No.

[0002] 63 / 718,848, filed November 11, 2024, which is incorporated by reference herein in its enthety.

[0003] GOVERNMENT SUPPORT CLAUSE

[0004] This invention was made with government support under Grant Nos. 5R01GM134042 and RO1HGO13861 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0005] BACKGROUND

[0006] In RNA synthesis, substrate ribonucleotides (rNTPs) and metal ions (e.g., magnesium ions) are often supplied to facilitate continuous RNA synthesis. Metal ions act as an essential cofactor for the certain RNA polymerases, stabilizing the transition state complex necessary for RNA synthesis. However, metal ions also pose a risk to RNA by catalyzing the hydrolysis of the RNA backbone, reducing the yield of full-length RNA and leading to truncated RNA impurities in a wide range of lengths. Hydrolysis of RNA occurs via two primary mechanisms: 1) contaminant nuclease (enzymatic) degradation and 2) intrinsic, or in-line, hydrolysis byhydroxide ions in water. The latter degradation increases with increasing pH.

[0007] In current RNA manufacturing, RNA is exposed to high concentrations of metal ions for many hours (or longer). To further stabilize RNA against degradation, there is a need to remove the metal ions (and other synthesis reagents) immediately after RNA synthesis is complete. These needs and others are at least partially satisfied by the present disclosure.

[0008] SUMMARY

[0009] In some aspects, disclosed herein is a method of filtering nucleic acids, the method including: a) providing a microfluidic device including: a first channel including a first inlet and a first outlet; a second channel adjacent and parallel to the first channel and including a second inlet and a second outlet; and a first permeable membrane disposed between and fluidically connecting the first channel and the second channel; b) flowing a crude mixture including synthesized nucleic acids and at least one nucleic acid synthesis reagent and / or byproduct through the first inlet; c) simultaneously flowing a first extraction solution through the second inlet; and d) recovering a processed mixture from the first outlet; wherein the processed mixture includes a lower concentration of the at least one nucleic acid synthesis reagent and / or byproduct than the crude mixture.

[0010] In some aspects, also disclosed herein is a method of filtering and / or concentrating nucleic acids, the method including: a) providing a microfluidic device comprising a first channel comprising a first inlet, wherein the first channel bifurcates into a first sub-channel having a first outlet and second sub-channel having a second outlet, b) positioning a positive electrode on the same side of the first channel as the first sub-channel and a negative electrode on the same side of the first channel as the second sub-channel, thereby creating an electric gradient perpendicular to the first channel, c) flowing a crude mixture comprising nucleic acids through the first inlet, and recovering a processed mixture from the first outlet, wherein the processed mixture comprises a higher concentration of the nucleic acid than the crude mixture. In some embodiments, the crude mixture further comprises at least one nucleic acid synthesis reagent and / or byproduct and wherein the processed mixture comprises a lower concentration of at least one positively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture.

[0011] In some aspects, also disclosed herein is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being downstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and including a 5’ terminus, wherein the 5’ terminus of the nontemplate strand extends past the 3’ terminus of the template strand, thereby forming a first 5’ overhang; b) enzymatically adding nucleotides to the 3' terminus of the template strand by filling in the first 5’ overhang, wherein a nucleotide added to the 3’ terminus of the template strand includes a first handle; c) functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0012] In some aspects, also disclosed herein is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and including a 5’ terminus, wherein the 3’ terminus of the template strand extends past the 5’ terminus of the nontemplate strand, thereby forming a first 3’ overhang; b) enzymatically adding nucleotides to the 3’ terminus of the template strand using a terminal transferase, wherein a nucleotide added to the 3’ terminus of the template strand includes a first handle; c) functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0013] In some aspects, also disclosed herein is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and including a 5’ terminus; b) ligating a first adapter, including a first handle, to the 3' terminus of the template strand and the 5’ terminus of the nontemplate strand, wherein an RNA polymerase is functionally coupled to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; c) producing RNA from the catalyst for RNA production; and d) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0014] In some aspects, also disclosed herein is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5’ direction, a promoter sequence and an RNA-encoding sequence, the RNA-encoding sequence being upstream of. and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, b) enzymatically nicking a site of the non template strand using a nicking endonuclease, thereby generating a gap in the nontemplate strand, the gap being a single stranded portion of the template strand without a corresponding complementary portion of the nontemplate strand, wherein the site is within a region of the nontemplate strand selected from the group consisting of: 1) a region of the nontemplate strand complementary to, and base paired with, the promoter sequence, ii) 1-100 nucleotides upstream of a sequence of the nontemplate strand complementary to, and base paired with, the promoter sequence, and iii) combinations thereof; c) hybridizing an invading nucleic acid molecule to the single stranded portion of the template strand, the invading nucleic acid molecule being complementary to the single stranded portion of the template strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA.from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0015] In some aspects, also disclosed herein is a method of RNA production, the method including: a.) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5' direction, a promoter sequence and an RNA-encoding sequence, the RN A-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, b) enzymatically nicking a site of the template strand using a nicking endonuclease, thereby generating a gap in the template strand, the gap being a single stranded portion of the nontemplate strand without a corresponding complementary portion of the template strand, wherein the site is within a region of the template strand selected from the group consisting of: i) a region of the promoter sequence, ii) 1—100 nucleotides upstream of the promoter sequence, and iii) combinations thereof; c) hybridizing an invading nucleic acid molecule to the single stranded portion of the nontemplate strand, the invading nucleic acid molecule being complementary to the single stranded portion of the nontemplate strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0016] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within tills description and be protected by the accompanying claims.

[0017] BRIEF DESCRIPTION OF DRAWINGS FIG. 1 depicts an example device in accordance with one embodiment of the present disclosure.

[0018] FIG. 2 depicts another example device in accordance with another embodiment of the present disclosure. FIG. 3 depicts yet another example device in accordance with yet another embodiment of the present disclosure.

[0019] FIG. 4 depicts a schematic of the in-line transcription reactor and desalting device. The crude RNA products exiting the reactor enter the purification stage, where salts and unreacted rNTPs are removed from the RNA stream. Applying an electric field across the membrane can accelerate the purification process.

[0020] FIG. 5A shows a schematic representation of the working principle of a semi-permeable hydrogel-integrated desalting device. FIG. 5B shows the chemical structures of the components in the monomer mixture used for polyacrylamide hydrogel. FIG. 5C shows that the mesh side of the hydrogel membrane can be tuned via both crosslinking density and total polymer wt%.

[0021] FIGS. 6A-6C depict schematic representations of multiple devices in series configuration (FIG.6A), dual-membrane device configuration (FIG.6B), and an electric-field assisted accelerated diffusion device (FIG. 6C).

[0022] FIG. 7 depicts schematics of electrode incorporation into the PDMS device.

[0023] FIG. 8 depicts a schematic of electrode wires incorporation into the PDMS device. FIG. 9 depicts a schematic of using a composite PDMS-Ag as electrodes in the desalting device.

[0024] FIG. 10 shows an optical microscope image showing the 10 wt% hydrogel membrane. FIG. 10B shows the brightfield microscope image of 10 wt% hydrogel membrane. FIG. 10C shows the hydrogel membrane thickness can be tuned by changing the width of the photomask used for the photolithography.

[0025] FIG. 11A shows the diffusion coefficients of Fluorescein and FITC-Dextran in hydrogels and in free solution. FIG. 11B is fluorescent images showing the (i) resistance of large molecules (FITC-Dextran) to pass through hydrogel membrane and (ii) passage of small molecules (rhodamine 6G).

[0026] FIG. 12A shows Mg2+separation using a 10 wt% hydrogel membrane microfluidic device. A 40 mM Mg2+solution was passed through the device at a 0.1 uL / min flow rate. The extraction flow was set at 4 different flow rates. FIGS. 12B-12E show the cumulative Mg2+separated into extraction streams at different flow rates is quantified using ICP-MS.

[0027] FIG. 13 shows schematics of the experimental set up to determine the maximum burst of pressure of gel membrane within the device. A graphical comparison shows the evolution of pressure inside the channel over time across gels with different total polymer concentrations. Snapshots from a video of gel show visual confirmation of gel rupture. Images were from a device with a gel membrane made with 15% total polymer concentration and is 300 um wide.

[0028] FIG. 14 shows an image of a device with dual gel membranes (indicated with dotted red boxes) delineating the channels for RNA stream and extraction buffer stream.

[0029] FIG. 15 shows a schematic of RNA concentration via electrophoresis. RNA migrates toward the positive electrode and is selectively extracted through an outlet channel, yielding a concentrated stream.

[0030] FIG. 16A shows a schematic of the experimental setup. The device consists of a simple rectangular channel with a hydrogel integrated in the center. Cut pipette tips serve as reservoirs at the inlet and outlet, and platinum wires are inserted into them to establish an electrical connection. FIG. 16B shows snapshots showing the migration of a FAM-labeled oligonucleotide (36 bases, green in the image) and a CY5-labeled DNA (-1 kbp, red in the image), respectively, under an electric field of E = 100 V / cm. After 3 min, the longer CY5-DNA shows negligible migration within the gel, while the shorter FAM-oligonucleotide has migrated significantly deeper into the gel section. After 7 min, most of the shorter DNA passed through the gel, but the large DNA has accumulated on the surface of gel, forming a band. The yellow lines indicate the gel boundary, and the white lines indicate the channel.

[0031] DETAILED DESCRIPTION

[0032] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0033] DEFINITIONS

[0034] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0035] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, nonlimiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0036] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0037] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0038] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0039] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0040] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically-stated otherwise.

[0041] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0042] The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which maybe single-stranded or double-stranded and may represent the sense or the antisense strand).

[0043] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length > 100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110).

[0044] A “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability.

[0045] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and / or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereof).

[0046] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or II), isoleucine (lie or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N -Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, P-alanine, P- Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2 ' -Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0047] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C -terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C-terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0048] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C -terminal truncation or both of a reference polypeptide or a 5 ' -terminal or 3!-terminal truncation or both of a reference polynucleotide).

[0049] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the enthe length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C -terminal region of a polypeptide or the ' -terminal region and / or the 3 ' terminal region of a polynucleotide. The term “ at least a fragment ” encompasses the full length polynucleotide or full length polypeptide.

[0050] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0051] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N-temiinus, the C -terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein ). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0052] A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3’ end of a first polynucleotide to a 5’ end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0053] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence. A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0054] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0055] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0056] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N. Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0057] A “nucleotide analog” is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, -methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties. There are many varieties of these types of molecules available in the art and available herein.

[0058] “Nucleotide substitutes” or “modified nucleotides” are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. There are many varieties of these types of molecules available in the art and available herein. Another example of a modified nucleotide is a “locked nucleotide” in which the sugar- moiety is constrained by a methylene bridge connecting the 2’ oxygen and the 4’ carbon of the pentose ring. Tills structural modification locks the ribose in the 3’-endo conformation, enhancing overall stability.

[0059] It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are many varieties of these types of molecules available in the art and available herein.

[0060] “Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RN A for limited periods of time.

[0061] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated. “Concentrate” or “concentrated” describes a process by which the proportion of a desired component of a mixture is increased, generally a solute. To concentrate a desired solute, the volume of the solution is reduced to lower the amount of solvent present thereby increasing the relative proportion of solute to solvent. DNA, RNA, and proteins are all examples of solutes that can be concentrated.

[0062] MICROFLUIDIC DEVICE FIG. 1 depicts an example microfluidic device 100. The device 100 is made up of a scaffold material, for example, polydimethylsiloxane (PDMS), polyethylene, Polymethylmethacrylate (PMMA), Polystyrene (PS), Polycarbonate (PC), Cyclic Olefin Copolymer (COC), polyethylene glycol, polymethyl methacrylate, polyethylene terephthalate, polyester, polystyrene, polycarbonate, polytetrafluoroethylene, or other suitable polymers or hydrogels. In other aspects, the scaffold material can include quartz or glass. The scaffold material additionally includes at least one photoinitiator, for example, benzophenone, alkylphenone, thioxanthone, peroxide, perester, iminosulphones, ketone, or any derivatives or combinations thereof. In some aspects, the photoinitiator can be coated on one or more surfaces of the scaffold material. Additionally or alternatively, in other aspects, the photoinitiator can be incorporated into or dispersed withi n the scaffold material. In yet other aspects, the scaffold material may not include a photoinitiator.

[0063] In some aspects, the scaffold material can include at least about 0.1 wt% photoinitiator (e.g,, at least about 0.15 wt%, at least about 0.2 wt%, at least about 0.25 wt%, at least about 0.3 wt%, at least about 0.35 wt%, at least about 0.4 wt%, at least about 0.45 wt%, at least about 0.5 wt%, at least about 0.55 wt%, at least about 0.6 wt%, at least about 0.65 wt%, at least about 0.7 wt%, at least about 0.75 wt%, at least about 0.8 wt%, at least about 0.85 wt%, at least about 0.9 wt%, at least about 0,95 wt%, at least about 1 wt%). In some aspects, the scaffold material can include up to about 1 wt% (e.g., up to about 0,95 wt%, up to about 0,9 wt%, up to about 0.85 wt%, up to about 0.8 wt%, up to about 0.75 wt%, up to about 0.7 wt%, up to about 0.65 wt%, up to about 0.6 wt%, up to about 0.55 wt%, up to about 0.5 wt%, up to about 0.45 wt%, up to about 0.4 wt%, up to about 0.35 wt%, up to about 0,3 wt%, up to about 0.25 wt%. up to about 0.2 wt%, up to about 0.1 wt%, up to about 0.1 wt%).

[0064] It is considered that the scaffold material can include an amount of photoinitiator ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the scaffold material can include from about 0.1 wt% to about 1 wt% photoinitiator (e.g., from about 0.15 wt% to about 0.95 wt%, from about 0.2 wt% to about 0.9 wt%, from about 0.25 wt% to about 0.85 wt%, from about 0, 3 wt% to about 0.8 wt%, from about 0,35 wt% to about 0.75 wt%, from about 0.4 wt% to about 0.7 wt%, from about 0.45 wt% to about 0.65 wt%, from about 0.5 wt% to about 0.6 wt%, from about 0.1 wt% to about 0.55 wt%, from about 0.15 wt% to about 0.5 wt%, from about 0.2 wt% to about 0.45 wt%, from about 0.25 wt% to about 0,4 wt%, from about 0.3 wt% to about 0.35 wt%, from about 0.55 wt% to about 1 wt%, from about 0.6 wt% to about 0.95 wt%, from about 0.65 wt% to about 0.9 wt%, from about 0.7 wt% to about 0.85 wt%, from about 0.75 wt% to about 0.8 wt%).

[0065] The device 100 includes a first channel 102 and a second channel 104 which are recessed into the scaffold material. The first channel 102 and the second channel 104 are immediately adjacent, however, in other aspects, they may be interspersed by, as an example, a connection region. A portion of the first channel 102 is parallel to a portion of the second channel 104. In some aspects, all of the first channel 102 may be parallel to all of the second channel 104. In other aspects, only a portion of the first channel 102 may be parallel to only a portion of the second channel 102. The first channel 102 and the second channel 104 are depicted as approximately the same size, however, in other aspects, the first channel 102 and the second channel 104 may have different dimensions.

[0066] Each of the first channel 102 and the second channel 104 includes an inlet 106a, 106b (e.g., a first inlet 106a and a second inlet 106b) and an outlet 108a, 108b (e.g., a first outlet 108a and a second outlet 108b). The inlets 106a, 106b are adjacent, and the outlets 108a, 108b are adjacent (i.e., the device is configured for parallel flow). In other aspects, the first inlet 106a may be adjacent to the second outlet 108b, and the second inlet 106b may be adjacent to the first outlet 108a (i.e., the device may be configured for counter flow).

[0067] A permeable membrane 110 is disposed between the first channel 102 and the second channel 104, thereby allowing exchange of fluid from the first channel 102 to the second channel 104 (or vice versa) across the permeable membrane 110 (i.e., fluidically connecting the first channel 102 and the second channel 104).

[0068] The permeable membrane 110 is a hydrogel includes a polymer, for example, polyacrylamide, polyethylene glycol, a zwitterionic polymer, or any combination thereof. The permeable membrane 110 is added to the device 100 by crosslinking a hydrogel precursor with the photoinitiator in the scaffold material (e.g., using a stereolithography photomask). This provides at least a partial fluidic seal between the permeable membrane 110 and the remainder of the device 100 to prevent fluid from flowing around the permeable membrane 110 instead of through the permeable membrane 110. In some aspects, the permeable membrane can be at least about 200 pm thick (e.g., at least about 220 pm, at least about 240 pm, at least about 260 pm, at least about 280 pm, at least about 300 pm, at least about 320 pm, at least about 340 pm, at least about 360 pm, at least about 380 pm, at least about 400 pm, at least about 420 pm, at least about 440 pm, at least about 460 pm, at least about 480 pm, at least about 500 pm). In some aspects, the permeable membrane can be up to about 500 pm thick (e.g., up to about 480 pm, up to about 460 pm, up to about 440 pm, up to about 420 pm, up to about 400 pm, up to about 80 pm, up to about 360 pm, up to about 340 pm, up to about 320 pm, up to about 300 pm, up to about 280 pm, up to about 260 pm, up to about 240 pm, up to about 220 pm, up to about 200 pm).

[0069] It is considered that the permeable membrane can have a thickness ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the permeable membrane can be from about 200 pm to about 500 pm thick (e.g., from about 220 pm to about 480 pm, from about 240 pm to about 460 pm, from about 260 pm to about 440 pm, from about 280 pm to about 420 pm, from about 300 pm to about 400 pm, from about 320 pm to about 380 pm, from about 340 pm to about 360 pm, from about 200 pm to about 360 pm, from about 220 pm to about 340 pm, from about 240 pm to about 320 pm, from about 260 pm to about 300 pm, from about 340 p m to about 500 pm, from about 360 pm to about 480 pm, from about 380 pm to about 460 pm, from about 360 pm to about 440 pm).

[0070] In some aspects, the permeable membrane 110 can include at least about 5 wt% total polymer (e.g., at least about 6 wt%, at least about 7 wt%, at least about 8 wt%, at least about 9 wt%, at least about 10 wt%, at least about 12 wt%, at least about 14 wt%, at least about 16 wt%, at least about 18 wt%, at least about 20 wt%, at least about 22 wt%, at least about 24 wt%, at least about 26 wt%, at least about 28 wt%, at least about 30 wt%, at least about 32 wt%, at least about 34 wt%, at least about 36 wt%, at least about 38 wt%, at least about 40 wt%). In some aspects, the permeable membrane 110 can include up to about 40 wt% total polymer (e.g., up to about 38 wt%, up to about 36 wt%, up to about 34 wt%, up to about 32 wt%, up to about 30 wt%, up to about 28 wt%, up to about 26 wt%, up to about 24 wt%, up to about 22 wt%, up to about 20 wt%, up to about 18 wt%, up to about 16 wt%, up to about 14 wt%, up to about 12 wt%, up to about 10 wt%, up to about 9 wt%, up to about 8 wt%, up to about 7 wt%, up to about 6 wt%, up to about 5 wt%).

[0071] It is considered that the permeable membrane 110 can include an amount of total polymer ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the permeable membrane 110 can include from about 5 wt% to about 40 wt% total polymer (e.g., from about 6 wt% to about 38 wt%, from about 7 wt% to about 36 wt%, from about 8 wt% to about 34 wt%, from about 9 wt% to about 32 wt%, from about 10 wt% to about 30 wt%, from about 12 wt% to about 28 wt%, from about 14 wt% to about 26 wt%, from about 16 wt% to about 24 wt%, from about 18 wt% to about 22 wt%, from about 5 wt% to about 20 wt%, from about 6 wt% to about 18 wt%, from about 7 wt% to about 16 wt%, from about 8 wt% to about 14 wt%, from about 9 wt% to about 12 wt%, from about 20 wt% to about 40 wt%, from about 22 wt% to about 38 wt%, from about 24 wt% to about 36 wt%, from about 26 wt% to about 34 wt%, from about 28 wt% to about 32 wt%).

[0072] FIG. 2 shows another example device 200. The device 200 is similar to the device 100 but includes a third channel 212, which includes a third inlet 206c and a third outlet 208c, and two permeable membranes 210a, 210b (e.g,, a first permeable membrane 210a and a second permeable membrane 210b). The first channel 202 and second channel 204 are described above (e.g., as the first channel 102 and the second channel 104). The first channel 202 and the third channel 212 are immediately adjacent, however, in other aspects, they may be interspersed by, as an example, a connection region. A portion of the first channel 202 is parallel to a portion of the third channel 21. In some aspects, all of the first channel 202 may be parallel to all of the third channel 212. In other aspects, only a portion of the first channel 202 may be parallel to only a portion of the third channel 212. The first channel 202 and the third channel 212 are depicted as approximately the same size, however, in other aspects, the first channel 202 and the third channel 212 may have different dimensions.

[0073] The first inlet 206a and the second inlet 206b are adjacent, and the first outlet 208a and the third outlet 208c are adjacent (i.e., the device is configured for parallel flow). In other aspects, the first inlet 206a can be adjacent to the second outlet 208b and / or the first outlet 208a can be adjacent to the third inlet 206c (i.e., the device can be configured for counter flow).

[0074] The first permeable membrane 210a is described above (e.g., as the permeable membrane 110). The second permeable membrane 210b is disposed between the first channel 202 and the third channel 12, thereby allowing exchange of fluid from the first channel 202 to the third channel 212 (or vice versa) across the second permeable membrane 210b (i.e., fluidically connecting the first channel 202 and the third channel 212).

[0075] In some aspects, the first permeable membrane 210a and the second permeable membrane 210b can have the same properties (e.g., composition, thickness, and / or pore size). In other aspects, the first permeable membrane 210a and the second permeable membrane 210b can have different properties (e.g., composition, thickness, and / or pore size). The two permeable membranes 210a, 210b are depicted as having the same length, however, in other aspects, they may have different lengths.

[0076] The first permeable membrane 210a is spaced from the second membrane 210b (i.e., the second membrane 210b begins and ends further along the first channel 202 than the first membrane 210a). In some aspects, the first permeable membrane 210a and the second permeable membrane 210b may be fully spaced apart (i.e., the second membrane 210b begins at or after the point along the first channel 202 where the first permeable membrane 210a ends). In other aspects, the first permeable membrane 210a and the second permeable membrane 210b may be only partially spaced apart (i.e., the second membrane 210b begins before the point along the first channel 202 where the first permeable membrane 210a ends).

[0077] In yet other aspects, the first permeable membrane 210a and the second permeable membrane 210b may be adjacent across the first channel 202 (i.e., the two permeable membranes 210a, 210b start and end at approximately the same points along the first channel 202, or, if one permeable membrane is longer than the other, the shorter permeable membrane starts after the point along the first channel 202 where the longer permeable membrane starts, and it ends before the point along the first channel 202 where the longer permeable membrane ends). For example, FIG. 3 depicts an example device 300 which is the same as the device 200 but has two permeable membranes 310a, 310b which are adjacent.

[0078] It is understood that the design of the device 200 and the device 300 are modular and may be combined. That is, the device 200 and / or the device 00 may further include additional channels disposed on the same or alternating sides of the first channel 202 / 302 (e.g., a fourth channel, a fifth channel, a sixth channel, a seventh channel, an eighth channel, etc.). Any additional permeable membranes connecting the first channel 202 / 302 to the additional channels may be spaced apart, partially or completely, or may be adjacent across the first channel 202 / 302.

[0079] Any of the disclosed devices 100 / 200 / 300 can further include at least one electrode pair, for example, to aid fluid flow across the permeable membrane(s). In some aspects, the at least one electrode pair can be placed on either side of the first channel. Additionally or alternatively, in other aspects, the at least one electrode pair can be placed underneath the microfluidic device. Additionally or alternatively, in yet other aspects, at least one pair of electrode pins can be inserted into any of the channels. In some aspects, the at least one electrode pair can be embedded within the microfluidic device. In some aspects, the at least one electrode pair can be arranged to provide an electrical gradient perpendicular to the first channel. In some aspects, particularly when the device includes at least one electrode pair providing an electrical gradient perpendicular to the first channel, the first channel can bifurcate downstream of the inlet in order to separate the contents of the crude mixture by electrical charge. FIG. 15 depicts an example first channel 402 which includes a positive electrode 414a, a negative electrode 414b, and a bifurcation point 416. The bifurcation point 416 can be positioned at any point along the length of the first channel 402. At the bifurcation point 416, the first channel 402 splits into a first sub-channel 418a and a second sub-channel 418b. The first sub-channel 418a includes a first outlet 420a, and the second sub -channel 418b includes a second outlet 420b.

[0080] The positive electrode 414a is positioned on the same side of the first channel 402 as the first sub-channel 418a. As the crude mixture flows through the first channel 402, any negatively charged components - namely, nucleic acids - are pulled towards the positive electrode 414a and directed into the first sub-channel 418a. As such, the processed mixture extracted from the first output 420a of the first sub-channel 418a has a higher concentration of nucleic acids than the crude mixture.

[0081] The negative electrode 414b is positioned on the same side of the first channel 402 as the second sub-channel 418b. As the crude mixture flows through the first channel 402, any positively charged components - for example, Mg2+or other positively charged metal ions -are pulled toward the negative electrode 414b and directed into the second sub-channel 418b. As such, if the crude mixture contains positively charged components, the processed mixture extracted from the first output 420a of the first sub-channel 418b has a lower concentration of said positively charged components than the crude mixture.

[0082] As depicted in FIG. 15, the first channel 402 includes a first permeable membrane 422a and a second permeable membrane 422b, which can include any of the permeable membranes disclosed herein. The first and second permeable membranes 422a, 422b can be positioned at any point along the length of the first channel 402, but generally are positioned upstream of the bifurcation point 416. The first and second permeable membranes 422a, 422b allow the first channel 402 to be used with any of the disclosed microfluidic devices having two adjacent permeable membranes, for example, the device 300 shown in FIG. 3. In such a configuration, the positive and negative electrodes 414a, 414b can pull negatively charged components and positively charged components, respectively, out of the first channel 402, thereby decreasing the concentration of said negatively and positively charged components in the processed mixture extracted from the first output 420a of the first sub-channel 418b. In some aspects, the first channel 402 may only include the first permeable membrane 422a or the second permeable membrane 422b. In such aspects, the first channel 402 can be used with any of the disclosed microfluidic devices including only one permeable membrane, for example, the device 100 shown in FIG. 1, or with any of the disclosed microfluidic devices including two, non-adjacent permeable membranes, lor example, the device 200 shown in FIG.

[0083] 2. As one example, when the first channel 402 is used in the device 200, the first sub-channel 418a can be fluidically connected to the third channel 212 as described above. In other aspects, the first channel 402 may include no permeable membranes, and be used merely to concentrate nucleic acids in a crude mixture or to remove positively charged components only.

[0084] METHODS

[0085] In one aspect, provided is a method of filtering nucleic acids, the method including: a) providing any of the disclosed microfluidic devices; b) flowing a crude mixture including synthesized nucleic acids and at least one nucleic acid synthesis reagent and / or byproduct through the first inlet; c) simultaneously flowing a first extraction solution through the second inlet; and d) recovering a processed mixture from the first outlet; wherein the processed mixture includes a lower concentration of the at least one nucleic acid synthesis reagent and / or byproduct than the crude mixture.

[0086] In some aspects, the processed mixture can include from about 70% to about 100% less of the at least one nucleic acid synthesis reagent and / or byproduct than the crude mixture (e.g., from about 75% to about 100%, from about 80% to about 100%, from about 81%’ to about 100%, from about 82% to about 100%, from about 83% to about 100%, from about 84% to about 100%, from about 85% to about 100%, from about 86% to about 100%, from about 87% to about 100%, from about 88% to about 100%, from about 89% to about 100%, from about 90% to about 100%, from about 91% to about 100%, from about 92% to about 100%, from about 93% to about 100%, from about 94% to about 100%, from about 95% to about 100%, from about 96% to about 100%, from about 97%> to about 100%, from about 98% to about 100%, from about 99% to about 100%).

[0087] In some aspects, the synthesized nucleic acids can include RNA, DNA, or any combination thereof. Specific examples of nucleic acid synthesis are described in further detail below. In some aspects, the at least one nucleic acid synthesis reagent and / or byproduct can include Mg2+, ribonucleotides, pyrophosphate, abortive nucleic acids, or any combination thereof. It is understood that the synthesized nucleic acids refer to nucleic acids of a target length or size (or within an acceptable range of length or size), whereas abortive nucleic acids refer to nucleic acids which are shorter than the target length or size (or which are shorter than the acceptable range of length or size). In some aspects the synthesized nucleic acids and nucleic acid synthesis reagent and / or byproduct are separated by a permeable membrane by size.

[0088] In some aspects, the first extraction solution can be a buffer, including, but not limited to, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES), a glutamate -based buffer, ethylenediaminetetraacetic acid (EDTA), Tween 20, or any combination thereof. In other aspects, the first extraction solution can be water.

[0089] In some aspects, the at least one nucleic acid synthesis reagent and / or byproduct can pass through the permeable membrane into the second channel and synthesized nucleic acids cannot. In some such aspects, the permeable membrane can separate the at least one nucleic acid synthesis reagent and / or byproduct from the synthesized nucleic acids by size.

[0090] In some aspects, the flow of the crude mixture can be parallel to the flow of the first extraction solution. In other aspects, the flow of the crude mixture can be counter to the flow of the first extraction solution.

[0091] In some aspects, when the microfluidic device includes a third channel, step c) can further include simultaneously flowing a second extraction solution through the third inlet. It is understood that, if the device includes more than three channels, an additional extraction solution can be flowed through each additional channel. In some aspects, the first extraction solution can be the same as the second extraction solution (and any additional extraction solutions). In other aspects, the first extraction solution may be different than the second extraction solution (and any additional extraction solutions).

[0092] In some aspects, when the microfluidic device includes at least one electrode pair, step c) can further include applying an electric gradient across the first permeable membrane and / or the second permeable membrane (and / or any additional membranes if the microfluidic devices includes more than three channels). In some aspects, the electric gradient can be perpendicular to the flow of the crude mixture. In some aspects, the at least one electrode pair can be placed on either side of the first channel. Additionally or alternatively, in other aspects, the at least one electrode pair can be placed underneath the microfluidic device. Additionally or alternatively, in yet other aspects, at least one pair of electrode pins can be inserted into the second channel (and / or any additional channels if the microfluidic device includes more than two channels). In some aspects, the at least one electrode pair can be embedded within the microfluidic device. In one aspect, provided is a method of filtering and / or concentrating nucleic acids, including a) providing any of the disclosed microfluidic devices including a first channel which bifurcates into a first sub-channel having a first outlet and a second sub-channel having a second outlet; b) positioning a positive electrode on the same side of the first channel as the first sub-channel and a negative electrode on the same side of the first channel as the second sub-channel, thereby creating an electric gradient perpendicular to the first channel; c) flowing a crude mixture including nucleic acids through the first inlet; and d) recovering a processed mixture from the first outlet.

[0093] In some aspects, the processed mixture can include a higher concentration of the nucleic acid than the crude mixture. For example, in some aspects, the processed mixture can include from about 10% to about 100% more of the nucleic acid than the crude mixture (e.g., from about 20% to about 95%, from about 30% to about 90%, from about 40% to about 85%, from about 50% to about 80%, from about 60% to about 70%, from about 10% to about 70%, from about 20% to about 60%, from about 30% to about 50%, from about 60%' to about 100%, from about 70% to about 95%, from about 80% to about 90%).

[0094] In some aspects, the crude mixture can further include at least one nucleic acid synthesis reagent and / or byproduct, and the processed mixture can include a lower concentration of at least one positively charged nucleic acid synthesis reagent and / or byproduct (e.g., Mg2+or other positively charged metal ions) than the crude mixture. For example, in some aspects, the processed mixture can include from about 70% to about 100% less of the at least one positively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture (e.g., from about 75% to about 100%, from about 80% to about 100%, from about 81% to about 100%, from about 82% to about 100%, from about 83% to about 100%, from about 84% to about 100%, from about 85% to about 100%, from about 86% to about 100%, from about 87% to about 100%, from about 88% to about 100%, from about 89% to about 100%, from about 90% to about 100%, from about 91% to about 100%, from about 92% to about 100%, from about 93% to about 100%, from about 94% to about 100%, from about 95% to about 100%, from about 96% to about 100%, from about 97% to about 100%, from about 98% to about 100%, from about 99% to about 100%).

[0095] In some aspects, particularly when the microfluidic device includes a second or third channel on the same side of the first channel as the first sub-channel, the processed mixture can include a lower concentration of at least one negatively charged nucleic acid synthesis reagent and / or byproduct (e.g., nucleotides or nucleosides) than the crude mixture. For example, in some aspects, the processed mixture can include from about 70% to about 100% less of the at least one negatively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture (e.g., from about 75% to about 100%, from about 80% to about 100%, from about 81% to about 100%, from about 82% to about 100%, from about 83% to about 100%, from about 84% to about 100%, from about 85% to about 100%, from about 86% to about 100%, from about 87% to about 100%, from about 88% to about 100%, from about 89% to about 100%, from about 90% to about 100%, from about 91% to about 100%, from about 92% to about 100%’, from about 93% to about 100%, from about 94%’ to about 100%, from about 95% to about 100%, from about 96% to about 100%, from about 97% to about 100%, from about 98% to about 100%, from about 99% to about 100%).

[0096] In another aspect, provided is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5 ’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being downstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and including a 5’ terminus, wherein the 5’ terminus of the nontemplate strand extends past the 3’ terminus of the template strand, thereby forming a first 5’ overhang; b) enzymatically adding nucleotides to the 3' terminus of the template strand by filling in the first 5’ overhang, wherein a nucleotide added to the 3’ terminus of the template strand includes a first handle; c) functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0097] In yet another aspect, provided is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5 ’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and including a 5’ terminus, wherein the 3’ terminus of the template strand extends past the 5’ terminus of the nontemplate strand, thereby forming a first 3’ overhang; b) enzymatically adding nucleotides to the 3’ terminus of the template strand using a terminal transferase, wherein a nucleotide added to the 3’ terminus of the template strand includes a first handle; c) functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA- encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0098] In yet still another aspect, provided is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence: and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and including a 5’ terminus; b) ligating a first adapter, including a first handle, to the 3' terminus of the template strand and the 5’ terminus of the nontemplate strand, wherein an RNA polymerase is functionally coupled to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; c) producing RNA from the catalyst for RNA production; and d) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0099] In yet still another aspect, provided is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5’ direction, a promoter sequence and an RNA-encoding sequence, the RN -encoding sequence being upstream of. and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, b) enzymatically nicking a site of the non template strand using a nicking endonuclease, thereby generating a gap in the nontemplate strand, the gap being a single stranded portion of the template strand without a corresponding complementary portion of the nontemplate strand, wherein the site is within a region of the nontemplate strand selected from the group consisting of: i) a region of the nontemplate strand complementary to, and base paired with, the promoter sequence, ii) 1-100 nucleotides upstream of a sequence of the nontemplate strand complementary to, and base paired with, the promoter sequence, and iii) combinations thereof; c) hybridizing an invading nucleic acid molecule to the single stranded portion of the template strand, the invading nucleic acid molecule being complementary to the single stranded portion of the template strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA.from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to any of the disclosed methods of filtering nucleic acids. In yet still another aspect, provided is a method of RNA production, the method including: a) providing a functional template DNA, the functional template DNA including: (i) a template strand, the template strand including, in a 3’ to 5’ direction, a promoter sequence and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, b) enzymatically nicking a site of the template strand using a nicking endonuclease, thereby generating a gap in the template strand, the gap being a single stranded portion of the nontemplate strand without a corresponding complementary portion of the template strand, wherein the site is within a region of the template strand selected from the group consisting of: i) a region of the promoter sequence, ii) 1-100 nucleotides upstream of the promoter sequence, and iii) combinations thereof: c) hybridizing an invading nucleic acid molecule to the single stranded portion of the nontemplate strand, the invading nucleic acid molecule being complementary to the single stranded portion of the nontemplate strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst tor RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to any of the disclosed methods of filtering nucleic acids.

[0100] In some aspects, the site can be within a region of the template strand that is at least 1 nucleotide (e.g., at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides) upstream of the promoter sequence. In some aspects, the site can be within a region of the template strand that is up to 100 nucleotides (e.g., up to 95 nucleotides, up to 90 nucleotides, up to 85 nucleotides, up to 80 nucleotides, up to 75 nucleotides, up to 70 nucleotides, up to 65 nucleotides, up to 60 nucleotides, up to 55 nucleotides, up to 50 nucleotides, up to 45 nucleotides, up to 40 nucleotides, up to 35 nucleotides, up to 30 nucleotides, up to 25 nucleotides, up to 20 nucleotides, up to 15 nucleotides, up to 10 nucleotides, up to 9 nucleotides, up to 8 nucleotides, up to 7 nucleotides, up to 6 nucleotides, up to 5 nucleotides, up to 4 nucleotides, up to 3 nucleotides, up to 2 nucleotides, up to 1 nucleotide) upstream of the promoter sequence. It is considered that the s ite can be within a region of the template strand that is a number of nucleotides upstream of the promoter sequence ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the site can be within a region of the template strand that is 1 to 100 nucleotides (e.g., 2 to 95 nucleotides, 3 to 90 nucleotides, 4 to 85 nucleotides, 5 to 80 nucleotides, 6 to 75 nucleotides, 7 to 70 nucleotides, 8 to 65 nucleotides, 9 to 60 nucleotides, 10 to 55 nucleotides, 15 to 50 nucleotides, 20 to 45 nucleotides, 25 to 40 nucleotides, 30 to 35 nucleotides, 1 to 35 nucleotides, 2 to 30 nucleotides, 3 to 25 nucleotides, 4 to 20 nucleotides, 5 to 15 nucleotides, 6 to 10 nucleotides. 7 to 9 nucleotides, 30 to 100 nucleotides, 35 to 95 nucleotides. 40 to 90 nucleotides, 45 to 85 nucleotides, 50 to 80 nucleotides, 55 to 75 nucleotides, 60 to 70 nucleotides) upstream of the promoter sequence.

[0101] In some aspects, the nicking endonuclease can be a Cas9 nickase.

[0102] In some aspects, the functional template DNA can be a plasmid DNA or an enzymatically produced DNA synthesized in vitro. In some aspects, the enzymatically produced DNA synthesized in vitro can be produced by enzymatic methods that do not utilize DNA amplification. The functional template DNA may be a digestion product.

[0103] The template strand may include a 5 ’ terminus and the nontemplate strand may include a 3’ terminus. In some aspects, the 5’ terminus of the template strand may extend past the 3’ terminus of the nontemplate strand thereby forming a second 5’ overhang, the method further including enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand thereby filling in the second 5’ overhang, wherein a nucleotide added to the 3’ terminus of the nontemplate strand can include a second handle. In some aspects, a 3’ terminal nucleotide added to the 3’ terminus of the nontemplate strand can be a nucleotide analog, for example, a dideoxynucleotide or a canonical nucleotide including any of the disclosed handles.

[0104] In some aspects, the method can further include enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand using terminal transferase, wherein a nucleotide added to the 3' terminus of the nontemplate strand can include a second handle.

[0105] In other aspects, the method can further include ligating a second adapter to the 3’ terminus of the nontemplate strand and the 5’ terminus of the template strand, the second adapter including a second handle.

[0106] The invading nucleic acid molecule may include a second handle. In some aspects, the invading nucleic acid molecule can include a modified nucleotide. The modified nucleotide may be a locked nucleic acid nucleotide or a peptide nucleic acid nucleotide. In some aspects, the method can further include — after hybridizing the invading nucleic acid molecule — -ligating a terminus of the invading nucleic acid molecule to an adjacent terminus of a nicked strand of the functional template DNA. In some aspects, the second handle is configured to be coupled to a solid support.

[0107] In some aspects, the first handle of the catalyst can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides upstream of a start of the RNA encoding sequence. The first handle of the catalyst may be 21 nucleotides upstream of the start of the RNA encoding sequence.

[0108] In some aspects, the RNA polymerase can be a T-phage RNA polymerase or related single subunit. The T-phage RNA polymerase may be a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, a SP6 RNA polymerase, a Syn5 RNA polymerase, or a variant of any of the foregoing.

[0109] The first handle may include a moiety selected from the group consisting of an alkyl halide, O6-benzylguanine, O2-benzylcytosine, organ oarsenic, trimethoprim ligand, and biotin. In some aspects, the first handle can be bifunctional.

[0110] The RNA polymerase may include a tag domain, configured to bind the first handle, selected from the group consisting of a HaloTag domain, an AviTag domain, a SNAP-Tag domain, FlAsH-Tag domain, ReAsH-Tag domain, a TMP-Tag domain, and a CLIP-Tag domain.

[0111] In some aspects, RNA-encoding sequence can be at least 50 nucleotides (e.g., at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1,000 nucleotides, at least 1,500 nucleotides, at least 2,000 nucleotides, at least 2,500 nucleotides, at least 3,000 nucleotides, at least 3,500 nucleotides, at least 4,000 nucleotides, at least 4,500 nucleotides, at least 5,000 nucleotides, at least 5,500 nucleotides, at least 6,000 nucleotides, at least 6,500 nucleotides, at least 7,000 nucleotides, at least 7,500 nucleotides, at least 8.000 nucleotides, at least 8,500 nucleotides, at least 9,000 nucleotides, at least 9,500 nucleotides, at least 10,000 nucleotides) in length. In some aspects, the RNA-encoding sequence can be up to 10,000 nucleotides (e.g., up to 9,500 nucleotides, up to 9,000 nucleotides, up to 8,500 nucleotides, up to 8,000 nucleotides, up to 7,500 nucleotides, up to 7,000 nucleotides, up to 6,500 nucleotides up to 6,000 nucleotides, up to 5,500 nucleotides, up to 5,000 nucleotides, up to 4,500 nucleotides, up to 4,000 nucleotides, up to 3,500 nucleotides, up to 3,000 nucleotides, up to 2,500 nucleotides, up to 2,000 nucleotides, up to 1,500 nucleotides, up to 1,000 nucleotides, up to 900 nucleotides, up to 800 nucleotides, up to 700 nucleotides, up to 600 nucleotides, up to 500 nucleotides, up to 450 nucleotides, up to 400 nucleotides, up to 350 nucleotides, up to 300 nucleotides, up to 250 nucleotides, up to 200 nucleotides, up to 150 nucleotides, up to 100 nucleotides, up to 50 nucleotides) in length.

[0112] It is considered that the RNA-encoding sequence can be a number of nucleotides in length ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the RNA-encoding sequence can be from 50 to 10,000 nucleotides (e.g., from 100 to 9,500 nucleotides, from 150 to 9,000 nucleotides, from 200 to 8,500 nucleotides, from 250 to 8,000 nucleotides, from 300 to 7,500 nucleotides, from 350 to 7,000 nucleotides, from 400 to 6,500 nucleotides, from 450 to 6.000 nucleotides, from 500 to 5,500 nucleotides, from 600 to 5,000 nucleotides, from 700 to 4,500 nucleotides, from 800 to 4,000 nucleotides, from 900 to 3,500 nucleotides, from 1,000 to 3,000 nucleotides, from 1.500 to 2,500 nucleotides, from 50 to 2.500 nucleotides, from 100 to 2,000 nucleotides, from 150 to 1,500 nucleotides, from 200 to 1,000 nucleotides, from 250 to 900 nucleotides, from 300 to 800 nucleotides, from 350 to 700 nucleotides, from 400 to 600 nucleotides, from 450 to 500 nucleotides, from 2,000 to 10,000 nucleotides, from 2,500 to 9,500 nucleotides, from 3,000 to 9,000 nucleotides, from 3,500 to 8,500 nucleotides, from 4,000 to 8,000 nucleotides, from 4.500 to 7,500 nucleotides, from 5,000 to 7,000 nucleotides, from 5,500 to 6,500 nucleotides) in length.

[0113] The methods disclosed herein for preparing a catalyst for RNA production from a functional template DNA are merely exemplary. It is contemplated as part of the present disclosure that the methods disclosed herein for introducing a first handle and a second handle into plasmid DNA may be used in various combinations suitable for preparing a catalyst for RNA production.

[0114] EXAMPLES

[0115] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. Example 1: In-Line Desalting, Buffer Exchange, RNA stabilization Disclosed herein is a microfluidic platform for the on-demand production of high-quality RNA with enhanced shelf life. The microfluidic chip features two segments: a reactor and a desalting unit (FIG. 4). The reactor segment contains the T7 RNA polymerase enzyme co-tethered with its template DNA. Substrate ribonucleotides (rNTPs) and magnesium ions are supplied to the chip to facilitate continuous RNA synthesis. Magnesium ions act as an essential cofactor for the T7 RNA polymerase, stabilizing the transition state complex necessary for RNA synthesis.

[0116] However, magnesium ions also pose a risk to RNA by catalyzing the hydrolysis of the RNA backbone, reducing the yield of full-length RNA and leading to truncated RNA impurities in a wide range of lengths. Hydrolysis of RNA occurs via two primary mechanisms: 1) contaminant nuclease (enzymatic) degradation and 2) intrinsic, or in-line, hydrolysis by hydroxide ions in water. The latter degradation increases with increasing pH.

[0117] In current RNA manufacturing, RNA is exposed to high concentrations of Mg2+at pH 7.8 for many hours (or longer). The continuous feed reactor allows for lower Mg2+concentrations, with shorter exposure times, but to further stabilize RNA against degradation, one desires to remove the Mg2+immediately after RNA synthesis is complete. In the configuration shown in FIG. 4, RNA synthesized in the reactor is immediately directed to the desal ting segment. This desalting segment, equipped with a semi -permeabl e membrane, removes excess magnesium ions from the RNA product. In addition, this membrane also removes the unreacted rNTP from the product, further improving the system's efficiency. Finally, since RNA is more stable at lower pH values, buffer exchange can also be used to reduce the pH of the medium (a common storage buffer is sodium citrate, pH 6.5). The simple filtration setup shown relies on the passive diffusion of the molecules and ions through the membrane.

[0118] Hydrogel membrane: Polyacrylamide hydrogel was employed as the semi-permeable membrane in the purification stage of the device due to its compatibility with diverse biomolecules and its tunable pore size. The semi-permeable hydrogel membrane allows the passage of magnesium ions and small molecules while selectively retaining larger RNA molecules (FIG.5A). This selectivity is dictated by the membrane's mesh (pore) size. The pore size of hydrogel membranes can be tuned by varying the crosslinker density and total polymer concentration. Increasing the density of crosslinkers and (or) the overall concentration of polymers reduces the size of pores within hydrogels (FIG. 5C). The total concentration of polymer was varied between 5 wt% to 40 wt% to optimize the membrane performance for separating Mg2+ions and rNTPs from crude RNA products.

[0119] The hydrogel was formed through the UV-initiated polymerization of a monomer mix containing acrylamide monomer, bis-acrylamide crosslinker, and LAP initiator (FIG. 5B).

[0120] Stereolithography methods were also optimized to fabricate thin hydrogel membranes within the microfluidic channels.

[0121] It is also possible to improve the desalting performance of the devices using several strategies:

[0122] A) Multiple devices in series: Using a configuration where multiple purification devices are connected in series can improve the desalting performance (FIG.6A). For instance, the outlet of the first device can be connected to the inlet of the next device, each having individual extraction streams. Such a configuration increases the residence time of crude RNA in contact with the membrane and maintains a sufficient concentration gradient to drive passive diffusion. The design can be optimized further to accommodate multiple devices

[0123] B) Dual-membrane device: In this configuration, two membranes are integrated within a microfluidics device with three channels, as shown in FIG. 6B. The device design is adapted to flow crude RNA products through the middle channel, allowing desalting from two adjacent extraction streams, thereby significantly improving the desalting performance.

[0124] C) Electric-field assisted accelerated diffusion devices: Through electrodeposition methods, it is possible to integrate microelectrodes into microfluidic devices. Such integration allows the application of electric fields across hydrogel membranes to speed up the diffusion of small molecules and ions into the extraction stream (FIG. 6C). In addition, the dual membrane device configuration allows for maintaining RNAs within the central channel, avoiding any potential electrochemical RNA degradation.

[0125] Electrodes on PDMS: Introduction of electrodes into the PDMS devices can be done using multiple ways:

[0126] 1. Coating a glass surface with electrode metals through vapor deposition or metal sputtering. The glass with metal can then be used to seal the PDMS microchannels. A general design of such a device is shown in FIG. 7.

[0127] 2. A cheaper alternative is to insert the electrode, in the form of thin wires, into the extraction stream channel, as shown in FIG. 8.

[0128] 3. Conductive PDMS composites can also be used. For example, PDMS mixed with silver (Ag) particles. This way, all the PDMS layers can be bonded seamlessly through plasma activation (refer to FIG. 9). PDMS-Hydrogel Interface: Since microfluidic devices are typically fabricated from polydimethylsiloxane (PDMS), integrating a semi -permeable membrane requires strong adhesion between the PDMS and the hydrogel. Such adhesion is essential to prevent solution leakage between the crude RNA and extraction streams, ensuring selective diffusion. A surface-bound polymerization approach using benzophenone (BP) was employed to achieve effective hydrogel-PDMS attachment. Benzophenone, a well-known photo-initiator, was infused into the PDMS matrix, and photo-polymerization was then performed using a monomer mixture.

[0129] To prepare BP-infused PDMS, 0.137 mmol of BP was added per 10 grains of PDMS part A during curing. The BP-infused PDMS was cured over the appropriate master mold to create the microfluidic channel features. Separately, BP-infused PDMS was spin-coated onto a glass slide, which was later used to seal the microfluidic channels. 'This approach resulted in channels with all walls including BP-infused PDMS.

[0130] Hydrogel Membrane: Stereolithography was used to fabricate a thin layer of polyacrylamide hydrogel within the microfluidic channels. The channels were filled with a monomer mix of appropriate total polymer concentrations (5 -40 wt%). A photomask of linear feature with a width of 500, 400, or 300 microns was placed onto the device on the glass side so that the line on the photomask aligned along the microfluidic channel. This setup was exposed to UV light at a wavelength of around 365 nm for 10 minutes to photoinitiate polymerization of a thin hydrogel membrane. The lowest thickness of the hydrogel membrane that was fabricated was 250 microns (FIGS. 10A-10C). The lower thickness gives rise to better / faster separation efficiency, but larger thicknesses will function adequately.

[0131] To evaluate the semi-permeable nature of the hydrogel membranes, Fluorescence Recovery After Photobleaching (FRAP) analysis was performed using small and large molecule fluorescent proxies. As shown in FIG. 11A, the diffusion coefficient for fluorescein (a small molecule proxy) in 10 wt% hydrogel is similar to that in free solution, implying that a molecule of similar size diffuses through the hydrogel network without significant hindrance. However, a large molecule (RNA) proxy (FITC-Dextran, 10 kDa) showed a considerable decrease in diffusion coefficients in 10 wt% hydrogels compared to free solutions. 'This sizedependent difference suggests the semi -permeable nature of the hydrogel. In addition, the diffusion of these probes through the hydrogel was monitored in real time using fluorescent microscopy (FIG. 11B). The large molecule proxy (FITC-Dextran) did not diffuse through the hydrogel membrane even after 2 hours, while the small molecule proxy readily diffused. Separation Efficiency: The efficiency of Mg2+ions separation using 10 wt% hydrogel membranes was also evaluated. The Mg2+separated from the high-yield transcription buffer was analyzed using spectrophotometric analysis and inductively coupled plasma mass spectrometry (ICP-MS). To demonstrate Mg2+separation using the hydrogel membrane, a 40 mM Mg2+solution was passed through the device at a flow rate of 0.1 pL / min, and the Mg2+concentration at the extraction stream was monitored. The extraction stream flow rate was varied between 0.25 pL / min and 1.0 pL / min. A 20 % Mg2+decrease at the low extraction flow rate was observed within two hours. However, as the extraction flow rate increased, the extraction efficiency increased to 95 % (FIGS. 12A-12E).

[0132] These results suggest a reduction of Mg2+concentration from 40 mM to 2 mM using a single membrane. Further reduction in Mg2+concentration can be achieved using multiple purification segments in series or by the application of an electric field across the membrane.

[0133] The results presented above demonstrate the utility of an in-line de-salting / buffer exchange system designed to immediately follow a flow chamber (reactor) generating RNA. These results show good exchange properties by simple, passive diffusion. For even faster and / or higher capacity separations, a small electric field can be applied.

[0134] Bonding Strength: As part of the characterization of the gel membrane, the maximum burst pressure of the gels was tested as a function of total polymer concentration in the crosslinked gel. The expectation was that the higher the polymer percentage, the higher the burst pressure would be, and the results aligned with our hypothesis. The evolution of pressure inside the device was tracked by connecting the inlet of one fluid channel to a syringe containing water with food coloring mounted on a syringe pump. The outlet of the same channel was connected to a pressure gauge. The pressure inside the channel steadily increased with the advancement of the syringe pump and dropped precipitously when the gel membrane ruptured and this pressure was recorded as the burst pressure. The burst pressures of the gels were ~1 bar (15% polymer), -0.63 bar (10% polymer), and -0.43 bar (7% polymer). For this set of experiments, the length and width of the gels were kept constant at 9 mm and 300 pm, respectively. This phenomenon was also visible; the colored solution was contained within the channel until the burst pressure was exceeded, at which point the enthe device flooded. This information on burst pressure is crucial for determining the allowable limits of the fluid flow rate inside the channel during operation (FIG. 13).

[0135] Improving the Extraction Rate: The rate of separation of small molecules using the gel membrane can be improved by increasing the available gel area for separation. To this end, a dual-gel-membrane device was developed. This device features a central channel for the crude RNA stream from the reactor, which is flanked by top and bottom channels containing the extraction stream / buffer. By positioning extraction channels on both sides of the RNA stream, this configuration also enhances the diffusional driving force, further promoting the migration of small molecules out of the central channel (FIG. 14).

[0136] Concentration of RNA using Electric Field: Additionally, this device can also be used to concentrate RNA through the electrophoretic movement of RNA. Under an electric field, the negatively charged RNA migrates towards the positive electrode but will be contained within the channel as the gel membrane’s pore size prevents its entry. This selective accumulation near the positive electrode allows the fluid channel to be pinched and isolate the RNA (FIG. 15). As a continuous process, this method is significantly more efficient in terms of time and effort than conventional batch concentration techniques.

[0137] Tests on Nucleic Acid Migration Under Electric Field: To test the migration of nucleic acids under an electric field in the microfluidic device, experiments were performed using DNA as a proxy for RNA. The migration of the DNA was monitored within a simple rectangular channel. A hydrogel was polymerized in the center of the device to prevent bulk liquid flow, thereby allowing only the movement of small molecules. These experiments were conducted under static conditions with no fluid flow. The experimental setup is shown in FIG.

[0138] 16A.

[0139] As an initial demonstration, the migration of a short F AM-labeled oligonucleotide (36 bases; proxy for abortive RNA) and a long CY5-labeled 1 kbp DNA fragment (proxy for full-length RNA) were evaluated within 15% (w / w) polyacrylamide hydrogels under an electric field. Results indicated that in this gel matrix, the 1 kbp DNA did not migrate appreciably, even under a strong electric field (E = 100 V / cm), whereas the 36-base oligonucleotide readily traversed the gel, as shown in FIG. 16B. Furthermore, the 1 kbp DNA molecules accumulated at the gel interface over time, which was indicated by the increased brightness of the band between 3 and 7 minutes.

[0140] The hydrogel's polymer content and thickness can be optimized to reduce the migration time for all species involved in RNA transcription-such as Mg2+, rNTPs, and both short and long RNAs. All experiments can incorporate a constant fluid flow, perpendicular to movement of species through the hydrogel, to enable continuous operation, as depicted in FIG. 15.

[0141] Manufacturing Versatility: The disclosed technologies are compatible with all commonly used materials for fabricating medical-grade microfluidic devices, including Polymethylmethacrylate (PMMA), Polystyrene (PS), Polycarbonate (PC), and Cyclic Olefin Copolymer (COC). For materials where surface chemistry modifications are not feasible, the device design can be altered to use the gel membrane as a liquid seal. Furthermore, in devices made from materials with low UV transparency, the gel can be cross-linked using traditional free radical initiators, such as Ammonium persulfate (APS).

[0142] Any patents, applications and publications as listed throughout this document are hereby incorporated by reference in their enthety herein.

[0143] EXAMPLE ASPECTS

[0144] In view of the described compositions, devices, systems, and methods, herein below are described certain more particularly described aspects of the inventions. The particularly recited aspects should not however, be interpreted to have any limiting effect on different claims containing different or more general teachings described herein or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein.

[0145] Example 1: A method of filtering nucleic acids, the method comprising: a) providing a microfluidic device comprising: a first channel comprising a first inlet and a first outlet; a second channel adjacent and parallel to the first channel and comprising a second inlet and a second outlet; a first permeable membrane disposed between and fluidically connecting the first channel and the second channel; b) flowing a crude mixture comprising synthesized nucleic acids and at least one nucleic acid synthesis reagent and / or byproduct through the first inlet; c) simultaneously flowing a first extraction solution through the second inlet; and d) recovering a processed mixture from the first outlet; wherein the processed mixture comprises a lower concentration of the at least one nucleic acid synthesis reagent and / or byproduct than the crude mixture.

[0146] Example 2: The method of any examples herein, particularly Example 1, wherein the microfluidic device comprises polydimethylsiloxane (PDMS) and at least one photoinitiator.

[0147] Example 3: The method of any examples herein, particularly Example 2, wherein the at least one photoinitiator comprises benzophenone, alkylphenone, thioxanthone, peroxide, perester, iminosulphones, ketone, or any derivatives or combinations thereof.

[0148] Example 4: The method of any examples herein, particularly Examples 1-3, wherein the permeable membrane comprises polyacrylamide, polyethylene glycol, a zwitterionic polymer, or any combination thereof.

[0149] Example 5: The method of any examples herein, particularly Examples 1-4, wherein the permeable membrane comprises from about 5 wt% to about 40 wt% total polymer. Example 6: The method of any examples herein, particularly Examples 1-5, wherein the synthesized nucleic acids comprise RNA.

[0150] Example 7: The method of any examples herein, particularly Examples 1-6, wherein the nucleic acid synthesis reagent and / or biproduct comprises Mg2+, ribonucleotides, pyrophosphate, abortive nucleic acids, or any combination thereof.

[0151] Example 8: The method of any examples herein, particularly Examples 1-7, wherein the first extraction solution is buffer.

[0152] Example 9: The method of any examples herein, particularly Examples 1-7, wherein the first extraction solution is water.

[0153] Example 10: The method of any examples herein, particularly Examples 1-9, wherein the at least one nucleic acid synthesis reagent and / or by product can pass through the permeable membrane into the second channel and synthesized nucleic acids cannot.

[0154] Example 11: The method of any examples herein, particularly Example 10, wherein the at least one nucleic acid synthesis reagent and / or byproduct can pass through the permeable membrane into the second channel and synthesized nucleic acids cannot.

[0155] Example 12: The method of any examples herein, particularly Examples 1-11, wherein the flow of the crude mixture is parallel to the flow of the first extraction solution.

[0156] Example 13: The method of any examples herein, particularly Examples 1-11, wherein the flow of crude mixture is counter to the flow' of the first extraction solution.

[0157] Example 14: The method of any examples herein, particularly Examples 1-13, wherein step c) further comprises applying an electric gradient across the first permeable membrane.

[0158] Example 15: The method of any examples herein, particularly Example 14, wherein the electric gradient is perpendicular to the flow of the crude mixture.

[0159] Example 16: The method of any examples herein, particularly Examples 14-15, wherein at least one electrode pair is placed on either side of the first channel.

[0160] Example 17: The method of any examples herein, particularly Examples 14-16, wherein at least one pair of electrode pins is inserted into the second channel.

[0161] Example 18: The method of any examples herein, particularly Examples 1-17, wherein die microfluidic device further comprises: a third channel adjacent and parallel to the first channel and comprising a third inlet and third outlet; a second permeable membrane disposed between and fluidly connecting the first channel and the third channel; wherein the second and third channel are on opposite sides of the first channel; and wherein step c) further comprises simultaneously flowing a second extraction solution through the third inlet. Example 19: The method of any examples herein, particularly Example 18, wherein the first permeable membrane and the second permeable membrane are adjacent across the first channel.

[0162] Example 20: The method of any examples herein, particularly Examplel8, wherein the first permeable membrane is spaced from the second permeable membrane.

[0163] Example 21: The method of any examples herein, particularly Examples 18-20, wherein the first extraction solution is the same as the second extraction solution.

[0164] Example 22: The method of any examples herein, particularly Examples 18-20, wherein the first extraction solution is different than the second extraction solution.

[0165] Example 23: The method of any examples herein, particularly Examples 18-22, wherein step c) further comprises applying an electric gradient across the first permeable membrane and / or the second permeable membrane.

[0166] Example 24: The method of any examples herein, particularly Example 23, wherein the electric gradient is perpendicular to the flow of the crude mixture.

[0167] Example 25: The method of any examples herein, particularly Examples 23-24, wherein at least one electrode pair is placed on either side of the first channel.

[0168] Example 26: The method of any examples herein, particularly Examples 23-25, wherein at least one pair of electrode pins is inserted into the second channel and / or the third channel.

[0169] Example 27: A method of filtering and / or concentrating nucleic acids, the method comprising: a) providing a microfluidic device comprising a first channel comprising a first inlet, wherein the first channel bifurcates into a first sub-channel having a first outlet and a second sub-channel having a second outlet; b) positioning a positive electrode on the same side of the first channel as the first sub-channel and a negative electrode on the same side of the first channel as the second sub-channel, thereby creating an electric gradient perpendicular to the first channel; c) flowing a crude mixture comprising nucleic acids through the first inlet; and d) recovering a processed mixture from the first outlet; wherein the processed mixture comprises a higher concentration of the nucleic acid than the crude mixture.

[0170] Example 28: The method of any examples herein, particularly Example 27, wherein the crude mixture further comprises at least one nucleic acid synthesis reagent and / or byproduct; and wherein the processed mixture comprises a lower concentration of at least one positively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture.

[0171] Example 29: The method of any examples herein, particularly Example 28, wherein the microfluidic device further comprises: a second channel adjacent and parallel to the first channel and comprising a second inlet and a third outlet; and a first permeable membrane disposed between and fluidicaliy connecting the first channel and the second channel; and wherein step c) further comprises simultaneously flowing a first extraction solution through the second inlet.

[0172] Example 30: The method of any examples herein, particularly Example 29, wherein the second channel is on the same side of the first channel as the second sub-channel.

[0173] Example 31: The method of any examples herein, particularly Example 29, wherein the second channel is on the same side of the first channel as the first sub-channel; and wherein the processed mixture comprises a lower concentration of at least one negatively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture.

[0174] Example 32: The method of any examples herein, particularly Examples 29-31, wherein the microfluidic device further comprises: a third channel adjacent and parallel to the first channel and comprising a third inlet and a fourth outlet; a second permeable membrane disposed between and fluidicaliy connecting the first channel and the third channel; wherein the second channel and the third channel are on opposite sides of the first channel; and wherein step c) further comprises simultaneously flowing a second extraction solution through the third inlet.

[0175] Example 33: The method of any examples herein, particularly Example 32, wherein the third channel is on the same side of the first channel as the second sub-channel.

[0176] Example 34: The method of any examples herein, particularly Example 32, wherein the third channel is on the same side of the first channel as the first sub-channel; and wherein the processed mixture comprises a lower concentration of at least one negatively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture.

[0177] Example 35: The method of any examples herein, particularly Examples 27-34, wherein the microfluidic device comprises polydimethylsiloxane (PDMS) and at least one photoinitiator.

[0178] Example 36: The method of any examples herein, particularly Example 35, wherein the at least one photoinitiator comprises benzophenone, alkylphenone, thioxanthone, peroxide, perester, iminosulphones, ketone, or any derivatives or combinations thereof.

[0179] Example 37: The method of any examples herein, particularly Examples 27-36, wherein the nucleic acids comprise RNA.

[0180] Example 38: The method of any examples herein, particularly Examples 29-37, wherein the permeable membrane comprises polyacrylamide, polyethylene glycol, a zwitterionic polymer, or any combination thereof. Example 39: The method of any examples herein, particularly Examples 29-38, wherein the permeable membrane comprises from about 5 wt% to about 40 wt% total polymer.

[0181] Example 40: The method of any examples herein, particularly Examples 28-39, wherein the at least one nucleic acid synthesis reagent and / or byproduct comprises Mg2+, ribonucleotides, pyrophosphate, abortive nucleic acids, or any combination thereof.

[0182] Example 41: The method of any examples herein, particularly Examples 29-40, wherein the first extraction solution is a buffer.

[0183] Example 42: The method of any examples herein, particularly Examples 29-41, wherein the first extraction solution is water.

[0184] Example 43: The method of any examples herein, particularly Examples 29-42, wherein the at least one nucleic acid synthesis reagent and / or byproduct can pass through the permeable membrane into the second channel and synthesized nucleic acids cannot.

[0185] Example 44: The method of any examples herein, particularly Examples 29-43, wherein the flow of crude mixture is parallel to the flow of the first extraction solution.

[0186] Example 45: The method of any examples herein, particularly Examples 29-43, wherein the flow of crude mixture is counter to the flow of the first extraction solution.

[0187] Example 46: The method of any examples herein, particularly Examples 32-45, wherein the first extraction solution is the same as the second extraction solution.

[0188] Example 47: The method of any examples herein, particularly Examples 32-45, wherein the first extraction solution is different than the second extraction solution.

[0189] Example 48: The method of any examples herein, particularly Examples 27-47, wherein at least one electrode pair is placed on either side of the first channel.

[0190] Example 49: The method of any examples herein, particularly Examples 29-48, wherein at least one electrode pins is inserted into the second channel and / or third channel.

[0191] Example 50: A method of RNA production, the method comprising: a) providing a functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being downstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus, wherein the 5’ terminus of the nontemplate strand extends past the 3’ terminus of the template strand, thereby forming a first 5’ overhang; b) enzymatically adding nucleotides to the 3’ terminus of the template strand by filling in the first 5’ overhang, wherein a nucleotide added to the 3’ terminus of the template strand comprises a first handle; c) functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to the method of any examples herein, particularly Examples 1-49.

[0192] Example 51: A method of RNA production, the method comprising: a) providing a functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus, wherein the 3’ terminus of the template strand extends past the 5’ terminus of the nontemplate strand, thereby forming a first 3’ overhang; b) enzymatically adding nucleotides to the 3’ terminus of the template strand using a terminal transferase, wherein a nucleotide added to the 3’ terminus of the template strand comprises a first handle; c) functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to the method of any examples herein, particularly Examples 1-49.

[0193] Example 52: A method of RNA production, the method comprising: a) providing a functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus; b) ligating a first adapter, comprising a first handle, to the 3’ terminus of the template strand and the 5’ terminus of the nontemplate strand, wherein an RNA polymerase is functionally coupled to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; c) producing RNA from the catalyst for RNA production; and d) filtering the RNA according to the method of any examples herein, particularly Examples 1-49.

[0194] Example 53: A method of RNA production, the method comprising: a) providing a functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3' to 5’ direction, a promoter sequence and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, b) enzymatically nicking a site of the nontemplate strand using a nicking endonuclease, thereby generating a gap in the nontemplate strand, the gap being a single stranded portion of the template strand without a corresponding complementary portion of the nontemplate strand, wherein the site is within a region of the nontemplate strand selected from the group consisting of: i) a region of the nontemplate strand complementary to, and base paired with, the promoter sequence, ii) 1-100 nucleotides upstream of a sequence of the nontemplate strand complementary to, and base paired with, the promoter sequence, and iii) combinations thereof; c) hybridizing an invading nucleic acid molecule to the single stranded portion of the template strand, the invading nucleic acid molecule being complementary to the single stranded portion of the template strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to the method of any examples herein, particularly Examples 1-49.

[0195] Example 54: A method of RNA production, the method comprising: a) providing a functional template DNA, the functional template DNA comprising: (i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a promoter sequence and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand, b) enzymatically nicking a site of the template strand using a nicking endonuclease, thereby generating a gap in the template strand, the gap being a single stranded portion of the nontemplate strand without a corresponding complementary portion of the template strand, wherein the site is within a region of the template strand selected from the group consisting of: i) a region of the promoter sequence, ii) 1-100 nucleotides upstream of the promoter sequence, and iii) combinations thereof; c) hybridizing an invading nucleic acid molecule to the single stranded portion of the nontemplate strand, the invading nucleic acid molecule being complementary to the single stranded portion of the nontemplate strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA; d) producing RNA from the catalyst for RNA production; and e) filtering the RNA according to the method of any examples herein, particularly Examples 1-49. Example 55: The method of any examples herein, particularly Examples 50-54, wherein the template strand comprises a 5’ terminus and the nontemplate strand comprises a 3’ terminus.

[0196] Example 56: The method of any examples herein, particularly Example 55, wherein the 5’ terminus of the template strand extends past the 3’ terminus of the nontemplate strand, thereby forming a second 5’ overhang, the method further comprising enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand, thereby filling in the second 5’ overhang, wherein a nucleotide added to the 3’ terminus of the nontemplate strand comprises a second handle.

[0197] Example 57: The method of any examples herein, particularly Example 56, wherein a 3’ terminal nucleotide added to the 3’ terminus of the nontemplate strand is a dideoxynucleotide.

[0198] Example 58: The method of any examples herein, particularly Example 55, the method further comprising enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand using terminal transferase, wherein a nucleotide added to the 3’ terminus of the nontemplate strand comprises a second handle.

[0199] Example 59: The method of any examples herein, particularly Example 55, the method further comprising ligating a second adapter to the 3’ terminus of the nontemplate strand and the 5’ terminus of the template strand, the second adapter comprising a second handle.

[0200] Example 60: The method of any examples herein, particularly Example 53, wherein the invading nucleic acid molecule comprises a second handle.

[0201] Example 61: The method of any examples herein, particularly Example 54, wherein the invading nucleic acid molecule comprises a second handle.

[0202] Example 62: The method of any examples herein, particularly Examples 50-61, wherein the first handle of the catalyst is X nucleotides upstream of a start of the RNA encoding sequence, X being an integer selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.

[0203] Example 63: The method of any examples herein, particularly Example 62, wherein the first handle of the catalyst is 21 nucleotides upstream of the start of the RNA encoding sequence.

[0204] Example 64: The method of any examples herein, particularly Examples 56-63, wherein the second handle is configured to be coupled to a solid support.

[0205] Example 65: The method of any examples herein, particularly Examples 53-64, wherein the nicking endonuclease is a Cas9 -nickase. Example 66: The method of any examples herein, particularly Examples 53-65, wherein the invading nucleic acid molecule comprises a modified nucleotide.

[0206] Example 67: The method of any examples herein, particularly Example 66, wherein the modified nucleotide is selected from the group consisting of a locked nucleic acid nucleotide and a peptide nucleic acid nucleotide.

[0207] Example 68: The method of any examples herein, particularly Examples 53-67, the method further comprising, after hybridizing the invading nucleic acid molecule, ligating a terminus of the invading nucleic acid molecule to an adjacent terminus of a nicked strand of the functional template DNA.

[0208] Example 69: The method of any examples herein, particularly Examples 50-68, wherein the RNA polymerase is a T-phage RNA polymerase.

[0209] Example 70: The method of any examples herein, particularly Example 69, wherein the T-phage RNA polymerase is selected from the group consisting of T7 RNA polymerase, a T3 RNA polymerase, a Kll RNA polymerase, a SP6 RNA polymerase, a Syn5 RNA polymerase, and a variant of any of the foregoing.

[0210] Example 71: The method of any examples herein, particularly Examples 50-70, wherein the first handle comprises a moiety selected from the group consisting of an alkyl halide, O6-benzylguanine, 02-benzylcytosine, organoarsenic, trimethoprim ligand, and biotin.

[0211] Example 72: The method of any examples herein, particularly Examples 50-71, wherein the RNA polymerase comprises a tag domain, configured to bind the first handle, selected from the group consisting of a HaloTag domain, an AviTag domain, a SNAP-Tag domain, FlAsH-Tag domain, ReAsH-Tag domain, a TMP-Tag domain, and a CLIP-Tag domain.

[0212] Example 73: The method of any examples herein, particularly Examples 50-55 or 61-72, wherein the first handle is bifunctional.

[0213] Example 74: The method of any examples herein, particularly Examples 50-73, wherein the functional template DNA is selected from the group consisting of a plasmid DNA and an enzymatically produced DNA synthesized in vitro.

[0214] Example 75: The method of any examples herein, particularly Examples 50-74, wherein the functional template DNA is a digestion product.

[0215] Example 76: The method of any examples herein, particularly Examples 50-75, wherein the RNA-encoding sequence is from 50 nucleotides to 10,000 nucleotides in length.

Claims

CLAIMSWhat is claimed is:

1. A method of filtering nucleic acids, the method comprising:a) providing a microfluidic device comprising:a first channel comprising a first inlet and a first outlet;a second channel adjacent and parallel to the first channel and comprising a second inlet and a second outlet;a first permeable membrane disposed between and fluidically connecting the first channel and the second channel;b) flowing a crude mixture comprising synthesized nucleic acids and at least one nucleic acid synthesis reagent and / or byproduct through the first inlet;c) simultaneously flowing a first extraction solution through the second inlet; and d) recovering a processed mixture from the first outlet;wherein the processed mixture comprises a lower concentration of the at least one nucleic acid synthesis reagent and / or byproduct than the crude mixture.

2. The method of claim 1, wherein the microfluidic device comprises polydi methyl siloxane (PDMS) and at least one photoinitiator.

3. The method of claim 2, wherein the at least one photoinitiator comprises benzophenone, alkylphenone, thioxanthone, peroxide, perester, iminosulphones, ketone, or any derivatives or combinations thereof.

4. The method of any one of claims 1-3, wherein the permeable membrane comprises polyacrylamide, polyethylene glycol, a zwitterionic polymer, or any combination thereof.

5. The method of any one of claims 1 -4, wherein the permeable membrane comprises from about 5 wt% to about 40 wt% total polymer.

6. The method of any one of claims 1-5, wherein the synthesized nucleic acids comprise RNA.

7. The method of any one of claims 1-6, wherein the at least one nucleic acid synthesis reagent and / or byproduct comprises Mg2+, ribonucleotides, pyrophosphate, abortive nucleic acids, or any combination thereof.

8. The method of any one of claims 1-7, wherein the first extraction solution is a buffer.

9. The method of any one of claims 1-7, wherein the first extraction solution is water.

10. The method of any one of claims 1-9, wherein the at least one nucleic acid synthesis reagent and / or byproduct can pass through the permeable membrane into the second channel and synthesized nucleic acids cannot.

11. The method of claim 10, wherein the permeable membrane separates the at least one nucleic acid synthesis reagent and / or byproduct from the synthesized nucleic acids by size.

12. The method of any one of claims 1-11, wherein the flow of the crude mixture is parallel to the flow of the first extraction solution.

13. The method of any one of claims 1-11, wherein the flow of the crude mixture is counter to the flow of the first extraction solution.

14. The method of any one of claims 1-13, wherein step c) further comprises applying an electric gradient across the first permeable membrane.

15. The method of claim 14, wherein the electric gradient is perpendicular to the flow of the crude mixture.

16. The method of any one of claims 14-15, wherein at least one electrode pair is placed on either side of the first channel.

17. The method of any one of claims 14-16, wherein at least one pair of electrode pins is inserted into the second channel.

18. The method of any one of claims 1-17, wherein the microfluidic device further comprises:a third channel adjacent and parallel to the first channel and comprising a third inlet and a third outlet;a second permeable membrane disposed between and fluidically connecting the first channel and the third channel;wherein the second channel and the third channel are on opposite sides of the first channel; andwherein step c) further comprises simultaneously flowing a second extraction solution through the third inlet.

19. The method of claim 18, wherein the first permeable membrane and the second permeable membrane are adjacent across the first channel.

20. The method of claim 18, wherein the first permeable membrane is spaced from the second permeable membrane.

21. The method of any one of claims 18-20, wherein the first extraction solution is the same as the second extraction solution.

22. The method of any one of claims 18-20, wherein the first extraction solution is different than the second extraction solution.

23. The method of any one of claims 18-22, wherein step c) further comprises applying an electric gradient across the first permeable membrane and / or the second permeable membrane.

24. The method of claim 23, wherein the electric gradient is perpendicular to the flow' of the crude mixture.

25. The method of any one of claims 23-24, wherein at least one electrode pair is placed on either side of the first channel.

26. The method of any one of claims 23-25, wherein at least one pair of electrode pins is inserted into the second channel and / or the third channel.

27. A method of filtering and / or concentrating nucleic acids, the method comprising: a) providing a microfluidic device comprising a first channel comprising a first inlet, wherein the first channel bifurcates into a first sub-channel having a first outlet and a second sub-channel having a second outlet;b) positioning a positive electrode on the same side of the first channel as the first subchannel and a negative electrode on the same side of the first channel as the second subchannel, thereby creating an electric gradient perpendicular to the first channel;c) flowing a crude mixture comprising nucleic acids through the first inlet; and d) recovering a processed mixture from the first outlet;wherein the processed mixture comprises a higher concentration of the nucleic acid than the crude mixture.

28. The method of claim 27, wherein the crude mixture further comprises at least one nucleic acid synthesis reagent and / or byproduct; andwherein the processed mixture comprises a lower concentration of at least one positively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture.

29. The method of claim 28, wherein the microfluidic device further comprises:a second channel adjacent and parallel to the first channel and comprising a second inlet and a third outlet; anda first permeable membrane disposed between and fluidically connecting the first channel and the second channel; andwherein step c) further comprises simultaneously flowing a first extraction solution through the second inlet.

30. The method of claim 29, wherein the second channel is on the same side of the first channel as the second sub-channel.

31. The method of claim 29, wherein the second channel is on the same side of the first channel as the first sub-channel; andwherein the processed mixture comprises a lower concentration of at least one negatively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture.

32. The method of any one of claims 29-31, wherein the microfluidic device further comprises:a third channel adjacent and parallel to the first channel and comprising a third inlet and a fourth outlet;a second permeable membrane disposed between and fluidically connecting the first channel and the third channel;wherein the second channel and the third channel are on opposite sides of the first channel; andwherein step c) further comprises simultaneously flowing a second extraction solution through the third inlet.

33. The method of claim 32, wherein the third channel is on the same side of the first channel as the second sub-channel.

34. The method of claim 32, wherein the third channel is on the same side of the first channel as the first sub-channel; andwherein the processed mixture comprises a lower concentration of at least one negatively charged nucleic acid synthesis reagent and / or byproduct than the crude mixture.

35. The method of any one of claims 27-34, wherein the microfluidic device comprises polydimethylsiloxane (PDMS) and at least one photoinitiator.

36. The method of claim 35, wherein the at least one photoinitiator comprises benzophenone, alkylphenone, thioxanthone, peroxide, perester, iminosulphones, ketone, or any derivatives or combinations thereof.

37. The method of any one of claims 27-36, wherein the nucleic acids comprise RNA and / or DNA.

38. The method of any one of claims 29-37, wherein the permeable membrane comprises polyacrylamide, polyethylene glycol, a zwitterionic polymer, or any combination thereof.

39. The method of any one of claims 29-38, wherein the permeable membrane comprises from about 5 wt% to about 40 wt% total polymer.

40. The method of any one of claims 28-39, wherein the at least one nucleic acid synthesis reagent and / or byproduct comprises Mg2+, ribonucleotides, pyrophosphate, abortive nucleic acids, or any combination thereof.

41. The method of any one of claims 29-40, wherein the first extraction solution is a buffer.

42. The method of any one of claims 29-41, wherein the first extraction solution is water.

43. The method of any one of claims 29-42, wherein the at least one nucleic acid synthesis reagent and / or byproduct can pass through the permeable membrane into the second channel and synthesized nucleic acids cannot.

44. The method of any one of claims 29-43, wherein the flow of crude mixture is parallel to the flow of the first extraction solution.

45. The method of any one of claims 29-43, wherein the flow of crude mixture is counter to the flow of the first extraction solution.

46. The method of any one of claims 32-45, wherein the first extraction solution is the same as the second extraction solution.

47. The method of any one of claims 32-45, wherein the first extraction solution is different than the second extraction solution.

48. The method of any one of claims 27-47, wherein at least one electrode pair is placed on either side of the first channel.

49. The method of any one of claims 29-48, wherein at least one electrode pins is inserted into the second channel and / or third channel.

50. A method of RNA production, the method comprising:a) providing a functional template DNA, the functional template DNA comprising:(i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being downstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus,wherein the 5’ terminus of the nontemplate strand extends past the 3’ terminus of the template strand, thereby forming a first 5’ overhang;b) enzymatically adding nucleotides to the 3’ terminus of the template strand by filling in the first 5' overhang, wherein a nucleotide added to the 3’ terminus of the template strand comprises a first handle;c) functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA;d) producing RNA from the catalyst for RNA production; ande) filtering the RNA according to the method of any one of claims 1-49.

51. A method of RNA production, the method comprising:a) providing a functional template DNA, the functional template DNA comprising:(i) a template strand, the template strand comprising, in a 3' to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus,wherein the 3’ terminus of the template strand extends past the 5’ terminus of the nontemplate strand, thereby forming a first 3’ overhang;b) enzymatically adding nucleotides to the 3’ terminus of the template strand using a terminal transferase, wherein a nucleotide added to the 3’ terminus of the template strand comprises a first handle;c) functionally coupling an RNA polymerase to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA;d) producing RNA from the catalyst for RNA production; ande) filtering the RNA according to the method of any one of claims 1-49.

52. A method of RNA production, the method comprising:a) providing a functional template DNA, the functional template DNA comprising:(i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a 3’ terminus, a promoter sequence, and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and (ii) a nontemplate strand, the nontemplate strand being complementary to the template strand and comprising a 5’ terminus;b) ligating a first adapter, comprising a first handle, to the 3’ terminus of the template strand and the 5’ terminus of the nontemplate strand, wherein an RNA polymerase is functionally coupled to the first handle, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA;c) producing RNA from the catalyst for RNA production; andd) filtering the RNA according to the method of any one of claims 1-49.

53. A method of RNA production, the method comprising:a) providing a functional template DNA, the functional template DNA comprising:(i) a template strand, the template strand comprising, in a 3’ to 5’ direction, a promoter sequence and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and(ii) a nontemplate strand, the nontemplate strand being complementary to the template strand,b) enzymatically nicking a site of the nontemplate strand using a nicking endonuclease, thereby generating a gap in the nontemplate strand, the gap being a single stranded portion of the template strand without a corresponding complementary portion of the nontemplate strand, wherein the site is within a region of the non template strand selected from the group consi sting of:i) a region of the nontemplate strand complementary to, and base paired with, the promoter sequence,ii) 1-100 nucleotides upstream of a sequence of the nontemplate strand complementary to, and base paired with, the promoter sequence, andiii) combinations thereof;c) hybridizing an invading nucleic acid molecule to the single stranded portion of the template strand, the invading nucleic acid molecule being complementary to the single strandedportion of the template strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA;d) producing RNA from the catalyst for RNA production; ande) filtering the RNA according to the method of any one of claims 1-49.

54. A method of RNA production, the method comprising:a) providing a functional template DNA, the functional template DNA comprising:(i) a template strand, the template strand comprising, in a 3' to 5’ direction, a promoter sequence and an RNA-encoding sequence, the RNA-encoding sequence being upstream of, and operably linked to, the promoter sequence; and(ii) a nontemplate strand, the nontemplate strand being complementary to the template strand,b) enzymatically nicking a site of the template strand using a nicking endonuclease, thereby generating a gap in the template strand, the gap being a single stranded portion of the nontemplate strand without a corresponding complementary portion of the template strand, wherein the site is within a region of the template strand selected from the group consisting of:i) a region of the promoter sequence,ii) 1-100 nucleotides upstream of the promoter sequence, andiii) combinations thereof;c) hybridizing an invading nucleic acid molecule to the single stranded portion of the nontemplate strand, the invading nucleic acid molecule being complementary to the single stranded portion of the nontemplate strand and having a first handle functionally coupled to an RNA polymerase, thereby forming the catalyst for RNA production configured to produce RNA from the RNA-encoding sequence of the functional template DNA;d) producing RNA from the catalyst for RNA production; ande) filtering the RNA according to the method of any one of claims 1-49.

55. The method of any one of claims 50-54, wherein the template strand comprises a 5’ terminus and the nontemplate strand comprises a 3’ terminus.

56. The method of claim 55, wherein the 5’ terminus of the template strand extends past the 3’ terminus of the nontemplate strand, thereby forming a second 5’ overhang,the method further comprising enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand, thereby filling in the second 5’ overhang, wherein a nucleotide added to the 3’ terminus of the nontemplate strand comprises a second handle.

57. The method of claim 56, wherein a 3’ terminal nucleotide added to the 3’ terminus of the nontemplate strand is a dideoxynucleotide.

58. The method of claim 55, the method further comprising enzymatically adding nucleotides to the 3’ terminus of the nontemplate strand using terminal transferase, wherein a nucleotide added to the 3’ terminus of the nontemplate strand comprises a second handle.

59. The method of claim 55, the method further compri sing ligating a second adapter to the 3’ terminus of the nontemplate strand and the 5’ terminus of the template strand, the second adapter comprising a second handle.

60. The method of claim 53, wherein the invading nucleic acid molecule comprises a second handle.

61. The method of claim 54, wherein the invading nucleic acid molecule comprises a second handle.

62. The method of any one of claims 50-61, wherein the first handle of the catalyst is X nucleotides upstream of a start of the RNA encoding sequence, X being an integer selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.

63. The method of claim 62, wherein the first handle of the catalyst is 21 nucleotides upstream of the start of the RNA encoding sequence.

64. The method of any one of claims 56-63, wherein the second handle is configured to be coupled to a solid support.

65. The method of any one of claims 53-64, wherein the nicking endonuclease is a Cas9 nickase.

66. The method of any one of claims 53-65, wherein the invading nucleic acid molecule comprises a modified nucleotide.

67. The method of claim 66, wherein the modified nucleotide is selected from the group consisting of a locked nucleic acid nucleotide and a peptide nucleic acid nucleotide.

68. The method of any one of claims 53-67, the method further comprising, after hybridizing the invading nucleic acid molecule, ligating a terminus of the invading nucleic acid molecule to an adjacent terminus of a nicked strand of the functional template DNA.

69. The method of any one of claims 50-68, wherein the RNA polymerase is a T-phage RNA polymerase.

70. The method of claim 69, wherein the T-phage RNA polymerase is selected from the group consisting of a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, a SP6 RNA polymerase, a Syn5 RNA polymerase, and a variant of any of the foregoing.

71. The method of any one of claims 50-70, wherein the first handle comprises a moiety selected from the group consisting of an alkyl halide, O6-benzylguanine, 02-benzylcytosine, organoarsenic, trimethoprim ligand, and biotin.

72. The method of any one of claims 50-71, wherein the RNA polymerase comprises a tag domain, configured to bind the first handle, selected from the group consisting of a HaloTag domain, an AviTag domain, a SNAP-Tag domain, FlAsH-Tag domain, ReAsH-Tag domain, a TMP-Tag domain, and a CLIP-Tag domain.

73. The method of any one of claims 50-55 or 61-72, wherein the first handle is bifunctional.

74. The method of any one of claims 50-73, wherein the functional template DNA is selected from the group consisting of a plasmid DNA and an enzymatically produced DNA synthesized in vitro.

75. The method of any one of claims 50-74, wherein the functional template DNA is a digestion product.

76. The method of any one of claims 50-75, wherein the RNA-encoding sequence is from 50 nucleotides to 10,000 nucleotides in length.

Images