Systems and methods for RNA transcription

Abstract

Systems and methods for performing RNA transcription are generally described. Some systems for performing RNA transcription described herein may be particularly well- suited for performing RNA transcription in small volumes of liquid. For instance, some systems may include components that facilitate handling small volumes of liquid, such as chambers with relatively small volumes and / or pumps that are capable of relatively precisely pumping and / or are configured to relatively precisely pump relatively small volumes of liquid. Performing RNA transcription in small volumes of liquid may be particularly well- suited to testing different RNA transcription conditions for efficacy in a manner that is relatively economical and / or for synthesizing relatively small batches of RNA for use in further research.

Description

[0001] SYSTEMS AND METHODS FOR RNA TRANSCRIPTION

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 633,574, filed April 12, 2024, and entitled “Systems and Methods for RNA Transcription,” which is incorporated herein by reference in its entirety for all purposes.

[0004] FIELD

[0005] Systems for performing RNA transcription, and associated methods, are generally described.

[0006] BACKGROUND

[0007] Systems may be employed to perform RNA transcription. However, many such systems are not capable of operating with small volumes of liquid and / or exhibit challenges associated with monitoring the progress of RNA transcription during operation.

[0008] Accordingly, new systems and methods that address these concerns would be beneficial.

[0009] SUMMARY

[0010] The present disclosure generally describes systems and methods. The subject matter described herein involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0011] Paragraph 1: In some embodiments, a system for performing RNA transcription is provided. The system comprises a pump, a valve, a first mixing chamber, and a second mixing chamber. The valve is in fluidic communication with a plurality of ports. The plurality of ports comprises a first port in fluidic communication with a source of a DNA template, a second port in fluidic communication with a source of RNA polymerase, and a third port in fluidic communication with a source of an NTP. The first mixing chamber is in fluidic communication with the pump and the valve. The second mixing chamber is in fluidic communication with the pump and the valve. The pump and the valve are positioned fluidically between the first and second mixing chambers.

[0012] Paragraph 2: In some embodiments, a method of performing RNA transcription is provided. The method comprises pumping a fluid from a first mixing chamber to a second mixing chamber, pumping the fluid from the second mixing chamber to the first mixing chamber, measuring an amount of RNA in the fluid, and, based on the measurement, maintaining the fluid composition or adjusting the fluid composition. Pumping the fluid causes the fluid to mix. The fluid comprises a DNA template. The fluid comprises an RNA polymerase. The fluid comprises an NTP. Adjusting the fluid composition comprises adding a reagent to the fluid from a port in fluidic communication with a valve. The valve is in fluidic communication with the first and second mixing chambers.

[0013] Paragraph 3: In some embodiments, a method of performing RNA transcription comprises pumping a fluid from a first mixing chamber to a second mixing chamber, pumping the fluid from the second mixing chamber to the first mixing chamber, adsorbing RNA from the fluid onto a filter positioned in the first mixing chamber, and pumping an elution buffer into the first mixing chamber. Pumping the fluid causes the fluid to mix. The fluid comprises a DNA template. The fluid comprises an RNA polymerase. The fluid comprises an NTP. The elution buffer causes the RNA to desorb from the filter.

[0014] Paragraph 4: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the fluid comprises a cofactor.

[0015] Paragraph 5: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein, prior to the measuring, the fluid is removed from the system.

[0016] Paragraph 6: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the measuring is performed while the fluid is in the system.

[0017] Paragraph 7: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the measuring comprises performing HPLC.

[0018] Paragraph 8: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the measuring comprises performing UV-vis spectroscopy.

[0019] Paragraph 9: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the system comprises a UV-vis sensor.

[0020] Paragraph 10: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the plurality of ports comprises a port in fluidic communication with a source of a cofactor. Paragraph 11: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the plurality of ports comprises a port in fluidic communication with a source of a sample.

[0021] Paragraph 12: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the sample comprises the DNA.

[0022] Paragraph 13: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the plurality of ports comprises a port in fluidic communication with a source of an elution buffer.

[0023] Paragraph 14: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the plurality of ports comprises a port in fluidic communication with a source of a wash buffer.

[0024] Paragraph 15: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the reagent comprises an NTP.

[0025] Paragraph 16: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the reagent comprises Mg2+

[0026] Paragraph 17: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the reagent comprises a capping reagent.

[0027] Paragraph 18: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the reagent comprises an RNA polymerase.

[0028] Paragraph 19: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the reagent comprises an acid.

[0029] Paragraph 20: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the reagent comprises a base.

[0030] Paragraph 21: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, further comprising pumping a wash buffer into the first mixing chamber.

[0031] Paragraph 22: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the first mixing chamber is capped by a 0.2 micron filter.

[0032] Paragraph 23: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the second mixing chamber is capped by a 0.2 micron filter. Paragraph 24: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the system comprises a channel fluidically connecting the first and second mixing chambers.

[0033] Paragraph 25: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein a surface of the channel is transparent to at least one wavelength of light.

[0034] Paragraph 26: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein at least a portion of the channel is transparent to at least one wavelength of light.

[0035] Paragraph 27: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the channel is transparent to at least one wavelength of light.

[0036] Paragraph 28: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the pump is a rotary membrane pump, a peristaltic pump, a rotary pump, a piston pump, or a diaphragm pump.

[0037] Paragraph 29: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the pump is a rotary membrane pump.

[0038] Paragraph 30: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the valve is a pneumatic valve, a turning valve, a rotary membrane valve, a pinch valve, a solenoid valve, and / or a shape-memory alloy valve.

[0039] Paragraph 31: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the valve is a rotary membrane valve.

[0040] Paragraph 32: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the valve is capable of placing two or more ports in fluidic communication with the first mixing chamber at the same time.

[0041] Paragraph 33: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the valve is capable of placing two or more ports in fluidic communication with the second mixing chamber at the same time.

[0042] Paragraph 34: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, further comprising pumping a fluid from a port through the valve and into the first mixing chamber.

[0043] Paragraph 35: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, further comprising simultaneously pumping a first fluid from a first port through the valve and into the first mixing chamber, and pumping a second fluid from a second port through the valve and into the first mixing chamber.

[0044] Paragraph 36: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, further comprising pumping a fluid from a port through the valve and into the second mixing chamber.

[0045] Paragraph 37: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, further comprising pumping a fluid from the second mixing chamber into the first mixing chamber.

[0046] Paragraph 38: In some embodiments, an instrument comprising the system of any preceding paragraph is provided.

[0047] Paragraph 39: In some embodiments, an instrument as in any preceding paragraph is provided, wherein the instrument comprises an incubator.

[0048] Paragraph 40: In some embodiments, an instrument as in any preceding paragraph is provided, wherein the first mixing chamber is positioned inside the incubator.

[0049] Paragraph 41: In some embodiments, an instrument as in any preceding paragraph is provided, wherein the first and second mixing chambers are positioned inside the incubator.

[0050] Paragraph 42: In some embodiments, an instrument as in any preceding paragraph is provided, wherein the channel is positioned inside the incubator.

[0051] Paragraph 43: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the system and / or the instrument further comprises a heater.

[0052] Paragraph 44: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the system is positioned on a substrate.

[0053] Paragraph 45: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the substrate is a chip.

[0054] Paragraph 46: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the chip is a microfluidic chip.

[0055] Paragraph 47: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein a surface of the chip is transparent to at least one wavelength of light.

[0056] Paragraph 48: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the chip is transparent to at least one wavelength of light. Paragraph 49: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the chip is formed from a material that is biocompatible.

[0057] Paragraph 50: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the chip is formed from a material that is sterilizable.

[0058] Paragraph 51: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the chip is formed from a plastic.

[0059] Paragraph 52: In some embodiments, a system, method, or instrument as in any preceding paragraph is provided, wherein the plastic comprises polystyrene and / or PMMA.

[0060] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0061] BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

[0063] FIG. 1 shows an exemplary system that comprises a mixing chamber, in accordance with some embodiments;

[0064] FIG. 2 shows an exemplary system that comprises two mixing chambers, in accordance with some embodiments;

[0065] FIG. 3 shows an exemplary system that comprises a valve, in accordance with some embodiments; FIG. 4 shows an exemplary system that comprises a pump, in accordance with some embodiments;

[0066] FIG. 5 shows one non-limiting example of a system comprising two mixing chambers, a valve, and a pump, in accordance with some embodiments;

[0067] FIGs. 6 and 7 show further views of the system depicted in FIG. 5, in accordance with some embodiments;

[0068] FIG. 8 shows one non-limiting example of an instrument in which a system is positioned, in accordance with some embodiments;

[0069] FIGs. 9 and 10 depict exemplary methods, in accordance with some embodiments;

[0070] FIGs. 11-17 show one non-limiting example of a valve having the above-described design and an associated system in which the valve is positioned, in accordance with some embodiments;

[0071] FIG. 18 shows one non-limiting embodiment of a system, in accordance with some embodiments;

[0072] FIG. 19 is a schematic depiction of the system employed in Example 1; and FIG. 20 is a photograph of the system employed in Example 1.

[0073] DETAILED DESCRIPTION

[0074] Systems and methods for performing RNA transcription are generally described.

[0075] Some systems for performing RNA transcription described herein may be particularly well-suited for performing RNA transcription in small volumes of liquid. For instance, some systems may include components that facilitate handling small volumes of liquid, such as chambers with relatively small volumes and / or pumps that are capable of relatively precisely pumping and / or are configured to relatively precisely pump relatively small volumes of liquid. Performing RNA transcription in small volumes of liquid may be particularly well- suited to testing different RNA transcription conditions for efficacy in a manner that is relatively economical and / or for synthesizing relatively small batches of RNA for use in further research.

[0076] Some systems for performing RNA transcription described herein may be particularly well-suited for performing RNA transcription in a manner that allows for facile monitoring of transcription progress during operation. For instance, some systems may comprise one or more surfaces that are transparent to at least one wavelength of light. In such instances, it may be possible for optical measurements to be performed through such surface(s) on fluid in which RNA transcription is occurring. As another example, some systems may allow for facile sampling of fluid therefrom. This may allow for fluid in which RNA transcription is occurring to be easily sampled and the associated progress of RNA transcription to be measured on such samples.

[0077] Some systems described herein are capable of performing one or more methods described herein and / or are configured to perform one or more methods described herein. Some methods described herein comprise one or more steps that are capable of being performed on one or more systems described herein and some methods comprise performing one or more steps on an instrument described herein. Some methods comprise performing RNA transcription entirely on a system described herein. Some methods comprise performing RNA transcription partially on a system described herein.

[0078] FIG. 1 shows one exemplary system 100. In FIG. 1, the exemplary system 100 comprises a mixing chamber 102. Mixing chambers present in the systems described herein, like the mixing chamber shown in FIG. 1, may be capable of mixing one or more fluids positioned therein and / or may be configured to mix one or more fluids positioned therein. For instance, a mixing chamber may comprise one or more mixing elements (e.g., one or more static mixing elements, one or more dynamic mixing elements) that are capable of mixing a fluid flowing therein, thereinto, and / or therethrough. Similarly, a mixing chamber may comprise one or more mixing elements that are configured to mix a fluid flowing therein, thereinto, and / or therethrough. It is also possible for a mixing chamber to be positioned at a location in the system such that fluid flowing thereinto may be turbulent and / or may be introduced into the mixing chamber in a manner that promotes turbulent mixing (e.g., at a high velocity). In such embodiments, the mixing chamber may be a location at which mixing occurs, but may not include any elements that themselves promote mixing.

[0079] In some embodiments, a system comprises two mixing chambers. FIG. 2 shows one non-limiting example of such a system. In FIG. 2, the system 200 comprises a first mixing chamber 202A and a second mixing chamber 202B. In such embodiments, the two mixing chambers may be in fluidic communication with each other, such as via the conduit 202C shown in FIG. 2. It is also possible for a system comprising two mixing chambers to comprise one or more configurations in which two mixing chambers present therein are in fluidic communication with each other and one or more configurations in which two mixing chambers present therein are not in fluidic communication with each other. For instance, a system may comprise a valve that may comprise one or more positions that place two mixing chambers present in the system in fluidic communication with each other and one or more positions that do not place two mixing chambers present in the system in fluidic communication with each other.

[0080] Mixing chambers may be placed in fluidic communication with each other in a variety of suitable manners. In some embodiments, such fluidic communication is accomplished by a conduit, such as a channel, fluidically connecting the first and second mixing chambers. In some embodiments, two mixing chambers may each be placed in fluidic communication with a common valve via conduits (e.g., channels). Such conduits may each be in fluidic communication with a port that itself is in fluidic communication with the valve.

[0081] FIG. 3 shows one example of a system 300 comprising a valve 304. The system shown in FIG. 3 also comprises a mixing chamber 302. As shown in FIG. 3, a valve may be in fluidic communication with a plurality of ports. For instance, with respect to FIG. 3, the valve 304 is in fluidic communication with a plurality of ports 306A-306D. This may be accomplished via conduits (e.g., channels) placing the valve in fluidic communication with the ports (e.g., as shown in FIG. 3, the fluidic conduits 308A-308D). It is also possible for the ports to be positioned on the valve and so directly contact the valve (not shown).

[0082] It is also possible for a valve positioned in a system to be in fluidic communication with a mixing chamber. With reference to FIG. 3, the valve 304 is in fluidic communication with the mixing chamber 302 via the conduit 310. It is also possible for a valve positioned in a system to be in fluidic communication with a mixing chamber in another manner (e.g., via a port that is in fluidic communication with the mixing chamber, via a port that is in fluidic communication with a conduit that is in fluidic communication with the mixing chamber).

[0083] In some embodiments, a valve is capable of placing and / or configured to place one or more ports and / or other system components with which it is in fluidic communication in fluidic communication with each other. For instance, a valve may be capable of placing and / or configured to have two or more positions, each of which places a unique subset of the ports in fluidic communication with each other, one or more (e.g., two) mixing chambers, a pump (as described in further detail below), and / or one or more other system components. In some embodiments, each position of the valve places exactly one port or zero ports in fluidic communication with either or both mixing chambers and / or a pump. In some embodiments, a pump comprises one or more positions that place two or more ports (e.g., each in fluidic communication with a source of a reagent or other fluid employed during RNA transcription) in fluidic communication with either or both mixing chambers and / or a pump at the same time.

[0084] A position of a valve may comprise the positioning of one or more components thereof. For instance, a valve may comprise a pathway that can be rotated among two or more configurations that place different combinations of ports in fluidic communication with each other through the valve. As another example, a valve may comprise multiple sub-valves that may be independently opened or closed, and the position of a valve may comprise the positions of each of the sub-valves.

[0085] Valves may allow for the composition of a liquid flowing into a mixing chamber (e.g., under the action of a pump) to be modified by modifying the position of the valve. For instance, a valve may be switched between positions in which it places different combinations of ports in fluidic communication with each other through the valve. A first such port may be in fluidic communication with a pump and / or a mixing chamber. Other such ports may be in fluidic communication with sources of liquids having different compositions (e.g., comprising different reagents, comprising a sample), with other ports, and / or with other portions of the system to which liquid from a mixing chamber can be directed (e.g., a different mixing chamber). In such instances, switching the valve position such that different ports are in fluidic communication the first port may switch the composition of the liquid that can be pumped by a pump into a mixing chamber.

[0086] In some embodiments, a system comprises a pump. FIG. 4 shows one non-limiting embodiment of a system 400 comprising a pump 412. The system 400 further comprises a mixing chamber 402. As shown in FIG. 4, a pump present in a system may be in fluidic communication with a mixing chamber therein. For instance, with respect to FIG. 4, the pump 412 is in fluidic communication with the mixing chamber 402 via the conduit 414. In some embodiments, a pump is in fluidic communication with a mixing chamber via a conduit that is a channel. It is also possible for a pump to be in fluidic communication with a mixing chamber via a valve that is positioned fluidically therebetween. Pumps present in the systems described herein may be capable of pumping one or more fluids into and / or through one or more components present in the systems described herein (e.g., one or more mixing chambers and / or one or more valves).

[0087] In some embodiments, a system comprises a combination of components shown in FIGs. 1-4. For instance, a system may comprise a mixing chamber and both a pump and a valve, two mixing chambers and a valve, two mixing chambers and a pump, and / or two mixing chambers, a valve, and a pump. When such combinations are present, they may be arranged in a variety of suitable manners.

[0088] FIG. 5 shows one non-limiting example of a system comprising two mixing chambers, a valve, and a pump. In FIG. 5, the pump and the valve are positioned fluidically between the two mixing chambers. In such embodiments, the valve may be in fluidic communication with all of these components and may determine whether the two mixing chambers are in fluidic communication with each other (and / or whether one of the mixing chambers is in fluidic communication with the pump). It is also possible for a valve to be positioned fluidically between a pump and both the first and second mixing chambers (in such embodiments, the valve may also be positioned fluidically between the two mixing chambers).

[0089] During operation of the system shown in FIG. 5, the direction of pumping and the position of the valve may be selected to supply fluid from one or more of the ports to one of the mixing chambers. The direction of pumping may determine whether fluid is flowed to or from a mixing chamber. The position of the valve may determine which fluid is flowed into a mixing chamber and / or where fluid from a mixing chamber is flowed to. In some embodiments, the valve may place a mixing chamber in fluidic communication with one or more other system components (e.g., a port, one, a source of a reagent in fluidic communication with a port, a source of a fluid employed during RNA transcription in fluidic communication with a port, a second mixing chamber in fluidic communication therewith) and the direction of pumping may determine which direction fluid is flowed between these system components. As one example, the direction of pumping and position of the valve may be selected to flow a fluid from a port in fluidic communication with a source of a fluid external to the system and / or external to the portion of the system depicted in FIG. 5 to one of the mixing chambers (e.g., the direction of pumping may be selected to flow a fluid from a source of a reagent to a mixing chamber, from a source of a wash buffer to a mixing chamber, from a source of an elution buffer to a mixing chamber, from a source of a sample to a mixing chamber). As another example, the direction of pumping and position of the valve may be selected to flow a fluid from one mixing chamber to another.

[0090] It should be appreciated that, the for the system depicted in FIG. 5, the mixing chamber and the valve have certain features that are present in some, but not all, embodiments having the depicted geometry (e.g., the filters associated with the mixing chamber, the identities of the ports in fluidic communication with the valve). Additionally, in some embodiments, a system has a design similar to that shown in FIG. 5 but includes a different number of ports.

[0091] FIGs. 6 and 7 shown further views of the system depicted in FIG. 5.

[0092] In some embodiments, like the embodiments shown in FIGs. 5-7, a system described herein is positioned on a substrate. It is also possible for some, but not all, components of a system described herein to be positioned on a substrate. For instance, a system may comprise one or more mixing chambers and a valve that is positioned on a substrate but a pump that is positioned off the substrate. As a second example, a system may comprise one or more mixing chambers that are positioned on a substrate and a valve and a pump that are positioned off the substrate. Non-limiting examples of suitable such substrates include chips, such as microfluidic chips.

[0093] Some systems described herein are part of an instrument, are positioned in an instrument, are associated with an instrument, are capable of being positioned in and / or associated with an instrument, and / or are configured to be positioned in and / or associated with an instrument. For instance, an instrument may comprise a system described herein. One non-limiting example of such an instrument is an instrument for performing RNA transcription. FIG. 8 shows one non-limiting example of an instrument in which a system described herein is positioned. As can be seen in FIG. 8, the system 800 is placed inside the instrument 818.

[0094] In some embodiments, a system described herein capable of performing RNA transcription and / or configured to perform RNA transcription (and / or one or more portions thereof). Similarly, some methods comprise performing RNA transcription (and / or one or more portions thereof). RNA transcription may occur in a mixing chamber and / or in another component of the systems and instruments described herein (e.g., in a channel in fluidic communication with a mixing chamber, such as a channel positioned between two mixing chambers, a channel positioned fluidically between a pump and / or a valve and a mixing chamber, and / or a channel positioned on the opposite side fluidically from a pump or a valve with respect to a mixing chamber).

[0095] Without wishing to be bound by any particular theory, RNA transcription is a process by which a DNA sequence is copied into a strand of messenger RNA. The process comprises three steps: initiation, elongation, and termination. Initiation comprises binding an RNA polymerase to a DNA promoter sequence on double-stranded DNA and then opening the DNA. Elongation comprises growing an RNA strand with the assistance of the RNA polymerase. During this step, one strand of the opened, double- stranded DNA serves as a template for the growing RNA strand. Additionally, nucleotides may be added to the RNA strand complementary to the template DNA strand. Termination occurs at the end of RNA transcription. Termination may be triggered by the formation of terminator sequences in the growing RNA strand. This causes the release of the RNA from the RNA polymerase.

[0096] The RNA transcription process may require the presence of one or more reagents. Such reagents may be introduced into a system and / or instrument described herein (and / or into one or more components thereof) from sources thereof. It is also possible for a system and / or instrument described herein comprises a source of a reagent. In some embodiments, a valve present in a system and / or instrument described herein is in fluidic communication with one or more ports, some or all of which are in fluidic communication with sources of reagents. Non-limiting examples of such reagents include DNA templates (i.e., doublestranded DNA that serves as a template for growing RNA), an RNA polymerase, nucleoside triphosphates (also referred to as "NTPs"), cofactors, Mg2+, capping reagents, acids, bases, RNase inhibitors, and pyrophosphatase. Without wishing to be bound by any particular theory, NTPs may serve as the precursors undergo a reaction during which they are incorporated into the growing RNA strand, cofactors are proteins that may bind to the DNA being transcribed and control the rate of RNA transcription, Mg2+(which may be provided in the form of a salt, such as MgCh) may assist with stabilizing the structure of the RNA polymerase during transcription, capping reagents may assist with forming a cap on the growing RNA, acids and bases may be employed to perform pH control, RNase inhibitors may reduce RNA degradation by inhibiting RNAse, and pyrophosphatase may remove pyrophosphates formed during RNA synthesis by catalyzing their hydrolysis to orthophosphates. In some embodiments, one or more reagents are provided in a buffer and / or are added to a fluid comprising a buffer (e.g., a transcription buffer).

[0097] One non-limiting example of an RNA polymerase is T7.

[0098] Non-limiting examples of suitable NTPs include adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP).

[0099] Non-limiting examples of suitable cofactors include general transcription factors (GTFs), mediator complexes, chromatin-modifying enzymes, RNA-binding proteins (RBPs), transcriptional activators, transcriptional repressors, and transcription elongation factors.

[0100] Non-limiting examples of suitable capping reagents include reagents suitable for enzymatic capping and / or post-transcriptional capping (e.g., vaccinia capping enzyme (VCE), faustovirus capping enzyme (FCE)), and co-transcriptional capping enzymes (e.g., cap analogs).

[0101] Non-limiting examples of suitable acids include acetic acid and citric acid.

[0102] Non-limiting examples of suitable bases include sodium hydroxide (NaOH), potassium hydroxide (KOH), and sodium bicarbonate (NaHCOa).

[0103] In some embodiments, a method of performing RNA transcription comprises performing one or more steps that involve allowing RNA to be synthesized from a DNA template, monitoring the progress of RNA synthesis, and / or collecting RNA synthesized during an RNA transcription process.

[0104] One exemplary method is shown in FIG. 9. The method 920 shown in FIG. 9 comprises the steps 922-928. The step 922 comprises pumping a fluid from a first mixing chamber to a second mixing chamber. The step 924 comprises pumping the fluid from the second mixing chamber to the first mixing chamber. The step 926 comprises measuring an amount of RNA in the fluid. The step 928 comprises maintaining the fluid composition or adjusting the fluid composition based on the measurement. For instance, the fluid composition may be maintained if the measured amount of RNA indicates that RNA transcription is proceeding in an appropriate and / or expected manner. As another example, the fluid composition may be adjusted if the measured amount of RNA indicates that RNA transcription is occurring at a rate that is too fast or too slow.

[0105] In some embodiments, a method like that shown in FIG. 9 may comprise, in addition to or instead of measuring the amount of RNA in the fluid and adjusting the fluid composition based on the measured amount of RNA, measuring the amount of a reagent present in the fluid and / or adjusting the fluid composition based on the measured amount of the reagent. For instance, a method may comprise measuring an amount of NTP, Mg2+, and / or a capping reagent and maintaining or adjusting the fluid composition based on the measurement. For instance, the fluid composition may be adjusted if the measurement indicates that the fluid includes too little or too much of such reagents for RNA synthesis to proceed at a desired rate.

[0106] When performed, adjusting the composition of a fluid may comprise adding a reagent to the fluid, such as from a source in fluidic communication with a valve present in the system (e.g., by flowing a fluid comprising the reagent from the source, through the valve, and into the fluid whose composition is being adjusted). For instance, the adjustment may comprise adding NTP, Mg2+, and / or a capping reagent to the fluid. It is also possible for an adjustment of the composition of a fluid to comprise adding a buffer (e.g., a transcription buffer) lacking any reagents to the fluid in order to dilute the concentration of reagents in the fluid.

[0107] Some or all of the above-described steps may be performed on a system and / or instrument described herein. For instance, the mixing chambers and / or pump may be positioned in a system and / or an instrument described herein, the measurement of the amount of RNA may be performed by a component of a system and / or an instrument described herein, and / or a system and / or an instrument described herein may be employed to adjust or maintain the fluid composition. It also possible for the fluid being pumped to flow through a valve positioned in a system and / or instrument described herein.

[0108] With respect to the types of steps outlined above, the steps 922 and 924 in FIG. 9 may assist with RNA transcription by mixing one or more reagents that may be present during RNA transcription and / or mixing a fluid in which RNA transcription is occurring. Pumping the fluid between the mixing chambers may cause the fluid to mix, thereby mixing any reagents therein. The step 926 in FIG. 9 may correspond to monitoring the progress of RNA synthesis because the amount of RNA present in the fluid may be indicative of the amount of RNA generated during transcription. The step 928 in FIG. 9 may also assist with RNA transcription by setting (either via maintenance or adjustment) the composition of a fluid in which RNA transcription is occurring (e.g., to have a composition that promotes RNA transcription at a desirable rate).

[0109] FIG. 10 shows another exemplary method. The method 1030 shown in FIG. 10 comprises the steps 1032-1040. Step 1032 comprises pumping a fluid from a first mixing chamber to a second mixing chamber. Step 1034 comprises pumping the fluid from the second mixing chamber to the first mixing chamber. Step 1036 comprises adsorbing RNA from the fluid onto a filter positioned in the first mixing chamber. Step 1038 comprises pumping an elution buffer into the first mixing chamber. Step 1040, which is optional, comprises pumping a wash buffer into the first mixing chamber. Some or all such steps may be performed on a system and / or instrument described herein. For instance, the mixing chambers and / or pump may be positioned in a system and / or an instrument described herein. It also possible for the fluid being pumped to flow through a valve positioned in a system and / or instrument described herein.

[0110] With respect to the types of steps outlined above, the steps 1032 and 1034 in FIG. 10 may assist with RNA transcription by mixing one or more reagents that may be present during RNA transcription and / or mixing a fluid in which RNA transcription is occurring. Pumping the fluid between the mixing chambers may cause the fluid to mix, thereby mixing any reagents therein. The steps 1036 and 1038 in FIG. 10 may correspond to collecting RNA synthesized during an RNA transcription process. For instance, RNA may be adsorbed onto a filter in a mixing chamber and then removed therefrom via release into an elution buffer. In other words, the elution buffer may cause the RNA to desorb from the filter. The elution buffer can then be removed from the system and / or the instrument, after which RNA may be recovered therefrom. Optional step 1040 in FIG. 10 may correspond to preparing the first mixing chamber for subsequent RNA collection. The wash buffer may wash away any elution buffer and / or RNA present in the first mixing chamber after elution (e.g., including any RNA not released from the filter). This may result in a clean mixing chamber and / or filter positioned therein ready for use in a further RNA transcription process (e.g., a process comprising some or all of steps 1032-1038 shown in FIG. 10).

[0111] As noted above, some methods of performing RNA transcription comprise pumping fluids. In some such embodiments, a fluid being pumped may be pumped through a valve. For instance, a fluid may be pumped from a port through a valve and into another system component (e.g., a first mixing chamber, a second mixing chamber). As one example, a fluid may be pumped from a port in fluidic communication with a source (e.g., a source of a reagent, a source of a fluid other than a reagent employed during RNA transcription), through a valve, and into a mixing chamber. It is also possible for two fluids to be simultaneously pumped through a valve into a common system component (e.g., into a single mixing chamber). For instance, a first fluid may be pumped from a first port (e.g., that is in fluidic communication with a source thereof) through a valve and into the first mixing chamber while a second fluid is pumped from a second port (e.g., that is in fluidic communication with a source thereof) through the valve and also into the first mixing chamber.

[0112] It should also be noted that some methods of performing RNA transcription comprise pumping fluids into different mixing chambers. For instance, a method of performing RNA synthesis may comprise pumping a first fluid through a valve and into a first mixing chamber (e.g., from a first port) and also comprise pumping a second fluid through the valve and into a second mixing chamber (e.g., from a second port). In such instances, it is possible for the first and second fluids (and ports) to be the same or for the first and second fluids (and ports) to be different. The former may be useful when RNA transcription is performed with the use of both mixing chambers and it is desirable to supply a fluid at various time points during RNA transcription (e.g., a fluid comprising a reagent that is consumed during RNA transcription). The latter may be useful when RNA transcription is performed with the use of both mixing chambers and it is desirable to supply different fluids at different points in time during RNA transcription and / or where the different mixing chambers are employed during different parts of the RNA transcription process.

[0113] Fluids being pumped pursuant to a method described herein may have a variety of compositions. In some embodiments, a fluid being pumped pursuant to a method described herein comprises one or more reagents (e.g., a DNA template, an RNA polymerase, an NTP, and / or a cofactor) and / or one or more other fluids employed during RNA transcription (e.g., a buffer, such as a transcription buffer, a wash buffer and / or an elution buffer).

[0114] In some embodiments, RNA transcription is performed on a sample (e.g., a sample obtained from a subject). In such embodiments, the RNA transcription may comprise transcribing the DNA present in the sample (i.e., the sample may comprise the DNA on which RNA transcription is performed).

[0115] As noted above, some systems may comprise pumps. A variety of suitable pumps may be employed, non-limiting examples of which include a rotary membrane pump, a peristaltic pump (e.g., a tubing pump), a rotary pump, a piston pump, a diaphragm pump, a motor pump (e.g., a syringe pump-like motor pump), and a roller pump. In some embodiments, a system comprises two or more pumps. In such embodiments, the two or more pumps may comprise two of the same pumps and / or two pumps that differ from each other in one or more ways.

[0116] As noted above, some systems may comprise valves. A variety of suitable valves may be employed. Non-limiting examples of suitable valves include pneumatic valves, turning valves, rotary membrane valves, pinch valves, solenoid valves, and shape-memory alloy valves.

[0117] In some embodiments, a valve employed in a system described herein has a design such that it places one port in fluidic communication with and upstream from an inlet to a pump and another port in fluidic communication with and downstream from an outlet of the pump. In some such embodiments, these ports can be selected arbitrarily (i.e., any port with which the valve is in fluidic communication may be placed in fluidic communication with and upstream from an inlet to a pump and any port with which the valve is in fluidic communication may be placed in fluidic communication with and downstream from an outlet of the pump, and / or any pairs of ports may be selected for these two positions). Without wishing to be bound by any particular theory, this may facilitate fluid flow through the system between a variety of components positioned therein that is driven by a pump that is only capable of pumping in a single direction. In other words, the direction of pumping of fluid through the system may be capable of being selected by the valve (e.g., even if the pump is only capable of pumping in a single direction).

[0118] FIGs. 11-17 show one non-limiting example of a valve having the above-described design and an associated system in which the valve is positioned. FIG. 11 depicts the valve pusher. From FIG. 11, it can be seen that the valve pusher has two open positions, which may allow for the selection of ports that are in fluidic communication with a pump (e.g., the fluidic communication may occur via the open positions). FIG. 12 shows one non-limiting embodiment of a portion of a system comprising such a valve and further comprising a pump. As can be seen from FIG. 12, the pusher may be capable of being rotated such that a variety of different ports are in fluidic communication with and upstream or downstream from the pump.

[0119] FIG. 13 shows one example of a position of the above-described valve that places a port (that is in fluidic communication with a source of reagents) in fluidic communication with and upstream from an inlet to the pump and places a port (that is upstream from and in fluidic communication with a mixing chamber) in fluidic communication with and downstream from an outlet of the pump. The arrow in FIG. 13 shows the flow of fluid from the port in fluidic communication with the source of reagents, through the valve, through the pump, through the valve again, and then to the mixing chamber. It is also possible for the valve to be employed to place two mixing chambers in fluidic communication with each other and / or to place a mixing chamber in fluidic communication with a waste chamber (not shown).

[0120] FIGs. 14-16 show further examples of manners in which fluid may flow through the valve depicted therein. FIGs. 14 and 15 depict employing the valve to cause flow that promotes mixing (i.e., by causing fluid to flow back and forth between two mixing chambers). FIG. 16 depicts employing the valve to cause flow that removes fluid from a mixing chamber to a waste chamber. As can be seen from FIGs. 13-16, the direction of flow through the valve and / or pump may be adjusted to cause flow in a number of different directions, to a number of different locations, and / or from a number of different locations.

[0121] FIG. 17 depicts the base of the system in which the above-described valve is positioned. As can be seen from FIG. 17, various components of the system may have static positions (e.g., they may not move and / or they may be mechanically coupled to a substrate present in the system) even though flow thereto, therethrough, and / or therefrom may be selected as desired.

[0122] The valves described herein may be in fluidic communication with a plurality of ports. In some embodiments, some or all of the ports are themselves in fluidic communication with sources of reagents. For instance, each port may be in fluidic communication with a different source of a different reagent and / or each port may be fluidically isolated from each other when not placed in fluidic communication via a valve with which they are in fluidic communication. Such reagents may be those described elsewhere herein with respect to RNA transcription. In some embodiments, the plurality of ports comprises a port in fluidic communication with a source of a DNA template, a port in fluidic communication with a source of RNA polymerase, a port in fluidic communication with a source of an NTP, and a port in fluidic communication with a source of a cofactor.

[0123] It is also possible for a valve to be in fluidic communication with ports that are in fluidic communication with a source of a fluid other than a reagent (e.g., a source of a buffer, such as a wash buffer and / or an elution buffer, a source of a sample) and / or a system component other than a source (e.g., with a mixing chamber, with a pump) and / or that are in fluidic communication with a system component other than a port (e.g., with a mixing chamber, with a pump).

[0124] The mixing chambers described herein may have a variety of suitable volumes. In some embodiments, a mixing chamber has a volume of greater than or equal to 1 microliter, greater than or equal to 2 microliters, greater than or equal to 5 microliters, greater than or equal to 7.5 microliters, greater than or equal to 10 microliters, greater than or equal to 20 microliters, greater than or equal to 50 microliters, greater than or equal to 75 microliters, greater than or equal to 100 microliters, greater than or equal to 200 microliters, greater than or equal to 500 microliters, or greater than or equal to 750 microliters. In some embodiments, a mixing chamber has a volume of less than or equal to 1000 microliters, less than or equal to 750 microliters, less than or equal to 500 microliters, less than or equal to 200 microliters, less than or equal to 100 microliters, less than or equal to 75 microliters, less than or equal to 50 microliters, less than or equal to 20 microliters, less than or equal to 10 microliters, less than or equal to 7.5 microliters, less than or equal to 5 microliters, or less than or equal to 2 microliters. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 1 microliter and less than or equal to 1000 microliters). Other ranges are also possible.

[0125] Each mixing chamber in a system described herein may independently have a volume in one or more of the above-referenced ranges.

[0126] In some embodiments, a mixing chamber (or two mixing chambers) are capped by a filter. Without wishing to be bound by any particular theory, this may allow for facile recovery of RNA from the system and / or facile removal of fluid from the system. For instance, a conduit (e.g., a channel) may be positioned on a side of the filter opposite the mixing chamber. In some such embodiments, fluid into which RNA has been desorbed (e.g., an elution buffer) may be flowed from the mixing chamber, through the filter, and into the conduit. Such fluid may then further flow into a component of the system and / or an instrument in which the system is positioned for recovery and / or analysis. It is also possible for fluid that it is otherwise desirable to remove from the system (e.g., a wash buffer, any fluid remaining after RNA transcription that is waste) through such a pathway and into a waste chamber or other appropriate destination.

[0127] In some embodiments, a mixing chamber is capped by a filter that has a pore size suitable for filtering out any aggregates that would be undesirable to transport out of the mixing chamber (e.g., any aggregates that it would be undesirable to have in RNA recovered therefrom). As one example, in some embodiments, a mixing chamber (e.g., a first mixing chamber, a second mixing chamber) is capped by a 0.2 micron filter.

[0128] As noted above, some systems described herein comprise conduits. Such conduits may place two or more components of the system in fluidic communication with each other. For instance, a system may comprise channels that place two or more of the following system components in fluidic communication with each other: a valve, a port, a source (e.g., of a reagent or other fluid employed during RNA transcription), a pump, and / or a mixing chamber. In some embodiments, a conduit may take the form of a channel, such as a channel positioned on a substrate in which one or more system components are disposed and / or with which one or more system components interface (e.g., one or more of the components that the channel may place in fluidic communication with each other).

[0129] In some embodiments, RNA transcription is performed partially or fully in a channel. It is also possible for RNA transcription to be performed in a portion of a system other than a channel but for an amount of RNA present in a fluid in which RNA transcription is being performed to be measured while the fluid is in the channel. In some embodiments, as described below, a channel may have one or more features that facilitate measuring the amount of RNA present in a fluid positioned therein. For instance, the channel may comprise a surface that is transparent to at least one wavelength of light (e.g., a transparent pathway may exist between an external surface of the system and an interior of the channel). When present, this surface may extend across the entirety of the channel or extend across only one or more portions thereof. In some embodiments, the channel as a whole is transparent to at least one wavelength of light (e.g., a transparent pathway may exist between two external surfaces, such as two opposing external surfaces, of the system that passes through an interior of the channel). In some embodiments, the entirety of a channel may be transparent to at least one wavelength of light. It is also possible for only one or more portions thereof to be transparent to at least one wavelength of light.

[0130] In some embodiments, the light to which a channel surface and / or channel is transparent has a wavelength of greater than or equal to 400 nm, greater than or equal to 450 nm, greater than or equal to 500 nm, greater than or equal to 550 nm, greater than or equal to 600 nm, or greater than or equal to 650 nm. In some embodiments, the light to which a channel surface and / or channel is transparent has a wavelength of less than or equal to 700 nm, less than or equal to 650 nm, less than or equal to 600 nm, less than or equal to 550 nm, less than or equal to 500 nm, or less than or equal to 450 nm. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 400 nm and less than or equal to 700 nm). Other ranges are also possible. In some embodiments, a channel surface and / or channel is transparent to all wavelengths of light within one or more of the abovereferenced ranges.

[0131] In some embodiments, a channel surface and / or channel is transparent to a wavelength of light in one or more of the above-referenced ranges with a transparency of greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 99%, or greater than or equal to 99.9%. In some embodiments, a channel surface and / or channel is transparent to a wavelength of light in one or more of the above-referenced ranges with a transparency of less than or equal to 100%, less than or equal to 99.9%, less than or equal to 99%, less than or equal to 95%, less than or equal to 90%, or less than or equal to 80%. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 70% and less than or equal to 100%). Other ranges are also possible. Each channel in a system described herein may be transparent to a wavelength of light in one or more of the above-referenced ranges with a transparency in one or more of the above-referenced ranges (and / or may comprise a surface having either or both of the foregoing properties).

[0132] Transparency may be assessed by UV-vis spectroscopy.

[0133] Channels described herein may have a variety of suitable volumes. In some embodiments, a channel has a volume of greater than or equal to 1 microliter, greater than or equal to 2 microliters, greater than or equal to 5 microliters, greater than or equal to 7.5 microliters, greater than or equal to 10 microliters, greater than or equal to 20 microliters, greater than or equal to 50 microliters, greater than or equal to 75 microliters, greater than or equal to 100 microliters, greater than or equal to 125 microliters, greater than or equal to 150 microliters, or greater than or equal to 175 microliters. In some embodiments, a channel has a volume of less than or equal to 200 microliters, less than or equal to 175 microliters, less than or equal to 150 microliters, less than or equal to 125 microliters, less than or equal to 100 microliters, less than or equal to 75 microliters, less than or equal to 50 microliters, less than or equal to 20 microliters, less than or equal to 10 microliters, less than or equal to 7.5 microliters, less than or equal to 5 microliters, or less than or equal to 2 microliters. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 1 microliter and less than or equal to 200 microliters). Other ranges are also possible.

[0134] Each channel in a system described herein may independently have a volume in one or more of the above-referenced ranges.

[0135] The sources of reagents described herein may have a variety of suitable volumes. In some embodiments, a source of a reagent has a volume of greater than or equal to 1 microliter, greater than or equal to 2 microliters, greater than or equal to 5 microliters, greater than or equal to 7.5 microliters, greater than or equal to 10 microliters, greater than or equal to 20 microliters, greater than or equal to 50 microliters, greater than or equal to 75 microliters, greater than or equal to 100 microliters, greater than or equal to 200 microliters, greater than or equal to 500 microliters, or greater than or equal to 750 microliters. In some embodiments, a source of a reagent has a volume of less than or equal to 1000 microliters, less than or equal to 750 microliters, less than or equal to 500 microliters, less than or equal to 200 microliters, less than or equal to 100 microliters, less than or equal to 75 microliters, less than or equal to 50 microliters, less than or equal to 20 microliters, less than or equal to 10 microliters, less than or equal to 7.5 microliters, less than or equal to 5 microliters, or less than or equal to 2 microliters. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 1 microliter and less than or equal to 1000 microliters). Other ranges are also possible.

[0136] Each source of a reagent in a system described herein may independently have a volume in one or more of the above-referenced ranges.

[0137] As noted above, some systems described herein may be positioned on substrates, such as chips (e.g., microfluidic chips). In some embodiments, one or more system components comprise volumes that have been removed (e.g., etched) from a substrate. Some or all such system components may be positioned in a single substrate and / or a system may comprise a single substrate from which one or more portions have been removed to form system component(s).

[0138] In some embodiments, a substrate (e.g., the chip, the microfluidic chip) may comprise a surface that is transparent to at least one wavelength of light. Such a surface may extend across an entirety of the substrate or may only extend across a portion of the substrate. In some embodiments, the substrate, may comprise one or more portions that, as a whole, are transparent to at least one wavelength of light. It is also possible for the entirety of the substrate to be transparent to at least one wavelength of light. The wavelengths of light to which the substrate may be transparent and the levels of transparency may be in one or more of the ranges provided above with respect to the transparency of a channel to wavelengths of light.

[0139] In some embodiments, a substrate on which a system is positioned has one or more features that facilitate performing RNA transcription therein. For instance, in some embodiments, a substrate may be biocompatible. For instance, it may be possible to culture cells on the substrate. Such cells may have a relatively high degree of viability as measured by dye (e.g., trypan blue), may exhibit adhesion to and / or proliferation on the substrate, and / or display cellular markers and / or proteins associated with cell health and function upon immunofluorescence staining.

[0140] In some embodiments, a substrate on which a system is positioned is sterilizable (e.g., via an autoclave, exposure to gamma radiation, exposure to x-ray radiation, exposure to ethylene oxide, dry heat sterilization, and / or chemical sterilization, such as via exposure to hydrogen peroxide vapor, bleach, and / or alcohol). Such substrates may change color and / or deform (or not change color and / or not deform) during sterilization. It is also possible for a substrate to be disposable. In such instances, some system components (e.g., those positioned off the substrate and / or those capable of being removed from the substrate, such as valves and / or pumps) may be reusable.

[0141] The substrates described herein may comprise and / or be formed from a variety of suitable materials. In some embodiments, a substrate is formed from and / or comprises a plastic, such as polystyrene, PMMA, a cyclic olefin copolymer, polycarbonate, polysulfone, polyetherimide (e.g., Ultem), polyurethane, and / or a 3D-printed resin. In some embodiments, a substrate is formed from and / or comprises PDMS. In some embodiments, a substrate is formed from and / or comprises glass.

[0142] In some embodiments, a system comprises a substrate and further comprises one or more additional components. Such components may be positioned above or below the substrate. For instance, a system may further comprise a bottom cap disposed beneath a substrate and / or a cover layer disposed on a substrate. In some such embodiments, an adhesive may be employed to adhere the substrate to such components. FIG. 18 shows one non-limiting embodiment of a system having this design. In some such embodiments, a cover layer disposed on the substrate comprises a membrane valve and pump and / or a silicone.

[0143] It is also possible for a system to comprise one or more components that fit into a volume that has been removed from a substrate (e.g., a pump that fits into such a volume, a sensor that fits into such a volume, an optical cable that fits into such a volume, an optical transmitter that fits into such a volume, and / or a valve that fits into such a volume).

[0144] It is also possible for a system to comprise one or more additional components that are not placed on a substrate and / or below a substrate. For instance, a system may comprise one or more motors (e.g., one or more motors for one or more pumps, one or more sensors, one or more optical cables, one or more optical transmitters, and / or one or more valves) that are placed in such locations.

[0145] As noted above, some embodiments relate to instruments that comprise a system described herein and / or are otherwise associated with an instrument described herein. One non-limiting example of such an instrument is an incubator (e.g., a carbon dioxide incubator). In some embodiments, a system is positioned inside an incubator that is itself positioned inside an optical detector (e.g., an optical microscope). In some embodiments, some or all of a system described herein is positioned inside an instrument that is an incubator. For instance, in some embodiments, a portion of a system in which RNA transcription is performed is positioned inside an incubator (e.g., when RNA transcription is being performed). As an example, an instrument comprises a first mixing chamber that is positioned inside an incubator, a second mixing chamber that is positioned inside an incubator, and / or a channel that is positioned inside an incubator (e.g., when RNA transcription is being performed in one or more such locations).

[0146] In some embodiments, a system and / or an instrument comprising the system and / or otherwise associated with the system comprises one or more components that may assist with maintaining a particular temperature in one or more components of the system. For instance, the system and / or instrument may comprise a component that maintains a particular temperature in one or more locations in the system in which RNA synthesis occurs (e.g., a mixing chamber, a channel). As one example, in some embodiments, a system and / or an instrument comprises a heater (e.g., a heater that heats a mixing chamber and / or a channel).

[0147] In some embodiments, a system and / or an instrument comprising the system and / or otherwise associated with the system comprises one or more sensors. Such sensors may be employed to sense one or more properties of a fluid present in the system (and / or instrument). Two non-limiting examples of suitable sensors include temperature sensors and optical sensors (e.g., UV sensors).

[0148] As noted above, some methods comprise measuring an amount of RNA in a fluid present in a system described herein. The measurement may be performed by removing the fluid from the system and then performing the measurement (e.g., via an outlet positioned in a measurement chamber) and / or may be performed while the fluid is in the system. It is also possible for one or features other than RNA content to be measured. For instance, the amount of one or more reagents present in the fluid may be measured (e.g., the amount NTP present in the fluid and / or the amount of a capping reagent present in the fluid). In some embodiments, the fluid composition may be maintained or adjusted based on such a measurement. For instance, the fluid composition may be maintained or adjusted based on a measured amount of RNA and / or the measured amount of one or more other reagents (e.g., NTP and / or a capping reagent).

[0149] When the measuring is performed while the fluid is in the system, it may be performed on a single sample or multiple samples may be obtained at multiple different points in time, each of which is subjected to measurement.

[0150] When the measuring is performed while the fluid is in the system, it may be performed by a component that is present in the system (e.g., a UV-vis sensor present in the system) and / or by a component that can interface with the system (e.g., a UV-vis sensor external to the system). Additionally, when the measuring is performed while the fluid is in the system, it may be performed in a part of the system that is transparent to at least one wavelength of light and / or that comprises a surface that is transparent to at least one wavelength of light. Without wishing to be bound by any particular theory, this may facilitate optical measurements in which light is transmitted from a light source, through the transparent surface, and into the fluid on which the optical measurement is being performed. Light that is reflected, scattered, or transmitted may then travel out of the fluid, through a transparent surface (e.g., the same transparent surface through which it was initially transmitted or a different transparent surface), and to an optical detector.

[0151] Non-limiting examples of methods by which the amount of RNA (and / or a reagent) may be measured include HPLC and UV-vis spectroscopy.

[0152] As noted above, some embodiments relate to fluid handling (e.g., pumping fluids, making measurements on fluids). A variety of suitable fluids may be employed. For instance, in some embodiments, one or more of the fluids described herein may be a liquid, such as an aqueous liquid. In some embodiments, a fluid described herein is and / or comprises a buffer. For instance, RNA transcription may be performed in a buffer, RNA may be recovered from an elution filter by an elution buffer, and / or one or more system components may be washed by a wash buffer.

[0153] EXAMPLE 1

[0154] This Example describes performing RNA transcription in a system comprising a plurality of ports, and a mixing chamber. A schematic depiction of the system is shown in FIG. 19 and a photograph of the system is shown in FIG. 20.

[0155] The system included four ports: a first port in fluidic communication with a source of a first plurality of reagents (comprising a buffer, acid, ATP, CTP, GTP, UTP, a capping reagent, Mg2+, and DTT), a second port in fluidic communication with a source of a second plurality of reagents (comprising DNA, RNase inhibitor, pyrophosphatase, and T7), a third port in fluidic communication with a source of a third plurality of reagents (comprising NTPs, a capping reagent, and Mg2+), and a fourth port in fluidic communication with a fourth plurality of reagents (comprising DNA and T7).

[0156] In a first experiment, the first and second pluralities of reagents were flowed through the first and second ports into the mixing chamber. The flow was stopped, and the mixing chamber was then incubated at 36.8 °C for three hours, after which a sample was extracted. The extracted sample was purified, and the concentration of RNA therein was determined to be over 4000 ng / microliter, indicating that RNA transcription occurred.

[0157] In a second experiment, the first and second pluralities of reagents were flowed into the mixing chamber as described above and the mixing chamber was incubated as described above, except that the mixing chamber was employed to cause mixing during incubation. After three hours, a sample was extracted and purified as described above. The concentration of the RNA therein was determined to be approximately 5000 ng / mL, indicating that the mixing enhanced RNA transcription.

[0158] The third and fourth ports may be employed in further experiments to supply additional TNPs, capping reagents, Mg2+, DNA template, and / or T7 as needed.

[0159] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0160] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0161] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0162] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0163] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0164] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0165] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS1. A system for performing RNA transcription, comprising: a pump; a valve, wherein the valve is in fluidic communication with a plurality of ports, wherein the plurality of ports comprises a first port in fluidic communication with a source of a DNA template, a second port in fluidic communication with a source of RNA polymerase, and a third port in fluidic communication with a source of an NTP; a first mixing chamber, wherein the first mixing chamber is in fluidic communication with the pump and the valve; and a second mixing chamber, wherein the second mixing chamber is in fluidic communication with the pump and the valve, and wherein the pump and the valve are positioned fluidically between the first and second mixing chambers.

2. A method of performing RNA transcription, comprising: pumping a fluid from a first mixing chamber to a second mixing chamber; pumping the fluid from the second mixing chamber to the first mixing chamber; measuring an amount of RNA in the fluid; and based on the measurement, maintaining the fluid composition or adjusting the fluid composition, wherein: pumping the fluid causes the fluid to mix, the fluid comprises a DNA template, the fluid comprises an RNA polymerase, the fluid comprises an NTP, adjusting the fluid composition comprises adding a reagent to the fluid from a port in fluidic communication with a valve, and the valve is in fluidic communication with the first and second mixing chambers.

3. A method of performing RNA transcription, comprising: pumping a fluid from a first mixing chamber to a second mixing chamber; pumping the fluid from the second mixing chamber to the first mixing chamber;adsorbing RNA from the fluid onto a filter positioned in the first mixing chamber; and pumping an elution buffer into the first mixing chamber, wherein: pumping the fluid causes the fluid to mix, the fluid comprises a DNA template, the fluid comprises an RNA polymerase, the fluid comprises an NTP, and the elution buffer causes the RNA to desorb from the filter.

4. A method as in claim 2, wherein the fluid comprises a cofactor.

5. A method as in claim 2, wherein, prior to the measuring, the fluid is removed from the system.

6. A method as in claim 2, wherein the measuring is performed while the fluid is in the system.

7. A method as in claim 2, wherein the measuring comprises performing HPLC.

8. A method as in claim 2, wherein the measuring comprises performing UV-vis spectroscopy.

9. A system as in claim 1, wherein the system comprises a UV-vis sensor.

10. A system as in claim 1, wherein the plurality of ports comprises a port in fluidic communication with a source of a cofactor.

11. A system as in claim 1, wherein the plurality of ports comprises a port in fluidic communication with a source of a sample.

12. A system as in claim 11, wherein the sample comprises the DNA.

13. A system as in claim 1, wherein the plurality of ports comprises a port in fluidic communication with a source of an elution buffer.

14. A system as in claim 1, wherein the plurality of ports comprises a port in fluidic communication with a source of a wash buffer.

15. A method as in claim 2, wherein the reagent comprises an NTP.

16. A method as in claim 2, wherein the reagent comprises Mg2+.

17. A method as in claim 2, wherein the reagent comprises a capping reagent.

18. A method as in claim 2, wherein the reagent comprises an RNA polymerase.

19. A method as in claim 2, wherein the reagent comprises an acid.

20. A method as in claim 2, wherein the reagent comprises a base.

21. A method as in claim 2, further comprising pumping a wash buffer into the first mixing chamber.

22. A system as in claim 1, wherein the first mixing chamber is capped by a 0.2 micron filter.

23. A system as in claim 1, wherein the second mixing chamber is capped by a 0.2 micron filter.

24. A system as in claim 1, wherein the system comprises a channel fluidically connecting the first and second mixing chambers.

25. A system as in claim 24, wherein a surface of the channel is transparent to at least one wavelength of light.

26. A system as in claim 24, wherein at least a portion of the channel is transparent to at least one wavelength of light.

27. A system as in claim 24, wherein the channel is transparent to at least one wavelength of light.

28. A system as in claim 1, wherein the pump is a rotary membrane pump, a peristaltic pump, a rotary pump, a piston pump, or a diaphragm pump.

29. A system as in claim 1, wherein the pump is a rotary membrane pump.

30. A system as in claim 1, wherein the valve is a pneumatic valve, a turning valve, a rotary membrane valve, a pinch valve, a solenoid valve, and / or a shape-memory alloy valve.

31. A system as in claim 1, wherein the valve is a rotary membrane valve.

32. A system as in claim 1, wherein the valve is capable of placing two or more ports in fluidic communication with the first mixing chamber at the same time.

33. A system as in claim 1, wherein the valve is capable of placing two or more ports in fluidic communication with the second mixing chamber at the same time.

34. A method as in claim 2, further comprising pumping a fluid from a port through the valve and into the first mixing chamber.

35. A method as in claim 2, further comprising simultaneously pumping a first fluid from a first port through the valve and into the first mixing chamber, and pumping a second fluid from a second port through the valve and into the first mixing chamber.

36. A method as in claim 2, further comprising pumping a fluid from a port through the valve and into the second mixing chamber.

37. A method as in claim 2, further comprising pumping a fluid from the second mixing chamber into the first mixing chamber.

38. An instrument comprising the system of claim 1.

39. An instrument as in claim 38, wherein the instrument comprises an incubator.

40. An instrument as in claim 39, wherein the first mixing chamber is positioned inside the incubator.

41. An instrument as in claim 39, wherein the first and second mixing chambers are positioned inside the incubator.

42. An instrument as in claim 39, wherein the channel is positioned inside the incubator.

43. An instrument as in claim 38, wherein the system and / or the instrument further comprises a heater.

44. A system as in claim 1, wherein the system is positioned on a substrate.

45. A system as in claim 44, wherein the substrate is a chip.

46. A system as in claim 45, wherein the chip is a microfluidic chip.

47. A system as in claim 45, wherein a surface of the chip is transparent to at least one wavelength of light.

48. A system as in claim 45, wherein the chip is transparent to at least one wavelength of light.

49. A system as in claim 45, wherein the chip is formed from a material that is biocompatible.

50. A system as in claim 45, wherein the chip is formed from a material that is sterilizable.

51. A system as in claim 45, wherein the chip is formed from a plastic.

52. A system as in claim 51, wherein the plastic comprises polystyrene and / or PMMA.

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