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WO2024073375A2 - Systèmes et procédés de criblage de banques de gènes de grande taille - Google Patents

Systèmes et procédés de criblage de banques de gènes de grande taille Download PDF

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Publication number
WO2024073375A2
WO2024073375A2 PCT/US2023/075065 US2023075065W WO2024073375A2 WO 2024073375 A2 WO2024073375 A2 WO 2024073375A2 US 2023075065 W US2023075065 W US 2023075065W WO 2024073375 A2 WO2024073375 A2 WO 2024073375A2
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Prior art keywords
droplets
activity
nucleic acids
nucleic acid
target substrate
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WO2024073375A3 (fr
Inventor
David A. Weitz
Karla MILCIC
Xinge Zhang
Anqi Chen
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Harvard University
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Harvard University
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Priority to EP23873810.8A priority Critical patent/EP4594516A2/fr
Publication of WO2024073375A2 publication Critical patent/WO2024073375A2/fr
Publication of WO2024073375A3 publication Critical patent/WO2024073375A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR

Definitions

  • a library of molecules may be screened based on activity of a library molecule, or on activity of an associated molecule, such as a protein expressed by the library molecule.
  • Conventional methods for library screening limit a number of variants that may be screened. Screening for protein expression (e.g., enzyme expression) by a library can be particularly difficult using conventional methods. Accurate methods of screening larger libraries are desirable.
  • Droplet-based systems and methods for library screening are provided.
  • the systems and methods may include steps of sorting droplets based on activity, amplifying nucleic acids of “activity droplets” having high activity, and separating nucleic acids from the activity droplets into new droplets.
  • the steps may be iterated, such that activity droplets from successive iterations tend to include fewer nucleic acids.
  • Such approaches may facilitate identification of active nucleic acids, or of nucleic acids encoding active proteins.
  • the subject matter of the present disclosure 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.
  • a method comprises: determining one or more activity droplets of a first plurality of droplets having an activity of a target substrate, wherein at least 50% of the droplets the first plurality of droplets each comprise at least 5 distinct nucleic acid sequences; separating nucleic acids from the one or more activity droplets of the first plurality of droplets into a second plurality of droplets; and amplifying the nucleic acids of the one or more activity droplets.
  • a method comprises: determining one or more activity droplets of a first plurality of droplets having an activity of a target substrate, wherein the first plurality of droplets comprises greater than or equal to 10 5 droplets containing the target substrate, and wherein at least 50% of the droplets the first plurality of droplets each comprise at least 10 5 distinct nucleic acid sequences; separating nucleic acids from the one or more activity droplets of the first plurality of droplets into a second plurality of droplets; and amplifying the nucleic acids of the one or more activity droplets.
  • a method comprises: in a first plurality of droplets comprising nucleic acids, translating proteins from the nucleic acids within the droplets, at least 50% of the droplets the first plurality of droplets containing therein at least 10 5 distinct nucleic acid sequences, and wherein the first plurality of droplets comprises greater than or equal to 10 5 droplets having distinct nucleic acid sequences contained therein; determining one or more activity droplets of the first plurality of droplets that contain activity of a target substrate; separating nucleic acids from the one or more activity droplets of the first plurality of droplets into a second plurality of droplets; and amplifying the nucleic acids of the one or more activity droplets.
  • an article comprises: a plurality of droplets, at least 50% of the droplets containing therein at least 10 5 distinct nucleic acid sequences and a target substrate, wherein the plurality of droplets comprises greater than or equal to 10 5 droplets having distinct nucleic acid sequences contained therein.
  • composition in another aspect, comprises: an amino acid sequence at least 70% identical to one of Seq. ID. Nos. 2-24, wherein the amino acid sequence is not Seq. ID. No. 25.
  • a composition in one aspect, comprises: a nucleic acid sequence at least 70% identical to one of Seq. ID. Nos. 26-48, wherein the nucleic acid sequence is not Seq. ID. No. 49.
  • FIG. 1A presents a schematic illustration of a nucleic acid, according to some embodiments
  • FIG. IB presents a schematic illustration of a nucleic acid, according to some embodiments.
  • FIG. 1C presents a schematic illustration of a nucleic acid, according to some embodiments.
  • FIG. 2A presents a schematic illustration of a method of screening a large library, according to some embodiments
  • FIG. 2B presents a schematic illustration of a method of screening a large library, according to some embodiments
  • FIG. 3A presents a schematic flow diagram of a method of screening a large library, according to some embodiments
  • FIG. 3B presents a schematic flow diagram of a method of screening a large library, according to some embodiments.
  • FIG. 3C presents a schematic flow diagram of a method of screening a large library, according to some embodiments.
  • FIG. 3D presents a schematic flow diagram of a method of screening a large library, according to some embodiments
  • FIG. 4 presents a schematic illustration of a circular nucleic acid and a plurality of primers suitable for preparing a nucleic acid library, according to some embodiments;
  • FIG. 5A presents a schematic illustration of a nucleic acid and pluralities of primers, according to some embodiments
  • FIG. 5B presents a schematic illustration of a nucleic acid and pluralities of primers, according to some embodiments
  • FIG. 5C presents a schematic illustration of a nucleic acid and pluralities of primers, according to some embodiments.
  • FIG. 5D presents a schematic illustration of nucleic acids representing libraries of nucleic acids, according to some embodiments.
  • FIG. 5E presents a schematic illustration of nucleic acids representing a library of nucleic acids, according to some embodiments.
  • FIG. 6A presents a confocal microscope image of a plurality of droplets including activity droplets, according to some embodiments
  • FIG. 6B presents a red fluorescence image of a plurality of droplets including activity droplets, according to some embodiments
  • FIG. 6C presents a green fluorescence image of a plurality of droplets including activity droplets, according to some embodiments
  • FIGS. 7A-7C present non-limiting schematic illustrations of microfluidic devices, according to some embodiments.
  • FIG. 8A presents a non-limiting, schematic illustration of cell-free protein synthesis in drops, according to some embodiments
  • FIG. 8B presents a non-limiting, schematic illustration of pico-injection of a substrate, followed by fluorescence-based droplet sorting, according to some embodiments
  • FIG. 8C presents a non-limiting, schematic illustration of co-encapsulation of one bacteria cell host expressing enzyme mutants, fluorescent substrate, and lysis buffer, followed by fluorescence-based droplet sorting, according to some embodiments;
  • FIG. 9 presents a non-limiting, schematic illustration of the sorting of activity droplets, according to some embodiments.
  • FIG. 10 presents the activity (in arbitrary units) of various sequences after thermal shock at 80 C as a function of the concentration of Tween, according to some embodiments.
  • Microfluidics offer a number of processing advantages for handling molecular libraries, but detection of dilute molecules in individual droplets can present certain challenges.
  • Conventional cell-based methods of library refinement such as phage display are also limiting, and may restrict the size of a molecular library to a number of molecules on the order of 10 8 .
  • the systems and methods described herein permit library refinement of large molecular libraries (e.g., libraries including a number of molecules on the order of 10 12 ) using microfluidic systems.
  • Disclosed herein are systems and methods for partitioning molecular libraries into microfluidic droplets and selecting for droplets based on activation of a target substrate.
  • a method comprises partitioning a library of ⁇ 10 12 DNA sequences into a plurality of ⁇ 10 4 droplets such that each droplet comprises at least 10 8 distinct DNA sequences.
  • the number of distinct DNA molecules per droplet may be controlled, for example, by controlling the DNA concentration and droplet size such that droplets are statistically likely to include the desired number of DNA sequences. In this way, each droplet may act as an independent library of sequences.
  • In vitro transcription and translation may be performed in the droplets, and a target molecule may be included in the droplets such that droplets produce a signal if they include an active protein encoded by the DNA library, and do not produce a signal if they do not include an active protein encoded by the DNA library.
  • An iterative process of selecting droplets for activity of the target substrate, amplifying the DNA sequences within the active droplets by PCR, and breaking the active droplets into a new plurality of droplets may be used to further differentiate nucleic acids expressing active proteins from nucleic acids that merely express inactive proteins.
  • active DNA strands may be refined from the library, and may be amplified, sequenced, or put to any other functional purpose for which protein activity is desired.
  • the present invention generally relates, in certain aspects, to droplet-based microfluidic devices and methods. It may be useful, in some embodiments, to identify activity of a target substrate within a droplet.
  • a method comprises determining one or more activity droplets of a plurality of droplets that have an activity of a target substrate.
  • the target substrate may be any of a variety of appropriate target substrates.
  • the target substrate may be a binding target (e.g., a viral or cancer cell antigen), a reaction target (e.g., a molecule that should react with or undergo a reaction catalyzed by a molecule in the droplet), or may be a target configured to activate as a result of any of a variety of other suitable biochemical processes, etc.
  • any of a variety of approaches may be used to determine the activity droplets having the activity of the target substrate.
  • some embodiments may comprise identifying activity droplets that include any activity of a target substrate.
  • some embodiments are directed to identifying a subset of droplets that includes activity exceeding a threshold value.
  • a target substrate may, upon activation, produce a signal (e.g., a colorimetric, fluorescent, or luminescent signal) exceeding a predefined minimum signal.
  • a signal e.g., a colorimetric, fluorescent, or luminescent signal
  • Such an approach may be useful in certain cases for identifying highly active molecules, e.g., while excluding less active molecules. It should, of course, be understood that the droplets with the most activity do not necessarily correspond to the droplets comprising the most active molecules.
  • droplets may stochastically include larger numbers of active sequences, thereby demonstrating higher apparent activity without necessarily including highly active variants. More generally, it should be understood that separation of active droplets may be performed by any of a variety of suitable systems and methods, e.g., as described herein, as the disclosure is not so limited.
  • Some aspects are generally directed to systems and methods of determining one or more nucleic acids in a sample (e.g., determining one or more nucleic acids that can cause activation of a target substrate).
  • the nucleic acids may be encapsulated into droplets.
  • the nucleic acids are encapsulated at relatively low concentrations, e.g., such that the droplets may, on the average contain less than 1 nucleic acid per droplet. This may be useful to ensure that most or all of the nucleic acids are transcribed, translated, or amplified, e.g., substantially evenly.
  • nucleic acids were to be transcribed, translated, or amplified in bulk solution, some nucleic acids could be transcribed, translated, or amplified without others being transcribed, translated, or amplified (or merely being transcribed, translated, or amplified to a much lesser degree).
  • nucleic acids are encapsulated into droplets, and manipulated therein.
  • a plurality of droplets may contain greater than or equal to 10 6 , greater than or equal to 10 7 , greater than or equal to 10 8 , greater than or equal to 10 9 , greater than or equal to IO 10 , greater than or equal to 10 11 , greater than or equal to 10 12 or more distinct nucleic acid sequences within the droplets.
  • a plurality of droplets contains less than or equal to 10 14 , less than or equal to 10 13 , less than or equal to 10 12 , less than or equal to 10 11 , less than or equal to IO 10 , less than or equal to 10 9 , or less distinct nucleic acid sequences within the droplets. Combinations of these ranges are possible.
  • a plurality of droplets contains greater than or equal to 10 6 and less than or equal to 10 14 distinct nucleic acid sequences within the droplets. Other ranges are also possible.
  • droplets of the first plurality of droplets contain greater than or equal to 5, greater than or equal to 10, greater than or equal to 10 2 , greater than or equal to 10 3 , greater than or equal to 10 4 , greater than or equal to 10 5 , greater than or equal to 10 6 , greater than or equal to 10 7 , or more distinct nucleic acid sequences.
  • droplets of the first plurality of droplets contain less than or equal to 10 8 , less than or equal to 10 7 , less than or equal to 10 6 , less than or equal to 10 5 , or less distinct nucleic acid sequences. Combinations of these ranges are possible.
  • droplets of the first plurality of droplets contain greater than or equal to 5 and less than or equal to 10 8 distinct nucleic acid sequences. Other ranges are also possible.
  • any appropriate fraction of the droplets of the first plurality may include a number of distinct nucleic acids, e.g., described in the preceding paragraph.
  • greater than or equal to 1%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 25%, greater than or equal to 50%, or more of the droplets of a plurality of droplets contains a number of distinct nucleic acids described above.
  • less than or equal to 100%, less than or equal to 99%, less than or equal to 95%, less than or equal to 75%, less than or equal to 50%, less than or equal to 25%, or less of the droplets of a plurality of droplets contains a number of distinct nucleic acids described above. Combinations of these ranges are possible.
  • greater than or equal to 1% and less than or equal to 100% of the droplets of a plurality of droplets may contain a number of distinct nucleic acids described above. Other ranges are also possible.
  • a sample containing nucleic acids may be contained within a plurality of droplets, e.g., contained within a suitable carrying fluid.
  • the nucleic acids may be present during formation of the droplets, and/or added to the droplets after formation. Any suitable method may be chosen to create droplets, and a wide variety of different droplet makers and techniques for forming droplets will be known to those of ordinary skill in the art. For example, a junction of channels may be used to create the droplets.
  • the junction may be, for instance, a T-junction, a Y-junction, a channel- within-a-channel junction (e.g., in a coaxial arrangement, or comprising an inner channel and an outer channel surrounding at least a portion of the inner channel), a cross (or “X”) junction, a flow-focusing junction, or any other suitable junction for creating droplets.
  • a T-junction e.g., a T-junction
  • Y-junction e.g., a channel- within-a-channel junction (e.g., in a coaxial arrangement, or comprising an inner channel and an outer channel surrounding at least a portion of the inner channel), a cross (or “X”) junction, a flow-focusing junction, or any other suitable junction for creating droplets.
  • nucleic acids may be added to droplet after the droplet has been formed, e.g., through picoinjection or other methods such as those discussed in Int. Pat. Apl. Pub. No. WO 2010/151776, entitled “Fluid Injection” (incorporated herein by reference), through fusion of the droplets with droplets containing the nucleic acids, or through other techniques known to those of ordinary skill in the art.
  • the nucleic acids may be natural nucleic acids, such as DNA or RNA.
  • a nucleic acid that activates the target substrate may be present at very low concentrations.
  • a nucleic acid that activates the target substrate may be present in a droplet containing other nucleic acids at a concentration of 1: 10 3 , 1: 10 4 , 1: 10 5 , 1: 10 6 , 1: 10 7 , 1: 10 8 , or even lower concentrations, versus the total number of nucleic acids in the droplet.
  • a nucleic acid that activates the target substrate may be totally non-existent in at least some droplets of the plurality.
  • a particular nucleic acid that activates the target substrate is present in multiple droplets of the plurality, such that multiple droplets contain activity of the target substrate resulting from action of a single nucleic acid common to the multiple droplets.
  • the nucleic acid may activate the target substrate directly (e.g., by direct action on the target substrate) or indirectly (e.g., by encoding a protein that, under the proper conditions, can be produced using the nucleic acid in order to activate the target substrate).
  • the target substrate may also be included in the droplet.
  • the target substrate may be present in the droplets initially, or may be introduced after their formation (e.g., using an aforementioned technique, such as picoinjection).
  • IVT methods produce (e.g., synthesize) an RNA transcript (e.g., mRNA transcript) by contacting a DNA template (e.g., an input DNA) with an RNA polymerase (e.g., a T7 RNA polymerase, a T7 RNA polymerase variant, etc.) under conditions that result in the production of the RNA transcript.
  • IVT conditions typically employ a DNA template containing a promoter, nucleoside triphosphates, a buffer system that includes dithiothreitol (DTT) and magnesium ions, and an RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application.
  • IVTT may be performed such that transcription and translation occur simultaneously for a given complex, or at least so that transcription and translation occur simultaneously within the same solution.
  • IVTT methods may produce (e.g., synthesize) a polypeptide (e.g., a protein) by contacting an mRNA produced in vitro with a ribosome under conditions that result in the production of the protein.
  • IVTT conditions typically employ a DNA template containing a promoter, a ribosome binding site, nucleoside triphosphates, a buffer system that includes dithiothreitol (DTT) and magnesium ions, an RNA polymerase, and a ribosome. The exact conditions used in the IVTT reaction may depend on the amount of protein needed for a specific application.
  • the first plurality of droplets comprises a nucleic acid sequence that encodes a mRNA or protein that activates the target substrate.
  • a method comprises translating proteins from one or more nucleic acids (e.g., mRNA molecules transcribed from nucleic acids of a library) present within the first plurality of droplets. As a corollary, according to some embodiments, proteins are translated prior to determining the one or more droplets of the first plurality of droplets that contain activity of the target substrate. Transcription and translation within a plurality of droplets may have certain advantages.
  • transcription and translation within a plurality of droplets may ensure that activity of a target substrate within a droplet is associated exclusively with the nucleic acids present within that droplet, and does not result from nucleic acids that are not present within that droplet.
  • the library may be used to transcribe any appropriate proteins or peptides, including enzymes or antigenic determinants.
  • FIG. 1A presents a schematic illustration of a nucleic acid 100 comprising a library nucleic acid 101.
  • the library nucleic acid 101 may be inserted into a vector for IVTT.
  • FIG. IB presents a schematic illustration of a nucleic acid 100 comprising library nucleic acid 101, promotor 103, and ribosome binding site 111.
  • Nucleic acid 101 may further comprise a terminator 105.
  • a leading and/or a trailing nucleic acid sequence may be used as a priming site for a primer.
  • a nucleic acid 100 may comprise priming sites 121 and 123 for primers.
  • primers may be added to a solution that recognize priming sites 121 and 123.
  • multiple priming sites may be used for successive iterations of amplification steps. For example, FIG.
  • nucleic acid 100 comprises a first pair of priming sites 121 and 123 for a first set of primers, used during a first amplification step; a second pair of priming sites 131 and 133 for a second set of primers, used during a second amplification step; and a third pair of priming sites 141 and 143 for a third set of primers, used during a third amplification step.
  • Nucleic acids contained within activity droplets containing activity of a target substrate may be amplified within the activity droplets. However, amplification may cause multiple nucleic acids to be amplified, which can make it difficult to identify the nucleic acids that activated the target substrate.
  • the amplified nucleic acids may be separated into another plurality of droplets.
  • the activity droplets may be broken and their contents pooled together, e.g., to create a pool of nucleic acids.
  • the nucleic acids may be amplified (e.g., in the plurality of activity droplets, or in the pool).
  • the pool of amplified nucleic acids may then be made into a new plurality of droplets for further analysis.
  • the nucleic acids of the first plurality may be made into a second plurality of droplets and amplified within the second plurality of droplets.
  • the amplified nucleic acids may be sequenced or determined (e.g., qualitatively or quantitatively) as discussed below.
  • FIG. 2A presents a non-limiting, schematic illustration of an example method described herein.
  • a droplet 201 comprises nucleic acids 205 and optional other reagents 207. Nucleic acids of the droplet are transcribed and translated in step 210, as described below, to produce proteins 211.
  • Target substrate 213 is included in droplet 201, and is inactive, as indicated in FIG. 2A by the fact that target substrate 213 is a white square.
  • target substrate 213 is activated (step 220) by at least some proteins translated from the library, as indicated by the color change of the target substrate to a black square.
  • Droplets 201 may then be sorted (step 230) into activity droplets 202 and non-activity droplets 203. Nucleic acids of the activity droplets can then be amplified and otherwise processed as described above.
  • FIG. 2B presents a non-limiting, schematic illustration of a portion of a method such as described herein.
  • a plurality of droplets (some of which are shown in dashed circle 241) are sorted into a plurality of activity droplets 202 and a plurality of non-activity droplets 203, also shown within dashed circles 241 in FIG. 2A.
  • Nucleic acids from activity droplets 202 can then be incorporated into a plurality of new droplets 204 (step 240).
  • the nucleic acids can be amplified in activity droplets 202, or can be amplified after separation into new droplets 204.
  • the new droplets 204 may contain fewer nucleic acids than the activity droplets 202 as a result of this separation.
  • step 250 may be performed, wherein new droplets 204 are treated (e.g., subjected to in vitro translation) to produce new activity droplets 202 and new nonactivity droplets 203.
  • a plurality of droplets containing amplified nucleic acids may be further refined by iterating one or more of the steps described above.
  • a starting plurality of droplets e.g., comprising amplified nucleic acids of activity droplets belonging to the first plurality of droplets
  • activity droplets comprising activity of the target substrate may be separated from the remaining droplets of the starting plurality of droplets.
  • Nucleic acids of the activity droplets may be separated into a new plurality of droplets.
  • the nucleic acids of the activity droplets may be amplified before or after separation into the new plurality of droplets. Referring again to FIG. 2B, in some embodiments, sorting and separating steps as shown are iterated to identify nucleic acids associated with activity droplets of successive pluralities of droplets.
  • the new plurality of droplets includes a smaller number of nucleic acids per droplet than the starting plurality of droplets.
  • an average number of nucleic acids in a droplet of a new plurality of droplets is less than or equal to 10 1 , less than or equal to 10’ 2 , less than or equal to 10’ 3 , less than or equal to 10’ 4 , or less than or equal to 10’ 5 or less times an average number of nucleic acids in a droplet of a starting plurality of droplets.
  • an average number of nucleic acids in a droplet of a new plurality of droplets is greater than or equal to 10’ 6 , greater than or equal to 10’ 5 , greater than or equal to IO -4 or more times an average number of nucleic acids in a droplet of a starting plurality of droplets. Combinations of these ranges are possible. For example, in some embodiments, an average number of nucleic acids in a droplet of a new plurality of droplets is greater than or equal to 10’ 6 and less than or equal to 10 1 times an average number of nucleic acids in a droplet of a starting plurality of droplets. Other ranges are also possible.
  • a method comprises repeating steps (a)-(c), where: step (a) is determining one or more droplets of a starting plurality of droplets that contain activity of a target substrate; step (b) is separating nucleic acids from the one or more droplets of the starting plurality of droplets into a new plurality of droplets; and step (c) is amplifying the new nucleic acids within the second plurality of droplets. Steps (a)-(c) may be repeated one or more times, depending on the desired attributes of an ultimate new plurality of droplets.
  • a method comprises repeating steps (a)-(c) until greater than or equal to 10%, 25%, 50%, 75%, 90%, 95%, 99%, or more of the droplets of the new plurality of droplets comprise less than or equal to 1 distinct nucleic acid sequence.
  • FIG. 3 A presents a non-limiting, schematic illustration of a method 301 of refining a nucleic acid library.
  • the method comprises a first step 305 of determining activity droplets of a starting plurality of droplets.
  • the method further comprises a step 307 of separating nucleic acids from the activity droplets to a new plurality of droplets, and a step 309 of amplifying nucleic acids from activity droplets.
  • step 309 occurs after step 307, this is not necessary.
  • FIG. 3B presents a non-limiting schematic illustration of a method 302 of refining a nucleic acid library.
  • Method 302 comprises steps 305, 307, and 309 as shown in FIG.
  • FIG. 3C presents a nonlimiting, schematic illustration of a method 303 of refining a nucleic acid library.
  • Method 303 is similar to method 302 of FIG. 3B.
  • method 303 further comprises step 310 of translating proteins from nucleic acids in the starting droplets (e.g., using in vitro translation). The translations step is not necessary to all embodiments, but may be useful for screening of libraries that can express active proteins.
  • FIG. 3D presents a non-limiting, schematic illustration of a method comprising iterative refinement of a nucleic acid library.
  • a first step 361 an original library is included in a plurality of droplets at a concentration of 5xl0 5 nucleic acids per droplet.
  • activity droplets are sorted from the first plurality and are used to form a second plurality of droplets including 1,500 nucleic acids per droplet.
  • activity droplets are sorted from the second plurality and are used to form a third plurality of droplets including 5 nucleic acids per droplet.
  • step 367 activity droplets are sorted from the third plurality and are used to form a fourth plurality of droplets including 1 nucleic acid per 10 droplets.
  • step 369 the droplets are sent to instrument 375 for sequencing.
  • a fluidic system may be used to perform some or all of the method steps described above.
  • emulsions are formed by flowing two, three, or more fluids through a system of channels of a fluidic system.
  • the fluidic system may be or comprise an article.
  • the system or article may be a microfluidic system or article.
  • Microfluidic refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension (measured perpendicular to the direction of fluid flow) of less than about 1 millimeter (mm), and in some cases, a ratio of length to largest cross-sectional dimension of at least 3: 1.
  • a “channel,” as used herein, means a feature on or in a system or article that at least partially directs flow of a fluid.
  • the channel can have any cross-sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered.
  • One or more of the channels may (but not necessarily), in cross section, have a height that is substantially the same as a width at the same point.
  • At least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlet(s) and/or outlet(s).
  • a channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2: 1, more typically at least 3: 1, 5: 1, 10: 1, 15: 1, 20: 1, or more.
  • An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid.
  • the fluid within the channel may partially or completely fill the channel.
  • the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus).
  • the channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm.
  • the dimensions of the channel may be chosen such that fluid is able to freely flow through the article or substrate.
  • the dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flowrate of fluid in the channel.
  • the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel or capillary may be used. For example, two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, positioned to intersect with each other, etc.
  • the fluidic droplets within the channels may have a cross-sectional dimension smaller than about 100% of an average cross-sectional dimension of the channel, and in certain embodiments, smaller than about 90%, smaller than about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 3%, about 1%, about 0.5%, about 0.3%, about 0.1%, about 0.05%, about 0.03%, or about 0.01% of the average cross-sectional dimension of the channel.
  • an article comprises at least some of a plurality of droplets described above.
  • the article may comprise all droplets of a plurality of droplets.
  • the droplets may be fluidic ally connected to one or more reservoirs of the fluidic system (e.g., to a pool used to form droplets, to a hydrophobic fluid used to form droplets, to a supply of a target substrate, to a supply of a detection agent, to a supply of in vitro transcription and translation reagents, or any of a variety of other fluids described herein) via the article.
  • the droplets may be connected to one or more reservoirs of a fluidic system via the microchannel.
  • the fluidic system comprises one or more additional components, such as a pressure source (for example, a pump), a detection tool (e.g., a sensor that may be used to detect fluorescence, luminescence, and/or colorimetric changes resulting from activity of a target substrate); and/or a waste stream.
  • a pressure source for example, a pump
  • a detection tool e.g., a sensor that may be used to detect fluorescence, luminescence, and/or colorimetric changes resulting from activity of a target substrate
  • a waste stream for example, a waste stream.
  • activity of a target substrate may be directly detectable (for example, when the activity causes a change in an optical property of a target substrate, such as fluorescence, luminescence, or a colorimetric change).
  • direct detection of target substrate activity is difficult or impossible. It may be advantageous, particularly when direct activity of a target substrate is difficult or impossible to detect, to include a detection agent for the purpose of detecting activity of the target substrate.
  • a detection agent is an indirect proxy for activity of a target substrate.
  • the detection agent may be an indicator that is configured to experience a signal change in the presence of target substrate activity.
  • the detection agent may be configured to produce a signal (e.g., an optical signal such as fluorescence, luminescence, or a colorimetric signal) when it encounters an activated target substrate.
  • a signal e.g., an optical signal such as fluorescence, luminescence, or a colorimetric signal
  • an activated target substrate inhibits a signal produced by a detection agent in the absence of the target substrate.
  • a detection agent may be configured to react with an activated target substrate via a reaction that consumes the detection agent, or that chemically modifies a detection agent to render it undetectable.
  • the nucleic acids within the droplets may be amplified. This may be useful, for example, to produce a larger number or concentration of nucleic acids, e.g., for subsequent analysis, sequencing, or the like.
  • PCR polymerase chain reaction
  • RT reverse transcriptase
  • IVT in vitro transcription amplification
  • MDA multiple displacement amplification
  • qPCR quantitative real-time PCR
  • the nucleic acids may be amplified within the droplets. Nucleic acid amplification within the droplets may allow amplification to occur “evenly” in some embodiments, e.g., such that the distribution of nucleic acids is not substantially changed after amplification, relative to before amplification.
  • the nucleic acids within a plurality of droplets may be amplified such that the number of nucleic acid molecules for each type of nucleic acid may have a distribution such that, after amplification, no more than about 5%, no more than about 2%, or no more than about 1% of the nucleic acids have a number less than about 90% (or less than about 95%, or less than about 99%) and/or greater than about 110% (or greater than about 105%, or greater than about 101%) of the overall average number of amplified nucleic acid molecules per droplet.
  • the nucleic acids within the droplets may be amplified such that each of the nucleic acids that are amplified can be detected in the amplified nucleic acids, and in some cases, such that the mass ratio of the nucleic acid to the overall nucleic acid population changes by less than about 50%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% after amplification, relative to the mass ratio before amplification.
  • certain primers are contained within the droplets to promote amplification. Such primers may be present during formation of the droplets, and/or added to the droplets after formation of the droplets. It should be noted that the manner in which the primers are added to the droplets may be the same or different from the manner in which the nucleic acids are added to the droplets.
  • a plurality of different types of primers may be added to the droplets. Different primers may be distinguishable due to their having different sequences, and/or may be able to amplify different potential targets. In some cases, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 5,000, or at least 10,000, etc., different primers may be used. This may allow, for example, a variety of different target nucleic acids to be amplified within different droplets.
  • Examples of techniques for forming droplets include those described above.
  • Examples of techniques for introducing primers after droplet formation include picoinjection or other methods such as those discussed in Int. Pat. Apl. Pub. No. WO 2010/151776, incorporated herein by reference, through fusion of the droplets with droplets containing primers, or the like. Other such techniques for either of these include, but are not limited to, any of those techniques described herein.
  • the primers may be present within the droplets at any suitable density.
  • the primers may have a density of greater than or equal to 0.1 micromolar, greater than or equal to 0.3 micromolar, greater than or equal to 0.5 micromolar, greater than or equal to 0.8 micromolar, greater than or equal to 1 micromolar, greater than or equal to 5 micromolar, or more.
  • the primers have a density of less than or equal to 100 micromolar, less than or equal to 50 micromolar, less than or equal to 20 micromolar, less than or equal to 10 micromolar, less than or equal to 5 micromolar, less than or equal to 1 micromolar, or less.
  • the density may be independent of the density of target nucleic acids.
  • an excess of primers are used, e.g., such that the target nucleic acids controls the reaction. For instance, if a large excess of primers are used, then substantially of the droplets will contain primer (regardless of whether or not the droplets also contain target nucleic acids).
  • At least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the droplets may contain at least one amplification primer.
  • Droplets containing both primer and a target nucleic acid may be treated to cause amplification of the nucleic acid to occur. This may allow a large amount or concentration of the target nucleic acids to be produced, e.g., without substantially altering the distribution of nucleic acids.
  • the primers are selected to allow substantially all, or only some, of the target nucleic acids suspected of being present to be amplified.
  • PCR polymerase chain reaction
  • other amplification techniques may be used to amplify nucleic acids, e.g., contained within droplets.
  • the nucleic acids are heated (e.g., to a temperature of at least about 50 °C, at least about 70 °C, or least about 90 °C in some cases) to cause dissociation of the nucleic acids into single strands, and a heat-stable DNA polymerase (such as Taq polymerase) is used to amplify the nucleic acid. This process is often repeated multiple times to amplify the nucleic acids.
  • a heat-stable DNA polymerase such as Taq polymerase
  • PCR amplification may be performed within the droplets.
  • the droplets may contain a polymerase (such as Taq polymerase), and DNA nucleotides (deoxyribonucleotides), and the droplets may be processed (e.g., via repeated heated and cooling) to amplify the nucleic acid within the droplets.
  • a polymerase such as Taq polymerase
  • DNA nucleotides deoxyribonucleotides
  • Suitable reagents for PCR or other amplification techniques such as polymerases and/or deoxyribonucleotides, may be added to the droplets during their formation, and/or afterwards (e.g., via merger with droplets containing such reagents, and/or via direct injection of such reagents, e.g., contained within a fluid).
  • primers may be added to the droplets, or the primers may be present on one or more of the nucleic acids within the droplets.
  • suitable primers many of which can be readily obtained commercially.
  • the primers may be distinguished, for example, using distinguishable fluorescent tags, barcodes, or other suitable identification tags.
  • distinguishable fluorescent tags include, but are not limited to, those described in U.S. Pat. Apl. Pub. No. 2018-0304222 or Int. Pat. Apl. Pub. No. WO 2015/164212, each incorporated herein by reference.
  • the nucleic acids may be amplified to any suitable extent.
  • the degree of amplification may be controlled, for example, by controlling factors such as the temperature, cycle time, or amount of enzyme and/or deoxyribonucleotides contained within the droplets.
  • a population of droplets may have at least about 50,000, at least about 100,000, at least about 150,000, at least about 200,000, at least about 250,000, at least about 300,000, at least about 400,000, at least about 500,000, at least about 750,000, at least about 1,000,000 or more molecules of the amplified nucleic acid per droplet.
  • the droplets are broken down after amplification, e.g., to allow the amplified nucleic acids to be pooled together.
  • a wide variety of methods for “breaking” or “bursting” droplets are available to those of ordinary skill in the art.
  • droplets contained in a carrying fluid may be disrupted using techniques such as mechanical disruption, chemical disruption, or ultrasound. Droplets may also be disrupted using chemical agents or surfactants, for example, 1H,1H,2H,2H- perfluorooctanol.
  • a method comprises purifying nucleic acids (e.g., nucleic acids pooled from a plurality of droplets). Purification may be used, for example, to extract the nucleic acids from unwanted reagents used in earlier steps. For example, purification may be used to extract the nucleic acids from proteins transcribed therefrom. Any of a variety of appropriate techniques may be used to purify the nucleic acids.
  • the nucleic acids may be purified using any of a variety of suitable methods, such as column- or gel-based methods (including electrophoretic and centrifuge-based methods). For example, nucleic acids may be purified using a PCR clean-up kit.
  • the nucleic acids may optionally be determined and/or sequenced, e.g., using techniques such as those described herein.
  • the droplets may be burst and the nucleic acids may be combined to facilitate determination and/or sequencing, although in some cases, the determination and/or sequencing may occur within the droplets.
  • the pool of amplified nucleic acids may be sequenced using droplet-based techniques, e.g., droplet-based PCR.
  • the amplified nucleic acids may be collected into droplets and the droplets exposed to certain primers.
  • the amplified nucleic acids may be collected into droplets at relatively low concentrations, e.g., such that the droplets may, on the average, contain less than 1 nucleic acid per droplet, as described herein.
  • the droplets may be divided into different groups of droplets, which are exposed to different primers. For instance, the droplets may be divided into at least 5, 10, 30, 100, etc.
  • the amplified nucleic acids may be present at relatively higher concentrations, e.g., at least 1 nucleic acid per droplet or at least 1 target per droplet. In some cases, more than one primer or one amplicon may be present within a droplet.
  • Examples of methods for determining and/or sequencing nucleic acids include, but are not limited to, chain-termination sequencing, sequencing-by-hybridization, Maxam-Gilbert sequencing, dye-terminator sequencing, chain-termination methods, Massively Parallel Signature Sequencing (Lynx Therapeutics), polony sequencing, pyrosequencing, sequencing by ligation, ion semiconductor sequencing, DNA nanoball sequencing, single-molecule real-time sequencing (e.g., Pacbio sequencing), nanopore sequencing, Sanger sequencing, digital RNA sequencing (“digital RNA-seq”), Illumina sequencing, etc.
  • a microarray such as a DNA microarray, may be used, for example, to determine, or to sequence, a nucleic acid.
  • the pool of amplified nucleic acids may be determined or identified, e.g., without any sequencing.
  • a droplet by applying (or removing) a first electric field (or a portion thereof), a droplet may be directed to a first region or channel; by applying (or removing) a second electric field to the device (or a portion thereof), the droplet may be directed to a second region or channel; by applying a third electric field to the device (or a portion thereof), the droplet may be directed to a third region or channel; etc., where the electric fields may differ in some way, for example, in intensity, direction, frequency, duration, etc.
  • certain embodiments comprise a droplet contained within a carrying fluid.
  • a first phase forming droplets contained within a second phase, where the surface between the phases comprises one or more proteins.
  • the second phase may comprise oil or a hydrophobic fluid, while the first phase may comprise water or another hydrophilic fluid (or vice versa).
  • a hydrophilic fluid is a fluid that is substantially miscible in water and does not show phase separation with water at equilibrium under ambient conditions (typically 25 °C and 1 atm).
  • hydrophilic fluids include, but are not limited to, water and other aqueous solutions comprising water, such as cell or biological media, ethanol, salt solutions, saline, blood, etc. In some cases, the fluid is biocompatible.
  • hydrophobic fluid is one that is substantially immiscible in water and will show phase separation with water at equilibrium under ambient conditions.
  • the hydrophobic fluid is sometimes referred to by those of ordinary skill in the art as the “oil phase” or simply as an oil.
  • oils such as hydrocarbons oils, silicon oils, fluorocarbon oils, organic solvents, perfluorinated oils, perfluorocarbons such as perfluoropolyether, etc.
  • hydrocarbons include, but are not limited to, light mineral oil (Sigma), kerosene (Fluka), hexadecane (Sigma), decane (Sigma), undecane (Sigma), dodecane (Sigma), octane (Sigma), cyclohexane (Sigma), hexane (Sigma), or the like.
  • Non-limiting examples of potentially suitable silicone oils include 2 cst polydimethylsiloxane oil (Sigma).
  • fluorocarbon oils include FC3283 (3M), FC40 (3M), Krytox GPL (Dupont), etc.
  • other hydrophobic entities may be contained within the hydrophobic fluid in some embodiments.
  • Non-limiting examples of other hydrophobic entities include drugs, immunologic adjuvants, or the like.
  • the hydrophobic fluid may be present as a separate phase from the hydrophilic fluid.
  • the hydrophobic fluid may be present as a separate layer, although in other embodiments, the hydrophobic fluid may be present as individual fluidic droplets contained within a continuous hydrophilic fluid, e.g. suspended or dispersed within the hydrophilic fluid. This is often referred to as an oil/water emulsion.
  • the droplets may be relatively monodisperse, or be present in a variety of different sizes, volumes, or average diameters. In some cases, the droplets may have an overall average diameter of less than about 1 mm, or other dimensions as discussed herein.
  • a surfactant may be used to stabilize the hydrophobic droplets within the hydrophilic liquid, for example, to prevent spontaneous coalescence of the droplets.
  • Non-limiting examples of surfactants include those discussed in U.S. Pat. Apl. Pub. No. 2010/0105112, incorporated herein by reference.
  • surfactants include Span80 (Sigma), Span80/Tween-20 (Sigma), Span80/Triton X-100 (Sigma), Abil EM90 (Degussa), Abil we09 (Degussa), polyglycerol polyricinoleate “PGPR90” (Danisco), Tween-85, 749 Fluid (Dow Coming), the ammonium carboxylate salt of Krytox 157 FSL (Dupont), the ammonium carboxylate salt of Krytox 157 FSM (Dupont), or the ammonium carboxylate salt of Krytox 157 FSH (Dupont).
  • the surfactant may be, for example, a peptide surfactant, bovine serum albumin (BSA), or human serum albumin.
  • the droplets may have any suitable shape and/or size.
  • the droplets may be microfluidic, and/or have an average diameter of less than about 1 mm.
  • the droplet may have an average diameter of less than about 1 mm, less than about 700 micrometers, less than about 500 micrometers, less than about 300 micrometers, less than about 100 micrometers, less than about 70 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 3 micrometers, less than about 1 micrometer, etc.
  • the average diameter may also be greater than about 1 micrometer, greater than about 3 micrometers, greater than about 5 micrometers, greater than about 7 micrometers, greater than about 10 micrometers, greater than about 30 micrometers, greater than about 50 micrometers, greater than about 70 micrometers, greater than about 100 micrometers, greater than about 300 micrometers, greater than about 500 micrometers, greater than about 700 micrometers, or greater than about 1 mm in some cases. Combinations of any of these are also possible; for example, the diameter of the droplet may be between about 1 mm and about 100 micrometers.
  • the diameter of a droplet, in a non- spherical droplet may be taken as the diameter of a perfect mathematical sphere having the same volume as the non- spherical droplet.
  • the droplets may be of substantially the same shape and/or size (i.e., “monodisperse”), or of different shapes and/or sizes, depending on the particular application.
  • the droplets may have a homogenous distribution of cross-sectional diameters, i.e., in some embodiments, the droplets may have a distribution of average diameters such that no more than about 20%, no more than about 10%, or no more than about 5% of the droplets may have an average diameter greater than about 120% or less than about 80%, greater than about 115% or less than about 85%, greater than about 110% or less than about 90%, greater than about 105% or less than about 95%, greater than about 103% or less than about 97%, or greater than about 101% or less than about 99% of the average diameter of the microfluidic droplets.
  • the coefficient of variation of the average diameter of the droplets may be less than or equal to about 20%, less than or equal to about 15%, less than or equal to about 10%, less than or equal to about 5%, less than or equal to about 3%, or less than or equal to about 1%.
  • the droplets may not necessarily be substantially monodisperse, and may instead exhibit a range of different diameters.
  • the droplets so formed can be spherical, or non-spherical in certain cases.
  • the diameter of a droplet, in a non-spherical droplet may be taken as the diameter of a perfect mathematical sphere having the same volume as the non-spherical droplet.
  • one or more droplets may be created within a channel by creating an electric charge on a fluid surrounded by a liquid, which may cause the fluid to separate into individual droplets within the liquid.
  • an electric field may be applied to the fluid to cause droplet formation to occur.
  • the fluid can be present as a series of individual charged and/or electrically inducible droplets within the liquid. Electric charge may be created in the fluid within the liquid using any suitable technique, for example, by placing the fluid within an electric field (which may be AC, DC, etc.), and/or causing a reaction to occur that causes the fluid to have an electric charge.
  • the electric field in some embodiments, is generated from an electric field generator, i.e., a device or system able to create an electric field that can be applied to the fluid.
  • the electric field generator may produce an AC field (i.e., one that varies periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that is constant with respect to time), a pulsed field, etc.
  • AC field i.e., one that varies periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.
  • a DC field i.e., one that is constant with respect to time
  • pulsed field etc.
  • an electric field is produced by applying voltage across a pair of electrodes, which may be positioned proximate a channel such that at least a portion of the electric field interacts with the channel.
  • the electrodes can be fashioned from any suitable electrode material or materials known to those of ordinary skill in the art, including, but not limited to, silver, gold, copper, carbon, platinum, copper, tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as well as combinations thereof.
  • droplets of fluid can be created from a fluid surrounded by a liquid within a channel by altering the channel dimensions in a manner that is able to induce the fluid to form individual droplets.
  • the channel may, for example, be a channel that expands relative to the direction of flow, e.g., such that the fluid does not adhere to the channel walls and forms individual droplets instead, or a channel that narrows relative to the direction of flow, e.g., such that the fluid is forced to coalesce into individual droplets.
  • the channel dimensions may be altered with respect to time (for example, mechanically or electromechanically, pneumatically, etc.) in such a manner as to cause the formation of individual droplets to occur.
  • the channel may be mechanically contracted (“squeezed”) to cause droplet formation, or a fluid stream may be mechanically disrupted to cause droplet formation, for example, through the use of moving baffles, rotating blades, or the like.
  • Some embodiments generally relate to systems and methods for fusing or coalescing two or more droplets into one droplet, e.g., where the two or more droplets ordinarily are unable to fuse or coalesce, for example, due to composition, surface tension, droplet size, the presence or absence of surfactants, etc.
  • the surface tension of the droplets, relative to the size of the droplets may also prevent fusion or coalescence of the droplets from occurring.
  • two droplets can be given opposite electric charges (i.e., positive and negative charges, not necessarily of the same magnitude), which can increase the electrical interaction of the two droplets such that fusion or coalescence of the droplets can occur due to their opposite electric charges.
  • opposite electric charges i.e., positive and negative charges, not necessarily of the same magnitude
  • an electric field may be applied to the droplets, the droplets may be passed through a capacitor, a chemical reaction may cause the droplets to become charged, etc.
  • the droplets in some cases, may not be able to fuse even if a surfactant is applied to lower the surface tension of the droplets.
  • the droplets are electrically charged with opposite charges (which can be, but are not necessarily of, the same magnitude), the droplets may be able to fuse or coalesce.
  • the droplets may not necessarily be given opposite electric charges (and, in some cases, may not be given any electric charge), and are fused through the use of dipoles induced in the droplets that causes the droplets to coalesce.
  • the two or more droplets allowed to coalesce are not necessarily required to meet “head-on.” Any angle of contact, so long as at least some fusion of the droplets initially occurs, is sufficient. See also, e.g., U.S. Patent Application Serial No. 11/698,298, filed January 24, 2007, entitled “Fluidic Droplet Coalescence,” by Ahn, et al., published as U.S. Patent Application Publication No. 2007/0195127 on August 23, 2007, incorporated herein by reference in its entirety.
  • a fluid may be injected into a droplet.
  • the fluid may be microinjected into the droplet in some cases, e.g., using a microneedle or other such device.
  • the fluid may be injected directly into a droplet using a fluidic channel as the droplet comes into contact with the fluidic channel.
  • Other techniques of fluid injection are disclosed in, e.g., International Patent Application No. PCT/US 2010/040006, filed June 25, 2010, entitled “Fluid Injection,” by Weitz, et al., published as WO 2010/151776 on December 29, 2010; or International Patent Application No.
  • WO 2005/021151 entitled “Electronic Control of Fluidic Species,” by Link et al:, Int. Pat. Apl. Pub. No. WO 2011/056546, entitled “Droplet Creation Techniques,” by Weitz, et al:, Int. Pat. Apl. Pub. No. WO 2010/033200, entitled “Creation of Libraries of Droplets and Related Species,” by Weitz, et al. U.S. Pat. Apl. Pub. No. 2012-0132288, entitled “Fluid Injection,” by Weitz, et al. Int. Pat. Apl. Pub. No.
  • WO 2008/109176 entitled “Assay And Other Reactions Involving Droplets,” by Agresti, et al.; and Int. Pat. Apl. Pub. No. WO 2010/151776, entitled “Fluid Injection,” by Weitz, et al.; and U.S. Pat. Apl. Ser. No. 62/072,944, entitled “Systems and Methods for Barcoding Nucleic Acids,” by Weitz, et al.
  • a library of nucleic acids as described herein may prepared by any of a variety of appropriate methods.
  • a library is prepared by error- prone PCR.
  • error-prone PCR is nonrandom; certain codon mutations are more likely than others, and some codon mutations are totally forbidden.
  • libraries may be generated by a more uniformly random process, without any forbidden mutations.
  • the libraries may then be screened using one or more of the systems or methods described above.
  • the systems and methods described herein may thereby surpass the performance of conventional methods of library refinement, such as refinement of libraries prepared by error-prone PCR, by screening more functionally robust and diverse nucleic acid libraries, with higher randomness, using fewer steps.
  • FIG. 4 provides a schematic illustration of a non-limiting common template nucleic acid, with nucleic acid 401 represented as a circular nucleic acid.
  • the white rectangles of nucleic acid 401 represent individual codons of nucleic acid 401 that are illustrated for emphasis — but the black curve is also part of nucleic acid 401 that is not shown in detail.
  • the common template nucleic acid may be selected by a user, and is not limiting. Any suitable template nucleic acid can be used.
  • a template nucleic acid may be chosen because it expresses a protein or other target of interest, of which a favorable improvement is desired.
  • the template nucleic acid may express an enzyme capable of performing a useful biological or chemical function.
  • the common template nucleic acid is a circular nucleic acid.
  • circular nucleic acids are used, which may be advantageous in some cases because only forward primers need be used.
  • the techniques provided herein are not so limited, and noncircular nucleic acids may be used in other embodiments.
  • certain primers are contained within a solution to promote amplification of a nucleic acid.
  • the method may comprise using a plurality of primers to synthesize a plurality of nucleic acids using the common template nucleic acid.
  • FIG. 4 shows plurality 405 of primers that are configured to synthesize a plurality of nucleic acids using nucleic acid 401.
  • a plurality of different types of primers may be added to the solution.
  • At least some primers of the plurality of primers may comprise a plurality of consecutive nucleotides that are complementary to a plurality of consecutive nucleotides of the common template nucleic acid.
  • a primer may be configured to bind to the common template nucleic acid, such that the primer can be used to synthesize a nucleic acid by an amplification technique (e.g., PCR), as described in greater detail below.
  • At least some primers comprising a plurality of consecutive nucleotides of the common template nucleic acid may include at least one codon that is not complementary to a codon of the template nucleic acid.
  • at least some primers comprising a plurality of consecutive nucleotides complementary to nucleotides of the common template nucleic acid include less than or equal to 2 codons that are not complementary to codons of the common template nucleic acid.
  • the primer may include exactly one codon that is not complementary to a codon of the template nucleic acid.
  • at least some primers comprising a plurality of consecutive nucleotides complementary to nucleotides of the common template nucleic acid include less than or equal to 4 codons (e.g., less than or equal to 3 codons, less than or equal to 2 codons) that are not complementary to codons of the common template nucleic acid.
  • the primer may include exactly one codon that is not complementary to a codon of the template nucleic acid.
  • plurality 405 of primers includes ten primers, each comprising 10 codons, represented as individual rectangles.
  • the nine white codons of each primer are complementary to portion 413 of nucleic acid 401; the colored codons represent a codon that is not complementary to codon 407 of nucleic acid 401.
  • a primer may be configured to bind to a common template nucleic acid, according to some embodiments.
  • each white codon of a primer of plurality of primers 405 includes 3 nucleotides that are complementary to 3 nucleotides of portion 413 of nucleic acid 401.
  • greater than or equal to 5, greater than or equal to 10, greater than or equal to 15, greater than or equal to 20, greater than or equal to 25, greater than or equal to 30, or more consecutive nucleotides of the common template nucleic acid are complimentary to complementary nucleotides of the common template primer.
  • less than or equal to 100, less than or equal to 75, less than or equal to 50, less than or equal to 40, less than or equal to 30, less than or equal to 25, or fewer consecutive nucleotides of the common template nucleic acid are complimentary to complementary nucleotides of the common template primer. Combinations of these ranges are possible. For example, in some embodiments, greater than or equal to 5 and less than or equal to 100 consecutive nucleotides of the common template nucleic acid are complimentary to complementary nucleotides of the common template primer. Other ranges are also possible.
  • the primers may be present within the solution at any suitable density.
  • the density may be independent of the density of nucleic acids. In some cases, an excess of primers is used, e.g., such that nucleic acids to be amplified by the primer control the reaction.
  • any of a variety of suitable numbers of primers may be used.
  • the plurality may include any appropriate number of primers.
  • at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, etc., different primers may be used. This may allow, for example, synthesis of a plurality of nucleic acids having one or more codons that differ from a common template nucleic acid. A variety of different nucleic acids may be amplified within different droplets.
  • the amplification may also be relatively selective, e.g., if library generation is centered on mutations of single, common template nucleic acid, by providing only certain primers.
  • one or only a relatively small number of primers e.g., less than or equal to 500, 400, 300, 200, 180, 160, 140, 120, 100, 80, 60, 40, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 primers
  • a relatively small number of primers e.g., less than or equal to 500, 400, 300, 200, 180, 160, 140, 120, 100, 80, 60, 40, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 primers
  • At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or at least 200 primers may be present. Combinations of the aforementioned ranges (e.g., at least 2 and less than or equal to 500) are also possible. In some embodiments, exactly one primer is used e.g., for the purpose of amplifying, rather than mutating, circular nucleic acids present in the solution.
  • primers that allow only certain mutations in a nucleic acid to be amplified may be used during amplification.
  • a plurality of primers may be used that have relatively small differences, e.g. such that the primers have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology, and/or such that the amplification primers are all substantially complementary to a common template primer, except for no more than 5, 4, 3, 2, or 1 nucleotide differences.
  • each primer of plurality 405 of primers includes exactly one non-complementary codon (including no more than 3, 2, or 1 nucleotide differences).
  • amplification using multiple pluralities of primers can be used to produce a plurality of nucleic acids.
  • the plurality of primers may represent variations on a common template primer, that is perfectly complementary to a portion of the nucleic acid.
  • FIG. 4 shows common template primer 403 that is perfectly complementary to portion 413 of nucleic acid 401, as illustrated by nucleotide bonds 420 between portion 413 and common template primer 403.
  • greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, or more primers of a plurality differ from a common template primer at greater than or equal to 1 and less than or equal to 2 codons (e.g., at exactly 2 codons). In some embodiments, less than or equal to 100%, less than or equal to 95%, or fewer primers of a plurality differ from a common template primer at greater than or equal to 1 and less than or equal to 2 codons (e.g., at exactly 2 codons). Combinations of these ranges are possible.
  • greater than or equal to 50% and less than or equal to 100% of primers of a plurality differ from a common template primer at greater than or equal to 1 and less than or equal to 2 codons (e.g., at exactly 2 codons). Other ranges are also possible.
  • greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, or more primers of a plurality differ from all other primers of the plurality at no more than two codons. In some embodiments, less than or equal to 100%, less than or equal to 95%, or fewer primers of a plurality differ from all other primers of the plurality at no more than two codons. Combinations of these ranges are possible. For example, in some embodiments, greater than or equal to 50% and less than or equal to 100% of primers of a plurality differ from all other primers of the plurality at no more than two codons. Other ranges are also possible.
  • Amplification using a plurality of primers including exactly one codon that is not complementary to the common template nucleic acid may thus produce a plurality of nucleic acids that differ from a common template nucleic acid. If the non- complementary codons of the primers occur in the same sequential position, amplification using the primers can produce a plurality of nucleic acids, each of which differs from the template nucleic acid at the non-complementary codon of the primer. Such a reaction may permit development of a library of nucleic acids via point-wise mutations, which may be particularly advantageous for library development, in some embodiments.
  • the common template nucleic acid may be amplified using 19 primers, each of which is non-complementary to the same codon, in order to produce a library of at least 19 nucleic acids (optionally including the common template nucleic acid, and additionally including 19 additional nucleic acids with a one- codon mutation) each of which would be identical, except for the changed codon.
  • a plurality of nucleic acids may comprise 19 primers for each codon of a common template primer (based on the 20 naturally-occurring amino acids) in certain embodiments as discussed herein.
  • a common template primer included 10 codons
  • the plurality could include up to 190 primers, reflecting every possible single-codon variation of the common template primer.
  • more or fewer than 19 primers for each codon may be used, for example, to include non-naturally-occurring amino acids or to omit certain amino acids, etc.
  • An advantageous feature of this approach is that a primer differing from the common template nucleic acid at exactly one codon can be amplified either using the common template nucleic acid or using a previously-synthesized nucleic acid. Even if (as is highly probable) a first primer of the plurality was amplified from a nucleic acid produced using second primer of the plurality, the mutation of the second primer would not be incorporated into the synthesized nucleic acid, since both primers have the same length and correspond to the same template primer.
  • the number of distinct nucleic acids included in the library may be controlled by the primers used, and generally cannot produce unexpected mutations.
  • Another advantage of this approach is that the library may be used in some embodiments to encode any or every natural amino acid at a given site, and there are no forbidden mutations.
  • mutations known to be detrimental may be deliberately excluded in certain embodiments, since the mutation process may be controlled, at least in part, by the primers used to amplify the nucleic acids.
  • primers within a first plurality may be configured to bind to a first portion of the common template nucleic acid (e.g., by corresponding to a first common template primer) and primers within a second plurality may be configured to bind to a second portion of the common template nucleic acid (e.g., by corresponding to a second common template primer, distinct from the first).
  • FIG. 5A presents an exemplary, schematic illustration of a non-limiting embodiment, where first plurality of primers 505 are complementary to the adjacent portion of nucleic acid 501, except at codons 507 indicated by black rectangles.
  • second plurality of primers 515 is complementary to nucleic acid 501 except at codons 517 indicated by black rectangles.
  • the pluralities 505 and 515 are represented as including only five primers, this this number was chosen arbitrarily and each primer may, in practice, include any of a variety of appropriate numbers of primers, as discussed above.
  • first portion of the common template nucleic acid and the second portion of the common template nucleic acid do not overlap.
  • first plurality of primers 505 does not complement the same portion of nucleic acid 501 as second plurality of primers 515.
  • the first portion of the common template nucleic acid and the second portion of the common template nucleic acid may at least partially overlap, as the disclosure is not so limited.
  • at least some (e.g., all) of the primers may be non-complementary to the common template nucleic acid at exactly one codon.
  • each primer of plurality of primers 505 is non-complementary to nucleic acid 501 only at codon 507 and plurality of primers 515 is non-complementary to nucleic acid 501 only at codon 517.
  • the first plurality of primers can be used to produce a mutation at a first sequential position of a common template nucleic acid and the second plurality of primers can be used to produce a mutation at a second sequential position of the common template nucleic acid, different from the first sequential position.
  • two pluralities of primers may be used to produce a library including every pairwise combination of the first mutation and the second mutation, producing a library including up to 20n x 20m distinct nucleic acid sequences, where “n” is the length of the primers of the first plurality of primers, and “m” is the length of the second plurality of primers. The lengths may be independent of each other.
  • a library of nucleic acids may be designed that differs from a common template nucleic acid at up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 150, up to 200, up to 250, up to 300, up to 400, up to 500, up to 1,000 or more codons, by using an appropriate number of primers differing at exactly one codon from the template nucleic acid.
  • FIG. 5A may represent the contents of a first pool, including common template nucleic acid 501, first plurality of primers 505, and second plurality of primers 515
  • FIG. 5B represents a second pool, including the common template nucleic acid 501, third plurality 525 of primers complementary to nucleic acid 501 except at one of codons 527, fourth plurality 535 of primers complementary to nucleic acid 501 except at one of codons 537, and fifth plurality 545 of primers complementary to nucleic acid 501 except at one of codons 547.
  • techniques such as the above example may produce a plurality of nucleic acids in a first pool, each differing from the common template nucleic acid at at least one codon, and a plurality of the nucleic acids in the first pool, each differing from the common template nucleic acid at at least one codon in a second pool.
  • Each of these pools would contain a plurality of nucleic acids differing from the common template nucleic acid by exactly one codon.
  • the use of different pools may be advantageous in some embodiments, since it may permit point mutations of nearby codons without using overlapping primers in the same pool.
  • nucleic acids of a plurality of nucleic acids prepared using a first plurality of common template primers may be mutated using a second plurality of primers also present in the solution.
  • FIG. 5C presents a nucleic acid 551, representative of a plurality of nucleic acids formed by amplification of nucleic acid 501 with first plurality of primers 505 as shown in FIG. 5A.
  • Codons 507, mutated by plurality of primers 505 shown in FIG. 5A are represented with a black circle representing any arbitrary mutation that could result from first plurality of primers 505 shown in FIG. 5A.
  • Nucleic acids 551 of the plurality of nucleic acids can then react with primers of the second plurality of primers 515, where each of the primers of second plurality of primers 515 differs from a second common template primer at exactly one codon.
  • Second plurality of primers 515 can introduce a second mutation corresponding to the second portion of the common template nucleic acid, producing nucleic acids differing from the common template nucleic acid by exactly two codons.
  • a plurality of primers includes primers that differ from the common template primer at two codons, depending on the embodiments.
  • the use of primers differing at more than two codons may be used, in some embodiments, to increase the mutation rate.
  • a first plurality of nucleic acids and a second plurality of nucleic acids may be used to synthesize a third plurality of nucleic acids, differing from the common template primer at more than one codon.
  • the first plurality of nucleic acids and the second plurality of nucleic acids may be mixed and shuffled using any of a variety of methods known to those of ordinary skill in the art.
  • the mixing and amplifying may be used to produce at third plurality of nucleic acids, wherein the third plurality of nucleic acids is identical to the common template nucleic acid except where the first plurality of nucleic acids or the second plurality of nucleic acids differs from the common template nucleic acid.
  • the third plurality of nucleic acids may be produced using any of a variety of suitable techniques, such as nucleic acid shuffling (e.g., DNA shuffling), overlap amplification (e.g., overlap PCR), or staggered extension (e.g., a process comprising or consisting of priming a nucleic acid of the plurality, followed by repeated cycles of denaturation and very short annealing and polymerase-catalyzed extension, wherein denaturation allows the partially-extended chains to melt away from their initial nucleic acids and bind to other nucleic acids, prior to a subsequent extension step).
  • nucleic acid shuffling e.g., DNA shuffling
  • overlap amplification e.g., overlap PCR
  • staggered extension e.g., a process comprising or consisting of priming a nucleic acid of the plurality, followed by repeated cycles of denaturation and very short annealing and polymerase-catalyzed extension, wherein den
  • the third plurality of nucleic acids is the library of nucleic acids. It should, of course, be understood that one or more additional steps of using an additional plurality of primers to synthesize an additional plurality of nucleic acids and/or of performing additional techniques such as DNA shuffling, overlap amplification, or staggered extension may also be performed, and that the steps of synthesis may be performed in any of a variety of appropriate orders, depending on the embodiment. The use of additional steps may contribute to the formation of even larger libraries.
  • FIGS. 5D-5E present a non-limiting example of formation of a third plurality of nucleic acids.
  • a first plurality of nucleic acids 521 which would be formed by the pool of FIG. 5 A
  • a second plurality of nucleic acids 531 which would be formed by the pool of FIG. 5E
  • Only one nucleic acid is shown for each plurality, but it represents a generic structure of nucleic acids of each plurality of nucleic acids. Codons 507, 517, 527, 537, and 547, mutated by pluralities 505, 515, 525, 535, and 545 of primers shown in FIGS.
  • FIGS. 5A-5B are represented with black circles, each representing any arbitrary mutation that could result from the associated pluralities of primers shown in FIGS. 5A-5B.
  • FIG. 5E represents the plurality nucleic acids 541 that would result from mixing and amplifying the pluralities of nucleic acids 521 and 531 shown in FIG. 5D.
  • Nucleic acids 541 are identical to the common template nucleic acid, except where the nucleic acids 521 or nucleic acids 531 differ from the common template nucleic acid 501 shown in FIGS. 5A-5B.
  • Libraries e.g., third pluralities of nucleic acids
  • a library of nucleic acid sequences produced by a method provided herein comprises greater than or equal to 10 5 , greater than or equal to 10 6 , greater than or equal to 10 7 , greater than or equal to 10 8 , greater than or equal to 10 9 , greater than or equal to IO 10 , greater than or equal to 10 11 , greater than or equal to 10 12 , greater than or equal to 10 13 or more sequences.
  • a library of nucleic acid sequences produced by a method provided herein comprises less than or equal to 10 14 , less than or equal to 10 13 , less than or equal to 10 12 , less than or equal to 10 11 , less than or equal to IO 10 , less than or equal to 10 9 , less than or equal to 10 8 , or less sequences. Combinations of these ranges are also possible.
  • a library of nucleic acids comprises greater than or equal to 10 5 and less than or equal to 10 14 sequences.
  • the methods provided herein may provide any of a variety of suitable rates of mutations per round. Notably, high mutation rates per round may be advantageous for preparing libraries as described herein, since high mutation rates may allow the preparation of larger nucleotide libraries with fewer rounds of mutation.
  • a method provided herein includes a step of synthesizing a plurality of nucleic acids with a maximum mutation rate less than or equal to 32 mutations per kb per round, less than or equal to 30 mutations per kb per round, less than or equal to 25 mutations per kb per round, less than or equal to 22 mutations per kb per round, less than or equal to 20 mutations per kb per round, less than or equal to 15 mutations per kb per round, less than or equal to 12 mutations per kb per round, less than or equal to 10 mutations per kb per round, less than or equal to 5 mutations per kb per round, less than or equal to 2 mutations per kb per round, or less.
  • a method provided herein includes a step of synthesizing a plurality of nucleic acids with a maximum mutation rate of greater than or equal to 1 mutations per kb per round, greater than or equal to 2 mutations per kb per round, greater than or equal to 5 mutations per kb per round, greater than or equal to 10 mutations per kb per round, greater than or equal to 12 mutations per kb per round, greater than or equal to 15 mutations per kb per round, greater than or equal to 20 mutations per kb per round, greater than or equal to 22 mutations per kb per round, greater than or equal to 25 mutations per kb per round, greater than or equal to 30 mutations per kb per round, or greater.
  • a method provided herein includes a step of synthesizing a plurality of nucleic acids with a maximum mutation rate of greater than or equal to 1 mutations per kb per round and less than or equal to 32 mutations per kb per round.
  • mutagenesis performed using a plurality of primers provided herein may have a very low or negligible likelihood of causing a frame-shift during mutagenesis. This may result in the preparation of libraries with relatively few early stop-codons, even when libraries are prepared using a high mutation rate as discussed above.
  • Libraries (e.g., third pluralities of nucleic acids) provided herein include a very small proportion of sequences with early stop codons. In some embodiments, less than or equal to 10%, less than or equal to 5%, less than or equal to 2.5%, less than or equal to 1% or less of the sequences of the library comprise an early stop codon.
  • a library of nucleic acids produced according to a method provided herein includes early stop codons in greater than or equal to 0.1% and less than or equal to 10 wt%. of its sequences.
  • sequential amplification steps may be used to produce a library.
  • a first plurality of nucleic acids may be synthesized, e.g., as described above.
  • at least some nucleic acids of the first plurality of nucleic acids may be amplified using a second plurality of primers to synthesize a second plurality of nucleic acids, e.g., as described above.
  • certain systems and methods comprise determining a subset of the first plurality of nucleic acids that is associated with favorable mutations of the common template nucleic acid.
  • a favorable subset may be identified by any of a variety of appropriate methods.
  • the subset may be identified by selecting for nucleic acids that bond to a target substrate.
  • each of the nucleic acids are used to express a protein, and the activity of that protein is measured by an appropriate method (e.g., by detecting activity of a probe, such as a change in fluorescence of a fluorescent probe, or a color change of a colorometric indicator).
  • the protein may be mixed with an enzyme substrate, and enzymatic activity of the protein may be detected, as described in the examples below.
  • the protein may be expressed by any of a variety of appropriate methods.
  • the protein may be expressed by performing a translation reaction to synthesize it, or by expressing the protein in a plasmid by inserting the nucleic acid into the plasmid. Other techniques are also possible, and the disclosure is not so limited.
  • any of a variety of appropriate numbers of nucleic acids of the first plurality of nucleic acids may be used to synthesize the second plurality of nucleic acids.
  • greater than or equal to 1, greater than or equal to 2, greater than or equal to 5, greater than or equal to 10, greater than or equal to 20, greater than or equal to 50, or more nucleic acids of the first plurality may be used to synthesize the second plurality of nucleic acids.
  • the less than or equal to 100%, less than or equal to 99%, less than or equal to 95%, less than or equal to 90%, less than or equal to 75%, less than or equal to 50%, or less of the nucleic acids of the first plurality may be used to synthesize the second plurality of nucleic acids. Combinations of the forgoing ranges are possible.
  • at least one nucleic acid of the first plurality and less than or equal to 100% of the nucleic acids of the first plurality of nucleic acids may be used to synthesize the second plurality of nucleic acids.
  • Other ranges are also possible.
  • the nucleic acids of the first plurality used to synthesize the second plurality of nucleic acids may be collected by any of a variety of suitable methods.
  • the first plurality of nucleic acids may be broken into droplets, as discussed in greater detail below.
  • One advantage of sequential mutation is that the determination of the favorable subset may be performed between amplification steps, and a subset of a first plurality of nucleic acids that is associated with favorable mutations of the common template nucleic acid may be selected for amplification.
  • a most favorable nucleic acid from the first plurality can be used to synthesize the second plurality of nucleic acids using the second plurality of primers.
  • the most favorable nucleic acid can be treated as a second common template nucleic acid, and primers may be designed to be complementary to the mutated codon, while introducing new mutations at the site of a different codon.
  • nucleic acids of the first plurality of nucleic acids are used to form the second plurality of nucleic acids.
  • the nucleic acids of the first plurality may be included in a single mixture, and may be amplified together within that mixture using the second plurality of primers.
  • the first plurality of nucleic acids may be partitioned, e.g., by breaking a mixture comprising the nucleic acids into a plurality of droplets and amplifying nucleic acids therein, as discussed below.
  • the second plurality of primers may be introduced into at least some of the plurality of droplets, in order to synthesize the second plurality of nucleic acids within the droplets.
  • Synthesis of the nucleic acids described herein may comprise an entirely sequential process of mutations, where one codon is mutated at a time.
  • the nucleic acids, one or more step may be used to introduce multiple mutations during the same reaction, as discussed above. Combinations of these steps may also be used.
  • a method may comprise a first step, where 5 pluralities of primers are used to prepare a first plurality of nucleic acids, each differing by up to 5 codons from a common template nucleic acid.
  • This method may further comprise using a sixth plurality of primers to introduce a mutation in exactly one additional codon of at least some nucleic acids of the first plurality.
  • Intermediate steps such as identifying a favorable subset of the first plurality of nucleic acids, may be used in concert with such a method.
  • Such a method may be useful for identifying useful mutations of proteins that are already favorably mutated relative to a wild type protein, permitting a directed process of library evolution.
  • a protein provided herein is a mutant of the Lipase A (“LipA”) enzyme.
  • a protein provided herein comprises a sequence that is at least 70%, at least 80%, at least 90%, or at least 95% identical to one of Seq. ID. Nos. 2-24 and is Seq. ID. No. 25.
  • a protein provided herein comprises one of Seq. ID. Nos. 2-24].
  • a protein provided herein may be one of Seq. ID. Nos. 2-24.
  • a nucleic acid provided herein is a nucleic acid that encodes a mutant of the LipA enzyme.
  • a nucleic acid provided herein comprises a sequence that is at least 70%, at least 80%, at least 90%, or at least 95% identical to one of Seq. ID. Nos. 26-48 and is not Seq. ID. No. 49.
  • a nucleic provided herein comprises one of Seq. ID. Nos. 26-48.
  • a protein provided herein may be one of Seq. ID. Nos. 26-48.
  • This example demonstrates that detection of an active nucleic acid in a droplet comprising a library of 100,000 distinct nucleic acid sequences is viable using a nonlimiting example of a method described herein.
  • the active nucleic acid sequence is able to express an active protein (wild-type Lipase A) that is capable of activating a target substrate (resorufin acetate) and was deliberately added to the library of at least 100,000 inactive nucleic acid sequences, to determine whether it could be identified.
  • a plurality of droplets was prepared, of which one in every ten droplets included the wild-type Lipase A gene (the “WT-gene”).
  • a positive control was also performed, wherein a control plurality of droplets was prepared that included only the WT-gene. In the control plurality of droplets, only one in every ten droplets included the WT-gene.
  • a gene library was prepared using a construct of the target nucleic acids and a pET28a+ vector. T7 specific primers were used for RNA transcription. A plurality of droplets was prepared using the gene library. The plurality of droplets had an average diameter of 30 microns, and included approximately 500,000 sequences.
  • IVTT in vitro transcription and translation
  • Droplet-based microfluidics was used to sort the droplets based on fluorescent activity resulting from the reaction of resorufin acetate with wild-type Lipase A. Droplets that demonstrated fluorescent activity were separated from the other droplets. 3. Nucleic acids present in the droplets demonstrating fluorescent activity were merged, and the nucleic acids therein were amplified using PCR and purified with a PCR clean-up kit.
  • the amplified nucleic acids were separated into a second plurality of droplets having a 15 micrometer diameter and including only 1,500 nucleic acid sequences.
  • the amplified nucleic acids were separated into a third plurality of droplets having a 15 micrometer diameter and including only 5 nucleic acid sequences per droplet.
  • the amplified nucleic acids were separated into a fourth plurality of droplets having a 15 micrometer diameter and including only 1 nucleic acid sequence per ten droplets (such that nine out of every ten droplets included no nucleic acids).
  • This prophetic example demonstrates screening a nucleic acid library for improved thermal stability of a synthesized protein.
  • a first plurality of droplets is prepared that includes a library of nucleic acids that expresses a variety of proteins.
  • nucleic acids in the droplets are transcribed and translated using IVTT.
  • the droplets are thermally shocked (e.g., by holding the droplets at 70 °C for 30 minutes).
  • an active substrate is pico-injected into the droplets, which are subsequently sorted for activity of the active substrate using microfluidics.
  • Steps of breaking the droplets, amplifying the nucleic acids therein, and sorting the droplets based on activity of the active substrate are iterated to identify one or more acids from the library capable of producing proteins that resist the thermal shock.
  • This prophetic example demonstrates the viability of the claimed methods for identifying favorably stable proteins based on droplet-based library screening.
  • nucleic acid library could be screened to identify variants that activate multiple target substrates.
  • a plurality of droplets was prepared as in Example 1; however, in addition to resorufin acetate (RA), pyranine (HTPS) was used as a target substrate. Wild-type Lipase A did not activate HTPS.
  • a library of 10 12 nucleic acids (“the LipA library”, included a plurality of about 2xl0 6 droplets, each including 500,000 nucleic acids) mutated from the WT-gene was prepared and screened for RA and HTPS activity. HTPS was a green fluorophore and RA was a red fluorophore, so activity of each fluorophore could be detected simultaneously.
  • FIGS. 6A-6C present confocal microscope images of the plurality of droplets by transmission (FIG. 6A) detection of red fluorescence (570-580 nm; FIG. 6B) and green fluorescence (450-510 nm; FIG. 6C).
  • FIG. 6A red fluorescence
  • FIG. 6B red fluorescence
  • FIG. 6C green fluorescence
  • Bottom figures are included in greyscale, but the relative brightness of various pixels indicates the relative fluorescent intensity within the specified range of wavelengths.
  • Droplets with high activity of both RA and HTPS, indicated by high fluorescent intensity in the red and green wavelengths, may be identified and sorted. This example demonstrates that a method such as that of Example 1 could be used to identify multifunctional proteins within a target library.
  • nucleic acid library may be screened to identify variants that cleave a target peptide.
  • the LipA library described in Example 3 was used along with the target substrate 5-FAM-PQPQLPYPQK-qxl (SEQ ID NO: 1), where 5- FAM was a fluorophore and qxl was a 5-FAM quencher.
  • the peptide itself had no fluorescent activity, but once cleaved, the 5-FAM was able to fluoresce, producing a fluorescent signal.
  • the following method was used.
  • the LipA library was prepared using a construct of the library nucleic acids and a pET28a+ vector. T7 specific primers were used for RNA transcription. A plurality of droplets was prepared using the LipA library. For the initial plurality of droplets, the following steps were performed:
  • IVTT in vitro transcription and translation
  • Droplet-based microfluidics was used to sort the droplets based on fluorescent activity resulting from the reaction of 5-FAM- PQPQLPYPQK-qxl (SEQ ID NO: 1) with translated proteins. Droplets that demonstrated fluorescent activity were separated from the other droplets.
  • the nucleic acids were separated into a second plurality of droplets having a 15 micrometer diameter and including only 1,500 nucleic acid sequences.
  • the nucleic acids were separated into a third plurality of droplets having a 15 micrometer diameter and including only 5 nucleic acid sequences per droplet.
  • partial sequencing of the nucleic acids in the third plurality of droplets was used to identify a plurality of variants that were enriched in the third plurality of droplets. At least three mutations of wild-type Lipase A were shown to be enriched in the sequenced populations.
  • the first variant (present in 5.53% of the nucleic acids in the third plurality of droplets) had the mutations K35R, V39A, and V96I.
  • the second variant (present in 2.61% of the nucleic acids in the third plurality of droplets) had the mutation K35R, but did not include any other identified mutations.
  • the third variant (present in 1.98% of the nucleic acids in the third plurality of droplets) included the mutations M8K, S16N, A81E, and N98S. These results demonstrate that library refinement may be used identify variants of interest in a library of nucleic acids, illustrating the utility of the methods described herein.
  • a DNA library is prepared that can express a library of peptides consisting of up to 10 amino acids.
  • the library comprises DNA sequences that include, in order: 4-5 primer binding sites for nested PCR, a T7 promotor, a ribosome binding site, a START codon, a peptide library sequence, a STOP codon, a T7 terminator, and 4-5 primer binding sites.
  • nucleic acids from the DNA library (which includes 10 12 nucleic acids) are encapsulated into each droplet of a first plurality of 10 6 droplets that further comprise an in vitro transcription and translation mixture and a reporter.
  • In vitro transcription and translation is performed for 4 h at 37 °C, and droplets are sorted based on a fluorescence increase from the reporter cell. Sorted droplets with high fluorescence are merged and nucleic acids therein are amplified using the primer binding sites, and purified using a PCR clean-up kit. Then, the nucleic acids present in the merged droplets of the first plurality are separated into a second plurality of droplets, including 1,000 droplets with around 10 4 nucleic acids per droplet.
  • IVTT is performed again and the droplets of the second plurality of droplets are sorted based on fluorescent activity, and the high fluorescence droplets are merged, and amplified.
  • the nucleic acids present in the merged droplets of the second plurality are separated into a third plurality of 1000 droplets, including about 10 nucleic acids per droplet.
  • the droplets of the third plurality of droplets are sorted based on fluorescent activity, and the high fluorescence droplets are merged, and amplified.
  • the amplified nucleic acids are then separated into a fourth plurality of droplets, including, on average, one nucleic acid per droplet.
  • the nucleic acids of the fourth plurality of droplets are then sequenced to identify active nucleic acids.
  • EXAMPLE 6 This example describes the preparation and screening of the LipA library initially described in Example 1.
  • Wild-type LipA (“WT-LipA”), originating from B. Subtilis, was chosen as a common template nucleic acid for preparation of the LipA library.
  • Each plurality of primers included 10x19 primers.
  • Each primer of a plurality was non-complementary to exactly one codon of the corresponding portion of the common template nucleic acid, and could thus be used to synthesize a nucleic acid differing from the common template nucleic acid at exactly one codon.
  • each plurality of primers could be used to synthesize a corresponding plurality of up to 190 nucleic acids — the number of nucleic acids necessary to encode every esterase mutant including a single amino acid mutation in the portion of the esterase encoded by the portion of the common template nucleic acid corresponding to the primers.
  • Primers were ordered through Integrated DNA Technology (IDT). Mutations of the first and last 5 codons were excluded. Pluralities of primers were designed to fully overlap in mutated regions, and 9, 6, or 3 nucleotides complementary to the wild-type sequence were added on the 5’ end when mutating the first, second, or third amino acid in that region, respectively, to ensure proper binding of every primer.
  • IDTT Integrated DNA Technology
  • pool 1 contained pluralities 1, 3, 5, 7, 9, 11, 13, 15, and 17, and pool 2 contained pluralities 2, 4, 6, 8, 10, 12, 14, 16, and 18.
  • Mutations were introduced into a wild-type sequence using Quick Change Lighting Multi Site Directed Mutagenesis kit (Agilent, CA, US) for each pool.
  • 10 pL of each pool was transformed into a 100 pL aliquot of E. coli Turbo cells (NEB, MA, US). Transformation was done according to the instruction manual and cultured overnight in 10 mL of LB-media supplemented with 50 pg/mL kanamycin at 37 °C. Only 50
  • Plasmids extracted from liquid culture were used for preparation of the LipA library. After the site-specific mutagenesis reactions and plasmid extractions, an additional layer of variability was introduced by executing a round of DNA shuffling.
  • the pluralities of nucleic acids were shuffled using the staggered extension process (“StEP”) by mixing 2.5 pL of the pluralities of LipA mutants (1 ng/pL), 1.5 pL of 10 pM T7 forward primer, 1.5 pL of 10 pM T7 reverse primer, 50 pL of 2 x Taq polymerase master mix (NEB, MA, US) and 44.5 pL of nuclease-free water.
  • StEP staggered extension process
  • PCR was done under the following conditions: 94 °C for 30 seconds, 55 °C for 5 seconds - 99 x.
  • PCR products were purified using PCR Clean up Kit (NEB, MA, US) and were for the second reaction: 5 pL of PCR product (5 ng/pL), 15 pL of 10 pM T7 forward primer, 15 pL of 10 pM T7 reverse primer, 500 pL of 2x Taq polymerase master mix, and 465 pL of nuclease-free water.
  • the mixture was split into 20 PCR tubes and PCR was done under the same conditions.
  • PCR reactions were purified separately and used as template DNA for 20 new reactions: 2.5 pL of template DNA (1 ng/pL), 1.5 pL of 10 pM T7 forward primer, 1.5 pL of 10 pM T7 reverse primer, 50 pL of 2 x Taq polymerase master mix (NEB, MA, US), and 44.5 pL of nuclease-free water. After PCR, all samples were mixed and purified using the same kit to produce the LipA library (which totaled around 10 12 nucleic acids).
  • FIG. 7A shows the design of the devices used to encapsulate and process the LipA library genes and the in vitro protein synthesis reagents. Labels 1 to 3 connote oil inlet 1, aqueous fluid inlet 2, and collection outlet 3.
  • FIG. 7B shows the design of the device used to pico-inject substrate into each water-in-oil droplet. Labels 4-7 connote spacing oil inlet 4, droplet reinjection inlet 5, substrate inlet 6, and collection outlet 7.
  • FIG. 7A shows the design of the devices used to encapsulate and process the LipA library genes and the in vitro protein synthesis reagents. Labels 1 to 3 connote oil inlet 1, aqueous fluid inlet 2, and collection outlet 3.
  • FIG. 7B shows the design of the device used to pico-inject substrate into each water-in-oil droplet. Labels 4-7 connote spacing oil inlet 4, droplet reinjection inlet 5, substrate inlet 6, and collection outlet 7.
  • FIG. 7A shows the design of the devices used to encapsulate and process
  • FIG. 7C shows the design of the device used to screen the droplets based on fluorescent intensities.
  • Labels 8-11 connote spacing oil inlet 8, droplet reinjection inlet 9, sorted droplet collection outlet 10, and waste outlet 11.
  • the devices (droplet generator, pico-injector, and droplet sorter) of FIGs. 7A-7C) were designed utilizing AutoCAD and were produced as photomasks (CAD/ Art Services, Inc.). The devices were fabricated through the well-established techniques of soft lithography, employing SU8-on-Silicon- wafer masters and PDMS-on-glass devices.
  • PDMS Polydimethylsiloxane
  • Sylgard 184 Polydimethylsiloxane
  • each PDMS device was cautiously peeled from the master and sealed to a pristine glass slide (Coming, 2947).
  • the electrodes were integrated into the design as channels within the microfluidic devices. These channels were filled with a low melting point metal alloy (Indalloy 19, 0.020 in. diameter) while the devices were heated on a hot plate. Terminal blocks were added to the punched holes in the devices to facilitate electrical connections during droplet processing.
  • Aquapel fluoroalkylsilanes
  • pressurized air was used to impel it through the microfluidic channel walls to render hydrophobic internal surfaces of the channels.
  • Excess Aquapel was expelled using compressed air, and the device was baked at 65 °C overnight.
  • FIG. 8A provides a non-limiting, schematic illustration of cell-free protein synthesis in drop.
  • FIG. 8B provides a non-limiting, schematic illustration of pico-injection of substrate, followed by fluorescence-based droplet sorting.
  • FIG. 8C shows schematics of co-encapsulation of one bacteria cell host expressing enzyme mutants, fluorescent substrate, and lysis buffer, followed by fluorescence-based droplet sorting.
  • the concentration of mutant genes per unit volume was precisely quantified, and the concentration of the previously established linear DNA gene library was diluted accordingly.
  • a known quantity of linear genes was then encapsulated, together with in vitro Protein Synthesis reagents (PURExpress, New England Biolabs), within water-in- oil microdroplets using the device shown in FIG. 7A.
  • the linear genes were combined with the in vitro protein synthesis reagents and loaded into a 1 mL syringe (Norm-Ject® Sterile Luer-Slip Syringes). This mixture was delivered to the droplet generator's aqueous inlet channel via a syringe needle attached to polyethylene tubing (BB31695- PE/2, Scientific Commodities, Inc.).
  • a surfactant solution containing 2% (w/v) fluorinated surfactant (RAN Biotechnologies, 008-FluoroSurfactant) in HFE 7500 (3M) was loaded into a 3 mL syringe (BD Luer-Lok 3-mL syringe) and introduced to the oil inlet channel of the droplet generator using a similar setup.
  • Harvard Apparatus Pumps were employed to exert controlled positive pressure on the syringes, yielding flow rates of 60 pL/h for the aqueous mixture and 300 pL/h for the oil-surfactant solution, resulting in the generation of monodisperse water-in-oil emulsions.
  • the emulsions were collected in an Eppendorf tube, over which 100 pL of mineral oil (MI499, Spectrum Chemical MFG Corp.) was carefully layered to prevent evaporation.
  • the tube was incubated in a thermostatic heat block at 37 °C for 4 hours to facilitate in-drop protein synthesis. Subsequently, the samples were subjected to heat shock at 70 °C for one hour to inactivate any non-thermostable variants.
  • the pressure was meticulously calibrated within the injector channel to minimize any dripping.
  • the droplets were close-packed and periodically introduced to the main flow channel by modulating the flow rates of the oil and droplets. Upon stabilization, a voltage was applied across electrodes, effecting the pico-injection of the substrate solution into each droplet at an approximate rate of 5 kHz.
  • the droplets were then collected into an Eppendorf tube placed in a thermostatic heat block setting at 65 °C for the reaction with resorufin acetate to complete.
  • a layer of 100 pF of mineral oil MI499, Spectrum Chemical MFG Corp.
  • the full LipA library of approximately 10 12 LipA mutants was screened by screening 2,103,839 droplets, each comprising an average of 500,000 mutants, of which 205 droplets were determined to be activity droplets and were sorted for subsequent processing, as indicated schematically in FIG. 9.
  • a 532 nm excitation laser was placed on the entrance of the sorter. If the fluorescent reaction product in one droplet was higher than the set fluorescence threshold, it was pulled by the electric field into an adjacent channel, which was then collected through polyethylene micro-tubing to an Eppendorf tube placed on ice.
  • the chip operated at 600-1000 drops- s’ 1 , probing -3- 10 6 cells-h’ 1 .
  • the emulsion was broken to forma second plurality of droplets by adding 200 pL of 20% (v/v) PFO (lH,lH,2H,2H-Perfhioro-l-octanol, 370533 Sigma Aldrich) in HFE 7500 (3M), and the aqueous phase was purified using a DNA clean-up kit (Monarch® PCR & DNA Cleanup Kit, New England Biolabs). The DNA was eluted in 5 pF of ddH2O. Using nested PCR primers, the sorted genes were amplified with a high-fidelity polymerase (NEB Q5® DNA Polymerase, New England Biolabs) following a standard thermocycling protocol.
  • a high-fidelity polymerase NEB Q5® DNA Polymerase, New England Biolabs
  • the second plurality of droplets including 808,052 droplets, each comprising an average of approximately 1,500 LipA mutants as indicated schematically in FIG. 9.
  • the second plurality of droplets was encapsulated with the in vitro Protein Synthesis reagents into 30 pm water-in-oil droplets.
  • the emulsion was incubated at 37 °C for 4 hours for in-drop protein synthesis, followed by a heat shock at 70 °C for 1 hour to inactivate the non-thermostable variants, RA picoinjection, and the second plurality of droplets was collected in mineral oil.
  • 402 activity droplets were identified.
  • a third plurality of 694,169 droplets was prepared, each comprising approximately 50 LipA mutants as indicated schematically in FIG. 9.
  • the sequences of the third plurality of plurality of droplets were amplified and screened to identify 329 activity droplets comprising an average of 1 LipA mutant per droplet, as indicated schematically by FIG. 9.
  • the fourth plurality of droplets was broken to form a fifth plurality of 3,513,257 droplets, which were amplified and sorted as previously described to ensure a high likelihood that each resulting activity droplet included exactly 1 LipA mutant.
  • the DNA plasmids were purified, and the plasmids were transformed into high-efficiency competent cells (Turbo Competent E. coli, New England Biolabs) for plasmid replication and extraction.
  • the sorted plasmids were linearized using a restriction enzyme (NruL HF, NEB # R3192S) and sequenced using PacBio Sequel II by commercial providers (Icahn Institute for Genomics and Multiscale Biology) to identify the active, thermostable sequences provided in Table 1.
  • the corresponding nucleic acid sequences are provided in Table 2.
  • Table 1 Amino acid sequences identified in final plurality of activity droplets.
  • Table 2 Coding nucleic acid sequences corresponding to amino acid sequences identified in final plurality of activity droplets.
  • thermostable mutants and WT-LipA were transformed on a pET28a+ vector backbone into BL21(DE3) competent cells (NEB C2527H) for overnight expression in liquid culture (Invitrogen, MagicMediA E. coli Expression Medium).
  • the protein was then purified and desalted.
  • the protein concentrations of the mutants were linearized to 0.1 mg/mL based on nanodrop measurements.
  • a spectroscopic analysis was conducted by measuring the absorbance of the purified protein at 280 nm to determine each thermostable mutant’s concentration. Each protein’s purity was confirmed by running an SDS-PAGE gel.
  • thermostable LipA mutants and the WT-LipA, were analyzed to determine their melting temperatures (T m ) and their heat inactivation resistance temperatures (T50 — the temperature at which each protein lost 50% of its activity).
  • thermostability of the identified enzyme mutants can be further augmented when exposed to detergent at elevated temperatures.
  • selected mutants from screening — specifically MT 5, 6, 7, 8, 10, and 20 — exhibited a significant increase in residual enzymatic activity following a 20-minute incubation at 80 °C in the presence of detergent (Tween 80) at concentrations of 1 mg/L, 10 mg/L, and 500 mg/L.
  • the wild-type enzyme (WT) showed no such increase in activity when subjected to the same detergent and temperature conditions.
  • Table 3 Melting temperatures and heat inactivation resistance temperatures of identified LipA mutants and WT-LipA.
  • 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 without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • 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.
  • “at least one of A and 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.
  • wt% is an abbreviation of weight percentage.
  • at% is an abbreviation of atomic percentage.
  • embodiments may be embodied as a method, of which various examples have been described.
  • the acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and/or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

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Abstract

Le criblage de banque est un instrument d'analyse important pour identifier des protéines ou des acides nucléiques fonctionnels. Dans certains aspects, l'invention concerne des systèmes et des procédés basés sur des gouttelettes pour le criblage de banques. Les systèmes et les procédés peuvent comprendre des étapes consistant à trier les gouttelettes sur la base de leur activité, à amplifier les acides nucléiques de "gouttelettes à activité" ayant une activité élevée, et à séparer les acides nucléiques des gouttelettes à activité en formant de nouvelles gouttelettes. Les étapes peuvent être itérées, de telle sorte que des gouttelettes à activité provenant d'itérations successives tendent à inclure moins d'acides nucléiques. De telles approches peuvent faciliter l'identification d'acides nucléiques actifs, ou d'acides nucléiques codant pour des protéines actives.
PCT/US2023/075065 2022-09-26 2023-09-25 Systèmes et procédés de criblage de banques de gènes de grande taille Ceased WO2024073375A2 (fr)

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