Systems and methods for high-throughput protein characterization

Abstract

The present disclosure relates to systems and methods for production and characterization of proteins. In particular, the present disclosure provides systems and methods for high-throughput in vitro transcription and translation and enzymatic analysis of enzymes.

Description

SYSTEMS AND METHODS FOR HIGH-THROUGHPUT PROTEIN CHARACTERIZATIONFIELD

[0001] The present disclosure relates to systems and methods for production and characterization of proteins. In particular, the present disclosure provides systems and methods for high-throughput in vitro transcription and / or translation and functional characterization of enzymes.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 672,426, filed July 17, 2024, the content of which is herein incorporated by reference in its entirety.BACKGROUND

[0003] Enzymes are the catalytic machines that drive chemical reactions. Thus, they have enormous value and potential in industry and medicine. Despite impressive and rapid progress in the ability to design protein sequences that reliably adopt desired low-energy structures, designing enzymes with specified activities, specificities, and catalytic efficiencies remains a challenge, involving rounds of costly and labor-intensive experimental directed evolution and analysis. Thus, new technologies capable of providing quantitative functional characterization of many enzyme variants in parallel are needed.SUMMARY

[0004] Embodiments of the present disclosure include methods and systems for high- throughput protein (e.g., enzyme) analysis.

[0005] In some embodiments, the methods comprise at least one or all of: patterning a library of polynucleotides encoding a plurality of enzymes in individual hydrophilic areas on a wettability patterned solid surface, wherein each hydrophilic area comprises a droplet comprising at least one polynucleotide encoding a different enzyme; contacting each droplet with in vitro transcription and / or translation reagents; and incubating under conditions for cell-free protein synthesis to produce the enzymes.

[0006] In some embodiments, the polynucleotides are plasmids or linear double stranded DNA templates encoding the enzyme.

[0007] In some embodiments, the plurality of enzymes comprises one or more enzyme variants or fragments thereof of an enzyme of interest. In some embodiments, the enzyme of interest is a wild-type enzyme. In some embodiments, the plurality of enzymes comprises variants or fragments of the wild-type enzyme having one or more amino acid substitutions, additions, or deletions as compared to the sequence of the wild-type enzyme. In some embodiments, the plurality of enzymes comprises one or more artificially generated enzymes.

[0008] In some embodiments, each droplet is less than 250uL in volume. In some embodiments, each droplet is less than 1 uL in volume.

[0009] In some embodiments, the contacting comprises stamping the solid support with a second wettability patterned surface containing the in vitro transcription and / or translation reagents.

[0010] In some embodiments, the method further comprises determining enzyme concentration in each droplet following incubation.

[0011] In some embodiments, the method further comprises immobilizing the enzymes. In some embodiments, the immobilizing comprises stamping a patterned solid surface comprising the enzymes with a third patterned surface comprising a surface-immobilized binding partner for each of the enzymes. In some embodiments, the binding partner is a nucleic acid, a protein, a peptide, a small molecule, or a combination thereof. In some embodiments, the binding partner comprises an antibody.

[0012] In some embodiments, the method further comprises washing immobilized enzymes. In some embodiments, the washing comprises stamping the solid surface comprising the immobilized enzymes with one or more fourth patterned solid surface comprising washing buffer and / or buffer of interest.

[0013] In some embodiments, the methods further comprise contacting each droplet comprising synthesized enzymes with enzymatic assay reagents; incubating under conditions for desired enzymatic reaction; and detecting enzymatic reaction at one or more timepoints during incubation.

[0014] In some embodiments, the contacting comprises stamping the solid support with a fifth wettability patterned surface containing the enzymatic assay reagents.

[0015] In some embodiments, the enzymatic assay reagents comprise a substrate for the enzyme. In some embodiments, the substrate is a fluorogenic, chromogenic, or luminescent substrate. In some embodiments, the enzymatic assay reagents further comprise a buffering system, a cofactor, a detection system, one or more inhibitors, or a combination thereof.

[0016] In some embodiments, detecting the enzymatic reaction comprises fluorescence, absorbance, mass spectrometry, or a combination thereof.

[0017] In some embodiments, the methods further comprise determining one or more enzymatic parameters for each enzyme.

[0018] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1E show an exemplary workflow for Droplet Array Microfluidic Enzyme Kinetics (DA-MEK). FIG. 1A is an exemplary experimental pipeline: (1) DNA templates encoding enzyme variants are printed onto wettability -patterned slides, (2) arrayed variants are ‘stamped’ to another patterned slide to add cell-free protein expression (CFPS) mix to each variant and express proteins, and then (3) expressed proteins are ‘stamped’ to fluorogenic substrates to begin reactions. Imaging over time allows quantification of protein expression and substrate turnover for all variants in parallel. FIG. IB shows the chemical steps for modifying the glass surface to introduce a dendrimer-modified vinyl surface via thiol-ene reactions with 1 -Thioglycerol (1-TG) and esterification with 4-Pentenoic acid. FIG. 1C shows patterning surface wettability using UV light and a mask through thiol-ene photoclick chemistry with 1 -Thioglycerol (1-TG) and perfluorodecanethiol (PFDT). FIG. ID shows examples of droplet array formation on slides with different spot sizes (left); images and contact angles of water and CFPS reagents on hydrophilic and hydrophobic surfaces (right). FIG. IE shows a table summarizing droplet array geometries and volumes for different droplet sizes (left) and bright field images of droplets on hydrophilic spots of different diameters (right); scale bar indicates 500 pm.

[0020] FIGS. 2A-2H show cell-free protein expression by stamping droplet microarrays. FIG. 2A is an exemplary experimental pipeline for cell-free protein expression via (i) printing template DNA encoding enzyme variants onto hydrophilic spots, (ii) stamping this template DNA DMA slide with a DMA slide containing cell-free protein expression mix in each droplet, and (iii)incubating to express the mutant protein library. FIG. 2B is a 3D rendering of microscope stage- mountable slide aligner to ‘stamp’ DMA slides together. FIG. 2C is a diagram illustrating the plasmid DNA template used to produce C-terminally eGFP-tagged PafA variants. FIG. 2D is a diagram illustrating how addition of cell-free protein synthesis reagents to template DNA yields mRNA and expressed eGFP-tagged protein. FIG. 2E is fluorescence microscopy images illustrating cell-free expression of PafA-eGFP variants at various timepoints. FIG. 2F is bright field microscopy images and a schematic showing gasket used to minimize droplet evaporation. Scale bar indicates 5 mm. FIG. 2G shows bright field microscopy images (top) and quantified droplet diameters (bottom) for droplets over time with and without the gasket. Scale bars indicate 500 pm. FIG. 2H is a graph of calibrated enzyme concentrations produced for droplets expressing 12 different eGFP-tagged PafA variants across 2 different DMA slides.

[0021] FIGS. 3A-3I show quantifying variant enzyme activity and kinetics by stamping. FIG. 3A is an exemplary experimental pipeline for quantifying enzyme activity via stamping and expressed DMA array to an array containing fluorogenic substrate. FIG. 3B is a schematic of fluorogenic reaction in which enzymatic removal of a phosphate group from a non-fluorescent substrate (4-MUP) generates a fluorescent product (4-MU). FIG. 3C is time-lapse images showing increased fluorescence over time for 4 example droplets either lacking DNA template (negative control, NC) or containing 3 different PafA constructs (WT, R164A, and T79). FIG. 3D is a graph of quantified fluorescence over time for the same droplets containing cither wild-type PafA (WT) or two mutant enzymes (R164A and T79S). Markers indicate median pixel intensity for a single droplet; lines indicate linear fits to initial rates. FIG. 3E is a graph of observed initial rates as a function of substrate concentration for WT, R164A, and T79S. Markers indicate single droplets; lines indicate Michaelis-Menten fits to the data. FIG. 3F is box plots showing measured corrected log(fct-a / / KM) for multiple enzyme variants obtained in DA-MEK. FIG. 3G is a graph of corrected log( '( !( / / K\i) values from DA-MEK versus those from well plates. FIG. 3H is a scatter plot comparing observed log(£raf / KM) values from DA-MEK versus prior literature results. FIG. 31 is a graph of corrected log(fcc<» / KM) values from DA-MEK versus prior literature results. The data across FIGS. 3F to 31 are based on 15 measurements for each variant.

[0022] FIG. 4 is an exemplary design of photomasks for thiol-ene photoclick chemistry.

[0023] FIG. 5 is a schematic diagram of automated dispenser for loading liquid reagents, built with motorized stages, a stage controller, and a mass flow controller (MFC), operated by python GUI script.

[0024] FIG. 6 is a graph of cell-free protein synthesis in DMA over time.

[0025] FIG. 7 is a comparison of enzyme expression as determined herein against catalytic efficiency (data from Markin and Moktari et al. Science, 2021 373 (6553), 411).

[0026] FIG. 8 is comparative heatmaps illustrating the reproducibility and reliability of expression over DMA slides.

[0027] FIG. 9A is a schematic of an exemplary workflow of the well-plate-based assay for PafA enzyme kinetics. FIGS. 9B-9E are representative data, as indicated, used for calculating Michaelis- Menten constants.

[0028] FIG. 10 is progress curves of PafA variants, as indicated, obtained in DMA.

[0029] FIG. 11 is the uncorrected Vi / [E] of PafA variants, as indicated, obtained in DMA.

[0030] FIG. 12 is the corrected Vi / [E] of PafA variants, as indicated, obtained in DMA.

[0031] FIG. 13 is the progress curves of PafA variants, as indicated, obtained in well plates.

[0032] FIG. 14 is the uncorrected Vi / [E] of PafA variants, as indicated, obtained in well plates.

[0033] FIG. 15 is the corrected Vi / [E] of PafA variants, as indicated, obtained in well plates.

[0034] FIG. 16 is the reproducibility assessment of kinetic analysis using DMA and well plate.

[0035] FIG. 17 shows simulations to determine the range of KM and kcat with 10% error in substrate concentrations for the indicated PafA variants. The boxes represent the maximum and minimum values obtainable with 10% error, while the dots represent the median of actual measurements.

[0036] FIG. 18 is a table of the comparison of cost for screening library with DMA and well plate.

[0037] FIGS. 19A-19H show the kinetics of immobilized enzymes by stamping. FIG. 19A is a schematic of an exemplary experimental workflow for immobilizing enzymes from crude in vitro transcription-translation (IVTT) products and subsequent purification through washing. The purification process involves applying fresh buffer droplets on each spot, followed by blotting with absorbent pads. FIG. 19B is a schematic illustration of adjusting the ratio of biotinylated anti-eGFP antibody to biotinylated BSA to control the amount of immobilized protein. FIG. 19C is fluorescent images of proteins immobilized on a DMA slide with varying amounts. FIG. 19Dis a calibration curve for protein binding by tuning the ratio of biotinylated BSA and anti-eGFP antibody. The Y-axis is scaled using log2 for visualization, while the values represent actual measurements (n = 8). FIG. 19E is a calibration curve correlating the amount of eGFP immobilized on the surface with initial reaction rates, measured using WT PafA and 1000 pM 4- MUP (n = 3). FIG. 19F is graph of initial rates as a function of substrate concentration for WT, R164A, and T79S PafA without immobilization. Markers indicate single droplets; lines indicate Michaelis-Menten fits to the data. FIG. 19G is a graph of the comparison of kcat / u values between immobilized DA-MEK experiments and corrected values from non-immobilized DA- MEK experiments. FIG. 19H is a graph of kCatf M values between immobilized DA-MEK experiments and prior literature values. The data across FIGS. 19G and 19H are based on 8 measurements for each variant. Error bars indicate standard deviation.

[0038] FIG. 20 shows detection of detecting samples on a DA-MEK slide using DESI-ToF mass spectrometry with different concentrations of ammonium bicarbonate buffer in combination with a representative detection molecule, 4-methylumbelliferone (4-MU), at four different concentrations. As the buffer concentration decreased, the signal clarity and sensitivity of 4-MU improved.

[0039] FIGS. 21A and 21B show DESI-ToF mass spectrometry for detection of enzymatic product formation over time using the DA-MEK slide platform. PafA was immobilized on the surface of a reaction slide. Droplets containing the substrate, 4-MUP, were deposited onto the enzyme-coated regions to initiate the reaction. At defined timepoints (1, 5, 10, and 60 minutes), the droplets were transferred to an enzyme-free readout slide, thereby terminating the reaction and enabling the capture of time-resolved enzymatic activity profiles (FIG. 21A). DESI-ToF analysis revealed a time-dependent conversion of substrate to product: the signal intensity of the substrate (4-MUP) decreased, while the signal from the enzymatic product (4-MU) increased over time (FIG. 21B).

[0040] FIG. 22 shows immobilization using Benzylguanine - SNAP-tag. eGFP-WT and eGFP-WT-SNAP proteins were each immobilized via the biotinylated BSA-NeutrAvidin-anti- eGFP antibody linkage, after which BG-549 (NEB SNAP-Surface® 549), a SNAP-reactive fluorophore, was applied to confirm the presence of the SNAP tag and successful BG-SNAP binding. Fluorescence Ch. eGFP shows the presence of protein, Cy3 shows the presence of BG- SNAP binding.DETAILED DESCRIPTION

[0041] The present disclosure relates to systems and methods for high-throughput functional characterization of proteins (e.g., enzymes).

[0042] Directed evolution campaigns are often costly and labor-intensive, as each round necessitates the synthesis of variant DNA libraries, expression of variant proteins, and functional characterization of enzymatic activity. At industrial scale, these steps often take place within multiwell plates that require approximately 10-20 pL of material for each reaction at each step. Microfluidic droplet assays enable ultra-high-throughput compartmentalization of reactions within very small volumes (e.g., ~ 1,000,000 droplets / hr and 6-pL / droplet), reducing reaction costs by up to 106-fold. However, the enrichment of only valiants with enhanced activities precludes gathering information about deleterious sequence changes that can be critical for building a protein design tool to predict activities from sequences. Furthermore, these approaches cannot directly link measured activity of individual variants with their sequences.

[0043] For commercial-directed evolution campaigns, the benefit of performing kinetic analysis was not considered as the focus was primarily on endpoint analysis. However, with the rapid development of protein design tools (e.g., based on artificial intelligence generation of new protein sequences and structures), there is a growing need for high-quality data to learn sequencefunction relationships for the functional design of proteins.

[0044] As described herein, the system and methods facilitate high-throughput cell-free expression and functional characterization of enzyme catalytic efficiency (herein referred to as DA-MEK, Droplet Array Microfluidic Enzyme Kinetics) within omniphobic / superhydrophilic droplet arrays. The capabilities of the platform were demonstrated using variants of PafA, a well- characterized model alkaline phosphatase. By printing DNA plasmids encoding the expression of C-terminally eGFP-tagged PafA variants onto hydrophilic spots, stamping these spots to a matched array containing cell-free protein expression mixture, incubating to produce protein, and imaging to quantify protein expression, the disclosed system and methods are able to reproducibly express >500 nM protein per droplet. By subsequently stamping these expressed protein arrays to a matched array containing a fluorogenic PafA substrate and imaging to quantify turnover over time, the disclosed system and methods facilitate quantification of reaction turnover and determination of Michaelis-Menten rate constants for each of the variants of the enzyme in parallel. Rate constants measured via DA-MEK agree well with values derived from traditional plate-basedassays (r2= 0.89 and RMSE = 0.2) for PafA variants spanning a catalytic efficiency range of over 5 orders of magnitude.

[0045] Droplet Array Microfluidic Enzyme Kinetics (DA-MEK) offers significant advantages over traditional methods in enzyme screening. By utilizing hydrophilic / omniphobic surfaces to create water-in-air droplet arrays, the disclosed methods and systems allow for high-throughput and cost-effective expression and kinetic characterization of enzyme variants. This method also provides accurate kinetic data, including both beneficial and deleterious mutations, facilitating a more comprehensive understanding of enzyme functionality by directly correlating sequencefunction relationships. The open format of DA-MEK simplifies reagent addition and sampling (e.g., sandwiching method), drastically reducing the effort to operate miniaturized systems like microfluidic chips. Additionally, the open format enables non-fluorescent analyses, allowing for directed evolution campaigns of target substances without reliance on fluorescence. This is particularly advantageous for industrial applications where working with the actual target compounds rather than Anorogenic substrate is critical.

[0046] Protein production and characterization are the costliest part of directed evolution campaigns. As this process often requires large volumes of expensive reagents, reduced reaction volumes directly translate into significant cost savings. As described elsewhere herein, DA-MEK enables parallelized production and functional characterization of enzymes within nL-to-pL volume droplets. Compared to conventional 384 well plate-based assays, this reduces pcr-rcaction costs by approximately 11 -fold for the 450 nL droplets used here for protein synthesis and enzymatic assays (Figure 18). As libraries grow in size, these savings can be substantial. Assuming 8 substrate concentrations per Michaelis-Menten curve and 3 replicates per measurement, characterizing a 1000-member library costs $12,744 using DA-MEK with 1500 pm diameter spots as compared to $141,600 for 384-well plates (Figure 18). Future experiments optimizing protein synthesis and enzymatic turnover measurements in 500 pm diameter spots could increase these savings further, reducing estimated costs for the same 1000-member library to $566, 250-fold less than 384-well plates. In total, DA-MEK enables high-throughput enzyme purification and iterative characterization, increasing the number of enzyme systems that can be profiled while simultaneously reducing expression costs.

[0047] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0048] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0049] In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.”

[0050] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The transitional phrase “consisting essentially of’ as used in claims in the present application limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention, as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition “consisting essentially of” recited elements may contain an unrecited contaminant at a level such that, though present, the contaminant does not alter the function of the recited composition as compared to a pure composition, i.e., a composition “consisting of’ the recited components. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0051] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. The term “one or more,” as used herein, refers to a number higher than one. For example, the term “one or more” encompasses any of the following: two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more,thirteen or more, fourteen or more, fifteen or more, twenty or more, fifty or more, 100 or more, or an even greater number.

[0052] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0053] “Polynucleotide” or “oligonucleotide” or “nucleic acid,” as used herein, means at least two nucleotides covalently linked together. The polynucleotide may be DNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. The nucleic acid, whether DNA or RNA may comprise non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”). Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. Polynucleotides may be single- or double- stranded or may contain portions of both double stranded and single stranded sequence. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.

[0054] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide” and “protein” are used interchangeably herein.

[0055] The term “detecting” as used herein generally refers to any form of measurement, and includes determining whether an element is present or not. This term includes quantitative and / or qualitative determinations.

[0056] “Omniphobic” refers to certain characteristics of a material that is both hydrophobic and oleophobic and capable of repelling various types of liquids such as water, oil, and as well as other contaminants. Omniphobic surfaces are those surfaces that repel (or are non-wetting to) almost all known liquids, polar or non-polar. Omniphobic surfaces are generally indicated to have contact angles OoiLand 0H2oof greater than 90°. While omniphobic wettability encompasses superomniphobic wettability, superomniphobic surfaces are typically considered to have SOIL and 9H2O of greater than or equal to about 150° up to about 180°, by way of example.

[0057] “Hydrophilic” refers to certain characteristics of a material which has affinity for water. Hydrophilic surfaces are generally indicated to have contact angles of less than 90°. While hydrophilic encompasses superhydrophilic, superhydrophilic surfaces are typically considered to have contact angles of less than 10°.

[0058] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity.

[0059] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein arc illustrative only and not intended to be limiting.2. Characterization Methods

[0060] The present disclosure provides systems and methods for characterizing proteins. In select embodiments, the systems and methods characterize a plurality of proteins (e.g., enzymes, or variants or fragments thereof) in a single assay. The systems and methods are not limited by the type of proteins or enzymes.

[0061] The protein (e.g., enzyme) variants or fragments thereof may be derived from a single starting amino acid sequence. The variants may comprise one or more amino acid substitutions, additions, or deletions as compared to the starting sequence. For example, the methods may characterize variants of a wild-type protein (e.g., enzyme) sequence, each variant with one or more substitutions, additions, or deletions as compared to the wild-type sequence. In some embodiments, the protein (e.g., enzyme) variant or fragments thereof may be derived from multiplestarting enzyme sequences. The valiants or fragments thereof may comprise a naturally occurring variants or fragments of a protein (e.g., enzyme) of interest.

[0062] In some embodiments, the systems and methods may be utilized to screen a plurality of artificially generated proteins or enzymes. The variants or fragments thereof may comprise artificially generated sequences, either from a known starting sequence or complete bespoke protein (e.g., enzyme) sequences based on various methods for de novo protein design.

[0063] In some embodiments, the protein (e.g., enzyme) variants comprises one or more control or reference sequences. For example, the plurality of proteins (e.g., enzymes, or variants or fragments thereof) may comprise a wild-type version of the protein (e.g., enzyme) of interest from which the variants are derived. Alternatively or in addition, the plurality of proteins (e.g., enzymes, or variants or fragments thereof) may include an enzyme which carries out the same or similar reaction as being characterized for the variants or fragments.

[0064] In some embodiments, the methods comprise one or more of all of patterning a library of polynucleotides encoding a plurality of enzymes in individual hydrophilic areas on a wettability patterned solid surface, wherein each hydrophilic area comprises a droplet comprising at least one polynucleotide encoding a different enzyme; contacting each droplet with in vitro transcription and / or translation reagents; and incubating under conditions for cell-free protein synthesis to produce the enzymes.

[0065] In some embodiments, the methods comprise one or more of all of: providing a wettability patterned solid surface comprising a plurality of hydrophilic areas separated by omniphobic regions; patterning a library of polynucleotides encoding a plurality of proteins (e.g., enzymes) or variants or fragments thereof in individual hydrophilic areas, wherein each hydrophilic area comprises a droplet comprising a polynucleotide encoding different protein (e.g., enzyme) variant or fragment thereof; contacting each droplet with in vitro transcription and translation reagents; and incubating under conditions for cell-free protein synthesis.

[0066] In some embodiments, the methods comprise providing or using a wettability patterned solid surface. The wettability patterned solid surface may comprise hydrophilic areas separated by omniphobic or hydrophobic regions. In general, such surfaces can be prepared by functionalizing the surface with compounds which can be easily modified under UV irradiation based on photoinduced thiol-yne or thiol-ene reactions, disulfide-yne or disulfide-ene reactions, or tetrazoleyne or tetrazole-ene reactions and then introducing groups which confer the desired degree ofhydrophilicity while masking those areas designated as omniphobic or hydrophobic regions, for example as shown in Figures IB, 1C and the related methods and examples provided herein.

[0067] The photomask can be developed to allow the hydrophilic areas to assume any size or shape. Thus, the disclosed methods are not limited by the patterning design. In some embodiments, the spot size is less than 10 mm, less than 2 mm, less than 1 mm, less than 750 pm, or less than 550 pm.

[0068] The droplet size or volume may be matched or configured to the size and shape of the hydrophilic areas. For example, the droplet volume may scale with the size of the hydrophilic area. In some embodiments, the droplet is less than 1 mL in volume (e.g., less than 900 pL, less than 800 pL, less than 700 pL, less than 600 pL, less than 500 pL, less than 400 pL, less than 300 pL, less than 200 pL, less than 100 pL, less than 50 pL, less than 10 pL, or less than 1 pL). In select embodiments, the droplet is less than 1 pL in volume (e.g., less than 900 nL, less than 800 nL, less than 700 nL, less than 600 nL, less than 500 nL, less than 400 nL, less than 300 nL, less than 200 nL, less than 100 nL, less than 50 nL, or less than 10 nL).

[0069] The polynucleotides in the library of polynucleotides encoding a plurality of proteins (e.g., enzymes) or variants or fragments may be DNA or RNA. In some embodiments, the polynucleotides comprise double stranded DNA. For example, the polynucleotides may be plasmids and / or linear double stranded DNA templates encoding the plurality of proteins (e.g., enzymes) or variants or fragments. Alternatively, the polynucleotides comprise RNA. For example, the polynucleotides may be mRNA templates, e.g., in vitro transcribed mRNA.

[0070] In some embodiments, the methods comprise contacting each droplet with in vitro transcription and / or translation reagents. The methods are not limited by the manner in which each droplet is contacted with in vitro transcription and / or translation reagents. For example, methods may include manual contacting, such as pipetting each droplet with in vitro transcription and / or translation reagents, or automatic or programmable contacting, such as using an injector or printer to deliver the in vitro transcription and translation reagents to each droplet.

[0071] In select embodiments, the contacting comprises stamping the solid surface comprising the library of polynucleotides with a second wettability patterned surface containing the in vitro transcription and translation reagents. “Stamping” as used herein refers to a process in which a second matched wettability patterned surface, comprising droplets with the desired materials (e.g., in vitro transcription and translation reagents), is brought in close proximity with the solid surfacepatterned with the library of polynucleotides, preferably vertically, forming a sandwich of the two surfaces with the droplets in the middle. The surfaces are brought in close enough proximity that the droplets comprising the library of polynucleotides and those droplets containing the in vitro transcription and translation reagents merge while adjacent droplets do not mix. This proximity can be determined visually and will be dependent upon the droplet size and shape, as well as the particular pattern of hydrophilic areas.

[0072] The delivery of the in vitro transcription and translation reagents to the droplet may be simultaneous, e.g., delivery of all the reagents for in vitro transcription and in vitro translation at a single time with a single step. Alternatively, the delivery of the in vitro transcription and translation reagents to the droplet may be sequential, e.g., delivery of the reagents for in vitro transcription followed by an incubation for in vitro transcription then delivery of the reagents for in vitro translation followed by an incubation cell-free protein synthesis. Such sequential delivery allows for different conditions for the transcription and translation reactions. Accordingly in some embodiments, the methods comprise contacting each droplet with in vitro transcription reagents, incubating under conditions for transcription, and contacting each droplet with in vitro translation reagents, incubating under conditions for cell-free protein synthesis. As described above the individual contacting steps can include manual contacting, such as pipetting each droplet with in vitro transcription and translation reagents, or automatic or programmable contacting, such as using an injector or printer to deliver the in vitro transcription and translation reagents to each droplet, or stamping as described in detail above.

[0073] The terms “in vitro transcription” and “cell-free transcription” are used interchangeably herein and are intended to refer to any method for cell-free synthesis of RNA from DNA without synthesis of protein from the RNA. A preferred RNA is messenger RNA (mRNA), which encodes proteins. The terms “in vitro transcription-translation” (IVTT) and “cell- free transcription-translation” are used interchangeably herein and are intended to refer to any method for cell-free synthesis of mRNA from DNA (transcription) and of protein from mRNA (translation).

[0074] Coupled or complementary transcription and translation systems, which carry out the synthesis of both RNA and protein in the same reaction, have been developed. In such in vitro transcription and translation (IVTT) systems, the reagents contain all the components for both transcription (to produce mRNA) and translation (to synthesize protein) in a single system.

[0075] In vitro transcription and translation reagents typically comprise an RNA polymerase that recognizes the promoter(s) to which the template nucleic acid encoding the enzyme of interest is operably linked and, optionally, one or more transcription factors directed to an optional regulatory sequence to which the template nucleic acid is operably linked; ribonucleotide triphosphates (rNTPs); ribosomes; transfer RNA (tRNA); optionally, other transcription factors and co-factors thereof; amino acids (optionally comprising one or more detectably labeled amino acids); one or more energy sources, (e.g., ATP, GTP); and other or optional translation factors (e.g., translation initiation, elongation and termination factors) and co-factors thereof. When using mRNA templates as the starting polynucleotides, the in vitro translation reagents may comprise ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation, and termination factors, energy sources, and transcription factors and co-factors thereof. In some embodiments, the in vitro transcription and translation reagents are purified components of the transcription and translation machinery from an organism.

[0076] In some embodiments, the methods comprise incubating the polynucleotides encoding a plurality of proteins (e.g., enzymes) and the in vitro transcription and translation reagents under conditions for cell-free protein synthesis. The terms “cell-free protein synthesis (CFPS),” “in vitro protein synthesis,” “in vitro translation,” “cell-free translation,” “RNA template-driven in vitro protein synthesis,” “RNA template-driven cell-free protein synthesis,” and “RNA template- driven cell-free protein synthesis,” arc used interchangeably herein and arc intended to refer to any method for cell-free synthesis of a protein.

[0077] The conditions for cell-free protein synthesis are dependent on the selected in vitro transcription and translation reagents. For example, the processivity and optimized temperature / pH parameters for the polymerase and the ribosomes will generally control the reactions conditions. In some embodiments the incubation is for at least 30 minutes and up to 12 hours. The incubation may be for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, or more. In some embodiments, the incubation temperature is between 20° C and 40° C.

[0078] In embodiments in which the polynucleotides encoding the plurality of proteins (e.g., enzymes, or variants or fragments thereof) are RNA molecules, the methods only utilize in vitro translation and the corresponding reagents for protein synthesis, as described above. Thus, in some embodiments, the methods comprise contacting each droplet with in vitro translation reagentsincubating the RNA encoding a plurality of proteins (e.g., enzymes) and the in vitro translation reagents under conditions for cell-free protein synthesis.

[0079] In some embodiments, the methods further comprise determining protein (e.g., enzyme) concentration in each droplet following incubation. Concentration can be measured by any of the known methods of measuring protein concentration, either directly in the droplet on the solid support or indirectly by utilizing an aliquot from the droplet to determine the concentration and calculating the concentration in the droplet accordingly. For example, concentration can be determined using a variety of spectrophotometric assays, as described in the examples. In some embodiments, protein concentration can be determined by measuring absorbance at 280 nm or another wavelength based on the properties of the protein (e.g., enzyme) of interest. Alternatively, a variety of protein interactive dyes or agents which result in colorimetric signal and / or absorbance change upon protein binding (e.g., Coomassie Blue, BCA, Folin-Ciocalteu reagent, ninhydrin, or lluorogenic dyes) can be used, preferably in indirect measurements.

[0080] In some embodiments, the methods further comprise immobilizing the in vitro translated proteins (e.g., enzymes). Immobilization can be based on known interactions, both chemical and physical, with supporting materials, functionalized or unfunctionalized. The association of the proteins (e.g., enzymes) with a solid support can be through covalent or non-covalent (e.g., ionic, hydrogen-bonding, Van der Waal, etc.) interactions bond formation and can be reversible or irreversible. One of skill in the art will appreciate that immobilization can be achieved by any method known in the art.

[0081] The proteins (e.g., enzymes) can be immobilized to a solid surface functionalized with a surface tethered specific binding partner. The specific binding partner can be any moiety or molecule which directly binds the proteins (e.g., enzymes) or variants or fragments thereof and the choice of binding partner will depend on the particular protein (e.g., enzyme) or variant or fragment thereof. In some embodiments, the binding partner comprises a nucleic acid, a protein, a peptide, a small molecule, or a combination thereof. In select embodiments, the binding partner comprises any antibody to the particular protein (e.g., enzyme) or variant or fragment thereof.

[0082] Alternatively, the in vitro translated proteins (e.g., enzymes) may include a domain or tag which can be used for immobilization. For example, the protein may include a domain or tag (e.g., self-labeling tags) known to specifically interact with a binding partner (e.g., a nucleic acid, a protein, a peptide, a small molecule, or a combination thereof) tethered to the surface of the solidsurface. Exemplary domains or tags include, but are not limited to, SNAP-tag, CLIP-tag, HaloTag, SpyTag / SpyCatcher system, AviTag, affinity tags (e.g., His tag, calmodulin binding peptide), and epitope tags (e.g., FLAG, myc and HA).

[0083] In some embodiments, immobilizing the in vitro translated proteins (e.g., enzymes), comprises stamping a patterned solid surface comprising the in vitro translated proteins (e.g., enzymes) with another wettability patterned surface comprising a surface-immobilized binding partner for each of the in vitro translated proteins (e.g., enzymes) in locations which align with the droplets on the solid surface used for protein expression. Thus, the proteins generated on a first solid surface will bind to the surface-immobilized binding partner and be immobilized the new solid surface.

[0084] Immobilizing the in vitro translated proteins (e.g., enzymes) provides the ability to wash or exchange solution or buffer components from those used for the in vitro transcription and / or translation. Washing or exchanging solution or buffer components may facilitate increased accuracy or sensitivity of the characterization and functional assessment, particularly for proteins (e.g., enzymes) which are sensitive to the conditions for in vitro transcription and / or translation. Washing or exchanging solutions or buffer components can also facilitate optimization of conditions for activity measurements, determination of optimized conditions, or characterization and functional assessment under a particular set of conditions (e.g., physiologically or commercially relevant conditions).

[0085] In some embodiments, the methods comprise washing the immobilized enzymes. Similarly to described above, the solid surface with the immobilized enzymes can be stamped with another patterned solid surface comprising washing buffer and / or buffer of interest. The immobilized enzyme can be washed any number of times to place the enzymes under the desired conditions. Between washing rounds, any residual washing buffer can be removed, e.g., with light blotting or air drying.

[0086] The methods further comprise contacting the proteins (e.g., enzymes) with reagents necessary for their characterization. In some embodiments, the methods further comprise contacting each droplet comprising synthesized enzymes or immobilized enzymes with enzymatic assay reagents; incubating under conditions for the desired enzymatic reaction; and detecting enzymatic reaction at one or more timepoints during incubation.

[0087] As described above in relation to the in vitro transcription and translation reagents, the methods are not limited by the methods used to contact the synthesized enzymes or immobilized enzymes with enzymatic assay reagents. In select embodiments, the contacting comprises stamping the solid support with another wettability patterned surface containing the enzymatic assay reagents.

[0088] The enzymatic assay may take on any variety of assay formats. The substrate can be detected in an assay when it is desired to observe substrate consumption during an enzymatic assay, while the product can be detected when it is desired to observe its formation during an enzymatic assay. Both substrate and product can be detected when it is desired to observe the enzymatic reaction from both perspectives, for example, to confirm that the amount of product produced correlates with the amount of substrate consumed.

[0089] In some embodiments, the enzymatic assay reagents comprise a substrate for the enzyme. The substrate may be natural substrate for the enzyme, or a synthetic substrate used for characterization.

[0090] The substrate may be configured for ease of detection. In some embodiments, the substrates are configured such that the product produced from such substrate can be detected. In some embodiments, the substrates are configured that the product produced from such substrate is not detectable, but the substrate itself is detectable. In some embodiments, the substrates are configured such that both the product and the substrate arc separate detectable.

[0091] For example, the substrate may be a fluorogenic, chromogenic, or luminescent substrate. Such substrates may be detectable (e.g., by absorbance, fluorescence, luminescence, or imaging techniques) prior to the enzymatic reaction or only after formation of the product (e.g., a detectable product). Alternatively, fluorogenic and chromogenic substrates may produce a product with a separately detectable signal.

[0092] The methods may further comprise the use of a detection system, e.g., a substrate detection system or a product detection system. For example, the substrate may produce a product which is not detectable. The detection system may interact with the product to produce a detectable signal, which then is used as a measure of the amount of product produced in the enzymatic reaction. Alternatively, a detection system may be used when the substrate is not detectable. A detection system may provide the components necessary for a coupled enzyme assay. Accordingly, in some embodiments, detection of the substrate or product may be indirectly measured.

[0093] In some embodiments, the enzymatic assay reagents comprise other components necessary for enzymatic reactions. For example, the enzymatic assay reagents may comprise a buffering system, a cofactor (e.g., a coenzyme, ion, etc.), a detection system, or a combination thereof.

[0094] The means of detection is dependent on the nature of the substrate or product, or detection system. Generally, substrate or products or detection systems suitable for use in the disclosed methods are those readily detected and / or quantified using various types of spectroscopies, e.g., fluorescence, absorbance, luminescence, mass spectrometry, or a combination thereof.

[0095] In some embodiments, the methods can also be used to analyze enzyme kinetics. Enzyme kinetics, as used herein, refers to the study of the rates of enzyme-controlled reactions. The methods comprise detecting enzymatic reaction at one or more timepoints during incubation. Thus, the methods and systems may be used to determine one or more kinetic parameters of any of the plurality of enzymes. Alternatively, the methods may comprise detecting an endpoint for an enzymatic reaction. Endpoint assays are those in which enzyme activity is measured via the quantity of substrate consumed, or the amount of product formed during the reaction over a fixed period of time.

[0096] Accordingly, the methods may further comprise determining one or more enzymatic parameters (kinetic or endpoint parameters) for each enzyme. For example, the methods may be used to determine maximum velocity, turnover numbers, Michaelis constants, catalytic efficiencies, and the like.

[0097] In some embodiments, the methods may further comprise adding a putative inhibitor of the enzymatic reaction to the assays. For example, the enzymatic assay reagents may further comprise a putative inhibitor of the enzymatic assay. Thus, the methods may be used to determine parameters for the inhibition of the enzyme, e.g., inhibition constants.

[0098] In some embodiments, the methods may further comprise adding a putative activator of the enzymatic reaction to the assays. For example, the enzymatic assay reagents may further comprise a putative activator of the enzymatic assay. Thus, the methods may be used to determine parameters for the activation of the enzyme, e.g., activator binding affinities.3. Systems and Kits

[0099] Also provided are kits or systems that can be used to conduct the methods described herein. Such kits or systems can be used to characterize a plurality of proteins or enzymes. The kits or systems may include any combination of: one or more wettability patterned solid surfaces, in vitro transcription and translation reagents, and enzymatic assay reagents. In some embodiments, the kits or systems further comprise a library of polynucleotides encoding proteins or enzymes.

[0100] In some embodiments, the kits or systems comprise a wettability patterned solid surface wherein each hydrophilic area comprises a polynucleotide (e.g., a plasmid, dsDNA, or mRNA) encoding a different enzyme.

[0101] The kits or systems may further comprise one or more control or reference samples and reagents for performing a desired characterization. For example, the subject kits may include reagents for characterizing known enzyme(s) for control or reference samples. The known enzyme may be a similar type of enzyme or carry out a similar reaction to those enzymes being characterized.

[0102] The systems may further comprise an incubation system or incubator, a means for stamping the wettability patterned surfaces, and or a detection system.

[0103] Individual member components of the kits may be physically packaged together or separately. The components of the kits may be provided in bulk packages (e.g., multi-use packages) or single-use packages. The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.

[0104] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kits. The materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the compositions, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kits or as a separate member component, either as a paper form or an electronic form which may be supplied on a computer readable memory device or downloaded from an internet website, or as a recorded presentation.

[0105] It is understood that the disclosed kits can be employed in connection with the disclosed methods.4. ExamplesMethods

[0106] Photomask design and production. Photomasks were designed using AutoCAD software (Autodesk, Inc.). Hydrophilic spots were designed to be transparent with diameters of 1500, 1000, 750, and 500 pm, and the spacing between adjacent spots was set to half the diameter of each spot. These designs were produced on a 5-inch chrome quartz mask (Front Range Photomask, Las Vegas, NV, USA). Details and specifications of all mask files are available for download.

[0107] Surface patterning. Nexterion glass B slides (25 x 75 mm, Schott) were activated using Piranha cleaning (H SCUH O = 1:3) and then treated with a three-cycle series of chemical reactions for dendrimer generation. For thiol-ene photoclick chemistry, a mixture of 1 wt% 2,2- Dimethoxy-2-phenylacetophenone (cat. no. 196118, Sigma-Aldrich) and 10 wt% 1 -Thioglycerol (cat. no. 88640, Sigma- Aldrich) in DI water / ethanol (1:1 vol) was applied to slide surfaces, prior to covering with a fluorinated quartz plate, and exposure to 254 nm UV light (36W, VirusLights, Delano, TN, USA) for 3 minutes at 3 cm distance. After exposure, slides were washed with ethanol and then dried using nitrogen gas and an 80°C convection oven for 5 min. For esterification, the dried slides were immersed in an esterification mixture consisting of 200 mg 4- (dimethylamino)pyridine (cat. no. W284300, Sigma- Aldrich), 444 pL pentenoic acid (cat. no. 8204990005, Sigma-Aldrich), and 180 pL N,N'-Diisopropylcarbodiimide (cat. no. 8036490010, Sigma- Aldrich) in 200 mL of acetone for 4 hours. After the final cycle, the thiol-ene reaction was initiated using a quartz chrome mask with clear spots (Figure 4) for hydrophilic spot patterning. To complete the functionalization, each slide was washed and dried, coated with a 10% lH,lH,2H,2H-perfluorodecanethiol (PFDT) (cat. no. 660493, Sigma- Aldrich) solution in acetone, covered with a fluorinated blank quartz plate, and again exposed to 254nm UV light (36 W at 3 cm distance for 3 min) to complete the functionalization of the surrounding area.

[0108] Contact angle measurement. Contact angles were measured using a goniometer (Model 290, rame-hart instrument, Succasunna, NJ, USA). 5 pL droplets of two different liquids (deionized water or cell-free protein expression (CFPS) reagent) were carefully applied onto hydrophilic or omniphobic-treated glass slides, respectively. For each type of treatment and liquid, images of 15 droplets were captured and analyzed using an ImageJ plugin (DropAnalysis).

[0109] Gasket design and assembly. To create a gasket that could prevent droplet evaporation during protein expression, one of two methods was employed: (1) hot embossing four layers of Parafilm at 80°C or (2) pouring and curing a mixture of polydimethylsiloxane (PDMS) (RTV-615, Momentive) in a 10:1 ratio between two fluorinated glass plates. Pieces of Whatman Thin Layer Chromatography Plates (cat. no. 4861-830, Thickness: 500 pm) were used as spacers. The thickness of the final product was inspected and then trimmed to create a rectangular window measuring 21 x 71 mm, ensuring a 2 mm overlap on the 25 x 75 mm slides.

[0110] Design and fabrication of Aligner. To accurately align and ‘stamp’ 2 DMA slides together, a manual aligner was designed that includes a lower holder in the form of a microscope- mountable insert and an upper holder with a feature for magnetic attachment and detachment to a 3-axis micromanipulator (Newport M-461). These components were fabricated using CNC machining and subsequently assembled and mounted to a motorized xy- stage (MS-2000, Applied Scientific Instrumentation) on a Nikon Ti-2 automated fluorescence microscope. All design files required to produce the aligner are available for download.

[0111] Immobilization of enzymes. Affinity-based surface immobilization technique was employed to attach eGFP-tagged enzymes onto spots. Biotinylated BSA (bBSA) (ThermoFisher Pierce, 29130) droplets (2 mg / mL) were stamped onto a slide and incubated at room temperature for 30 minutes to allow non-specific adsorption. After incubation, the spots were washed three times with fresh IX PBS buffer droplets. NcutrAvidin (NA) (Thermo Scientific, 31000) droplets (1 mg / mL) were then stamped onto the slide and incubated for 30 minutes to bind to the bBSA on the surface, followed by washing with fresh PBS buffer droplets. Subsequently, biotinylated anti- eGFP antibody (b-anti-eGFP) (Abeam, ab6658) droplets (100 pg / mL) containing a proper ratio of bBSA were introduced to adjust the density of eGFP-tagged protein on the surface (Figures 19C and 19D), and the slide was washed with PBS buffer. Finally, droplets of crude eGFP-tagged enzyme CFPS products were stamped onto the slides to form the top layer of the stack, followed by washing with MOPS reaction buffer.

[0112] Washing slides. Two washing protocols were used for cleaning the slides. During the series of processes for enzyme immobilization, an array of fresh buffer droplet was merged and blotted using an absorbent pad to prevent cross-contamination. This process was repeated three times. Slides with immobilized enzymes were floated enzyme side-down on a Petri dish containingMOPS buffer (100 mM MOPS, 500 mM NaCl, 100 pM ZnCL, pH 8.0) for at least 3 minutes to remove any remaining products to reuse the slides.

[0113] Printing of plasmid libraries onto droplet array slides. Plasmids encoding expression of C-terminally eGFP-tagged PafA variants were produced via Gibson assembly and QuickChange mutagenesis (Agilent) as previously described (Markin, C. J., et al., Science 2021, 373 (6553), 411). To print plasmids onto the slides, a SciFlexArrayer S3 (Scienion) equipped with a PDC70 piezoelectric capillary nozzle (P2030-S6041 , Scienion) was used to print 150 nL of plasmid DNA (at a concentration of 100-300 ng / pL) in deionized water (DIW) (-100 ng / ml) onto each hydrophilic spot of the slide. Between the printing of each variant, the nozzle was washed three times and dried for 15 seconds in an ambient environment.

[0114] Cell-free protein expression. To produce protein, either cell-free expression mix provided by Codexis (Redwood City, CA, USA) or the PURExpress kit from New England Biolabs (USA) was used. Mixtures were prepared according to the manufacturer's protocol with the addition of RNase inhibitor and then loaded onto DMA slides using a custom-built automated liquid dispenser (Figure 5). After stamping slides to add cell-free protein expression mix to printed DNA templates, the slides were incubated at 23°C for 4 hours to express proteins (unless otherwise stated).

[0115] Microscopy setup. All images were obtained using a Nikon Ti-S Microscope which includes a motorized XY stage from Applied Scientific Instrumentation (MS-2000 XY stage), a CMOS camera by Oxford Instruments (Andor Zyla 4.2), a solid-state light source by Lumencor (SOLA SE Light Engine), and an automated filter turret. This turret was outfitted with eGFP and DAPI filter sets from Chroma Technology Corp, (part no. 49002) and Semrock Inc. (catalog no. DAPI-1160B-NTE), respectively. Imaging was carried out with a 2x objective lens (CFI Plan Apo 1 2x NA 0.10, Nikon) using 2x2 binning settings (resulting in an image resolution of 1024x1024 pixels). The exposure times were set to 500 ms for eGFP, with DAPI exposures at 50 ms.

[0116] Generation of eGFP and 4-MU calibration curves. The concentration of eGFP produced in CFPS buffer was determined by comparing its absorbance at 488 nm to that of a blank CFPS reagent, utilizing a molar extinction coefficient of 56,000 M-1cm_1. This measurement was conducted using a UV-vis spectrophotometer (DeNovix, Wilmington, DE, USA). To correlate the fluorescent signal intensities of eGFP product observed under the microscope with the measured eGFP concentration, the eGFP product droplets were imaged at 2x magnification, droplet heightsof 500 pm, and exposure times of 500 ms. A linear fit of measurements of observed intensity vs. known concentration were performed for each condition and used to estimate eGFP concentrations from images of expressed protein. For the calibration of 4-methylumbelliferone (4-MU, Sigma- Aldrich), solutions of 4-MU in concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.6, and 7.8 pM were prepared and imaged to quantify droplet intensities. A linear fit of observed intensities vs. known concentrations was performed. To estimate substrate turnover, the lineal' fit parameters were used to estimate concentrations from observed fluorescence. The representative fluorescence signal intensity for each droplet was determined by calculating the median signal intensity measured within a circular- region that has a diameter 0.7 times that of the droplet.

[0117] Enzyme kinetics measurements. 4-MUP was loaded onto the substrate slide at varying concentrations (2000, 1000, 500, 250, 125, 62.5, 31.25, and 15.63 pM), each mixed with an equal volume of enzyme droplets to achieve halved concentrations. On a separate slide with the expressed enzyme, dilutions ranging from 8 to 32 times were performed using a merging-and- splitting process with a reaction buffer (100 mM MOPS, 500 mM NaCl, 100 pM ZnCh, pH 8.0) slide. Enzyme and substrate slides were then placed on the microscope stage. The aligner was used to facilitate the merging of droplets from both slides. Time-lapse imaging was conducted in 30 second intervals for one hour.

[0118] Enzyme kinetics measurements with immobilization. 4-MUP was loaded onto the substrate slides at varying concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.63, and 7.81 pM). Substrate slides were mounted on the holder, aligned with the enzyme slides placed on the microscope, and merged to initiate the reaction. Time-lapse imaging was performed at 30-second intervals for one hour. Between each reaction, excessive washing was conducted by floating the enzyme slide on the MOPS buffer solution for its reused. The remaining buffer droplets on the slide were kept intact and the slides are stored in a wet chamber to maintain enzyme hydration. The buffer droplets on the enzyme slide were blotted just before merging with the substrate slide for the next reaction.

[0119] Image processing and intensity quantification. Images were analyzed using a custom Python script. Briefly, images captured at each timepoint were stitched together to form a single composite image. After stitching, outlines and centroids of droplets captured in bright field images were identified, a region of interest (ROI) for each droplet (consisting of a circle with a diameterequaling 0.7 times that of the droplet, centered on the droplet's centroid) was defined, and the median intensity within each ROI was computed at each timepoint.

[0120] Data analysis to estimate rate constants. A custom Python script was used to analyze the initial velocity of the process curve for each droplet. The initial velocity was determined by calculating the slope of the linear region of the curve up to the maximum time that the goodness of fit of the lineal' slope remained equal to or above 0.97. The parameters of the Michaelis-Menten model, fcat and KM, were estimated using the curve_fit function from the SciPy library with initial estimates based on the substrate concentration and reaction rate data from experiments.

[0121] Plate-based assays to quantify enzymatic activity. Concentrations of proteins expressed in tubes, using either Codexis CFPS mix or PURExpress, were initially quantified using a spectrophotometer. Subsequently, these protein solutions were appropriately diluted to become 2x the final concentration. These diluted solutions were dispensed into a 384-well plate, allocating 10 pL to each well. 4-MUP solutions were prepared at various concentrations (2000, 1000, 500, 250, 125, 62.5, 31.25, and 15.63 pM) and introduced into the enzyme-containing wells using a multichannel pipette. A Tecan Infinite M Plex spectrophotometer (Mannedorf, Switzerland) was employed to measure fluorescent intensities every 30 seconds for one hour at Ex355 / Em460.

[0122] Malachite green assay for quantifying inorganic phosphate concentration. The malachite green assay kit (MAK307, Sigma-Aldrich) was purchased and used according to the manufacturer's protocol. A scries of phosphate standard solutions with final concentrations of 40, 32, 24, 16, 12, 8, 4, and 0 pM, containing malachite green, were prepared, and their absorbance was measured at 644 nm. The protein crude product, prepared using the previously described recipe, was diluted to fall within the appropriate measurement range, and its absorbance was measured. The measured values were used to determine the concentrations through a standard curve and then the original concentrations were obtained by multiplying the dilution factors.Example 1Pipeline for high-throughput recombinant enzyme expression and kinetic characterization within droplet arrays

[0123] To collect enzyme kinetics data, a library of DNA templates encoding expression of C- terminally eGFP-tagged variants was printed onto hydrophilic spots patterned within a larger omniphobic surface (either manually or using a robotic printer). To express protein variants, asecond patterned slide containing reagents for cell-free protein expression (CFPS) was ‘stamped’ onto this printed library slide to add CFPS reagents to each spot containing a DNA template. Following the addition of CFPS reagents, slides are incubated for 4 hours at 23°C, allowing each droplet to act as a miniaturized protein-producing bioreactor. Expressed protein can be quantified via eGFP-associated fluorescence, allowing for simultaneous, non-contact quantification of protein expression over time and across variants. To quantify reaction kinetics for each variant in parallel, a third patterned slide was prepared with fluorogenic substrate onto the slide containing expressed protein to start enzymatic reactions and then image over time. Quantifying substrate fluorescence over time makes it possible to quantify reaction progress over time and ultimately determine functional kinetic parameters e.g., kcat, KM, and feat / KM) (Figure 1A).Example 2Patterned slide surfaces drive robust formation of uniform water-in-air droplet arrays

[0124] To fabricate droplet microarray (DMA) slides, a surface-tethered dendrimer modification scheme that proceeds via thiol-ene photoclick reactions and Steglich esterification (Benz, M.; et al., Nat Commun 2020, 11 (1) and Munawar, S.; et al., Heliyon 2024, 10 (1)) was adopted (Figure IB). First, 1 -thioglycerol is covalently coupled to vinylsilanes attached to the glass surface via exposure to UV light, leading to the formation of two hydroxyl groups; subsequent esterification with 4-pcntcnoic acid reintroduces vinyl groups to each hydroxyl. Repeating this process three times expands a single vinyl group to eight vinyl groups, simultaneously increasing the density of chemical moieties and enhancing the surface area of the smooth glass to yield improved hydrophilic and omniphobic properties. Coupling 1 -thioglycerol to the slide surface and covering it with a designed photomask that only allows light to penetrate in circular spots across the slide surface yields an array of hydrophilic spots (Figures 1C and 4); subsequently treating the remaining area with lH,lH,2H,2H-perfluorodecanethiol creates omniphobic barriers with a low surface free energy surrounding each hydrophilic spot. This results in the formation of a virtual well plate consisting of invisible hydrophilic patches and omniphobic walls (Figure ID, left), facilitating the organization and segregation of different chemical or biological samples.

[0125] Aqueous liquids deposited onto hydrophilic and omniphobic surfaces spread out or round up to form droplets, respectively, with the contact angle of the droplet determined by the relative hydrophilicity or hydrophobicity of the surface and properties of the aqueous fluid.Measured contact angles for deionized water pipetted onto hydrophilic and omniphobic regions were 6.1 ± 0.7° and 106.53 ± 1.6°, respectively; the difference between contact angles for cell-free protein expression product was slightly lower (13.4 ± 1.3° and 99.5 ± 2.1° on hydrophilic and omniphobic regions, respectively), which is thought to be due to the adsorption of the expressed protein in the cell-free protein expression product onto the surface or the reduced surface tension of the droplet caused by the expressed protein (Figure. ID, right). Water-in-air droplets remained well-separated over time, enabling the creation of arrays with varying numbers of droplets containing different fluid volumes (Figure IE). To determine the maximum droplet volume that can be accommodated on each hydrophilic spot, the largest volume at which the droplet contact angle stabilized at 100° or less for a 1500 pm droplet was empirically determined and used as a reference point to estimate the other volumes for the various spot sizes (Figure IE).Example 3Water-in-air droplet arrays enable reproducible high-throughput recombinant protein expression

[0126] A stamping process in which two arrays are brought in close proximity vertically such that droplets merge while hydrophobic forces prevent adjacent droplets from mixing was leveraged for high-throughput protein expression by: (1) printing plasmids encoding expression of protein variants on one DMA slide, (2) preparing a second DMA slide with droplets of identical spacing containing reagents required for cell-free protein expression with an automated dispenser (Figure 5), (3) loading DMA slides into a custom-built micro-manipulatable aligner to bring them together to mix reagents, and then (4) incubating to allow protein expression (Figures 2A and B).

[0127] To evaluate DMA protein expression, twelve plasmid templates were generated encoding C-terminally eGFP-tagged variants of the phosphate monoesterase PafA, a model enzyme with previously characterized active site mutants whose catalytic efficiencies span >5 orders of magnitude (Figures 2C and 2D). Slides bearing these templates were stamped with slides containing cell-free expression mixtures and then quantified eGFP fluorescence over time via microscopy to estimate expressed protein concentrations (Figure 2E). Initial trials of protein expression revealed significant evaporation and droplet shrinkage during the 3-hour required incubation at 23°C, altering buffer concentrations and reducing protein expression (Figure 2G). To ameliorate this, a gasket that sits between the two glass slides was designed to prevent dropletevaporation during incubations or enzyme turnover processes, serving as both a sealant and a spacer (Figure 2F). The gasket was made via hot embossing of multiple layers of Parafilm and is cut to occupy approximately 2 mm along the outline of the slides (25 x 75 mm). Before gasket addition, droplets showed a 50% reduction in diameter during a 3-hour incubation at 23°C; after gasket addition, droplet diameters remained unchanged over the same timeframe (Figure 2G).

[0128] Measured intensities for droplets containing printed plasmids encoding expression of either wild-type PafA or single-nucleotide variants showed steady increases in fluorescence over time while negative control droplets lacking plasmids remained dark, as expected (Figure 2E). To quantify protein yields, calibration curves were generated by adding the same volumes of purified PafA-eGFP (86.4 kDa) at known concentrations and quantifying intensity (Figure 6). Calibrated protein intensities across multiple droplets on a given slide showed strongly reproducible variant expression with final concentrations ranging between 500-800 nM protein in 900-nL droplet volumes (38.9-62.2 ng per variant) (Figure 2H). Measured fluorescence intensities were uncorrelated with previously determined catalytic efficiencies for each variant, confirming that phosphate monoesterase activity does not impact expression (Figure 7). Protein expression was relatively constant across different variants and did not correlate with either droplet position within a slide or experimental replicate (Figures 2H and 8).Example 4Water-in-air droplet arrays enable quantitative kinetic measurements of enzymatic turnover with a wide dynamic range within low volumes

[0129] Next, DMA stamping was used to enable high-throughput and quantitative measurements of enzyme catalysis by: (1) merging a DMA array containing expressed enzyme variants with a second array containing fluorogenic substrate, and then (2) imaging over time to detect the production of a fluorescent product (Figure 3A). To monitor PafA phosphatase activity, a coumarin-based fluorogenic 4-Methylumbelliferyl Phosphate (4-MUP) substrate was employed that yields a fluorescent 4-Methylumbelliferone (4-MU) product upon hydrolysis (Figure 3B and Equation 1). Fluorescent images of PafA- variant-containing droplet arrays after stamping to fluorogenic 4-MUP substrates showed variant-dependent differences in measured fluorescence intensities over time consistent with enzymatic turnover (Figure 3C). As expected, wild- type PafA yielded the highest enzymatic activity; the R164A and T79S mutant constructs showed slightlyand dramatically lower enzymatic activities, respectively. Negative control droplets containing all of the reagents required for cell-free protein synthesis except for plasmid template showed no increased fluorescence with time, establishing little to no background hydrolysis in the absence of expressed enzyme (Figure 3C).

[0130] Using calibration curves to convert measured intensities to the amount of product generated allowed quantitative estimation of substrate turnover over time within each droplet (Figures 10 and 13); plotting initial rates as a function of substrate concentration and fitting to the Michaelis-Menten equation then allowed estimation of kinetic parameters (fcat, KM, and kcai / K ) (Figures 11 and 14). As cell-free expression mixtures contain substantial inorganic phosphate (a known ground state inhibitor for PafA). (1) the amount of inorganic phosphate within cell-free expression mixtures was quantified using a standard malachite green assay (Figure 6), and then (2) both the initial velocities at each substrate concentration and the fitted KM were corrected using competitive inhibition model equations (Equations 2, 3, and Figure 9) For each mutant, previously- measured inhibition constants were used for each variant and inorganic phosphate levels were measured to yield accurate values. Measured kcat / K values were highly reproducible across mutants and spanned nearly 5 orders of magnitude (Figures 3F, 12, and 15), with the lowest resolvable activity of ~ 14.6 M^s'1for a catalytically compromised K162A variant.Equation 1 : 4-MUP reaction catalyzed by PafAEquation 2: Correction of KM with inhibition constant (Ki) and inhibitor concentration ([I])Equation 3: Correction of initial velocities (Vi) at substrate concentration ([S]) with inhibition constant (Ki) and inhibitor concentration ([I])Example 5Water-in-air droplet array measurements recapitulate traditional plate-based values

[0131] To assess the accuracy and precision of DA-MEK catalytic activity measurements, activities for the same set of variants were measured catalytic via a traditional plate-based assay using the same cell-free expression mix and values obtained via DA-MEK and plate-based assays were directly compared (Figures 3G-I and 16). DA-MEK measurements of 'cai / K i agreed extremely well with plate-based measurements (r2= 0.89 and RMSE = 0.20) (Figure 3G), establishing the ability to acquire high-quality estimates of catalytic activity while consuming 11- fold fewer reagents per reaction. Although DA-MEK experiments quantified turnover for enzymes in solution and in the presence of cell-free expression mix while prior HT-MEK experiments quantified turnover for surface-immobilized enzymes following washing and purification, the results from both techniques also showed strong agreement with one another (r2= 0.893 and RMSE = 0.663), as shown in Figure 3H. The largest measured disagreement was for PafA N100A (3.37 vs 4.89); as N100A is strongly inhibited by inorganic phosphate ions, this discrepancy likely results from uncertainty in measurements of the amount of background inorganic phosphate within the DA-MEK arrays following cell-free protein expression.Example 6Immobilization of enzymes enables efficient and reliable measurement of the kinetics of various variants across a range of inhibitor affinities

[0132] Many enzymes of interest cannot be accurately characterized within cell-free expression mixtures due to either the presence of enzymes with competing activities (e.g., polymerases and tRNA synthetases) or components that inhibit hydrolysis (e.g., high amounts of inorganic phosphate). To enable high-throughput immobilization and purification of expressed enzymes, hydrophilic surfaces were iteratively patterned with antibodies to enable an affinitybased immobilization and purification strategy (Figure 4A). Specifically, hydrophilic spots were sequentially coated with biotinylated BSA (bBSA), NeutrAvidin (NA), and biotinylated anti- eGFP antibody (b-anti-eGFP Ab); between each coating step, the patterned slides were washed by ‘stamping’ patterned slides to slides containing buffer alone and blotted using an absorbent pad. To evaluate and optimize this washing method, the remaining fluorescence intensity of 1000 pM 4-MU droplets was measured after applying different numbers of washing cycles. Twowashing cycles effectively removed > 99% of 4-MU; for all subsequent reactions, the slides were washed > 3 cycles per step.

[0133] Next, the ability to immobilize and accurately quantify kinetics for 20 PafA variants (A80V, D163V, G458V, G537V, H83V, 1496G, K162A, L527G, N100A, N 100A / R164A, R164A, R164V, S110V, S487V, T321G, T79G, T79S, W102V, WT, Y435V) was evaluated. As several variants are strongly inhibited by inorganic phosphate (e.g., T79S: 13 pM, H83V: 94 pM, G458V: 200 pM), the measured enzymatic activities after washing provide a quantitative readout of elimination of cell-free protein synthesis reagents via immobilization and washing. To precisely control the concentration of immobilized enzyme, surfaces were exposed to systematically varied ratios of bBSA and b-anti-eGFP during the final patterning step (thereby tuning the surface density of presented anti-eGFP antibodies) (Figures 19B and 19C). After washing, measured eGFP intensities for surface-immobilized enzymes decreased with decreasing anti-eGFP surface density (R2= 0.9838, RMSE = 58.70) (Figure 19D). After ‘stamping’ to a slide bearing 4-MUP substrate, measured initial rates for eGFP-tagged and immobilized WT PafA varied linearly with eGFP intensity (R2= 0.9718, RMSE = 0.4845), confirming that immobilized enzymes were properly folded and active (Figure 19E). Finally, the ability to fully remove cell-free protein synthesis reagents was assessed by iteratively ‘stamping’ the slide with immobilized enzyme variants to 8 substrate concentrations, quantifying initial velocities, and washing between concentrations (Figure 19F). Michaclis-Mcntcn fits to initial velocities as a function of substrate concentration yielded kM / K\\ values in strong agreement with solution DA- MEK values after correction for phosphate inhibition (R2= 0.9311, RMSE = 0.3931) and previously reported HT-MEK values for purified enzymes (R2= 0.9226, RMSE = 0.3276) (Figures 19G and 19H).

[0134] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0135] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0136] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMSWhat is claimed is:

1. A method for high-throughput enzyme analysis comprising: patterning a library of polynucleotides encoding a plurality of enzymes in individual hydrophilic areas on a wettability patterned solid surface, wherein each hydrophilic area comprises a droplet comprising at least one polynucleotide encoding a different enzyme; contacting each droplet with in vitro transcription and / or translation reagents; and incubating under conditions for cell-free protein synthesis to produce the enzymes.

2. The method of claim 1, wherein the polynucleotides are plasmids or linear double stranded DNA templates.

3. The method of claim 1 or 2, wherein the plurality of enzymes comprises one or more enzyme variants or fragments thereof of an enzyme of interest.

4. The method of claim 3, wherein the enzyme of interest is a wild-type enzyme.

5. The method of claim 4, wherein the plurality of enzymes comprises variants or fragments of the wild-type enzyme having one or more amino acid substitutions, additions, or deletions as compared to the sequence of the wild-type enzyme.

6. The method of any of claims 1-5, wherein the plurality of enzymes comprises one or more artificially generated enzymes.

7. The method of any of claims 1-6, wherein each droplet is less than 250 uL in volume.

8. The method of any of claims 1-7, wherein each droplet is less than 1 uL in volume.

9. The method of any of claims 1-8, wherein the contacting comprises stamping the solid surface comprising the library of polynucleotides with a second wettability patterned surface comprising the in vitro transcription and / or translation reagents.

10. The method of any of claims 1-9, wherein the method further comprises determining enzyme concentration in each droplet following incubation.

11. The method of any of claims 1-10, wherein the method further comprises immobilizing the enzymes.

12. The method of claim 11, wherein the immobilizing comprises stamping a patterned solid surface comprising the enzymes with a third patterned surface comprising a surface-immobilized binding partner for each of the enzymes.

13. The method of claim 12, wherein the binding partner is a nucleic acid, a protein, a peptide, a small molecule, or a combination thereof.

14. The method of claim 12 or 13, wherein the binding partner comprises an antibody.

15. The method of any of claims 11-14, wherein the method further comprises washing immobilized enzymes.

16. The method of claim 15, wherein the washing comprises stamping the solid surface comprising the immobilized enzymes with one or more fourth patterned solid surface comprising washing buffer and / or buffer of interest.

17. The method of any of claims 1-16, further comprising: contacting the enzymes or immobilized enzymes with enzymatic assay reagents; incubating under conditions for desired enzymatic reaction; and detecting enzymatic reaction at one or more timepoints during incubation.

18. The method of claim 17, wherein the contacting comprises stamping a patterned solid surface comprising the enzymes or immobilized enzymes with a fifth patterned surface comprising the enzymatic assay reagents.

19. The method of claim 17 or 18, wherein the enzymatic assay reagents comprise a substrate for the enzyme.

20. The method of claim 19, wherein the substrate is a fluorogenic, chromogenic, or luminescent substrate.

21. The method of any of claims 17-20, wherein the enzymatic assay reagents comprise a buffering system, a cofactor, a detection system, one or more inhibitors, one or more activators, or a combination thereof.

22. The method of any of claims 17-21, wherein detecting enzymatic reaction comprises fluorescence, absorbance, mass spectrometry, or a combination thereof.

23. The method of any of claims 17-22, further comprising determining one or more enzymatic parameters for each enzyme.

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