Use of DNA encoded libraries

Abstract

Provided herein are methods using DNA encoded libraries. The methods do not require a heat treatment step and are suitable for use on devices such as electrowetting-on-dielectric (EWoD) devices which may have surfaces that are not stable to heat treatment.

Description

[0001] Use of DNA encoded libraries

[0002] FIELD OF THE INVENTION

[0003] DNA-encoded libraries (DELs) are a revolutionary technology in the field of drug discovery and chemical biology. These libraries use nucleic acid sequences as a unique and versatile tag to encode information about individual chemical compounds, allowing for the rapid and efficient screening of millions to billions of small molecules for their ability to bind to specific biological targets. DNA-encoded libraries have gained popularity in the pharmaceutical industry due to their efficiency, cost-effectiveness, and the potential to uncover new drug candidates. They offer a powerful tool for identifying lead compounds and advancing the drug discovery process, ultimately contributing to the development of novel therapeutics and drug candidates.

[0004] Key features of DNA-encoded libraries include:

[0005] 1. DNA Tagging: Each chemical compound in a DEL is covalently linked to a DNA fragment, which serves as a unique identifier for that compound.

[0006] 2. High Diversity: DELs can contain vast numbers of different compounds, providing a highly diverse set of molecules for screening.

[0007] 3. Rapid Hit Identification: DELs enable the identification of hit compounds that bind to a target of interest in a high-throughput manner, significantly speeding up the drug discovery process. Researchers can simultaneously test a large number of compounds by exposing them to a target of interest, followed by DNA-based identification of the binding molecules.

[0008] 4. Iterative Screening: DELs allow for iterative rounds of screening, in which identified hits can be further optimized and tested for improved binding affinity and selectivity.

[0009] 5. Data-Driven Drug Discovery: The encoded DNA tags enable the generation of extensive structure-activity relationship (SAR) data, which helps in understanding the interactions between compounds and targets.

[0010] 6. Reduction in Sample Handling: Since the DNA tags store information about compound identity, researchers can work with smaller quantities of each compound, reducing the need for extensive sample handling and storage.

[0011] A DEL is a mixture of large numbers (millions to billions) of drug-like molecules of small molecular weight, where each molecule is conjugated to a specific and unique DNA-barcode that encodes its chemical structure. The composition of a DEL mixture can be readily interrogated before or after interacting with a protein of choice following an amplification step and sequencing. The identification of DEL labels associated with the protein of interest allows deduction of the identity of the corresponding binding molecules which selectively bind to the target. A typical DEL molecule is a hybrid molecule harbouring a DNA label linked with a chemical linker to a small molecular weight molecule that was generated by serial addition of building blocks (BBs) onto a scaffold, using a combinatorial approach that most often is based on a split-and-pool protocols. The combinatorial approach gives rise to libraries containing millions to billions of compounds within a few synthetic chemical steps.

[0012] Screening of DNA encoded libraries is used to determine the members in the library with high affinity for binding to a biological target, typically a protein, usually bearing an affinity tag. The component steps of the screen are listed below:

[0013] 1. Binding of DNA-encoded library to the biological target, typically a protein, to form a complex with a subset of the library members.

[0014] 2. Binding of complex through the affinity tag to a solid support, e.g. magnetic beads.

[0015] 3. Washing of solid support to remove unbound library members.

[0016] 4. Elution of bound library members from the bound complex.

[0017] Steps 1 and 2 can be carried out in either order.

[0018] Typically, multiple selection rounds (2-3) are carried out to enhance the discrimination of the selection. Determination of the highest affinity library members (“hits”) is carried out via amplification of the DNA codes into a form amenable to sequencing and sequencing of the amplified DNA to reveal the most abundant library members.

[0019] LIS2013288929 discloses methods for creating and using display libraries. The libraries are bound to supports and then the bound material identified by using heat based amplification (PCR) to recover the immobilised sequences. The amplification using thermocycling is not an applicable technique for recovery of material on a digital microfluidic device as the device is not sufficiently stable to tolerate exposure to the high temperatures (typically > 90 °C) used to denature DNA.

[0020] SPIE BiOS Vol 10061 2017 describes a Lab-on-a-chip platform for high throughput drug discovery with DNA encoded chemical libraries. As with the above technique, library members retained after washing away unbound members are PCR amplified and identified using sequencing. Methods using high temperatures for amplification are not amenable to digital microfluidic devices.

[0021] EP4257978 relates to a method of screening molecules. The screening uses a library which can contain suitable sequence specific peptide cleavage sites for releasing part of the peptides and thus cleaving the tags from the library members. In order to be screened the library must contain the peptide for cleavage in addition to the variable tags and the binding members.

[0022] WO2023281273 involves selectively illuminating microdroplets on a electrowetting array in order to screen interactions between a biological entity and a molecule. Whilst the application refers to the use of a tagged library, the method used for the analysis of the tags is not disclosed.

[0023] The inventors herein have improved the screening and identification process to overcome limitations in the prior art, particularly in relation to the use of DELs on microfluidic devices.

[0024] SUMMARY OF THE INVENTION

[0025] It is desirable to carry out the DEL / target screening protocol using a microfluidic device to conserve the scarce and valuable DNA encoded library and to automate the protocol to improve reliability and reproducibility, making the screening less labour intensive. To carry out the procedure using a microfluidic device, all steps must be fully compatible with the device and its operation. This is most important if multiple selection rounds are carried out using the device.

[0026] After binding to the target, the binding interaction is denatured in order to recover and identify the bound material. Typically the denaturing / elution is carried out by thermal denaturation of the bound protein / target interaction. However a thermal denaturation step may be incompatible with the construction and operation of the microfluidic device and also with reservoirs of thermally unstable materials used in the screen e.g. the protein held on the device. Hence a method for ambient elution of the DNA encoded library is highly desirable if a microfluidic device is used for the screen.

[0027] Provided herein are methods and kits suitable for the use of DNA Encoded Libraries (DEL’s) on digital microfluidic devices. Disclosed is a method of screening interactions between a DNA Encoded Library of chemical compounds (DEL) and a protein target using droplets on a digital microfluidic device comprising the steps of: a. binding the DNA encoded library to beads via the protein target in one or more droplets on the device; b. removing the unbound library from the beads; c. washing the beads; d. elution of the DNA encoded library from the beads by enzymatically digesting the protein target using a protease; and e. removing, denaturing or inhibiting the protease.

[0028] Disclosed is a method of screening interactions between a DNA Encoded Library of chemical compounds (DEL) and a protein target using droplets on a digital microfluidic device comprising the steps of: a. binding the DNA encoded library to beads via the protein target in one or more droplets on the device; b. removing the unbound library from the beads by holding the beads and reducing the volume of the droplet; c. washing the beads by adding additional droplets to the beads; d. elution of the DNA encoded library from the beads by enzymatically digesting the protein target using a protease; and e. denaturing or inhibiting the protease.

[0029] The DEL may be exposed to the beads having the immobilised protein target. Alternatively the DEL may be exposed to the protein target which is then immobilised on the beads. The binding and selection steps may be repeated. Steps a-e may be repeated one or more times. The steps may be repeated in further droplets on the digital microfluidic device.

[0030] Droplets may be manipulated on the device using electrowetting. Further droplets can be added in order to dilute reagents or wash particles. Particles can be separated from droplets, either by holding the droplets and removing the particles, or by holding the particles and moving the droplets (or both the particles and droplets can be moved in opposing directions). After selection, the nucleic acid, which may be DNA, is typically amplified and sequenced to identify the most abundant library members.

[0031] One of the benefits in the use of droplets on digital microfluidic devices is the small droplet volume (typically less than 1 iL per droplet). A further benefit is the ability to simultaneously handle large numbers of droplets in parallel. Thus a DEL can be screened against a variety of targets at the same time. The DEL may be exposed to a variety of protein targets. For example the protein targets may be a selection of length variants or truncations. The protein targets may be sequence variants or isoforms.

[0032] The selection process may involve screening against functionally related protein targets in order to identify selective binders. For example the selection may be performed against functional proteins, for example protein kinases in order to identify binders which are selective for a particular kinase. Screening may be performed in the presence of a known protein binding moiety along with the DEL to look for competitive binding at a particular site. The protein target may be pre- loaded with the binder or performed as a competitive assay.

[0033] The binding may be tested at a variety of conditions, for example ionic strength or pH. The screening may be performed in parallel at a variety of different conditions in different droplets, for example a variety of screening conditions including pH, buffers, salts, detergents or temperature. The use of electrodes allows localised heating, hence some droplets can be operated at different temperatures to other droplets.

[0034] Purified proteins may be made added to the device. Alternatively the protein targets may be expressed and purified on the digital microfluidic device.

[0035] After the binding step has been performed, the beads, which may optionally be magnetic beads, can be manipulated on the device. The beads may be held in one location whilst the surrounding droplet is moved, thus separating and purifying the immobilised material. The droplets can be held on one location and the beads moved, again thus separating and purifying the immobilised material. The beads may be exposed to further droplets to dilute and / or wash the bead to remove loosely or non-adhered material. The majority of the library does not adhere to the beads and is thus removed. After binding and washing, the beads are treated to remove the selected DEL components.

[0036] Where the beads are magnetic, the beads may be immobilised using localised magnets, which may be external to the digital microfluidic device.

[0037] In order to identify the bound material, the selected DEL members are removed from the supports. Whilst prior art DEL selection generally relies on thermal treatments or the use of chemical denaturants, such treatments are typically incompatible with digital microfluidic devices. The use of high temperatures or chemical treatments generally destroys the hydrophobic layers needed for droplet handling operations. The inventors herein have therefore developed the use of a mild denaturing step based on the use of one or more proteases to digest or cleave the protein targets, thereby releasing the DEL members from the supports. The released DEL can be removed from the device for amplification and sequencing as required.

[0038] In order to amplify the nucleic acid, a nucleic acid polymerase is required. If the protease is not removed or made inactive prior to amplification, the protease inhibits amplification of the nucleic acid tags. Thus the protease should be removed or rendered inactive prior to amplification. The protease must also be removed or rendered inactive if further library selection steps are used, otherwise the protease digests the target and thus prevents selection of the DEL. The chosen protease may be rendered inactive using a protease inhibitor. Alternatively if the final library members are removed from the device, the protease may be thermally or chemically denatured. Thus step e may be performed on or off device. Where the denaturing or inhibiting the protease is performed on device, a chemical protease inhibitor is used which is compatible with the device. Alternatively the protease may contain a binding moiety allowing physical removal, for example using an affinity support.

[0039] Any particular protease and inhibitor combination may be used. For example the protease may be a serine protease such as trypsin or proteinase K. The protease may be an ATP- dependent protease (e.g., Lon protease or CIpXP protease) The protease inhibitor may be a serine protease inhibitor such as for example (4-(2-aminoethyl)benzoylsulfonyl fluoride, AEBSF).

[0040] The added protease may cleave the protein of interest from the support. The protease may be a serine, threonine, cysteine or methionine protease. The protease may be a metalloprotease. The protease may be trypsin. The protease may be proteinase K. The protease may be a TEV or 3C protease. The inhibitor may be a serine, threonine, cysteine or methionine protease inhibitor. The protease may be an ATP dependent protease. CIpXP is an typical AAA+ protease, consisting of CIpX and CIpP. CIpX is an ATP-dependent protein unfoldase and polypeptide translocase, whereas CIpP is a self-compartmentalized peptidase.

[0041] The TEV protease may act upon the amino acid sequence ENLYFQS (SEQ ID No 1). The template for expression of the POI may include the nucleic acid sequence GAGAACCTGTACTTCCAGAGC (SEQ ID No 2).

[0042] The 3C protease may act upon the amino acid sequence LEVLFQGP (SEQ ID No 3). The template for expression of the POI may include the nucleic acid sequence CTCGAGGTTCTGTTCCAAGGACCT (SEQ ID No 4).

[0043] The digital microfluidic device may be an electrowetting-on-dielectric (EWoD) device. The device may comprise a planar array of electrodes. The device may comprise an array of active-matrix thin-film transistors.

[0044] The method may comprise the steps of: a. expressing a variety of proteins in droplets on a digital microfluidic device; b. binding the expressed protein to magnetic beads to purify the expressed proteins; c. binding a DNA encoded library to the magnetic beads via the expressed protein; d. removing the unbound library from the beads by holding the beads and reducing the volume of the droplet; e. washing the beads by adding additional droplets to the beads; f. elution of the DNA encoded library from the beads by enzymatically digesting the protein target using a protease; and g. denaturing or inhibiting the protease.

[0045] Disclosed is a kit comprising: i. a DNA Encoded Library of chemical compounds (DEL); ii. magnetic beads; iii. an electrowetting device; and iv. a protease;

[0046] The kit may further comprise reagents for cell-free protein expression to express protein targets having capture moieties to enable attachment to the magnetic beads.

[0047] The kit may further comprise a protease inhibitor.

[0048] The kit may further comprise a variety of nucleic acid template sequences coding for a variety of target proteins.

[0049] FIGURES

[0050] Figure 1 shows an outline scheme of DNA Encoded Library (DEL) selection for proteins

[0051] Figure 2 shows the distribution of sequences in a synthesised library.

[0052] Figures 3a & b show two-dimensional plots of two sections through the three-dimensional dataset of the unselected library at cycle 1 , synthon 3 and cycle 2, synthon 3.

[0053] Figure 4 shows the distribution of sequences containing all three tag sequences is a selected library using thermal elution.

[0054] Figures 5a & b show two-dimensional plots of two sections through the three-dimensional dataset of DNA sequences at cycle 1 , synthon 3 and cycle 2, synthon 3. These illustrate the high degree of selection towards certain members of the test library when compared with the corresponding plots for the unselected library.

[0055] Figure 6 shows the distribution of sequences after one round of selection with trypsin elution.

[0056] Figure 7 shows the distribution of sequences after two rounds of trypsin elution.

[0057] Figure 8 shows the distribution of sequences after 1 round of trypsin and one round of thermal elution, which is similar to that after two rounds of trypsin elution.

[0058] Figures 9a & b show two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after two rounds of trypsin elution at cycle 1 , synthon 3 and cycle 2, synthon 3, showing a high degree of enrichment of selection for certain sequences.

[0059] Figures 10a & b show two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after one rounds of trypsin elution and one round of thermal elution at cycle 1 , synthon 3 and cycle 2, synthon 3.

[0060] Figure 11 shows the distribution of sequences from sequencing of five replicates run in parallel on a microfluidic device, each identified with unique indices.

[0061] Figures 12 a & b shows two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after microfluidic device selection, indicating a high degree of selection for certain sequences at cycle 1 , synthon 3 and cycle 2, synthon 3.

[0062] Figures 13a-c show the distribution of sequences after one round of selection with proteinase K elution (Figure 13a), two rounds of proteinase K elution (Figure 13b) and one round of proteinase K elution and one round of thermal elution (Figure 13c).

[0063] Figures 14a & b show two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after two rounds of proteinase K elution at cycle 1 , synthon 3 and cycle 2, synthon 3, showing a high degree of enrichment of selection for certain sequences.

[0064] Figures 15a & b show two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after one rounds of proteinase K elution and one round of thermal elution at cycle 1 , synthon 3 and cycle 2, synthon 3. Figure 16 shows the distribution of sequences from a selection experiment using four replicates run in parallel on the device, each identified with unique indices, and pooling of the data.

[0065] Figure 17a & b show two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after microfluidic device selection, indicating a high degree of selection for certain sequences at cycle 1, synthon 3 and cycle 2, synthon 2.

[0066] DETAILED DESCRIPTION OF THE INVENTION

[0067] DNA-encoded libraries (DELs) are a revolutionary technology in the field of drug discovery and chemical biology. These libraries use nucleic acid sequences as a unique and versatile tag to encode information about individual chemical compounds, allowing for the rapid and efficient screening of millions to billions of small molecules for their ability to bind to specific biological targets. Disclosed herein are methods to enable DEL screening on a digital microfluidic device. One problem solved by the inventors herein is the ambient elution of the selected library members from the immobilised proteins, and the subsequent processing of the library members in order to identify hits from the screen.

[0068] The invention is described in the claims. The invention applies to any encoded library. The library is typically a DNA encoded library. The library members are screened for binding to target proteins and the binding members selected. In order to identify the binding members of the library, the encoding tags need to be recovered.

[0069] Disclosed is a method of screening and recovering a DEL in a digital microfluidic device. The droplets having the components required DEL screening, can be manipulated by electrokinesis in order to effect and improve binding, washing and recovery.

[0070] The use of EWoD devices can screen large numbers of closely related conditions in parallel in droplets on the device. The droplets can be blended on the device in order to prepare the reagents. For example a DEL can be supplemented with a variety of additional components at a selection of concentrations. For example a salt screen, buffer screen or pH screen can be performed across a range of conditions and at variable concentrations. Described herein is the preparation of a variety of different conditions on a single device for the purposes of simultaneously screening a variety of binding conditions.

[0071] Disclosed herein is a method for the handling of a DEL screening assay on an electrowetting- on-dielectric (EWoD) device. The method comprises exposing the DEL to the protein (either before or after the protein is immobilised on a solid support), washing the support to remove the unbound members and then recovering the bound members from the support without damaging the device. The method may use a protease to denature the protein target and thereby recover the selected DEL members. The protease activity is removed prior to either further rounds of selection or amplification of the library to decode the identity of the binding members. The protease activity can be removed on or off device. Where the protease is removed off device an suitable treatment can be used, including heat denaturation or chemical denaturation. Where the activity is removed on device, an ambient method such as the addition of inhibitors may be used.

[0072] Pharmacophore libraries are most commonly constructed using DNA-recorded synthesis, which relies on the use of split-and-pool procedures. Libraries are built up through a series of chemical transformations in multiple steps, each of which is encoded by the addition of either double-stranded or single-stranded DNA fragments (dsDNA or ssDNA) that uniquely identify them. In a typical construction procedure, n different chemical building blocks are encoded using the same number of DNA fragments. In general, DNA fragments only differ by a short sequence, typically 6-7 base pairs, which serve as a barcode to identify each chemical building block. A 6 base sequence of the 4 nucleic acid bases can code up to 4A6 (4096) varieties of n. After the first step, all the individual DNA-conjugated small molecules are pooled together, which can be subsequently split into m number of different wells, allowing a second cycle of chemical transformation and DNA tag elongation to yield mxn library compounds. Again if a 6 base tag sequence is used for 4096 variations of m, the resulting 12 base sequence can code 4096x4096 (>16 million) library members. The split-and-pool procedure can be iterated multiple times (most of the DELs typically involve 2-3 cycles). Typical library sizes are at least 1 million discreet chemical species.

[0073] In the case of double stranded (ds) DNA, DNA fragments can be ligated using overhang tags, while single stranded (ss) DNA fragments can be assembled using splint-mediated ligations. If desired, ssDNA DEL encoding can be converted into the corresponding dsDNA format by the use of polymerase extension from a complementary oligonucleotide primer. Alternative strategies such as chemical ligation have also been developed. Either ss or ds molecules can be used as either can be recovered and amplified.

[0074] Solid-phase affinity-based selections rely on the immobilization of the proteins of interest on solid supports, which are successively incubated with a DNA-encoded library. Preferentially enriched binders are separated from other library members after affinity capture by washing steps. Suitable solid supports for affinity capture include magnetic beads. Proteins may be immobilized either by covalent modification of lysine residues or by non-covalent capture of suitable tags (e.g., biotin, His-tag, or other peptide binding tags). Washing conditions and the use of detergents may significantly influence the selection output. In certain cases, immobilization of the protein of interest on a solid support may impair its folding, thus contributing to the isolation of false positives. Non-specific binding with the matrix may also lead to an increase in the noise level of the selections, thus hindering the discrimination between real binders and artifacts. Non-specific binding events can be avoided using competing double stranded DNA, proteins such as bovine serum albumin, biotin, or imidazole as blocking agents. The simplicity of affinity capture methodologies has made them attractive for many screening campaigns and they have successfully been used for the identification of DEL-derived hits.

[0075] The protein targets may be attached to beads. The protein beads may be prepared off device then dispersed into droplets on device. Alternatively the protein targets may be expressed on the device. If the expressed protein is produced using a suitable binding tag, the proteins can be captured onto solid supports to allow for further interactions with the DEL’s to be probed on the same device. Thus disclosed herein are methods to probe the interactions between proteins expressed on electrowetting devices and DEL’s.

[0076] Disclosed is a method for the synthesis of a protein on an electrowetting-on-dielectric (EWoD) device, the method comprising taking a reaction system having at least one template nucleic acid encoding a protein of interest, blending droplets to form a series of droplets having varying cell-free reagent compositions, including enzymes for protein synthesis and the template nucleic acid and monitoring the synthesis of the protein of interest in the various compositions, thereby identifying a composition suitable for expression of the protein of interest, followed by probing binding using a DEL.

[0077] Electrowetting is the modification of the wetting properties of a surface (which is typically hydrophobic) with an applied electric field. Microfluidic devices for manipulating droplets or magnetic beads based on electrowetting have been extensively described. In the case of droplets in channels this can be achieved by causing the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic channel defined by the walls of a cartridge or microfluidic tubing. Embedded in the walls of the cartridge or tubing are electrodes covered with a dielectric layer each of which are connected to an A / C biasing circuit capable of being switched on and off rapidly at intervals to modify the electrowetting field characteristics of the layer. This gives rise to the ability to steer the droplet along a given path. As an alternative to microfluidic channel systems, droplets can also be generated and manipulated on planar surfaces using digital microfluidics (DMF). In contrast to channel based microfluidics, DMF utilizes alternating currents on an electrode array for moving fluid on the surface of the array. Liquids can thus be moved on an open-plan device by electrowetting. Digital microfluidics allows precise control over the droplet movements including droplet fusion and separation.

[0078] Digital microfluidics can be carried out in an air-filled system where the liquid drops are manipulated on the surface in air. However, at elevated temperatures or over prolonged periods, the volatile aqueous droplets simply dry onto the surface by evaporation. This issue is compounded by the high surface area to volume ratio of nanoliter and microliter sized drops. Hence air-filled systems are generally not suitable for protein assays where the temperature of the system needs to be maintained at a temperature suitable for binding activity and the duration of the synthesis needs to be prolonged for synthesized protein levels to be detectable.

[0079] Protein expression typically requires an ample supply of oxygen. The most convenient and high yielding way to power CFPS is via oxidative phosphorylation where O2 serves as the final electron acceptor; however, there are other ways that involve replenishing with energy molecules not involved in oxidative phosphorylation. In a confined microfluidic or digital microfluidic system of droplets, insufficient oxygen is available to enable efficient protein synthesis. The requirement for oxygen is highest when a cellular lysate system is used for expression. Reconstituted systems do not require as much oxygen and do not consume oxygen via metabolic pathways.

[0080] Described herein are improved methods allowing for the cell-free expression of peptides or proteins in a digital microfluidic device. Included is a method for the cell-free expression of peptides or proteins in a microfluidic device wherein the method comprises one or more droplets containing a nucleic acid template (i.e. , DNA or RNA) and a cell-free system having components for protein expression in an oil-filled environment, and moving said droplets using electrokinesis. The components for the cell-free protein synthesis droplet can be pre-mixed prior to introduction to or mixed on the digital microfluidic device.

[0081] The droplet can be repeatedly moved for at least a period of 30 minutes whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows oxygen to be supplied to the droplet and dispersed throughout the droplet. The act of moving improves the level of protein expression over a droplet which remains static.

[0082] The droplet can be moved using any means of electrokinesis. The droplet can be moved using electrowetting-on-dielectric (EWoD). The electrical signal on the EWoD or optical EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors, or digital micromirrors.

[0083] The filler liquid may be a hydrophobic or non-ionic liquid. For example the filler liquid may be decane or dodecane. The filler fluid may be a silicone oil such as dodecamethylpentasiloxane (DM PS). The filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85. The oil can be oxygenated prior to or during the expression process. Alternatively, the device can be an air-filled device where droplets containing cell-free protein synthesis reagents are rapidly moved into position and fixed into an array under a humidified gas to prevent evaporation. Humidification can be achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs. Additionally, humidification can be achieved by connecting an aqueous reservoir to an enclosed or sealed digital microfluidic device. The aqueous reservoir can have a defined temperature or solute concentration in order to provide specific relative humidities (e.g., a saturated potassium sulfate solution at 30 °C).

[0084] The droplets can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid with a second droplet containing a cell-free extract having the components for protein expression to form a combined droplet capable of cell-free protein synthesis.

[0085] The droplets can be split on the device either before or after expression. Included herein is a method further comprising splitting the aqueous droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one or more of the split droplets are merged with the DEL library droplets for screening.

[0086] To date, digital microfluidics, electrowetting-on-dielectric (EWoD), and electrokinesis in general have only found limited uses in cell-free biological-based applications, mostly due to biofouling, where biological components such as proteins, nucleic acids, crude cell extracts and other bioproducts adsorb and / or denature to hydrophobic surfaces. Biofouling is well known in the art to limit the ability of EWoD devices to manipulate droplets containing biomacromolecules. Wheeler and colleagues report that the maximum actuation time for droplets on EWoD devices containing biological media is 30 min before biofouling inhibits EWoD-based droplet actuation (Z_angmt / / r 2011 , 27, 13, 8586-8594).

[0087] Reconstituted reagents for cell-free protein synthesis are commercially available. For example PUREfrex® kit is a reconstituted in vitro Coupled Transcription / Translation Systems, completely different from an E.coli extract S30 system. By adding DNA or mRNA that encodes the target protein to the reaction solution, proteins can be synthesized easily and quickly without using living cells (https: / / purefrex.genefrontier.com / ).

[0088] The cells used for expression may be of different origin from the cells from the reconstituted expression system is derived. The cells may be for example mammalian cells, insect cells, prokaryotic cells, yeast cells, plant cells or protozoa. The cells may be derived from human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO), HeLa cells, BHK21 cells, NSO cells, Sp2 / 0 cells, Escherichia coli cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, tobacco cells, wheat cells, or Leishmania tarentolae cells.

[0089] Where the synthesis is performed on a microfluidic device, the mixture can be optimised by combining reagents on the device. For example concentrations of reagents that give optimal expression can be identified by blending components at different ratios in a large number of droplets and the expression monitored in parallel to identify optimal compositions. For example a variety of tRNA compositions can be used to express each of the one or more sequences.

[0090] The expressed protein may be fused to a peptide tag. The peptide tag may be one component of a fluorescent protein and the further polypeptide a complementary portion of the fluorescent protein. The fluorescent protein could include sfGFP, ccGFP, GFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmllRFP, miRFP670nano. For example the peptide tag may be GFPn and the further polypeptide GFP1.10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherrymo. The peptide tag may be CFASTn or CFAST10 and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog.

[0091] The binding tag or GFPn or GFP1.10 can be fused to the protein of interest through an amino acid linker. In one embodiment, the oligopeptide, peptide, or polypeptide linker can be 0 - 50 amino acids. The protein targets may be eukaryotic proteins The eukaryotic proteins may be mammalian or human.

[0092] The droplets can be formed before entering the microfluidic device and flowed into the device. Droplets can be formed from reagent reservoirs on the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing DNA Encoded Library with a second droplet containing protein target.

[0093] The droplets can be split on the device either before or after binding. Included herein is a method further comprising splitting the aqueous droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one or more of the split droplets are merged with additive droplets for screening.

[0094] In order to optimise or study binding, the binding system can be supplemented with additional components. The additional components may include salts, co-factors, buffers, surfactants, chaperones or additional protein components.

[0095] The expressed protein may contain a variety of flanking tags to enhance solubility or purification. The expressed protein may contain a sequence acting as a solubility enhancer, for example selected from:

[0096]

[0097] The expressed protein may contain a sequence acting as binding moiety for purification. The binding moiety for purification may contain four or more amino acids. The binding sequences may contain 4-30 amino acids. The binding moiety may be selected from: Alfa-tag (SRLEEELRRRLTE) (SEQ ID No 5)

[0098] Avi-tag (GLNDIFEAQKIEWHE) (SEQ ID No 6)

[0099] C-tag (EPEA) (SEQ ID No 7)

[0100] Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID No 8)

[0101] Dogtag (DIPATYEFTDGKHYITNEPIPPK) (SEQ ID No 9)

[0102] E-tag (GAPVPYPDPLEPR) (SEQ ID No 10)

[0103] FLAG (DYKDDDDK) (SEQ ID No 11)

[0104] G4T (EELLSKNYHLENEVARLKK) (SEQ ID No 12)

[0105] HA (YPYDVPDYA) (SEQ ID No 13)

[0106] His (HHHHHH) (SEQ ID No 14)

[0107] Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID No 15) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID No 16)

[0108] Myc (EQKLISEEDL) (SEQ ID No 17)

[0109] NE-Tag (TKENPRSNQEESYDDNES) (SEQ ID No 18)

[0110] Poly Glutamate-tag (EEEEEEE) (SEQ ID No 19)

[0111] Poly Arginine-tag (RRRRRRR) (SEQ ID No 20)

[0112] Rho1 D4-tag (TETSQVAPA) (SEQ ID No 21)

[0113] SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID No 22)

[0114] Sdytag (DPIVMIDNDKPIT) (SEQ ID No 23)

[0115] SH3 (STVPVAPPRRRRG) (SEQ ID No 24)

[0116] SNAC (GSHHW) (SEQ ID No 25)

[0117] Snooptag (KLGDIEFIKVNK) (SEQ ID No 26)

[0118] Softag 1 (SLAELLNAGLGGS) (SEQ ID No 27)

[0119] Softag 3 (TQDPSRVG) (SEQ ID No 28)

[0120] Spot-tag (PDRVRAVSHWSS) (SEQ ID No 29)

[0121] Spytag (AHIVMVDAYKPTK) (SEQ ID No 30)

[0122] S-tag (KETAAAKFERQHMDS) (SEQ ID No 31)

[0123] Strep-tag (AWAHPQPGG) (SEQ ID No 32) (AWRHPQFGG) (SEQ ID No 33)

[0124] Strep-tag II (WSHPQFEK) (SEQ ID No 34)

[0125] T7tag (MASMTGGQQMG) (SEQ ID No 35)

[0126] TC-tag (EVHTNQDPLD) (SEQ ID No 36)

[0127] Ty-tag (CCPGCC) (SEQ ID No 37)

[0128] VSV-tag (YTDIEMNRLGK) (SEQ ID No 38)

[0129] Xpress-tag (DLYDDDDK) (SEQ ID No 39)

[0130] The expression composition may be assembled on the device from mixing a variety of droplets in order to screen a variety of compositions in parallel. By way of example, the screening reagents may include,

[0131] ■ Chaperone mix (e.q.,PUREfrex GrpE mix)

[0132] ■ Kinase 1 (e.g., EB CK2)

[0133] ■ Kinase 2 (e.g., NEB PKA)

[0134] ■ Protease 1 (e.g., EB.TEV)

[0135] ■ Protease 2 (e.g., Merck HR 3C)

[0136] ■ N-acetyl transferase

[0137] ■ Common metal ions cocktail

[0138] ■ Common co-factors cocktail

[0139] The compositions can be blended by the user and the level of expression of the protein of interest monitored in each of the blended conditions.

[0140] Metal ions may include one or more of the following: MgCh, CuCh, ZnCh, CaCh, MnCh, NiCh, CoCI2.

[0141] Co-factors may include one or more of Nicotinamide adenine dinucleotide (NAD), Flavin adenine dinucleotide (FAD), S-adenosyl methionine (SAM), pyridoxal phosphate (PLP) Coenzyme A (CoA), thiamine pyrophosphate (TPP) or haem.

[0142] Chaperones may include one or more of DnaK, DnaJ, GroE, GrpE, heat shock proteins, protein disulfide isomerase (PDI), human protein disulfide isomerase (hPDI), disulfide bond C (DsbC), a thioredoxin, such as TRXB1 , Caseinolytic peptidase B protein homolog (CLPB) or FK506 binding proteins (FKBPs).

[0143] The additive may be for example one or more reducing agents. The additive may be selected from DTT, glutathione (GSH) or glutathione disulfide (GSSG).

[0144] The added protease may cleave the protein of interest from flanking regions and therefore from the support. The flanking regions may include tags used for detection or solubility or other buffer regions. The flanking regions may be cleaved by any protease. The protease may be a TEV or 3C protease.

[0145] The TEV protease may act upon the amino acid sequence ENLYFQS (SEQ ID No 1). The template for expression of the POI may include the nucleic acid sequence GAGAACCTGTACTTCCAGAGC (SEQ ID No 2). The 3C protease may act upon the amino acid sequence LEVLFQGP (SEQ ID No 3). The template for expression of the POI may include the nucleic acid sequence CTCGAGGTTCTGTTCCAAGGACCT (SEQ ID No 4).

[0146] The method may be used to perform a surfactant screen to identify the best surfactant for expression of a particular protein. The surfactant may be ionic, nonionic, or zwitterionic.

[0147] Surfactant molecules are composed of a hydrophobic region and a hydrophilic region. This amphiphilic structure enables surfactants to obtain a discoidal conformation in the solution, known as micelles. Micelles solubilize membrane proteins by encompassing the transmembrane domains of integral membrane proteins, with the loops and hydrophilic regions exposed to solvent. The minimum concentration of a surfactant necessary to form micelles and extract membrane proteins is called critical micelle concentration or CMC.

[0148] Depending on the charge of hydrophilic group, surfactants are classified into three groups: ionic, nonionic, and zwitterionic surfactants. Ionic surfactants carry a charged group, either negative (anionic) or positive (cationic), and historically have been the most efficient group of detergents in extracting membrane proteins from lipid bilayers. However, ionic detergents can have deleterious effects on protein-protein interactions and often lead to protein denaturation. Sodium dodecyl sulfate (SDS) and sodium cholate are two common examples of ionic detergents.

[0149] Nonionic surfactants are currently the most popular and successful group of surfactants in solubilizing membrane proteins for both functional and structure determination purposes. This is due to their nondisruptive nature, which enables them to preserve the native structure of the target protein by breaking protein-lipid interactions instead of protein-protein interactions. Alkyl glycoside surfactants such as n-dodecyl-B-D-maltoside (DDM), n-decyl-B-D-maltoside (DM), n-Octyl-B-D-Glucopyranoside (OG), and n-Nonyl-B-D-Glucopyranoside (NG) by contributing to the purification and crystallization of about 70% of membrane proteins are the most common nonionic surfactants for protein studies. Another advantage of nonionic surfactants is that they do not interfere with optical measurements, which enables fluorescence-based experiments on expressed proteins.

[0150] Zwitterionic surfactants typically have an intermediate level of harshness between ionic and nonionic detergents. They carry both positive and negative charged groups in their polar regions with an overall net charge of zero. An example of a Zwitterionic surfactant is lauryldimethylamine-N-oxide or LDAO. Membrane mimetic systems, such as nanodiscs and styrene malic acid lipid particles (SMALPs), provide an alternative platform for stabilization of membrane proteins and hence eliminate the deleterious effects of detergents on these macromolecules. Nanodiscs are composed of phospholipid patches surrounded by two copies of membrane scaffold protein (MSP), a genetically engineered version of human serum apolipoprotein A-l.

[0151] Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates, which can be produced either in cells or synthetically. LETs can be made via PCR. While LETs are easier and faster to make, plasmid yields are usually higher in CFPS. mRNA can be produced through in vitro transcription systems. The methods use a single nucleic acid template per droplet. The methods can use multiple droplets having a different nucleic acid template per droplet.

[0152] An energy source is an important part of a cell-free reaction. Usually, a separate mixture containing the needed energy source, along with a supply of amino acids, is added to the extract for the reaction. Common sources are phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. The energy source can be replenished during the expression process by adding further reagents to the droplet during the process.

[0153] The cell-free system is assembled from the required reagents. E Coli derived systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production (e.g. PURE-FREX). The PURE system is composed of all the enzymes that are involved in transcription and translation, as well as highly purified 70S ribosomes. The protein synthesis reaction of the PURE system lacks proteases and ribonucleases, which are often present as undesired molecules in cell extracts.

[0154] The term digital microfluidic device refers to a device having a two-dimensional array of planar microelectrodes. The term excludes any devices simply having droplets in a flow of oil in a channel. The droplets are moved over the surface by electrokinetic forces by activation of particular electrodes. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface. A digital microfluidic (DMF) device set-up is known in the art, and depends on the substrates used, the electrodes, the configuration of those electrodes, the use of a dielectric material, the thickness of that dielectric material, the hydrophobic layers, and the applied voltage. Once the CFPS reagents have been enclosed in the droplets, additional reagents can be supplied by merging the original droplet with a second droplet. The second droplet can carry any desired additional reagents, including for example oxygen or ‘power’ sources, or test reagents to which it is desired to expose to the expressed protein.

[0155] The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces by merging droplets on the device in order to screen a selection of expression compositions in parallel, the expression conditions optimally including screening compositions having varying tRNA populations. The compositions may include varying template nucleic acid sequences which express a desired amino acid sequence.

[0156] The droplets can be aqueous droplets. The droplets can contain an oil immiscible organic solvent such as for example DMSO. The droplets can be a mixture of water and solvent, providing the droplets do not dissolve into the bulk oil.

[0157] The droplets can be in a bulk oil layer. A dry gaseous environment simply dries the bubbles onto the surface during the expression process, leaving comet type smears of dried material by evaporation. Thus the device is filled with liquid for the expression process. Alternatively, the aqueous droplets can be in a humidified gaseous environment. A device filled with air can be sealed and humidified in order to provide an environment that reduces evaporation of CFPS droplets.

[0158] The droplets containing the cell-free extract having the components for protein expression will therefore typically be in the oil filled environment before the nucleic acid templates are added to the droplets. The templates can be added by merging droplets on the microfluidic device. Alternatively, the templates can be added to the droplets outside the device and then flowed into the device for the expression process. For example the expression process can be initiated on the device by increasing the temperature. The expression system typically operates optimally at temperatures above standard room temperatures, for example at or above 29 °C.

[0159] The expression process typically takes many hours. Thus the process should be left for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be left for at least 12 hours. During the process of expression the droplets should be moved within the device. The moving improves the process by mixing the reagents and ensuring sufficient oxygen is available within the droplet. The moving can be continuous, or can be repeated with intervening periods of non-movement. Thus the aqueous droplet can be repeatedly moved for at least a period of 30 minutes or one hour whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows mixing within the droplet, and allows oxygen or other reagents to be supplied to the droplet. The act of moving improves the level of protein expression over a droplet which remains static.

[0160] Digital microfluidics (DMF) refers to a two-dimensional planar surface platform for lab-on-a- chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.

[0161] The droplet can be moved using any means of electrokinesis. The aqueous droplet can be moved using electrowetting-on-dielectric (EWoD). Electrowetting on a dielectric (EWoD) is a variant of the electrowetting phenomenon that is based on dielectric materials. During EWoD, a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface.

[0162] The electrical signal on the EWoD or optically-activated amorphous silicon (a-Si) EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors or digital micromirrors. Optically-activated s-Si EWoD devices are well known in the art for actuating droplets ( . Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0163] A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the aqueous droplets during the protein expression. Alternatively the source of oxygen can be a molecular source which releases oxygen. Alternatively the droplets can be moved to an air / liquid boundary to enable increased diffusion of oxygen from a gaseous environment. Additionally, a source of supplemental oxygen can be found by oxygenating the oil that is used as the filler medium. It is well-known in the art that oils such as hexadecane, HFE-7500, and others can be oxygenated to support the oxygen requirements of cell growth, especially E. coli cell growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air. Alternatively the oil can be oxygenated. Alternatively the droplets can be presented in a humidified air filled device.

[0164] Through an affinity tag, such as a FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag, or other form of affinity tag, CFPS-expressed proteins can be immobilized to a solid-support affinity resin and fresh batches of CFPS reagent can be delivered over the said resin. Thus, renewed reagents can be used to carry out protein synthesis, closely mimicking industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF- and CE-CFPS, users can scale up their CFPS production methods.

[0165] The droplets can be actuated on a hydrophobic surface on the digital microfluidic device (ACS Nano 2018, 12, 6, 6050-6058). The hydrophobic surface can be a hydrophobic surface such as polytetrafluoroethylene (PTFE), Teflon AF (DuPont Inc), CYTOP (AGC Chemicals Inc), or FluoroPei (Cytonix LLC). The hydrophobic surface may be modified in such a way to reduce biofouling, especially biofouling resulting from exposure to CFPS reagents or nucleic acid reagents. The hydrophobic surface may also be superhydrophobic, such as NeverWet (NeverWet LLC) or Ultra-Ever Dry (Flotech Performance Systems Ltd). Superhydrophobic surfaces prevent biofouling compared with typical fluorocarbon-based hydrophobic surfaces. Superhydrophobic surfaces thus prolong the capability of digital microfluidic devices to move CFPS droplets and general solutions containing biopolymers (RSC Adv., 2017, 7, 49633- 49648). The hydrophobic surface can also be a slippery liquid infused porous surface (SLIPS), which can be formed by infusing Krtox-103 oil (DuPont) with porous PTFE film (Lab Chip, 2019, 19, 2275).

[0166] Such hydrophobic surfaces are sensitive to temperature and chemical denaturants, which damage the surface and prevent electrowetting. In extreme cases the surfaces break down completely causing water ingress to the electrodes and therefore electrolysis. Such devices are no longer suitable for electrowetting.

[0167] Droplets can also contain additives to reduce the effects of biofouling on digital microfluidic surfaces. Specifically, droplets containing CFPS components can also contain additives such as surfactants or detergents to reduce the effects of biofouling on the hydrophobic or superhydrophobic surface of a digital microfluidic device (Langmuir 2011 , 27, 13, 8586-8594). Such droplets may use antifouling additives such as TWEEN 20, Triton X-100, and / or Pluronic F127. Specifically, droplets containing CFPS components may contain TWEEN 20 at 0.1% v / v, Triton X-100 at 0.1 % v / v, and / or Pluronic F127 at 0.08% w / v. For electrowetting on dielectrics (EWoD), the change in contact angle of reagent upon the application of electric potential is an inverse function of surface tension. Thus, for low voltage EWoD operations, reduction in surface tension is achieved by addition of surfactants to reagents, which for CFPS reactions means to the lysate and to the DNA. This results in a dilution of the lysate, and it has been seen, in experiments, that diluting or otherwise adulterating the lysate results in a decrease in expression level of the protein of interest. Thus performing CFPS on DMF where the surfactants are added to the solutions being moved will necessarily result in a dilution and adulteration of the lysate and thus a decrease in the level of protein expression. In addition to being a problem in its own right, this further complicates extrapolation of on-DMF results to in-tube predictions of protein yield. An additional detriment of having to add surfactants to the samples is that this increases the time required for sample preparation, as well as increasing the potential for inconsistent results due to ‘user error,’ as there is more handling of reagents. An additional detriment of having to add surfactants to the samples is that certain downstream operations are hindered. For example, if a protein of interest is expressed in a cell-free system with a GFPn (or similar) peptide tag, it’s downstream complementation with a GFP1.10 (or similar) detector polypeptide is hindered in the presence of surfactant. Removal of the surfactant from the aqueous phase is therefore advantageous.

[0168] Rather than adding surfactants to the aqueous sample, it is instead possible to add surfactant, such as a sorbitan ester such as Span85 (e.g. Sorbitan trioleate, Sigma Aldrich, SKU 8401240025), to the oil. This has the advantages of enabling CFPS reactions to proceed on- DMF without dilution or adulteration. Additionally, it simplifies the sample preparation procedure for setting up the reactions, increasing the ease of use and the consistency of results. Using 1 % w / w Span85 in dodecane allows for dilution-free CFPS reactions on-DMF, as well as dilution-free detection of the expressed non-fluorescent proteins. Other surfactants besides Span85, and oils other than dodecane could be used. A range of concentrations of Span85 could be used. Surfactants could be nonionic, anionic, cationic, amphoteric or a mixture thereof. Oils could be mineral oils or synthetic oils, including silicone oils, petroleum oils, and perfluorinated oils. Surfactants can have a detrimental effect on (1) the CFPS reactions and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and detector). For example, by performing the CFPS reaction on- DMF with oil-surfactant mix, the detection of the expressed protein can also proceed without dilution and without adding aqueous surfactant. It has been shown that surfactants reduce the efficiency of some detection systems, including but not limited to the Split GFP (e.g. GFP11 / GFP1.10) system, so removing surfactants from the reagent mix and instead adding them to the oil can be beneficial. The peptide tag can be attached to the C or N terminus of the protein. The peptide tag may be one component of a green fluorescent protein (GFP). For example the peptide tag may be GFPn and the further polypeptide GFP1.10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi- .

[0169] The protein may be fused to multiple tags. For example the protein may be fused to multiple GFP11 peptide tags and the synthesis occurs in the presence of multiple GFP1.10 polypeptides. For example the protein may be fused to multiple sfCherryn peptide tags and the synthesis occurs in the presence of multiple sfCherryi- polypeptides. The protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPn peptide tags and the synthesis occurs in the presence of one or more GFP1.10 polypeptides and one or more sfCherryi- polypeptides.

[0170] Devices

[0171] The manipulation of droplets by the application of electrical potential can be achieved on electrodes covered with an insulator or a dielectric or a series of insulators or dielectrics. Droplet manipulation as a result of an applied electrical potential is known as electrowetting. Electrokinesis occurs as result of a non-uniform electric field that influences the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on solid surface (electrowetting-on-dielectric or EWoD). DEP can also be used to create forces on polarizable particles to induce their movement. The electrical signal can be transmitted to a discrete electrode, a transistor, an array of transistors, or a sheet of semiconductor film whose electrical properties can be modulated by an optical signal.

[0172] EWoD phenomena occur when droplets are actuated between two parallel electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle. The electrowetting effect can be quantitatively treated using Young- Lippmann equation: cos0 - cos0o= (1 / 2 / LG) c.V2where 0o is the contact angle when the electric field across the interfacial layer is zero, yLG is the liquid-gas tension, c is the specific capacitance (given as sr. so / t, where sris dielectric constant of the insulator / dielectric, so is permittivity of vacuum, t is thickness) and V is the applied voltage or electrical potential. The change in contact angle (inducing droplet movement) is thus a function of surface tension, electrical potential, dielectric thickness, and dielectric constant.

[0173] When a droplet is actuated by EWoD, there are two opposing sets of forces that act upon it: an electrowetting force induced by electric field and resistant forces that include the drag forces resulting from the interaction of the droplet with filler medium and the contact line friction. The minimum voltage applied to balance the electrowetting force with the sum of all drag forces (threshold voltage) is variably determined by the thickness-to-dielectric contact ratio of the insulator / dielectric, (t / Sr )1 / 2. Thus, to reduce actuation voltage, it is required to reduce (t / Sr )1 / 2(i.e., increase dielectric constant or decrease insulator / dielectric thickness). To achieve low voltage actuation, thin insulator / dielectric layers must be used. However, the deposition of high quality thin insulator / dielectric layers is a technical challenge, and these thin layers are easily damaged before the desired electrowetting contact angle is large enough to drive the droplet is achieved. Most academic studies thus report the use of much higher voltages >100 V on easily fabricated, thick dielectric films (>3 pm) to effect electrowetting.

[0174] High voltage EWoD-based devices with thick dielectric films, however, have limited industrial applicability largely due to their limited droplet multiplexing capability. The use of low voltage devices including thin-film transistors (TFT) and optically-activated amorphous silicon layers (a-Si) have paved the way for the industrial adoption of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplex fashion. The driving voltage for TFTs or optically-activated a-Si are low (typically <15 V). The bottleneck for fabrication and thus adoption of low voltage devices has been the technical challenge of depositing high quality, thin film insulators / dielectrics. Hence there has been a particular need for improving the fabrication and composition of thin film insulator / dielectric devices.

[0175] Typically, the electrodes (or the array elements) used for EWoD are covered with (i) a hydrophilic insulator / dielectric and a hydrophobic coating or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings comprise of fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of this material as a hydrophobic coating on the dielectric is typically <100 nm and can have defects in the form of pinholes or a porous structure; hence, it is particularly important that the insulator / dielectric is pinhole free to avoid electrical shorting. Teflon has also been used as an insulator / dielectric, but it has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole free. Other hydrophobic insulator / dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy (e.g. Sll-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Due to minimal contact angle hysteresis and a higher contact angle with aqueous solutions, Teflon is still used as a hydrophobic topcoat on these insulator / dielectric polymers. However, there are difficulties in reliably producing <1 micron pinhole-free coatings of parylene or Sll-8; thus, the thickness of these materials is typically kept at a 2-5 microns at the cost of increased voltage requirements for electrowetting. It has also been reported that traditional EWoD devices with parylene C are easily broken and unstable for repeated droplet manipulation with cell culture medium. Multi-layer insulator devices deposited with metal-oxide and parylene C films have been used to produce a more robust insulator / dielectric and enable operations with lower applied voltages. Inorganic materials, such metal oxides and semiconductor oxides, commonly used in the CMOS industry as “gate dielectrics”, have been used as insulator / dielectric for EWoD devices. They offer the advantage of utilizing standard cleanroom processes for thin film depositions (<100 nm). These materials are inherently hydrophilic, requiring an additional hydrophobic coating, and can be prone to pinhole formation as a result of thin film layer deposition process. Together with the need for lower voltage operations of EWoD, recent developmental work has focused on (1) using materials with improved dielectric properties (e.g., using high-dielectric constant insulators / dielectrics), (2) optimizing the fabrication process to make the insulator / dielectric pinhole free to avoid dielectric breakdown.

[0176] Operation of EWoD devices suffers from contact angle saturation and hysteresis, which is believed to be brought about by either one or combination of these phenomena: (1) entrapment of charges in the hydrophobic film or insulator / dielectric interface, (2) adsorption of ions, (3) thermodynamic contact angle instabilities, (4) dielectric breakdown of dielectric layer, (5) the electrode-electrode-insulator interface capacitance (arising from the double layer effect), and (6) fouling of the surface (such as by biomacromolecules). One of the adverse effects of this hysteresis is reduced operational lifetime of the EWoD-based device.

[0177] Contact angle hysteresis is believed to be a result of charge accumulation at the interface or within the hydrophobic insulator after several operations. The required actuation voltage increases due to this charging phenomenon resulting in eventual catastrophic dielectric breakdown. The most probable explanation is that pinholes at the insulator / dielectric may allow the liquid to come into contact with the electrode causing electrolysis. Electrolysis is further facilitated by pinhole-prone or porous hydrophobic insulators.

[0178] Most of the studies to understand contact angle hysteresis on EWoD have been conducted on short time scales and with low conductivity solutions. Long duration actuations (e.g., >1 hour) and high conductivity solutions (e.g., 1 M NaCI) could produce several effects other than electrolysis. The ions in solution can permeate through the hydrophobic coat (under the applied electric field) and interact with the underlying insulator / dielectric. Ion permeation can result in (1) change in dielectric constant due to charge entrapment (which is different from interfacial charging) and (2) change in surface potential of a pH sensitive metal oxide. Both can result in reduction of electrowetting forces to manipulate aqueous droplets, leading to contact angle hysteresis. The inventors have previously found that the damage from high conductivity solutions reduces or disables electrowetting on electrodes by inhibiting the modulation of contact angle when an electric field is applied.

[0179] An electrokinetic device includes a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes.

[0180] The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminum oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials.

[0181] The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation / dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 pm thick. The conformal layer may be between 100 nm and 200 nm thick.

[0182] The hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid-infused porous coating.

[0183] The elements may comprise one or more of a plurality of array elements, each element containing an element circuit; discrete electrodes; a thin film semiconductor in which the electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light.

[0184] The functional coating may include a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer. This has been found to be a particularly advantageous combination.

[0185] The electrokinetic device may include a controller to regulate a voltage provided to the individual matrix electrodes. The electrokinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each of the thin film transistors is coupled to a scan line and a gate line, and the plurality of gate lines are operatively connected to the controller. This allows all the individual elements to be individually controlled.

[0186] The second substrate may also comprise a second hydrophobic layer disposed on the second electrode. The first and second substrates may be disposed so that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining the electrokinetic workspace between the hydrophobic layers.

[0187] The method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.

[0188] The EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on “Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing”, US patent application no 2019 / 0111433, incorporated herein by reference.

[0189] Described herein are electrokinetic devices, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;

[0190] Described herein is an electrokinetic device, including: a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminum oxide in contact with the matrix electrodes, a conformal layer comprising parylene in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer; a second substrate comprising a top electrode; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes;

[0191] The electrokinetic devices as described may be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for the introduction of reagents as needed.

[0192] Disclosed is a kit comprising: i. a DNA Encoded Library of chemical compounds (DEL); ii. magnetic beads; iii. an digital microfluidic device; and iv. a protease.

[0193] The kit may further comprising reagents for cell-free protein expression to express protein targets having capture moieties to enable attachment to the magnetic beads.

[0194] Use of DNA encoded libraries

[0195] EXAMPLES

[0196] Example 1 : Structure of Test Library

[0197] The structure of the test library was as follows, where M is a small-molecule component assembled in three cycles, each of 20 unique synthons and Nxis a sequence of 46 base pairs containing a combination of three unique tag sequences which code for the synthons of the small-molecule component.

[0198] H > TGACTCCCAAATCGATGTGGT[N!xCACACGTCTGAACTCCAGTCAC-3‘

[0199] My - ACTGAGGGTTTAGCTACACCA[N]xGTGTGCAGACTTGAGGTCAGTG-5'

[0200] 6 o

[0201] Example 2: Structure of Positive Control

[0202] The structure of the positive control was as for the test library, where M is shown below and Nxis a unique tag sequence.

[0203] Example 3: Sequencing of Test Library

[0204] A sample of DNA-encoded test library (3 cycle, 8000 members, 18 nM, 1 pL) was amplified in a PCR reaction with 2X Phire Hot Start II PCR Master Mix (Thermo Scientific™ Product Code 15371732, 25 pL), primer 1 (10 pM, 0.5 pL) and primers 2 and 3 (10 pM, 5 pL) required for Illumina sequencing, where [i5] and [i7] are unique 8-base indices. The reaction was made up with water to 50 pL.

[0205] TACACGACGCTCTTCCGATCTTGACTCCCAAATCGATGTGGT (Primer 1) (SEQ ID No 40) CAAGCAGAAGACGGCATACGAGAT[i7]GTGACTGGAGTTCAGACGTG*T (Primer 2) (SEQ ID No 41)

[0206] AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCC GATC*T (Primer 3) (SEQ ID No 42)

[0207] The following PCR method was used; 95 °C (2 min) then 20 cycles of 95 °C (20 sec), 63 °C (20 sec), 72 °C (20 sec) then 12 °C (infinite). The DNA product was purified using a Thermo Fisher GeneJET PCR Purification Kit K0702. The DNA amplicons were sequenced using an Illumina iSeq DNA sequencer with 20% PhiX v2 control library to improve library diversity. All 8,000 DEL sequences were identified. The almost symmetrical histogram distribution of sequences is shown in Figure 2 (total reads 234912, minimum, 2 reads, maximum 62, median 29, mean 29.4, standard deviation 9.3).

[0208] Two-dimensional plots of two sections through the three-dimensional dataset of the unselected library at cycle 1 , synthon 3 and cycle 2, synthon 3 are shown in Figures 3a & b.

[0209] Example 4: In-Tube Two-Round Affinity Selection with Aurora Kinase A, Thermal Elution.

[0210] The following components were combined at ambient temperature; test library (10 pL, 5 nmol), positive control (solution in 10 mM Tris, 0.1 mM EDTA, pH 8.5, 7 pL, 0.625 pmol), his-tag aurora kinase A (Invitrogen part number PV 3612, solution in 50 mM Tris, 150 mM NaCI, 0.5 mM EDTA, 0.05% Triton® X-100, pH 7, 11.4 pL, 50 pmol), 2X selection buffer (100 mM sodium phosphate, 600 mM NaCI, 2 mg / mL bovine serum albumin, 2 mg / mL sheared salmon sperm DNA, 0.1% Pluronic F-127, pH 8.0, 30 pL) and water (1.6 pL). The solution was allowed to stand for 30 minutes. Dynabeads™ His-Tag Isolation and Pulldown (Invitrogen Part No. 10104D) were resuspended, a 20 pL aliquot was magnetised and the supernatant was removed. The beads were washed twice with elution buffer (50 mM sodium phosphate, 300 mM NaCI, 0.05% Pluronic F-127, pH 8, 100 pL), suspended in 1X selection buffer (20 pL) and added to the affinity selection. The suspension was agitated gently for 30 minutes, when the beads were magnetised, the supernatant was removed and the beads were washed eight times with wash buffer (50 mM, Tris, 150 mM NaCI, 0.05% Pluronic F-127, pH 7.5, 100 pL). The beads were suspended in elution buffer (30 pL) and heated at 72 °C for 5 minutes. The beads were magnetised, and the supernatant was removed. Additional fresh magnetic beads (20 pL) were washed as described above, suspended in 2X selection buffer (15 pL) and added to the supernatant. The suspension was allowed to stand for 30 minutes with occasional mixing, then the supernatant was separated. The treatment with fresh magnetic beads was repeated once. Fresh his-tag aurora kinase A (5.7 pL, 25 pmol) was added, the solution was allowed to stand for 1 h at ambient temperature, then fresh magnetic beads (20 pL), washed as described above and suspended in 1X selection buffer (15 pL) were added. The suspension was agitated gently for 30 minutes, then the beads were magnetised and the supernatant was separated. The beads were washed eight times with wash buffer (200 pL), suspended in elution buffer (30 pL) and heated at 72 °C for 5 minutes. The supernatant was separated, a 1 pL aliquot was amplified using the PCR method described previously, and the DNA was sequenced as described previously. The histogram distribution of sequences containing all three tag sequences is shown in Figure 4. The skewed nature of the distribution, compared to the unselected library, in which there are a small number of abundant sequences illustrates the high degree of selection (8000 sequences, total reads excluding positive control, 1067435, minimum 1 read, maximum 26891 reads, mean 133.4, median 23, mode 14). The positive control was present at a higher abundance than any individual library member (165997 reads, 13.5% of total + positive control).

[0211] Two-dimensional plots of two sections through the three-dimensional dataset of DNA sequences at cycle 1 , synthon 3 and cycle 2, synthon 3 are given in Figures 5a & b. These illustrate the high degree of selection towards certain members of the test library when compared with the corresponding plots for the unselected library.

[0212] Example 5: In-Tube Two-Round Affinity Selection with Aurora Kinase A, Trypsin Elution The following components were combined at ambient temperature; test library (10 pL, 5 nmol), his-tag aurora kinase A (Invitrogen part number PV 3612, solution in 50 mM Tris, 150 mM NaCI, 0.5 mM EDTA, 0.05% Triton® X-100, pH 7, 11.4 pL, 50 pmol), 2X selection buffer (100 mM sodium phosphate, 600 mM NaCI, 2 mg / mL bovine serum albumin, 2 mg / mL sheared salmon sperm DNA, 0.1% Pluronic F-127, pH 8.0, 30 pL) and water (8.6 pL). The solution was allowed to stand for 30 minutes. Dynabeads™ His-Tag Isolation and Pulldown (Invitrogen Part No. 10104D) were resuspended, a 20 pL fraction was magnetised and the supernatant was removed. The beads were washed twice with elution buffer (50 mM sodium phosphate, 300 mM NaCI, 0.05% Pluronic F-127, pH 8, 100 pL) then suspended in 1X selection buffer (20 pL) and added to the affinity selection. The suspension was agitated gently for 30 minutes, when the beads were magnetised, the supernatant was removed and the beads were washed with wash buffer (50 mM, Tris, 150 mM NaCI, 0.05% Pluronic F-127, pH 7.5, 100 pL *8). The beads were suspended in elution buffer (11.5 pL), Trypsin Ultra™ (NEB Part No. P8101S made up at 1 .1 mg / mL in water, 1.1 pL) was added and the suspension was agitated by gentle repeated inversion at ambient temperature for 1 h. 4-(2-Aminoethyl)benzoylsulfonyl fluoride hydrochloride, AEBSF, (10 mM, 2 pL) was added, then additional elution buffer (15.2 pL) was added. The suspension was agitated by gentle repeated inversion at ambient temperature for 1 h, then the beads were magnetised and the supernatant was removed. A 1 pL aliquot was amplified by PCR and sequenced as described above. Additional fresh magnetic beads (20 pL) were washed as above, suspended in 2X selection buffer (15 pL) and added to the supernatant. The suspension was allowed to stand for 30 minutes with occasional mixing, then the supernatant was separated. The treatment with fresh magnetic beads was repeated once. Fresh his-tag aurora kinase A (5.7 pL, 25 pmol) was added, the solution was allowed to stand for 1 h at ambient temperature, then fresh magnetic beads (20 pL), washed as described above and suspended in 1X selection buffer (15 pL) were added. The suspension was agitated gently for 30 minutes. The beads were magnetised and the supernatant was removed. The beads were washed eight times with 1X wash buffer (100 pL), then suspended in elution buffer (30 pL). The suspension was split into two equal portions. To the first, Trypsin Ultra™ (1 mg / mL, 1.1 pL) was added, and the suspension was agitated by gentle inversion at ambient temperature for 1 h. 4-(2-Aminoethyl)benzoylsulfonyl fluoride hydrochloride, AEBSF, (10 mM, 2 pL) was added, the suspension was agitated by gentle inversion at ambient temperature for 1 h, then the beads were magnetised and the supernatant was removed. A 1 pL aliquot was amplified by PCR and sequenced as described previously. The second portion of magnetic beads was heated at 72 °C for 5 minutes, the beads were magnetised and the supernatant was removed. A 1 pL aliquot was amplified by PCR and sequenced as described in Example 3.

[0213] The distribution of sequences after one round of selection with trypsin elution was skewed and is shown in Figure 6 (8000 sequences, total reads 231689, minimum 1 read, maximum 826 reads, mean 29.0, median 23, mode 18).

[0214] The distribution of sequences after two rounds of trypsin elution was more highly skewed and is shown in Figure 7 (7985 sequences, total reads 207375, minimum 0 reads, maximum 1468 reads, mean 26.0, median 10, mode 7).

[0215] The distribution of sequences after 1 round of trypsin and one round of thermal elution, which is similar to that after two rounds of trypsin elution is shown in Figure 8 (8000 sequences, total reads 274767, minimum 0 read, maximum 2403 reads, mean 34.4, median 13, mode 9).

[0216] Two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after two rounds of trypsin elution at cycle 1 , synthon 3 and cycle 2, synthon 3, showing a high degree of enrichment of selection for certain sequences are given in Figures 9a & b.

[0217] Two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after one rounds of trypsin elution and one round of thermal elution at cycle 1 , synthon 3 and cycle 2, synthon 3 are given in Figures 10a & b.

[0218] Example 6: Microfluidic Two Round Affinity Selection with Aurora Kinase A Using Trypsin Elution.

[0219] The microfluidic device was loaded with the following reagents, all containing 0.05% Pluronic F-127.

[0220] 1. Wash Buffer (50 mM Tris pH 7.5, 150 mM NaCI), 108 pL.

[0221] 2. Elution Buffer (50 mM sodium phosphate, 300 mM NaCI, pH 8.0), 6 pL. 3. Selection buffer (3 mg / mL bovine serum albumin, 3 mg / mL sheared salmon sperm DNA, 150 mM pH 8 phosphate, 810 mM NaCI), 6 pL.

[0222] 4. Aurora Kinase A enzyme, from dilution of Invitrogen part number PV 3612 (138 nM, 31 mM Tris, 94 mM NaCI, 0.31 mM EDTA, 0.031% Triton X-100), 6 pL.

[0223] 5. Trypsin Ultra™ (NEB Part Number P8101S) made up at 0.4 mg / mL in water), 3 pL.

[0224] 6. 4-(2-Aminoethyl)benzoylsulfonyl fluoride hydrochloride, AEBSF (10 mM), 6 pL.

[0225] 7. DNA-encoded library (0.25 mM), 3 pL.

[0226] 8. Dynabeads™ His-Tag Isolation and Pulldown (Invitrogen Part No. 10104D) suspended in 50 mM sodium phosphate, 300 mM NaCI, pH 8.0 at 35 mg / mL, 12 pL.

[0227] The following operations were automatically orchestrated on the microfluidic device in replicate, according to a pre-defined script:

[0228] 1. Aurora kinase A (180 nL), DNA-encoded library (180 nL) and selection buffer (180 nL) were combined.

[0229] 2. The solution was incubated at ambient temperature for 30 minutes.

[0230] 3. Dynabeads™ (245 nL) were magnetised to pellet them, the supernatant was removed and the solution was combined with the beads.

[0231] 4. The suspension was incubated at ambient temperature for 30 minutes.

[0232] 5. The beads were pelleted and the supernatant was removed.

[0233] 6. The beads were washed with wash buffer (8 x 490 nL).

[0234] 7. The beads were suspended in elution buffer (245 nL).

[0235] 8. Trypsin Ultra™ (80 nL) was combined with the resuspended magnetic beads.

[0236] 9. The suspension was incubated at ambient temperature for 30 minutes.

[0237] 10. The suspension was combined with AEBSF (80 nL).

[0238] 11. The suspension was incubated at ambient temperature for 30 minutes.

[0239] 12. The beads were pelleted and the supernatant was removed.

[0240] 13. The supernatant was combined with pelleted beads (ex 245 nL).

[0241] 14. The suspension was incubated at ambient temperature for 30 minutes.

[0242] 15. The beads were pelleted and the supernatant was removed.

[0243] 16. The supernatant was combined with pelleted beads (ex 245 nL).

[0244] 17. The suspension was incubated at ambient temperature for 30 minutes.

[0245] 18. The beads were pelleted and the supernatant was removed.

[0246] 19. The supernatant was combined with aurora kinase A (180 nL) and incubated at ambient temperature for 30 minutes.

[0247] 20. The solution was combined with pelleted beads (ex 245 nL).

[0248] 21. The suspension was incubated at ambient temperature for 30 minutes.

[0249] 22. The beads were pelleted and the supernatant was removed. 23. The beads were washed with wash buffer (8 x 490 nL).

[0250] 24. The beads were resuspended in elution buffer (180 nL).

[0251] 25. The suspension was combined with AEBSF (80 nL).

[0252] 26. The beads were removed from the microfluidic device manually using a P200 pipette and transferred to a microcentrifuge tube for downstream processing.

[0253] The beads were suspended in elution buffer (10 pL), heated to 72 °C for 5 minutes, the beads were magnetised, the supernatant was removed, input into a PCR reaction and sequenced, as described in Example 3. The distribution of sequences from sequencing of five replicates run in parallel on the device, each identified with unique indices, and pooling of the data shown in Figure 11 was skewed indicating a high degree of selection (8000 sequences, total reads 1153055, minimum 12 reads, maximum 4901 reads, mean 144, median 97, mode 90).

[0254] Two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after microfluidic device selection, indicating a high degree of selection for certain sequences at cycle 1 , synthon 3 and cycle 2, synthon 3 are shown in Figures 12a & b.

[0255] Example 7: In-Tube Two-Round Affinity Selection with Aurora Kinase A, Proteinase K Elution

[0256] The following components were combined at ambient temperature; test library (10 pL, 5 nmol), his-tag aurora kinase A (Invitrogen part number PV 3612, solution in 50 mM Tris, 150 mM NaCI, 0.5 mM EDTA, 0.05% Triton® X-100, pH 7, 11.4 pL, 50 pmol), 2X selection buffer (100 mM sodium phosphate, 600 mM NaCI, 2 mg / mL bovine serum albumin, 2 mg / mL sheared salmon sperm DNA, 0.1% Pluronic F-127, pH 8.0, 30 pL) and water (8.6 pL). The solution was allowed to stand for 30 minutes. Dynabeads™ His-Tag Isolation and Pulldown (Invitrogen Part No. 10104D) were resuspended, a 20 pL fraction was magnetised and the supernatant was removed. The beads were washed twice with elution buffer (50 mM sodium phosphate, 300 mM NaCI, 0.05% Pluronic F-127, pH 8, 100 pL) then suspended in 1X selection buffer (20 pL) and added to the affinity selection. The suspension was agitated gently for 30 minutes, when the beads were magnetised, the supernatant was removed and the beads were washed with wash buffer (50 mM, Tris, 150 mM NaCI, 0.05% Pluronic F-127, pH 7.5, 200 pL x8). The beads were suspended in 75 mM CAPSO, 300 mM NaCI, 0.05% Pluronic F-127, pH 10.63, 13.4 pL), proteinase K (NEB part No. P8107S diluted to 2 U / mL in 0.05% Pluronic F-127, 4.3 pL) was added and the suspension was agitated gently at ambient temperature for 1 h. 4-(2- Aminoethyl)benzoylsulfonyl fluoride, AEBSF (100 mM in 128 mM sodium phosphate monobasic, 0.05% Pluronic F-127, 4.3 pL) was added and the suspension was agitated by gentle repeated inversion at ambient temperature for 1 h. Elution buffer (50 mM sodium phosphate, 300 mM NaCI, 0.05% Pluronic F-127, pH 8, 8 pL) was added, the beads were magnetised and the supernatant was removed. A 1 pL aliquot was amplified by PCR and sequenced as described above. Additional fresh magnetic beads (20 pL) were washed as above, suspended in 1X selection buffer (15 pL) and added to the supernatant. The suspension was allowed to stand for 30 minutes with occasional mixing, then the supernatant was separated. The treatment with fresh magnetic beads was repeated once. Fresh his-tag aurora kinase A (5.7 pL, 25 pmol) was added, the solution was allowed to stand for 1 h at ambient temperature, then fresh magnetic beads (20 pL), washed as described above and suspended in 1X selection buffer (15 pL) were added. The suspension was agitated gently for 30 minutes. The beads were magnetised and the supernatant was removed. The beads were washed eight times with 1X wash buffer (7 x 200 pL), then the sample was split into two equal portions. The beads were magnetised, the supernatants were removed and combined. To the first sample of beads, 75 mM CAPSO, 300 mM NaCI, 0.05% Pluronic F-127, pH 10.63 (13.4 pL) and proteinase K (2 U / rnL in 0.05% Pluronic F-127, 4.3 pL) were added. The suspension was agitated by gentle inversion at ambient temperature for 1 h. 4-(2- Aminoethyl)benzoylsulfonyl fluoride hydrochloride, AEBSF, (100 mM in 128 mM sodium phosphate monobasic, 0.05% Pluronic F-127, 4.3 pL) was added. The suspension was agitated gently at ambient temperature for 1 h, then the beads were magnetised and the supernatant was removed. A 1 pL aliquot was amplified by PCR and sequenced as described previously. The second portion of magnetic beads was suspended in elution buffer (22 microlitres) and heated at 72 °C for 5 minutes. The beads were magnetised and the supernatant was removed. A 1 pL aliquot was amplified by PCR and sequenced as described above. The distribution of sequences after one round of selection with proteinase K elution was skewed and is shown in Figure 13a (7999 sequences, total reads 1595792, minimum 0 reads, maximum 7020 reads, mean 199.5, median 134, mode 131). The distribution of sequences after two rounds of proteinase K elution was more highly skewed and is shown in Figure 13b (7999 sequences, total reads 832351 , minimum 0 reads, maximum 9675, reads, mean 107.4, median 42, mode 37). The distribution of sequences after 1 round of proteinase K elution and one round of thermal elution was similar to that after two rounds of proteinase K elution and is shown in Figure 13c (8000 sequences, total reads 1094587, minimum 1 read, maximum 6454 reads, mean 142.8, median 65, mode 59).

[0257] Two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after two rounds of proteinase K elution at cycle 1 , synthon 3 and cycle 2, synthon 3, showing a high degree of enrichment of selection for certain sequences are given in Figures 14 a & b. Two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after one rounds of proteinase K elution and one round of thermal elution at cycle 1 , synthon 3 and cycle 2, synthon 3 are given in Figures 15 a & b.

[0258] Example 8: Two Round Affinity Selection with Aurora Kinase A Using Microfluidic Device (2)

[0259] The selection experiment was conducted as for Example 6, but with a different composition of wash buffer; 50 mM phosphate buffered saline (PBS); pH7.4, 0.1 mg / mL sheared salmon sperm DNA, 150 mM NaCI, 10 mM imidazole. The distribution of sequences from sequencing of four replicates run in parallel on the device, each identified with unique indices, and pooling of the data shown in Figure 16 was skewed indicating a high degree of selection (7999 sequences, total reads 1544930, minimum 0 reads, maximum 10109 reads, mean 198, median 109, mode 96). Two-dimensional plots of sections through the three-dimensional dataset of DNA sequences after microfluidic device selection, indicating a high degree of selection for certain sequences at cycle 1 , synthon 3 and cycle 2, synthon 2 are shown in Figures 17 a & b.

Claims

Claims1. A method of screening interactions between a DNA Encoded Library of chemical compounds (DEL) and a protein target using droplets on a digital microfluidic device comprising the steps of: a. binding the DNA encoded library to beads via the protein target in one or more droplets on the device; b. removing the unbound library from the beads; c. washing the beads; d. elution of the DNA encoded library from the beads by enzymatically digesting the protein target using a protease; and e. removing, denaturing or inhibiting the protease.

2. The method according to claim 1 comprising the steps of: a. binding the DNA encoded library to beads via the protein target in one or more droplets on the device; b. removing the unbound library from the beads by holding the beads and reducing the volume of the droplet; c. washing the beads by adding additional droplets to the beads; d. elution of the DNA encoded library from the beads by enzymatically digesting the protein target using a protease; and e. denaturing or inhibiting the protease.

3. The method according to claim 1 or claim 2, wherein the DEL is exposed to the beads having the immobilised protein target.

4. The method according to claim 1 or claim 2, wherein the DEL is exposed to the protein target which is then immobilised on the beads.

5. The method according to any one of claims 1-4, wherein steps a-e are repeated one or more times.

6. The method according to any one of claims 1-5, wherein the DNA is amplified and sequenced to identify the most abundant library members.

7. The method according to any one of claims 1-6, wherein the DEL is exposed to a variety of protein targets.

8. The method according to claim 7, wherein the variety of protein targets are length variants or truncations.

9. The method according to claim 7, wherein the variety of protein targets are sequence variants, homologs, orthologs or isoforms.

10. The method according to claim 7, wherein the variety of protein targets are kinases.

11. The method according to any one of claims 1-10, wherein the droplets contain a known protein binding moiety with the DEL to look for competitive binding at a particular site.

12. The method according to any one of claims 1-10, wherein the droplets contain a variety of screening conditions.

13. The method according to any one of claims 1-10, wherein the variety of screening conditions include pH, buffers, salts, detergents or temperature.

14. The method according to any one of claims 1-11, wherein the protein targets are expressed and purified on the digital microfluidic device.

15. The method according to any one of claims 1-14, wherein the digital microfluidic device comprises a planar array of electrodes.

16. The method according to any one of claims 1-15, wherein the device comprises active-matrix thin-film transistors.

17. The method according to any one of claims 1-16, wherein the method comprises the steps of: a. expressing a variety of proteins in droplets on a digital microfluidic device; b. binding the expressed protein to magnetic beads to purify the expressed proteins; c. binding a DNA encoded library to the magnetic beads via the expressed protein; d. removing the unbound library from the beads by holding the beads and reducing the volume of the droplet; e. washing the beads by adding additional droplets to the beads; f. elution of the DNA encoded library from the beads by enzymatically digesting the protein target using a protease; and g. denaturing or inhibiting the protease.

18. The method according to any one of claims 1-17, wherein the protease is trypsin.

19. A kit comprising: i. a DNA Encoded Library of chemical compounds (DEL); ii. magnetic beads; iii. an digital microfluidic device; and iv. a protease.

20. The kit according to claim 19 further comprising reagents for cell-free protein expression to express protein targets having capture moieties to enable attachment to the magnetic beads.

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