High-throughput cell-free expression and screening microfluidic platform for gpcr drug discovery

Abstract

A method for screening a library of plasmids encoding peptides to screen for one or more peptides that functionally activate a G protein-coupled receptor in a cell by contacting the peptides with a cell expressing a G protein-coupled receptor that functions as a sensor and isolating cells that functionally activate the G protein-coupled receptor to determine the sequence encoding the peptide.

Description

Technical Field

[0001] The present invention relates to a method for screening plasmid libraries encoding peptides that functionally activate G protein-coupled receptors in cells, and an apparatus configured to perform said method. Background Technology

[0002] Cell-free Directed Evolution of a Protease in Microdroplets at Ultrahigh Throughput, J Holstein, C Gylstorff, and F Hollfelder, ACSSynth. Biol. 2021, 10, 252-257 discloses a three-step workflow that separates three previously incompatible steps that could not be performed simultaneously until now. In the first step, plasmids are amplified by rolling circle amplification in microfluidic water-in-oil droplets. In a subsequent step, reagents for in vitro protein expression are injected into the droplets via microinjection to express the protease variant, and then, in the final step, the substrate for the protease is also added via microinjection. If the fluorescent casein substrate is hydrolyzed, a fluorescent signal is emitted, which is used to sort the droplets and determine the sequence of the active protease. However, the three-step workflow is complex and requires precise control, especially during the microinjection step, to ensure the delivery of the correct amount of polymerase and protein expression system.

[0003] A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution is disclosed in Fallah-Araghi, J Baret, M Ryckelynck, and A Griffiths Lab Chip, 2012, 12, 882. A method is described in which a single gene encoding β-galactosidase is compartmentalized in an aqueous droplet, dispersed in an inert carrier oil, and amplified using polymerase chain reaction (PCR). After amplification, the droplet is fused one-to-one with a droplet containing a cell-free coupled transcription-translation (IVTT) system and a substrate for fluorescence assay (i.e., fluorescein-di-β-D-galactopyranoside). The gene is then selectively recovered from the droplet containing the β-galactosidase active variant using fluorescence-activated electropolymerization with an aqueous flow.

[0004] Drug development is a process requiring significant upfront capital investment with no guarantee of success. Therefore, to remain competitive, the pharmaceutical industry is under pressure to reduce the overall cost of drug development while discovering new active pharmaceutical ingredients (APIs). With the advent of genomics and combinatorial chemistry, and the adoption of high-throughput screening (HTS) methods, data on potential therapeutic targets and libraries of molecules to be screened have surged. For example, testing large libraries of chemicals for specific biological activities is typically a lengthy and laborious task. This necessitates the development of even more cost-effective methods to efficiently screen large numbers of compounds targeting a variety of therapeutic targets.

[0005] Screening for compounds with activity against specific therapeutic targets has traditionally been performed manually at typical throughputs of approximately 1,000 assays per day. Today, high-throughput drug screening assays are conducted in microtiter plates, where drug solutions are added to target cells or molecules. Using robotics, reagent and material reductions can be achieved by increasing the number of wells per microtiter plate and correspondingly decreasing the well volume.

[0006] The use of droplet-based microfluidics dramatically increases the number of units that can be screened, as the volume decreases to picoliters and sample consumption is significantly reduced. Drug screening in droplets appears to be a promising solution because they enable high-throughput screening of multiple drugs in a single experiment, leading to rapid discovery of targeted therapeutic candidates. Despite the enormous potential of droplet-based microfluidics for cheaper and faster drug screening, microfluidics have not lived up to expectations. This gap arises from several limitations encountered when converting drug screening to droplet-based microfluidics. Different candidate compounds must be loaded into the microfluidic device, a process that is extremely slow (minutes or hours) compared to droplet generation itself (thousands per second), thus limiting throughput. When the number of chemically different drugs is in the thousands or millions, loading all these compounds into different droplets becomes impossible. To achieve continuous and automated production of droplets containing different candidate compounds (e.g., peptides or proteins), it has been proposed to encapsulate single plasmids from plasmid libraries within single microfluidic water-in-oil droplets, which can then be amplified via rolling circle amplification through individual microinjection into an in vitro transcription and translation (IVTT) system. However, these strategies do not offer the possibility of monitoring the interaction between candidate peptide compounds and their targets. Summary of the Invention

[0007] Therefore, the object of the present invention is to provide a solution to the above-mentioned problems, namely, to provide a method in which the agonist or antagonist effects of a large number of candidate compounds encoded in a plasmid library on a target can be tested in vivo, even when living cells are in contact with the candidate compounds.

[0008] In its first objective, the present invention therefore provides a method for screening plasmid libraries encoding peptides to screen for one or more peptides that functionally activate G protein-coupled receptors in cells, the method comprising the following steps:

[0009] - Provides a first set of water-in-oil droplets, each containing a single plasmid encoding a candidate peptide or its precursor, a polymerase capable of plasmid rolling circle replication, an in vitro transcription / translation system, and optionally a protease.

[0010] - Within the water-in-oil droplets, the plasmid is replicated using a polymerase capable of plasmid rolling circle replication, and simultaneously, a candidate peptide or propeptide encoded on the plasmid is expressed using the in vitro transcription / translation system to form droplets each containing the candidate peptide. If the propeptide is expressed, it is hydrolyzed to form droplets each containing the candidate peptide.

[0011] - A second set of water-in-oil droplets is provided, wherein each droplet contains one or more cells having G protein-coupled receptors expressed on their surface, wherein the G protein-coupled receptors have a reporter portion coupled thereto, and the reporter portion is capable of emitting a signal, preferably an electromagnetic radiation signal, when the G protein-coupled receptors are functionally activated by a suitable candidate peptide.

[0012] - Droplets containing candidate peptides or propeptides from the first group are combined one-to-one with droplets from the second group containing one or more cells with G protein-coupled receptors expressed on their surface to form a third group of combined droplets.

[0013] - In the third group of combined droplets, droplets emitting signals above a predetermined signal strength threshold are separated from droplets emitting signals below a predetermined signal strength threshold to form a fourth group of combined droplets.

[0014] - In the fourth set of combined droplets, the sequence of the peptide or propeptide encoded on the plasmid is determined.

[0015] In a second objective, the invention also provides a microfluidic device configured to perform the method according to the first objective, the device comprising...

[0016] - A microfluidic forming chamber for forming a first set of water-in-oil droplets, each droplet containing a single plasmid encoding a candidate peptide or its precursor peptide, a polymerase capable of plasmid rolling circle replication, an in vitro transcription / translation system, and optionally a protease.

[0017] The formation chamber is fluidly connected to a reservoir for oil and a reservoir for an aqueous liquid containing plasmids from a plasmid library, each plasmid containing a sequence encoding a candidate peptide or its precursor peptide, a polymerase and an in vitro transcription / translation system capable of plasmid rolling circle replication, and optionally a protease.

[0018] - A microfluidic merging chamber, fluidly connected to the formation chamber, for receiving water-in-oil droplets from the formation chamber, for merging a first group of droplets containing candidate peptides with a second group of droplets in a one-to-one manner, the droplets containing one or more cells having G protein-coupled receptors expressed on their surface, to form a third group of combined droplets.

[0019] The merging chamber preferably includes electrodes capable of generating an electric field across the merging chamber.

[0020] - A sorting chamber, which is fluidly connected to the merging chamber, is used to receive combined droplets and separate combined droplets that emit signals above a predetermined signal strength threshold from combined droplets that emit signals below a predetermined signal strength threshold, the former forming a fourth group of droplets.

[0021] Further embodiments of the invention are given in the dependent claims. Attached Figure Description

[0022] Some preferred embodiments of the invention are described below with reference to the accompanying drawings, which are for illustrative purposes only and not for limiting the invention. In the drawings,

[0023] Figure 1 Figure 1A shows the relative mean fluorescence intensity of cells expressing PACLight1 after being combined with droplets containing the corresponding expression product of 100 fg / μL plasmid, relative to PACAP. 6-38 The fluorescence intensity of the antagonists was normalized, and sequence information for each of the 25 pentapeptides from the library that lead to the functional activation of PCLight1 is shown in 1B.

[0024] Figure 3 An example of a screening workflow according to the present invention is shown, wherein in 3A, the process of binding the peptide agonist PACAP is depicted. 1-38The conformational changes of the PACLight1 G-protein coupled receptor are described in section 3B, which outlines the steps of a screening method: providing droplets containing a single plasmid, IVTT reagent, RCA polymerase, and TEV protease; replicating and expressing the protein encoded on the plasmid; merging the droplets with cells containing cells expressing the G-protein coupled receptor with a fluorescent moiety; sorting droplets containing combinations of peptides that functionally activate the receptor; and finally sequencing the plasmid to determine the sequence encoding the activating peptide.

[0025] Figure 2 The upper left of 2A shows a schematic diagram of an apparatus for screening plasmid libraries encoding peptides that functionally activate G protein-coupled receptors in cells, wherein combined droplets flow through a fluorescence-activated droplet sorting (FADS) chamber and are separated based on whether they emit signals above or below a predetermined signal intensity. Figure 2The lower left magnified view of A shows the sorting chamber, depicting a fluorescence detection device equipped with a laser operating at 488 nm, coupled to control circuitry that allows the combined droplets to be deflected via a positive electrode placed immediately before the bifurcation in a microfluidic Y-junction, based on the measured fluorescence. The upper right shows two microscopic images of the sorting chamber: one showing droplets discarded (“unsorted droplets”, top), and the other showing droplets retained for further plasmid analysis (e.g., sequencing) (“sorted droplets”, bottom). In 2B, graphs were plotted for fluorescence intensity versus droplet count in a fluorescence-activated droplet sorting (FADS) chamber for three experiments: where only combination droplets containing a plasmid encoding a G protein-coupled receptor antagonist and one or more cells having the G protein-coupled receptor with a fluorescent reporter portion were used; where combination droplets containing a plasmid encoding a G protein-coupled receptor antagonist and one or more cells having the G protein-coupled receptor with a fluorescent reporter portion were used with plasmids containing a plasmid encoding a G protein-coupled receptor agonist and one or more cells having the G protein-coupled receptor with a fluorescent reporter portion. Combination droplets containing more cells were introduced at a ratio of 10:1, as well as combination droplets containing only plasmids encoding antagonists of G protein-coupled receptors and one or more cells having the G protein-coupled receptor with a fluorescent reporter portion, wherein combination droplets containing plasmids encoding antagonists of G protein-coupled receptors and one or more cells having the G protein-coupled receptor with a fluorescent reporter portion were introduced at a ratio of 10000:1 with combination droplets containing plasmids encoding agonists and one or more cells having the G protein-coupled receptor with a fluorescent reporter portion. It can be seen that when only plasmids encoding antagonists are used, almost no positive combination droplets (fluorescence intensity greater than 0.4) are detected, while in the case of using a mixture of plasmids encoding agonists and antagonists at different ratios, combination droplets containing plasmids encoding agonists are recognized (fluorescence intensity greater than 0.4) because the expressed peptides of the plasmids activate the G protein-coupled receptor with the fluorescent reporter portion. It should be noted that even at a very high dilution ratio of 10,000:1, a relatively small number of positive combination cells can still be reliably detected, and therefore, the relevant sequences encoded on the plasmids can be isolated and recovered, such as... Figure 2 As shown in C. Detailed Implementation

[0026] In its first objective, the present invention therefore provides a method for screening plasmid libraries encoding peptides that functionally activate G protein-coupled receptors in cells.

[0027] In the context of this invention, a plasmid library is a genomic library in which a target polynucleotide fragment is inserted into a plasmid vector such that the target polynucleotide fragment encoding a peptide can be expressed in the presence of the plasmid vector in a system capable of transcription and translation. Plasmid vectors are well known and commercially available, as are methods for generating genomic libraries from them. For example, a suitable plasmid for use in this invention is the pBEST plasmid.

[0028] In the context of this invention, a peptide is a chain of amino acid residues linked by peptide bonds. It should be noted that, generally speaking, the number of amino acid residues in a peptide is not limited in the context of this invention, but is typically from 4 to 600, and preferably from 10 to 200. As an example, a peptide may contain four to several hundred amino acid residues. This invention is applicable to both post-translational modified peptides and unmodified peptides. In cases where post-translational modifications are intended, such as enzymatic cleavage or glycosylation, the appropriate reactants, such as proteases or (glycosyl)transferases, can be added to an aqueous liquid forming droplets.

[0029] G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, 7TM receptors, seven-helix receptors, curved receptors, and G protein-linked receptors (GPLRs), form a large class of evolutionarily related proteins that are cell surface receptors that detect extracellular molecules and activate cellular responses. They cross the cell membrane seven times in the form of six loops (three extracellular loops that interact with ligand molecules, three intracellular loops that interact with G proteins, an N-terminal extracellular region of amino acid residues, and a C-terminal intracellular region), which is why they are sometimes called seven-transmembrane receptors. Their ligands can bind to the extracellular N-terminus and loops (e.g., glutamate receptors) or to binding sites within the transmembrane helix (rhodopsin-like families), and they are typically activated by ligands (i.e., agonists).

[0030] The method according to the present invention includes the following steps:

[0031] - A first set of water-in-oil droplets is provided, each containing a single plasmid encoding a candidate peptide or its precursor, a polymerase capable of plasmid rolling circle replication, and an in vitro transcription / translation system. In other words, the contents of the droplets contain all the elements for the replication and translation of the gene encoding the candidate peptide, as well as the final hydrolysis of the peptide, as commonly referred to as a "one-pot reaction".

[0032] To form and provide a first set of water-in-oil droplets, the first set of water-in-oil droplets contains a single plasmid from a plasmid library, the single plasmid containing a sequence encoding a candidate peptide. The first set of water-in-oil droplets also contains a polymerase capable of plasmid rolling circle replication and an in vitro transcription / translation system. All the aforementioned components are contained in an aqueous liquid, such as an aqueous buffer, such that when water-in-oil droplets are formed from the aqueous liquid, the majority of the droplets containing plasmids are single plasmids from the plasmid library, and a sufficient amount of polymerase and in vitro transcription / translation system to express the peptide encoded in the plasmid. To ensure that the majority of the formed droplets contain single plasmids from the plasmid library, or no more than single plasmids from the plasmid library, the plasmid concentration in the aqueous liquid forming the droplets must be selected within a suitable range. It has been found that plasmid concentrations of 10 to 100 fg / μl in the aqueous liquid yield high yields of droplets containing a single plasmid. The inclusion of plasmids in droplets is controlled by a Poisson distribution, and therefore it should be understood that the term "the majority of droplets containing plasmids are droplets containing a single plasmid of a plasmid library" means that at least 65%, preferably at least 75%, and more preferably at least 80% of droplets are droplets containing a single plasmid of a plasmid library, while the remaining droplets contain two or more plasmids of a plasmid library.

[0033] During droplet formation, preferably performed in a microfluidic device, a Poisson distribution is achieved, wherein approximately 10% of the droplets contain one plasmid, approximately 2% of the droplets contain two or more plasmids, and approximately 88% of the droplets do not contain plasmids. Therefore, a preferred embodiment is the formation of a first set of water-in-oil droplets in which the ratio of droplets containing a single plasmid from a plasmid library to droplets containing two or more plasmids from a plasmid library is greater than 3, preferably greater than 4, or more preferably greater than 5.

[0034] In a preferred embodiment, a first set of water-in-oil droplets is formed in the microfluidic device.

[0035] In a preferred embodiment, the first set of water-in-oil droplets formed in the microfluidic device can be pre-sorted by staining the plasmid DNA inside the droplets with a dye such as SYBR dye to exclude droplets that do not contain plasmids. Droplets that do not contain plasmids do not retain the dye and can be discarded, leaving droplets containing one, two, or more plasmids. Since more plasmid DNA would mean more staining, for example by SYBR, pre-sorting can preferably be performed after the step of replicating the plasmid with a polymerase capable of enabling plasmid rolling circle replication, rather than immediately after droplet formation.

[0036] The concentrations of the polymerase capable of performing plasmid rolling circle replication and the concentration of the in vitro transcription / translation system must be carefully selected to ensure the expression of the peptide encoded in the plasmid. It has been found that peptide or propeptide expression is ensured when the polymerase concentration in the aqueous solution is at least 0.5 U / μl or at least 1 U / μl, and when the concentration of the in vitro transcription / translation system is at least 20% v / v, preferably at least 25% v / v, based on the total volume of the aqueous solution.

[0037] To form the first set of water-in-oil droplets in the microfluidic device, the oil can be selected from any suitable synthetic oil, such as fluorinated oil, such as HFE-7500 which is commercially available from 3M, and optional surfactants, such as fluorinated surfactants CAS297730-93-9.

[0038] A microfluidic device suitable for forming the first set of water-in-oil droplets can be a microfluidic flow focusing device (with a height of 20 μm and a width of 20 μm at the flow focusing junction), wherein at the junction where the fluid is connected to the aqueous solution reservoir, the flow of the aqueous solution is introduced into a fluorinated oil containing about 5% by weight (w / w) of a fluorinated surfactant, which enables the formation of the first set of water-in-oil droplets.

[0039] The method according to the present invention includes the following steps:

[0040] - Within the water-in-oil droplet, the plasmid is replicated using a polymerase capable of performing plasmid rolling circle replication, and simultaneously the candidate peptide or propeptide encoded by the plasmid is expressed using the in vitro transcription / translation system to form droplets each containing the candidate peptide, wherein if the propeptide is expressed, the propeptide is hydrolyzed to form droplets each containing the candidate peptide.

[0041] Any polymerase capable of performing plasmid rolling circle replication can be used in the context of this invention, and preferably is a polymerase with proofreading activity. It should be understood that the aqueous liquid should also contain reactants required for polymerase activity, such as dNTPs and suitable primers. As an example of a suitable polymerase, the EquiPh i29™ DNA polymerase, commercially available from Thermo Fisher Scientific, can be used in combination with exonuclease-resistant random primers also commercially available from Thermo Fisher Scientific. Ideally, the polymerase is allowed to replicate the plasmid for at least two hours, preferably at least three hours, or three to twenty-four hours or three to four hours. In a preferred embodiment, the polymerase capable of performing plasmid rolling circle replication is one that can perform it at a temperature of 30°C to 45°C, and more preferably at a temperature of 30°C to 42°C. In a preferred embodiment, plasmid rolling circle replication is achieved by collecting water-in-oil droplets and placing them at a temperature of 30°C to 45°C (e.g., in a thermal cycler) for three to twenty-four hours or three to four hours.

[0042] The in vitro transcription / translation system used for expressing candidate peptides encoded on plasmids is preferably a cell-free in vitro transcription / translation system, such as the IVTT (in vitro transcription and translation) reagent commercially available from the PUREfrex kit (GeneFrontier), a newly developed recombinant cell-free protein synthesis reagent based on the PURE system technology published by Professor Takuya Ueda of the University of Tokyo (Shimizu et al., 2001, Nat. Biotechnol., 19, 751). Ideally, the expression of the candidate peptide encoded on the plasmid is allowed to proceed for at least two hours, preferably at least three hours, or three to twenty-four hours or three to four hours. Therefore, the in vitro transcription / translation system used for expressing candidate peptides encoded on plasmids is preferably an in vitro transcription / translation system derived from prokaryotes (e.g., from *Escherichia coli*). In a preferred embodiment, the expression of the candidate peptide encoded on the plasmid is achieved by collecting water-in-oil droplets and placing them at a temperature of 30°C to 45°C for three to twenty-four hours or three to four hours (e.g., in a thermal cycler).

[0043] The method according to the present invention includes the following steps:

[0044] - Provide a second set of water-in-oil droplets, wherein each droplet contains one or more cells having G protein-coupled receptors expressed on their surface, wherein the G protein-coupled receptors have a reporter portion coupled thereto, and the reporter portion is capable of signaling, preferably an electromagnetic radiation signal, when the G protein-coupled receptors are functionally activated by a suitable candidate peptide.

[0045] To form and provide a second set of water-in-oil droplets, each droplet containing at least one or more cells having G protein-coupled receptors expressed on their surface, the cells having G protein-coupled receptors expressed on their surface are contained in an aqueous liquid, such as an aqueous cell culture buffer, such that when the water-in-oil droplets are formed, most droplets contain a single cell having G protein-coupled receptors expressed on their surface and a certain amount of aqueous liquid. To ensure that substantially each formed droplet contains at least a single cell, the cell concentration in the cell-containing aqueous liquid forming the droplets must be selected within a suitable range. Therefore, the term "most droplets contain at least a single cell having G protein-coupled receptors expressed on their surface" should be understood to mean that at least about 65%, preferably at least about 75%, of the droplets contain one cell, while the remaining droplets contain two or more cells. Each droplet containing one or two cells is acceptable because this ultimately means more sensors are available. In this way, throughput can be further increased because droplets containing one or two cells can subsequently be incorporated into water-in-oil droplets containing candidate peptides.

[0046] G protein-coupled receptors (GPCRs) have a reporter moiety coupled thereto that is capable of emitting a signal when the GPCR is functionally activated by a suitable candidate peptide. This allows for determination of whether the binding of a suitable candidate peptide can induce functional activation of the GPCR by detecting the signal using appropriate means. In a preferred embodiment of the method according to the invention, the GPCR reporter moiety is capable of emitting an optical signal, and particularly a fluorescent signal when illuminated by a light source. As an example of a GPCR having a fluorescent reporter moiety coupled thereto, a GPCR is disclosed in EP3 544 992 A4, the sequence of which is incorporated herein by reference. It should be understood that the reporter moiety capable of emitting a signal can also be a reporter moiety capable of emitting a non-fluorescent signal, such as a reporter moiety capable of inducing a change in the state of cells expressing the GPCR on its surface, such as the expression of a reporter protein or programmed cell death. While the inventors have implemented a screening method using a G protein-coupled receptor (GPCR) reporter portion in the context of this invention, said GPCR reporter portion emitting a fluorescent signal upon ligand binding and illumination with a light source, it should be understood that this invention is not limited to reporter portions that emit a fluorescent signal upon ligand illumination, but may also encompass reporter portions that quench the fluorescent signal upon ligand binding. Therefore, a GPCR reporter portion capable of emitting an optical signal should be understood to mean a reporter portion capable of increasing or decreasing the optical signal upon ligand binding.

[0047] The method according to the present invention includes the following steps:

[0048] - Droplets containing candidate peptides from the first group are combined one-to-one with droplets from the second group containing one or more cells with G protein-coupled receptors expressed on their surface to form a third group of combined droplets.

[0049] In a microfluidic device, each droplet containing a candidate peptide can be merged one-to-one with a droplet containing one or more cells having G protein-coupled receptors expressed on their surface to form a third set of combined droplets. For example, merging can be achieved by exposing two droplets to be merged to each other when they are brought close together in a microfluidic device, to pulses of an electric field provided by electrodes.

[0050] In a preferred embodiment of the method according to the invention, the step of combining each droplet containing the candidate peptide with a droplet containing one or more cells having G protein-coupled receptors expressed on their surface in a one-to-one manner to form a third set of combined droplets is preferably temperature-controlled, and more preferably performed at about 4°C to 20°C, and most preferably at about 4°C. At 4°C, the metabolic rate of the single cells containing the G protein-coupled receptors expressed on their surface in the droplets is slowed, which avoids the decrease in viability observed in less than one hour when the step is performed at about 20°C or higher.

[0051] Suitable microfluidic devices for droplet merging may include flow focusing junctions (e.g., 50 μm wide) for single-cell encapsulation, droplet following junctions (e.g., 20 μm wide), droplet merging structures (e.g., including a pressurized region of 40 μm width and a depressurized region of 60 μm width), and electrode channels (e.g., 100 μm wide). The electrode channels can be filled with a 5 M NaCl salt solution to form electrodes. Droplet merging can be triggered in an electric field, which can be applied by electrodes connected to a high-voltage amplifier and a power supply, wherein the voltage output is 500 V at a frequency of 1 kHz. The typical pressures within the microfluidic device for oil, cell suspension, and droplets are approximately 800 mbar, 700 mbar, and 850 mbar, respectively.

[0052] The method according to the present invention includes the following steps:

[0053] - In the third group of combined droplets, the combined droplets that emit signals above a predetermined signal strength threshold are separated from the combined droplets that emit signals below a predetermined signal strength threshold to form a fourth group of combined droplets.

[0054] Detection of the signal emitted by the reporter portion in a composite droplet depends on the specific type of signal emitted by the reporter portion. For example, when the reporter portion is capable of emitting an optical signal, such as a fluorescence signal, detection can be performed by exposing the composite droplet to radiation of a first wavelength emitted by a radiation source of the microfluidic device, and subsequently detecting radiation of a different second wavelength emitted by the fluorescent reporter portion via a radiation sensor of the microfluidic device. For example, such radiation of the first wavelength can be selected from VIS, UV, or IR radiation. Detecting the signal emitted by the reporter portion in the composite droplet by the first radiation that induces a fluorescence signal from the fluorescent reporter portion has the advantage of allowing high throughput of the composite droplet because the response of the fluorescent reporter portion to radiation of the first wavelength is almost instantaneous. In particular, when the signal portion is activated by a conformational change due to the binding of a candidate peptide ligand, there is no hysteresis before the composite droplet can be detected. Nevertheless, when the reporter portion is capable of emitting a non-optical signal, such as the expression of a reporter protein or programmed cell death, the composite droplet can also be detected by a suitable detection scheme. For example, dyes for staining dead cells that can be detected by fluorescence are known. In addition, reporter proteins that can be detected by fluorescence are also known.

[0055] In any case, a predetermined signal intensity threshold is defined to ensure the identification of peptides that identify functionally activating G protein-coupled receptors expressed on the cell surface. For example, the predetermined signal intensity threshold may be defined as a signal intensity higher than that observed for wild-type agonist peptide ligands of a specific G protein-coupled receptor for which alternative agonist peptide ligands are being investigated.

[0056] When the reporting section is capable of emitting an optical signal, such as a fluorescence signal, detection can be performed by exposing the combined droplets to radiation having a first wavelength and subsequently detecting radiation emitted by the fluorescent reporting section with a different second wavelength. Suitable devices for detecting the combined droplets include a radiation source emitting radiation with the first wavelength and a radiation detector detecting radiation emitted by the fluorescent reporting section with the second wavelength. For example, such a device is a cell sorting device, such as a fluorescence-activated cell sorting (FACS) device.

[0057] Separating droplets emitting signals above a predetermined signal strength threshold from a third group of droplets can be done in several ways, such as by deflecting the droplets emitting signals above the predetermined signal strength threshold as they pass through an electric field, preferably orthogonal to the direction of the droplets' passage. While droplets can be deflected, for example, as in a fluorescence-activated cell sorting device (FACS), as they fall vertically through an electric field generated by two electrodes, droplets emitting signals above the predetermined signal strength threshold are preferably separated by an electric field spanning a microfluidic channel through which the droplets flow. In the latter case, the droplets move with the flow of an oil-in-water emulsion containing the droplets. Droplets not emitting signals above the predetermined signal strength threshold can also be separated from the droplets using the same steps described above. For example, in cases where the electric field deflects cells in the microfluidic device, a Y-junction can be used immediately downstream of the electric field.

[0058] It should be understood that the separation of the combined droplets can result in: when the combined droplets deflect into the same container, all “positive” combined droplets pool into a separate container, or when the combined droplets deflect into separate containers, each “positive” combined droplet can be placed in a separate container.

[0059] The method according to the present invention includes the following steps:

[0060] - In the fourth set of combined droplets, the sequence of the peptide or propeptide encoded on the plasmid is determined.

[0061] Following separation, plasmids derived from combined droplets emitting signals exceeding a predetermined signal intensity threshold are isolated and can be further amplified in a dedicated polymerase chain reaction (PCR). This can be achieved, for example, through rolling circle amplification of the entire plasmid or by amplifying the plasmid DNA in the presence of primers designed for selective amplification of sequences encoding candidate peptides. In either case, the amplified plasmid or fragment can then be cloned into an expression vector to generate candidate peptides for further investigation into their role in the functional activation of the investigated GPCR and / or sequencing can be performed to determine the coding sequences of the candidate peptides or propeptides.

[0062] The sequence encoding the candidate peptide in the plasmid can be determined using sequencing methods known in the art, such as Sanger sequencing, NGS sequencing, nanopore sequencing, and pyrosequencing. When all "positive" combination droplets aggregated into a single container are sequenced in bulk, the sequence encoding the candidate peptide in the plasmid will be a shared sequence reflecting conserved and variable positions within the candidate peptide sequence. When "positive" combination droplets are placed individually in separate containers, the sequences encoding candidate peptide clones can be sequenced individually, for example, providing sequences that explicitly encode candidate peptides.

[0063] In a preferred embodiment of the invention's first objective, the G protein-coupled receptor is a G protein-coupled receptor comprising a fluorescent reporter portion, and particularly, wherein the reporter portion is an intracellular reporter portion. The fluorescent reporter portion may be a fluorescent reporter portion integrated into a third intracellular loop of the G protein-coupled receptor, similar to the G protein-coupled receptor reported in US20210101960A1.

[0064] In a preferred embodiment of the invention, the cells are human cells, such as HEK cells. An exemplary HEK cell line is HEK 293T.

[0065] In a preferred embodiment of the invention, which is the first objective of the invention, the detection of signals emitted by the reporting portion within the combined droplets and the separation of combined droplets emitting signals above a predetermined signal intensity threshold are performed by fluorescence-activated droplet sorting (FADS). FADS is a technique for sorting droplets encapsulating individual cells or particles based on the fluorescence properties of the droplets or their contents. FADS is not a proprietary technology. FADS is described in Baret et al., Lab Chip, 2009, 9, 1850.

[0066] In a preferred embodiment of the invention's first objective, plasmids from combined droplets emitting signals above a predetermined signal strength threshold are collected after separation and used as contiguous sequences to determine the sequences encoding candidate peptides within the plasmids. In other words, combining droplets emitting signals above a predetermined signal strength threshold are collected in a container and batch sequenced, yielding sequences that exhibit both conserved and variable portions.

[0067] In a preferred embodiment of the invention's first objective, plasmids from combined droplets emitting signals above a predetermined signal strength threshold are individually isolated, and the sequences encoding candidate peptides in said plasmids are individually determined. In other words, each plasmid is sequenced individually, and thus each sequence obtained can be definitively attributed to a candidate peptide from the corresponding combined droplet that caused the signal above the predetermined signal strength threshold.

[0068] In a preferred embodiment of the invention's first objective, the formed water-in-oil droplet further comprises a protease capable of hydrolyzing a precursor of the candidate peptide and releasing the candidate peptide. When the candidate peptide is expressed in the droplet, the peptide can be initially expressed as a precursor, which is subsequently hydrolyzed to produce the actual candidate peptide itself. For example, the precursor may contain a target sequence (typically located on the N-terminal side) or an affinity tag sequence, which is preferably removed from the precursor if the interaction between the candidate peptide and the G protein-coupled receptor is to be unaffected by the target sequence or affinity tag sequence and identical to the natural interaction.

[0069] In a preferred embodiment of the first objective of the invention, the precursor peptide of the candidate peptide is a fusion peptide having a tobacco etch virus (TEV) protease cleavage site on its n-terminal side.

[0070] In a preferred embodiment of the invention's first objective, the formed water-in-oil droplet further comprises a tobacco etch virus protease capable of hydrolyzing a precursor peptide (e.g., a fusion protein) and releasing the candidate peptide. The tobacco etch virus protease exhibits high sequence specificity and is therefore suitable for the controlled hydrolysis of fusion proteins, such as removing an N-terminal affinity tag from the fusion peptide, provided that the peptide sequence is contained in the correct position within the peptide and is the only peptide sequence accessible to the TEV protease.

[0071] In a preferred embodiment of the invention, the diameter of the water droplets is 20 to 25 μm. The diameter of the water droplets can be influenced by changing the flow rates of the aqueous and / or oil phases during droplet formation.

[0072] In a preferred embodiment of the invention's first objective, the reporter portion is capable of emitting a light signal above a predetermined threshold within 0.001 to 1 second, preferably within 0.001 to 0.1 seconds, of functional activation of the G protein-coupled receptor by a suitable candidate peptide, allowing detection within this timeframe. This is significantly faster than a reporter portion capable of emitting non-optical signals (e.g., programmed cell death or reporter protein expression), and it avoids the lag time following droplet consolidation, allowing for immediate signal detection and sorting.

[0073] In a preferred embodiment of the invention’s first objective, the G protein-coupled receptor is PACLight1, a modified G protein-coupled receptor according to sequence SEQ ID1, which is capable of emitting a fluorescent signal when functionally activated with a ligand.

[0074] Example

[0075] To identify peptide agonists that functionally activate G protein-coupled receptors expressed in cells, methods were employed to screen complete plasmid libraries encoding peptides according to the invention. In this case, the G protein-coupled receptor was PACLight1 expressed in HEK cells. PACLight1 has the sequence according to SEQ ID1.

[0076] The plasmid library encodes random pentapeptides, and thus provides approximately 3 million (20) [unclear] ... 5 The library size of ) peptides.

[0077] This method combines rolling circle amplification (RCA) of a single plasmid, in vitro transcription and translation (IVTT), and peptide cleavage using tobacco erosion virus protease into a simplified approach.

[0078] In the first step, an aqueous liquid containing plasmid DNA, etc., is provided, and oil-in-water droplets are formed from this aqueous liquid in a continuous phase of fluorinated oil (HFE-7500) in a microfluidic device. The DNA concentration is adjusted to 100 fg / μL, at which the desired 10% single-cell occupancy and 2% two-cell occupancy are achieved within the droplets according to the Poisson distribution. The aqueous liquid also contains appropriate concentrations of RCA polymerase and IVTT for the replication and transcription of plasmid DNA within the droplets, and appropriate concentrations of TEV protease to allow for the proteolysis of the propeptide and the release of the pentapeptide under investigation.

[0079] To achieve functional droplet assays, a droplet merging device is used in microfluidics to merge previously generated droplets containing expressed pentapeptides with droplets containing the G protein-coupled receptor PACLight1 expressed in HEK cells.

[0080] The droplet merging device is made of PDMS and is 35 μm high. It includes a flow focusing junction (50 μm wide) for single-cell encapsulation, a droplet synchronization region (20 μm wide), a droplet merging section (including a pressurized region of 40 μm wide and a depressurized region of 60 μm wide), and an electrode channel (100 μm wide).

[0081] Electrodes were prepared by filling the channels with a 5 M NaCl salt solution. Droplet coalescence occurred at the pressurization and depressurization structures in the main channel. Droplet coalescence was triggered by an electric field applied by electrodes connected to a high-voltage amplifier and a power supply, with a voltage output of 500 V and a frequency of 1 kHz. The standard pressures used for oil, cell suspension, and droplets after incubation were 800 mbar, 700 mbar, and 850 mbar, respectively, resulting in a droplet dilution of approximately 10-fold after the reaction.

[0082] The fluorescence intensity was set to 1.0, equal to that of the antagonist PACAP. 6-38 The fluorescent signal generated by incubation with cells expressing PACLight1 was used to detect, identify, and subsequently separate combined droplets emitting signals higher than that signal intensity using a fluorescence activated droplet sorting (FADS) device.

[0083] PACAP 6-38 It has the sequence according to SEQ ID2. PACAP represents pituitary adenylate cyclase activating polypeptide.

[0084] PACAP 1-38 It has the sequence according to SEQ ID3. PACAP represents pituitary adenylate cyclase activating polypeptide.

[0085] Based on the above criteria, a total of 25 candidates were identified and isolated from the library. For each candidate, plasmids from combined droplets emitting signals above a predetermined signal intensity threshold were isolated, and the sequences encoding the candidate peptides in the plasmids were determined by Sanger sequencing. (Q5) ® High-fidelity DNA polymerase amplifies plasmid DNA, which is then purified by DNA electrophoresis, gel extraction, and PCR clean-up, followed by Sanger sequencing.

[0086] To determine the potency of the 25 selected peptides, sequences encoding candidate peptides were expressed in separate experiments and co-incubated with cells expressing the PACLight1 receptor. It was observed that 100% of the peptide candidates thus identified effectively and functionally activated the G protein receptor, resulting in recorded fluorescence similar to that observed against the agonist PACAP. 1-38 Recorded fluorescence. For each pentapeptide, 100 fg / μL of DNA encoding the corresponding pentapeptide was combined with the IVTT system to express the pentapeptide.

[0087] Figure 1 Peptide sequences of twenty-five pentapeptides are shown in A and 1B, along with the recorded relative average fluorescence of the merged droplets.

[0088] Therefore, the screening method according to the present invention can rapidly and efficiently isolate pentapeptides capable of functionally activating specific G protein-coupled receptors from a large pentapeptide library. It should be understood that, in principle, plasmids can be modified to represent even larger libraries of possible ligands for any type of receptor, which generates a detectable signal upon interaction with the ligand. In other words, the present invention can be applied to any ligand-receptor pair in animal cells, provided that the receptor is expressed on the surface of the animal cell and contains a reporter portion capable of signaling upon functional activation of the receptor.

[0089] List of reference numerals

[0090] none

Claims

1. A method for screening plasmid libraries encoding peptides to identify one or more peptides that functionally activate G protein-coupled receptors in cells, the method comprising the following steps: - Provides a first set of water-in-oil droplets, each droplet comprising a single plasmid encoding a candidate peptide or its precursor peptide, a polymerase capable of plasmid rolling circle replication, an in vitro transcription / translation system, and optionally a protease. - Within the water-in-oil droplet, the plasmid is replicated using the polymerase capable of plasmid rolling circle replication, and simultaneously, a candidate peptide or propeptide encoded on the plasmid is expressed using the in vitro transcription / translation system to form droplets each containing the candidate peptide, wherein if the propeptide is expressed, the propeptide is hydrolyzed to form droplets each containing the candidate peptide. - A second set of water-in-oil droplets is provided, wherein each droplet contains one or more cells having G protein-coupled receptors expressed on their surface, wherein the G protein-coupled receptors have a reporter portion coupled thereto, and the reporter portion is capable of emitting a signal, preferably an electromagnetic radiation signal, when the G protein-coupled receptors are functionally activated by a suitable candidate peptide. - Droplets containing candidate peptides from the first group are combined one-to-one with droplets from the second group containing cells with one or more G protein-coupled receptors expressed on their surface to form a third group of combined droplets. - In the third group of combined droplets, droplets emitting signals above a predetermined signal strength threshold are separated from droplets emitting signals below a predetermined signal strength threshold to form a fourth group of combined droplets. - In the fourth set of combined droplets, the sequence of the peptide or propeptide encoded on the plasmid is determined.

2. The method for screening plasmid libraries according to claim 1, wherein the G protein-coupled receptor is a G protein-coupled receptor comprising a fluorescent reporter portion, and particularly, wherein the reporter portion is an intracellular fluorescent reporter portion, such as green fluorescent protein.

3. The method for screening plasmid libraries according to claim 1 or 2, wherein the cells having G protein-coupled receptors expressed on their surface are mammalian cells, preferably human cells such as HEK cells.

4. The method for screening plasmid libraries according to any one of claims 1 to 3, wherein the separation of combined droplets emitting signals above a predetermined signal intensity threshold is performed by fluorescence activated droplet sorting (FADS).

5. The method for screening a plasmid library according to any one of claims 1 to 4, wherein the sequence of a peptide or propeptide encoded on the plasmid is determined by aggregating a combination of droplets emitting signals above the predetermined signal intensity threshold, and the sequence of a candidate peptide encoded in the plasmid is determined as a common sequence.

6. The method for screening a plasmid library according to any one of claims 1 to 4, wherein the sequence of a peptide or propeptide encoded on the plasmid is determined by individually separating a combination of droplets emitting a signal above the predetermined signal intensity threshold, and the sequence of a candidate peptide encoded in the plasmid is determined individually.

7. The method for screening plasmid libraries according to any one of claims 1 to 6, wherein the first group of water-in-oil droplets contains a protease capable of hydrolyzing the precursor peptide of a candidate peptide and releasing the candidate peptide.

8. The method for screening plasmid libraries according to claim 7, wherein the protease capable of hydrolyzing the precursor peptide of the candidate peptide and releasing the candidate peptide.

9. The method for screening plasmid libraries according to any one of claims 1 to 8, wherein the diameter of the water droplets is 20 to 25 μm.

10. The method for screening plasmid libraries according to any one of claims 1 to 9, wherein the reporter portion is capable of emitting an electromagnetic radiation signal, particularly a fluorescence signal, within 0.001 to 1 second, preferably within 0.001 to 0.1 seconds, after the G protein-coupled receptor is functionally activated by a suitable candidate peptide.

11. The method for screening plasmid libraries according to any one of claims 1 to 10, wherein the G protein-coupled receptor is based on the human PAC1 receptor (hPAC1R), and preferably has the sequence according to SEQ ID 1.

12. A microfluidic device configured to perform the method according to any one of claims 1 to 11, said microfluidic device comprising - A microfluidic forming chamber for forming a first set of water-in-oil droplets, each droplet containing a single plasmid encoding a candidate peptide or its precursor peptide, a polymerase capable of plasmid rolling circle replication, an in vitro transcription / translation system, and optionally a protease. The formation chamber is fluidly connected to a reservoir for oil and a reservoir for an aqueous liquid containing plasmids from a plasmid library, each plasmid containing a sequence encoding a candidate peptide or its precursor peptide, a polymerase capable of plasmid rolling circle replication, an in vitro transcription / translation system, and optionally a protease. - A microfluidic merging chamber, fluidly connected to the formation chamber, for receiving water-in-oil droplets from the formation chamber, for merging a first group of droplets containing candidate peptides with a second group of droplets in a one-to-one manner, the droplets containing one or more cells having G protein-coupled receptors expressed on their surface, to form a third group of combined droplets. The merging chamber preferably includes electrodes capable of generating an electric field across the merging chamber. A sorting chamber, fluidly connected to the merging chamber, is used to receive combined droplets and separate combined droplets emitting signals above a predetermined signal strength threshold from those emitting signals below a predetermined signal strength threshold, forming a fourth group of droplets. - and optional collection chamber, which is used to receive separated combined droplets that emit signals above a predetermined signal strength threshold.

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