Chemical libraries encoded in oligonucleotides

Abstract

We provide a system for screening compounds. [Solution] This application provides beads with covalently attached compounds and covalently attached DNA barcodes, as well as methods for using such beads. The beads have many substantially identical copies of compounds and many substantially identical copies of DNA barcodes. The compounds consist of one or more chemical monomers, and the DNA barcodes take the form of barcode modules, each module enabling the identification of corresponding and corresponding chemical monomers. The nucleic acid barcodes may have a chain-like or orthogonal structure. Methods for sequencing bead-bound nucleic acid barcodes, methods for cleaving compounds from beads, and methods for evaluating the biological activity of released compounds are provided.

Description

[Technical Field]

[0001] This disclosure relates to high-throughput screening using a library of compounds, wherein the compounds are conjugated to or contained within beads, and each bead contains multiple copies of a single compound, and further, the beads also include a DNA tag encoding the identity or synthetic history of the compound contained within or on the beads. This disclosure also relates to a high-throughput assay performed in a picowell, the picowell comprising compound-loaded beads and assay material. This disclosure further relates to releasing bead-bound compounds and screening them for biological activity. In general, this disclosure envisions assays in which beads are used as a compound delivery vehicle, and methods for preparing such compound-loaded beads.

[0002] This disclosure relates to bead-bound compounds, each compound being constructed from one or more monomers belonging to a chemical library. This disclosure also relates to bead-bound DNA barcodes, i.e., nucleic acids (not related to genetic coding) whose sequences are the codes, pointing to a specific chemical library monomer. This disclosure further relates to releasing bead-bound compounds and subsequently screening the released compounds for biological activity.

[0003] This disclosure also relates, in general, to methods for perturbing cells or a small number of cells using dose-controlled compounds and analyzing changes in the cellular state by RNA and / or protein analysis. The methods disclosed herein may be applied at the single-cell level or to multiple cells for purposes of high-throughput screening, target discovery, or diagnostics, and other similar applications.

[0004] Cross-reference of related applications This application claims the interests and priority of U.S. Provisional Patent Application No. 62 / 562,905, filed on September 25, 2017, and U.S. Provisional Patent Application No. 62 / 562,912, further filed on September 25, 2017, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0005] For example, combinatorial chemistry, including split-pool chemistry, can be used to synthesize large quantities of compounds. Compounds produced in this way have found use in the field of medicinal chemistry, and the compounds can be screened for various biochemical activities. These activities include binding to one or more proteins, which are known at the time the screening test is performed. Alternatively, the protein to which the compound being tested binds is identified only after the binding event is detected. Compounds can also be screened for their activity in inhibiting or activating known proteins (this is not simply screening for "binding" activity). Alternatively, compounds can be screened for their activity in inhibiting or activating cellular functions, where the molecular target is unknown to the researcher at the time of screening.

[0006] Screening compounds, such as those belonging to a vast library of chemicals prepared by the split-pool method, can be easily performed using arrays of thousands of microwells, nanowells, or picowells. Furthermore, screening can be facilitated by providing different compounds to each picowell via beads, with each bead containing hundreds of copies of the same compound, and each bead containing hundreds more copies of its "DNA barcode," which can be used to identify compounds bound to the same bead. Additionally, the use of cleavable linkers further simplifies compound screening, allowing for controlled release of compounds from the beads, which can then be used in biochemical or cell line assays within the same picowell.

[0007] Assaying compounds in very small, confined volumes, such as droplets, picowells, or microfluidic environments, is broadly beneficial, for example, due to the small amount of assay reagents required, and therefore not limited to compounds produced in combinatorial chemistry. Any method by which compounds can be loaded onto beads and eluted from the beads at a later time can be used to deliver bead-bound compounds to assays in small, confined volumes. Adding nucleic acid barcodes to the beads allows the identity of compounds present within the beads to be carried along the assay volume. The method allows for very fast throughput assays without requiring robotics or spatial indexing of compounds in microtiter plates. Millions to billions of compounds can be held in a single small vial, and the identity of the compounds is tagged to the same beads containing each individual compound.

[0008] A common method of drug discovery involves selecting a target of interest and monitoring the interaction between the target protein or enzyme and a large library of compounds. Often, numerous initial hits are toxic to the body or cross-react with other proteins in the body, demonstrating that target-based selection is an inefficient method of drug screening. The need for pre-selected targets is also an inherent limitation, as the biological basis of the disease must be well known and understood. Screening compounds against an entire organism is difficult, costly, and a very low-throughput operation.

[0009] Traditional phenotypic screening in cells involves creating disease-state cell models, contacting cells with various drug libraries, and monitoring whether the disease phenotype is modified by measurable assays. While such screening methods are called phenotypic screening because the underlying biological mechanisms are not always understood initially, measurable phenotypic changes indicating a healing response are considered relevant metrics. Currently, a vast number of cell lines and disease models reflecting various baseline and diseased cell states are available. Numerous compound libraries and biological drug candidates are also available. However, because assays are limited to microtiter plate formats and imaging techniques, overt screening campaigns combining different cell models with different drug candidates to search for phenotypic responses face technical limitations, both of which significantly limit throughput.

[0010] One way to overcome throughput limitations is to employ high-throughput single-cell screening approaches in drug discovery (see, e.g., Heath et al., Nat Rev Drug Discov. 15:204-216, 2016). These approaches allow single cells to be separated and isolated in compartments, and individual assays to be performed on each cell. Genomic analysis by single-cell mRNA sequencing using methods such as droplet encapsulation is a common method for revealing complex details hidden in ensemble measurements (see, e.g., Macosko et al., Cell 161:1202-1214, 2015, and Ziegenhain et al., Mol Cell 65:631-643, 2017, whose disclosures are incorporated herein by reference in their entirety). Current single-cell analysis platforms are said to be capable of quantifying mRNA transcripts at single-cell resolution, characterizing and fingerprinting cells based on their transcriptional state. This approach allows for comparison between tissue samples extracted from controls or prepared experimentally, and examination of single-cell transcription and, consequently, protein expression status. Measurement of single-cell mRNA by transcriptome sequencing and profiling is a crucial approach for investigating the molecular mechanisms of drug efficacy, resistance, and therapeutic target discovery, as well as the phylogenetic phenotype of cells in disease progression (see, e.g., Chu et al., Cell Biol and Toxicol 33:83-97, 2017; Wang, Cell Biol Toxicol 32:359-361, 2016; and Wang et al., Cell Biol Toxicol 33:423-427, 2017). Applications of single-cell RNA sequencing are used to define intercellular heterogeneity, demonstrated by intercellular variability of the transcriptome, which is highly relevant to drug efficacy and specificity, transcriptional probability, transcriptome plasticity, and genomic evolution. Encapsulation within picowells has also been demonstrated (see, for example, Gierahn et al., Nat Methods 14:395-398, 2017).Using a similar isolation method, single-cell proteins can also be measured (Butnik et al., BioRxiv, Jan. 2017, Su et al., Proteomics 17:3-4, 2017).

[0011] Despite the rapid increase in high-throughput single-cell RNA sequencing (RNA-seq) methods, including commercialized versions of automated platforms such as Fluidigm C1, 10XGenomics, or the 1CellBiO system, the application of single-cell RNA profiling for target-agnostic high-throughput drug screening and target discovery is constrained by the lack of a method that can efficiently separate different drugs into different cells. While it is possible to incubate cells or tissues under different perturbations in well plates and subsequently perform single-cell analysis and comparison between transcription profiles, the number of drugs that can be tested is limited by the plate capacity. Furthermore, a significant bottleneck arises as barcoded mRNA must be isolated and prepared from each sample, and comprehensive RNA profiling must be performed for all samples. [Overview of the project]

[0012] In short, the present disclosure is a system for screening compounds, comprising: (a) a picowell array plate comprising a plurality of picowells, each picowell having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, and a wall existing between the upper hole and the floor; (b) beads disposed within the picowells, comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds; and (c) beads comprising bead-bound DNA barcodes taking the form of either a chained DNA barcode or an orthogonal DNA barcode, wherein the DNA barcode takes the form of a chained DNA barcode, the chained DNA barcode is prepared by (i) using click chemistry, or (ii) using a repeating cycle of steps, the repeating cycle of steps being a partially prepared bead-bound DNA barcode. The system provides a method comprising: (d) using a code and hybridizable sprint oligonucleotide (sprint oligo), wherein hybridization is mediated by an annealing site on the sprint oligo and a corresponding complementary annealing site on a partially constructed bead-bound DNA barcode, the annealed sprint oligo being used as a template for extending the partially constructed DNA barcode using DNA polymerase, the sprint oligo containing a base complementary to the DNA barcode module which is polymerized onto the partially constructed DNA barcode; and (d) each of several substantially identical bead-bound compounds containing one or more chemical library monomers, each bead-bound DNA barcode module identifying a corresponding chemical library monomer, the term “compound” is used to refer to a finished product containing one or more chemical library members, and the finished DNA barcode identifying a compound.

[0013] The floor of a microwell, nanowell, or picowell need not be flat. The floor can be curved, such as the bottom of a glass test tube or a metal centrifuge tube. Also, the floor can be conical in shape, such as a conical centrifuge tube. The floor can be flat, but can have a notch, for example, a notch that facilitates the behavior of an assay solution or a cell culture solution near the bottom of any bead located in a picowell. In embodiments with a flat floor, the system and method may require a flat floor.

[0014] Linked DNA barcodes can be fully fabricated by methods of organic chemistry, such as click chemistry. Also, orthogonal DNA barcodes can be fully fabricated by methods of organic chemistry, including, for example, click chemistry.

[0015] Further provided is the above-described system, further including a plurality of caps, each cap being capable of fitting over the opening of a different picowell, each cap being capable of minimizing or preventing evaporation of fluid inside the picowell, and each cap being capable of minimizing or preventing leakage of fluid inside the picowell.

[0016] Further included is the above-described system, wherein the linked DNA barcode is made by a method that uses (i) both iterative cycles of steps using click chemistry and split oligos, (ii) both click chemistry and chemical methods other than the click chemistry method, (iii) only click chemistry, or (iv) only iterative cycles of steps using split oligos. In the case of this particular embodiment, the "linked DNA barcode" in question does not include any chemical coupler used to directly bind nucleic acids to beads.

[0017] In an embodiment of the spherical cap, provided is the above system further comprising a plurality of spherical caps, each cap being capable of fitting into a picowell hole, the hole being circular, each cap being capable of minimizing or preventing evaporation of the fluid inside the picowell, and each cap being capable of minimizing or preventing leakage of the fluid inside the picowell.

[0018] In an embodiment of the response element, provided is the above system, wherein at least one bead disposed in at least one picowell comprises at least one response capture element coupled to the at least one bead. Also contemplated is the above system, wherein at least one bead disposed in at least one picowell comprises at least one response capture element coupled to the at least one bead, and the at least one response capture element comprises (a) poly(dT) or (b) an exon-targeted RNA probe.

[0019] Also contemplated is the above system, wherein the DNA barcode is either a concatenated DNA barcode or an orthogonal DNA barcode, the DNA barcode comprises one or more DNA barcode modules, each of the one or more DNA barcode modules encodes information identifying a chemical library monomer, and the concatenated DNA barcode or the orthogonal DNA barcode further comprises one or both of (a) one or more functional nucleic acids and (b) one or more nucleic acids encoding information of a type other than the identity of the chemical library monomer.

[0020] The following discloses embodiments consisting "only" and embodiments including, which apply to the number of bead-bound DNA barcode modules constituting the DNA barcode. Provided are embodiments of a DNA barcode consisting of only one DNA barcode module, or only two DNA barcode modules, or only three DNA barcode modules, or four DNA barcode modules, etc., or a DNA barcode including at least one DNA barcode module, or at least two DNA barcode modules, or at least three DNA barcode modules, or at least four DNA barcode modules, etc.

[0021] Furthermore, the bead-linked chain DNA barcodes include (i) a first DNA barcode module, or (i) a first DNA barcode module, a first annealing site, and a second DNA barcode module, or (ii) a first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, and a third DNA barcode module, or (iii) a first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, a third annealing site, and a fourth DNA barcode module, or (iv) a first (v) a system comprising a DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, a third annealing site, a fourth DNA barcode module, a fourth annealing site, and a fifth DNA barcode module, or (v) a system comprising a first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, a third annealing site, a fourth DNA barcode module, a fourth annealing site, a fifth DNA barcode module, a fifth annealing site, and a sixth DNA barcode module.

[0022] Furthermore, the system is intended to further include a primer binding site capable of binding to a DNA sequencing primer, the primer binding site capable of directing sequencing of one or more first, second, third, fourth, fifth, or sixth DNA barcode modules, wherein the primer binding site is located 3 primes relative to the first DNA barcode module, 3 primes relative to the second DNA barcode module, 3 primes relative to the third DNA barcode module, 3 primes relative to the fourth DNA barcode module, 3 primes relative to the fifth DNA barcode module, or 3 primes relative to the sixth DNA barcode module, or the primer binding site is located between the first and second DNA barcode modules, or between the second and third DNA barcode modules, or between the third and fourth DNA barcode modules, or between the fourth and fifth DNA barcode modules, or between the fifth and sixth DNA barcode modules.

[0023] Furthermore, the system provided includes the above, wherein the primer binding site is located between the first and second DNA barcode modules, or between the second and third DNA barcode modules, or between the third and fourth DNA barcode modules, or between the fourth and fifth DNA barcode modules, or between the fifth and sixth DNA barcode modules. In embodiments relating to the location of primer binding sites associated with upstream DNA barcode modules and downstream DNA barcode modules, the system provided is the above, wherein the primer binding site is located between each pair and all pairs of consecutive DNA barcode modules.

[0024] Furthermore, the system provided includes a bead comprising a DNA barcode which is an orthogonal DNA barcode, the bead comprising an outer surface, the orthogonal DNA barcode comprising (a) a first nucleic acid comprising an annealing site for a first DNA barcode module and sequencing primer, which binds to the bead at a first position, (b) a second nucleic acid comprising an annealing site for a second DNA barcode module and sequencing primer, which binds to the bead at a second position, and (c) a third nucleic acid comprising an annealing site for a third DNA barcode module and sequencing primer, to which the second nucleic acid binds to the bead at a third position, wherein the first, second, and third positions on the bead are respectively located at different positions on the outer surface of the bead.

[0025] In an embodiment of the coding, the provided system includes one or more nucleic acids in which the DNA barcode does not identify any chemical library monomer, but instead identifies (a) the class of compound to be cleavably attached to the beads, (b) the number of steps in a multi-step organic synthesis pathway, (c) the date the bead-bound compound was synthesized, (d) the disease to which the bead-bound compound is intended to be treated, (e) the cellular event to which the bead-bound compound is intended to be stimulated or inhibited, or (f) the reaction conditions used to bind a given chemical library monomer to the beads.

[0026] In embodiments of the linker, the system provided is one in which each of a plurality of substantially identical bead-binding compounds is bonded to a bead via a cleavable linker. Also provided is one in which each of a plurality of substantially identical bead-binding compounds is bonded to a bead via a photocleavable linker. Also provided is one in which each of a plurality of substantially identical bead-binding compounds is bonded to a bead via an incleavable linker.

[0027] In embodiments of TentaGel®, the provided system comprises a grafted copolymer in which at least one bead consists of a low-crosslinked polystyrene matrix grafted with polyethylene glycol (PEG).

[0028] In an embodiment of the release monitor, the disclosure provides the above-described system in which at least one picowell includes release monitor beads and does not include any other types of beads.

[0029] The emission monitor bead comprises a bead-binding quencher and a bead-binding fluorophore, the bead-binding quencher being quenchingly positioned immediately adjacent to the bead-binding fluorophore and capable of quenching at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or at least 99.5%, or at least 99.9%) of the fluorescence of the bead-binding fluorophore, the bead-binding fluorophore being bound via a first photocleavable linker, and the picowell containing the emission monitor bead is the first picowell, the first picowell contains the first solution, Exposure of the first picowell to cleavage conditions enables the cleavage of the photocleavable linker, releasing the fluorophore into the first solution in the first picowell. Exposure results in the diffusion of the fluorophore throughout the first solution within the first picowell. The fluorescence signal obtained by shining light on the first picowell containing the first solution with the diffused fluorophore is used by the user to calculate the release rate of the bead-bound fluorophore from the release monitor beads, and the second picowell contains a bead-bound compound bound to the same type of photocleavable linker as the first photocleavable linker, and the second picowell contains the second solution.

[0030] The release rate value calculated from the release monitor beads in the first picowell allows us to calculate the concentration of the released compound in the second solution in the second picowell.

[0031] In embodiments relating to the identity of all compounds bound to a given bead, or to the identity of all DNA barcodes bound to a given bead, the system provided is one in which at least one bead contains a plurality of substantially identical bead-bound DNA barcodes, the plurality being 10 million to 100 million copies of substantially identical bead-bound DNA barcodes. Further provided is the system in which at least one bead contains a plurality of substantially identical bead-bound compounds, the plurality being 10 million to 100 million copies of substantially identical bead-bound compounds.

[0032] In embodiments relating to cells (e.g., mammalian cells, cancer cells, bacterial cells), the system provided is such that at least one picowell contains at least one cell, and a plurality of substantially identical bead-bound compounds are bound to at least one bead via a cleavable linker, and cleaving the cleavable linker releases the bead-bound compound from the bead, producing the released compound, which can come into contact with at least one cell. In other cellular embodiments, the provided system comprises at least one picowell containing at least one cell, wherein multiple substantially identical bead-binding compounds are bound to at least one bead via a cleavable linker, and cleaving the cleavable linker releases the bead-binding compound from the bead, producing the released compound, which is capable of contacting at least one cell, the at least one cell being (i) a mammalian cell that is not a cancer cell, (ii) a mammalian cancer cell, (iii) a dead mammalian cell, (iv) an apoptotic mammalian cell, (v) a necrotic mammalian cell, (vi) a bacterial cell, (vii) a malaria parasite cell, (vii) a metabolically active cell that has a cross-linked genome and is incapable of cell division, or (ix) a virus-infected mammalian cell.

[0033] In an embodiment of the device, the provided system is such that each picowell has an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, a wall existing between the upper hole and the floor, the hole being circular, the floor being circular, the wall being in the shape of a frustocone, the hole having a first diameter, the floor having a second diameter, and the first diameter being greater than the second diameter.

[0034] In embodiments relating to other devices, each picowell is provided having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, a wall existing between the upper hole and the floor, the hole being circular, the floor being circular, the wall taking the form of a frustocone, the hole having a first diameter, the floor having a second diameter, the first diameter being greater than the second diameter, and further comprising a cap that fits snugly into the hole, the hole being composed of a polymer having a larger durometer (harder), and the cap being made of a polymer having a smaller durometer (softer), the relative durometers of the cap and the hole being such that the cap reversibly and snugly fits into the hole. The system provides the following, which allows the cap to: (i) be a cap intended solely to stop the picowell and prevent leakage; (ii) be a passive cap capable of absorbing metabolites released by cells while cells in cell culture medium are being cultured in the picowell; (iii) be an active cap taking the form of beads containing multiple essentially identical compounds, each of which is linked to the beads by a cleavable linker; and (iv) be an active cap taking the form of beads containing multiple identical reagents, each of which is linked to the beads by a cleavable linker. The system provided is either spherical or non-spherical.

[0035] In an embodiment of the mat, the system comprises a picowell array plate having a general top plane, a plurality of picowells, each picowell having a top hole defining an opening at the top of the picowell, a bottom defined by a floor, the top hole being separated from the floor by a wall, the wall being located between the top hole and the floor, and optionally, beads placed in at least one of the plurality of picowells, each bead containing a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds, and the picowell array plate having a plurality of picowells The present invention further includes a mat capable of reliably covering at least one or all of the top openings, or substantially reliably covering at least one or all of the top openings of a plurality of picowells, wherein the reliably covering is reversible, and the mat optionally includes one or all of the following: (a) an absorbent surface capable of absorbing any metabolites, biochemicals, or proteins that may be contained in one or more of the plurality of picowells when placed in contact with the general plane of the top of the picowell array plate; and (b) an adhesive surface capable of maintaining reversible adhesion to the general plane of the top of the picowell array plate.

[0036] In embodiments of the biochemical assay, the system comprises at least one picowell, the at least one picowell comprising beads containing a plurality of substantially identical compounds and a plurality of substantially identical barcodes, and the at least one picowell comprising an assay medium containing a substrate for cereblon E3 ubiquitin ligase such as cereblon E3 ubiquitin ligase, Ikaros, or Aeolus, the system being capable of reducing the intracellular concentration of Ikaros or Aeolus to enable screening of compounds that activate the E3 ubiquitin ligase activity of cereblon.

[0037] In another embodiment of the biochemical assay, the system described above is intended to include at least one picowell, the at least one picowell containing beads containing a plurality of substantially identical compounds and a plurality of substantially identical barcodes, and the at least one picowell containing an assay medium containing a substrate for MDM2 E3 ubiquitin ligase, such as MDM2 E3 ubiquitin ligase, p53, and the system is capable of increasing the intracellular concentration of p53 to enable screening of compounds that activate the E3 ubiquitin ligase activity of MDM2.

[0038] In more barcoding embodiments, the provided system comprises one or more nucleic acids that, instead of encoding any chemical library monomer, (a) a class of compounds cleavably attached to the beads, (b) a step in a multi-step organic synthesis pathway in which the bead-binding nucleic acid corresponds to a given chemical monomer used to construct the bead-binding compound, and the bead-binding nucleic acid corresponding to the given chemical monomer identifies that chemical monomer, (c) the date the bead-binding compound was synthesized, (d) a disease for which the bead-binding compound is intended to be treated, and (e) a cellular event for which the bead-binding compound is intended to be stimulated or inhibited.

[0039] In embodiments lacking any headpiece, the system provided is such that at least one bead comprises a plurality of substantially identical bead-binding compounds, further comprising a plurality of substantially identical bead-binding DNA barcodes, and there is no headpiece that links any of the bead-binding compounds to any of the bead-binding DNA barcodes.

[0040] Furthermore, the intended system is one in which at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of substantially identical bead-bound DNA barcodes have the same structure. Furthermore, the intended system is one in which at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of substantially identical bead-bound compounds have the same structure.

[0041] Furthermore, the supplied system is one in which the linked DNA barcode includes at least one nucleic acid that is a DNA barcode module, or one in which the linked DNA barcode includes only one nucleic acid that is a DNA barcode module.

[0042] In embodiments of the sequencing primer annealing site, the provided system comprises a chain of DNA barcodes comprising at least one nucleic acid which is a DNA barcode module, and (a) a hairpin structure which is usable as an annealing site for a sequencing primer, (b) a hairpin structure which is usable to form a hairpin structure which comprises a sequencing primer, an annealing site for the sequencing primer, and a bead in the hairpin structure which is usable to form a hairpin structure which is 5-prime with respect to the sequencing primer and 3-prime with respect to the annealing site for the sequencing primer, or (c) a spacer nucleic acid which comprises at least one functional nucleic acid which is a spacer nucleic acid.

[0043] In other embodiments of the sequencing primer, the system provided includes an orthogonal DNA barcode comprising a plurality of DNA barcode modules, each of which is bound to a different site on a bead, either directly or via a linker, and each of the plurality of DNA barcode modules being (a) usable as an annealing site for a sequencing primer, (b) capable of forming a hairpin structure, the hairpin structure comprising a sequencing primer, an annealing site for the sequencing primer, and a bead, the bead being 5-prime to the sequencing primer and 3-prime to the annealing site for the sequencing primer, or (c) a spacer nucleic acid, comprising at least one functional nucleic acid.

[0044] In embodiments that enumerate the functional language for sprint oligos, provided are beads containing a chain of DNA barcodes, the chain of DNA barcodes comprising (a) a sprint oligo comprising three nucleic acids, the three nucleic acids being a capture that hybridizes to a first annealing site, a capture that hybridizes to a second DNA barcode module, and a nucleic acid that is a second annealing site, comprising a first DNA barcode module and a first annealing site of a first sprint oligo (sprint oligo), and (b) a second sprint oligo comprising three nucleic acids, the three nucleic acids being a capture that hybridizes to a second annealing site, a nucleic acid that is a third DNA barcode module, and a nucleic acid that is a third annealing site, comprising a second DNA barcode module and a second annealing site of a second sprint oligo.

[0045] In another embodiment, which includes a functional language related to sprint oligos, the provided bead further comprises a third DNA barcode module and a third annealing site of the third sprint oligo, wherein the third sprint oligo comprises three nucleic acids, the three nucleic acids being nucleic acids that are captures to hybridize to a third annealing site, a fourth DNA barcode module, and a fourth annealing site.

[0046] Furthermore, in yet another embodiment including a functional language related to sprint oligos, provided are the beads described above, further comprising (i) a fourth sprint oligo comprising three nucleic acids, the three nucleic acids being a capture that hybridizes to a fourth annealing site, a nucleic acid that is a fifth DNA barcode module, and a nucleic acid that is a fifth annealing site; (ii) a response capture element; and (iii) an emission monitor.

[0047] In embodiments of the linker, the beads are those to which a chain of DNA barcodes is attached, but (i) not attached to the beads via any photocleavable linker, (ii) not attached to the beads by any enzymatically cleavable linker, or (iii) not attached to the beads by any cleavable linker of any kind.

[0048] In embodiments relating to separate binding sites, the provided beads include a compound to which a chained DNA barcode is bound at a first position on the beads, and the beads also include a compound to which the first position is bound at a second position on the beads, wherein the first position is not the same as the second position.

[0049] In the surface embodiment (internal and external), the provided bead comprises an external and an internal surface, and the bead comprises at least 10,000 substantially identical linked DNA barcodes bound to the bead, with at least 90% of the at least 10,000 substantially identical linked DNA barcodes bound to the external surface.

[0050] In an exclusionary embodiment that can distinguish this disclosure from other embodiments, provided are the above-mentioned beads, which do not contain any polyacrylamide, and the linked DNA barcodes (i) do not contain any nucleic acid that is a promoter, (ii) do not contain any nucleic acid that is poly(A), or (iii) do not contain any nucleic acid that is a promoter and do not contain any nucleic acid that is poly(A).

[0051] In embodiments of emission monitor beads, the disclosure provides emission monitor beads capable of functioning in an aqueous medium, the emission monitor beads comprising a bead-binding quencher and a bead-binding fluorophore, wherein the bead-binding quencher is quenchingly positioned very close to the bead-binding fluorophore and capable of quenching at least 50% of the fluorescence of the bead-binding fluorophore, the bead-binding fluorophore is bound via a first photocleavable linker, the picowell containing the emission monitor beads is a first picowell, the first picowell contains a first solution, and exposure of the first picowell to cleavage conditions allows for cleavage of the photocleavable linker, the fluorophore is bound via the first picowell Release into the first solution in the well and exposure results in the diffusion of the fluorophore throughout the first solution in the first picowell. The fluorescence signal obtained by shining light on the first picowell containing the first solution with the diffused fluorophore is used by the user to calculate the release rate of the bead-bound fluorophore from the release monitor beads. The second picowell contains a bead-bound compound bound to the same type of photocleavable linker as the first photocleavable linker, and the second picowell contains a second solution. The release rate value calculated from the release monitor beads in the first picowell allows for the calculation of the concentration of the released compound in the second solution in the second picowell. In other embodiments of the emission monitor, the provided release monitor beads are those in which the fluorophore is TAMRA and the quencher is QSY7, and release monitor beads having the structure shown in Figure 9, and release monitor beads having the structure shown in Figure 10, and release monitor beads capable of quenching at least 90%, at least 98%, at least 99%, or at least 99.9%.

[0052] Embodiments of the manufacturing method include a method for synthesizing release monitor beads, the release monitor beads comprising beads, a quencher, a fluorophore, and a photocleavable linker that binds the fluorophore to the beads, the method comprising, in this order: (i) providing a resin; (ii) coupling a lysine linker to the resin, wherein the reagent containing the lysine linker is L-Fmoc-Lys(4-methyltrityl)-OH; (iii) removing the Fmoc protecting group; (iv) coupling a quencher using a reagent which is quencher-N-hydroxysuccinimide (quencher-NHS) as the source of the quencher; (v) removing the 4-methyltrityl protecting group using a reagent which contains trifluoroacetic acid; (vi) coupling a photocleavable linker to the epsilon-amino group of lysine, wherein the photocleavable linker is provided by a reagent which is Fmoc-photocleavable-linker-OH; and (vii) coupling the fluorophore. Embodiments described above are also provided, but the order of the steps is not considered. In another embodiment of the method, the above method is provided in which the fluorophore is TAMRA and the quencher is QSY7.

[0053] A method relating to the usefulness of release monitor beads is provided, which is a method for controlling the concentration of a compound in a solution present in a picowell, the method being applied to a bead-bound compound in a picowell, the picowell containing a solution, the bead-bound compound being bound to beads via a cleavable linker, the method comprising (a) exposing the bead-bound compound to conditions causing the cleavable linker to break, thereby releasing the bead-bound compound from the beads and producing the released compound, (b) conditions including light capable of breaking the cleavable linker, (c) conditions adjusted to produce a determined concentration of substantially uniform concentration, and (d) a determined concentration, carried out taking into account the concentration of the released fluorophore released from the bead-bound release monitor. Further provided are the above method wherein the conditions are adjusted by adjusting one or more of the wavelength of light, the intensity of light, and the duration of exposure, as well as the above method wherein the concentration of the emitted fluorophore emitted from the bead-bonded emission monitor is determined at the same time as it results in the emission of the bead-bonded compound from the beads and the generation of the emitted compound, as well as the above method wherein the concentration of the emitted fluorophore emitted from the bead-bonded emission monitor is determined at a point substantially prior to the emission of the bead-bonded compound from the beads and the generation of the emitted compound.

[0054] The term "determined" can mean a concentration that is determined in advance and is set to the desired concentration before the beads are exposed to light. Alternatively, the term "determined" can mean a concentration that is determined "in real time," i.e., the concentration that is determined simultaneously with the exposure of the beads to light.

[0055] In embodiments of the cap, the cap is included in combination with a picowell plate containing a plurality of picowells, the cap is usable with the picowell plate containing a plurality of picowells, each of the plurality of picowells is defined by a hole, a floor, and a wall, the wall is defined by an upper hole and a bottom floor, the hole is circular, the floor is circular, the wall takes the form of a frustoconical surface, the hole has a first diameter, the floor has a second diameter, the first diameter is greater than the second diameter,

[0056] The cap is a spherical cap capable of fitting snugly into the hole, the hole is made of a polymer having a larger durometer (harder), and the cap is made of a polymer having a smaller durometer (softer), and the relative durometers of the cap and hole allow the spherical cap to fit reversibly and snugly into the hole, and the cap is capable of (i) sealing the picowell to prevent leakage, (ii) being a passive cap capable of absorbing metabolites released from cells in a situation where cells in cell medium are cultured in the picowell, (iii) being an active cap taking the form of beads containing multiple essentially identical compounds, each of which is bound to the bead by a cleavable linker, and upon cleavage of the cleavable linker, at least a portion of the multiple compounds are released from the beads, and (iv) being an active cap taking the form of beads containing multiple identical reagents, each of which is bound to the bead by a cleavable linker, and upon cleavage of the cleavable linker, at least a portion of the multiple reagents are released from the beads.

[0057] In embodiments of the porous caps, provided are a plurality of porous caps combined with a picowell plate and a solid polymer coating, each of the plurality of porous caps comprising an upper and lower surface, the picowell plate comprising a plurality of picowells, at least one porous cap in contact with a picowell and reversibly and tightly fitting to the picowell, each of the picowell plate and the upper surface of the plurality of porous caps being covered with a solid polymer coating, the solid polymer coating in contact with at least a portion of the upper surface of each cap and adhesively adhering to at least a portion of the upper surface, and (i) each of the plurality of picowells being capable of holding an aqueous solution, the product of the reaction being formed in the solution and generated (ii) at least a portion of the substance is absorbed by the underside of each of the multiple porous caps, (ii) a polymerizable reagent solution is poured over the multiple porous caps in combination with a picowell plate, the polymerizable reagent polymerizes to form a substantially flat surface that coats substantially all of the upper surface of the picowell plate, thereby fixing the polymerized reagent to each of the multiple porous caps, (iii) all of the multiple porous caps are removable by peeling from the multiple picowells, the adhesion is maintained between the multiple porous caps and the polymerizing reagent, resulting in an array of caps partially adhered to the upper surface of each cap embedded in the polymerizing reagent, and the underside of each cap is available for analysis of the absorbed reaction product.

[0058] This provides an embodiment of a manufacturing method for inducing the enzymatic synthesis of DNA barcodes using sprint oligos. Provided is a method for producing bead-linked ligatures DNA barcodes, the bead-linked ligatures DNA barcodes comprising a plurality of DNA barcode modules, optionally one or more functional nucleic acids, and optionally one or more identity-coding nucleic acids encoding identity other than identity of chemical library monomers, wherein (a) a first annealing site is a first sprint oligonucleotide (sprint oligo), a first sprint oligo capable of functioning as a template for DNA polymerase that catalyzes polymerization to a linked polynucleotide, a hybridized first sprint oligo capable of hybridizing with complementary nucleotides to those of the hybridized first sprint oligo after polymerization, and the polymerized complementary nucleotides to those of the hybridized first sprint oligo are bead-linked to a second The method comprises: (b) providing a bead with a conjugated polynucleotide comprising a first DNA barcode module and a first annealing site, comprising a DNA barcode module and a second annealing site; (c) providing the bead with a polynucleotide conjugated with the first sprint oligo, thereby hybridizing the first sprint oligo with the conjugated polynucleotide; (d) adding a DNA polymerase and deoxynucleotide triphosphate (dNTP) to which the conjugated polynucleotide has a free 3' end and polymerization is relative to the free 3' end, thereby enabling the DNA polymerase to catalyze the polymerization of the dNTP into the conjugated polynucleotide; and (d) washing away the first sprint oligo. The method is also intended to be one in which the first sprint oligo comprises a first annealing site, a second DNA barcode module, and a second annealing site.

[0059] A further method of the manufacturing embodiment is provided, wherein the first sprint oligo comprises nucleic acids encoding a first annealing site, a second DNA barcode module, a second annealing site, and a first sequencing primer annealing site, the first sequencing primer annealing site being capable of hybridizing to a sequencing primer, resulting in a hybridized sequencing primer capable of directing sequencing of the second DNA barcode module and the first DNA barcode module.

[0060] Furthermore, the method described above is intended to either add the first sprint oligo, DNA polymerase, and dNTPs all simultaneously, or add them separately at different times.

[0061] With respect to the internal versus external position of the beads, the provided method is such that the beads consist of external and internal positions, the bead-bound chain DNA barcode is substantially external to the beads and slightly internal to the beads, the beads also contain multiple bound compounds, all of which have substantially identical structures when compared to one another, and the beads are composed of substantially hydrophobic polymers.

[0062] In a further embodiment of the method, provided is a first DNA barcode module, a first annealing site, a second DNA barcode, and a second annealing site, the second annealing site comprising a second sprint oligo, a second sprint oligo capable of functioning as a template for DNA polymerase catalyzing polymerization to a second sprint oligo, a second sprint oligo hybridizable with complementary nucleotides of the hybridized second sprint oligo, and the polymerized complementary nucleotides of the hybridized second sprint oligo after polymerization, comprising a bead-bound third DNA barcode module and a third annealing site. The method further comprises: (b) providing a bead with a conjugated first longer polynucleotide; (c) providing the bead with a polynucleotide conjugated with a second sprint oligo, thereby hybridizing the second sprint oligo with the conjugated first longer polynucleotide; (d) adding a DNA polymerase and a deoxynucleotide triphosphate (dNTP), wherein the conjugated longer polynucleotide has a free 3' end and polymerization is relative to the free 3' end, thereby enabling the DNA polymerase to catalyze the polymerization of the dNTP into the conjugated longer polynucleotide; and (d) washing away the second sprint oligo.

[0063] This relates to the sequential numbering of a first DNA barcode module, a second DNA barcode module, a third DNA barcode module, and so on, for manufacturing an entire DNA barcode. This also relates to the repeated cycle of the steps of the method many times in manufacturing an entire DNA barcode. Provided is the above method, in which each of the plurality of DNA barcode modules is identified or named by number, and the method further comprises repeating the enumerated steps, in which, as a first iteration, the name of the DNA barcode module is incremented by adding one number to an existing name, the name of the annealing site is incremented by adding one number to an existing name, the name of the sprint oligo is incremented by adding one number to the name of an existing distal end DNA barcode module, the name of the "first longer polynucleotide" is changed by adding one number to an existing name, and the repetition of the enumerated steps is in which one, or two, or three, or four, or five, or more than five, or more than ten, the above method.

[0064] Furthermore, the proposed method involves the above, which includes multiple sprint oligos, each of which includes a sequencing primer annealing site, the sequencing primer annealing site being capable of hybridizing to a sequencing primer, resulting in a hybridized sequencing primer, the hybridized sequencing primer being capable of directing sequencing of at least one bead-bound DNA barcode module and at least one bead-bound DNA barcode module.

[0065] This relates to embodiments relating to sprint oligos that guide DNA polymerase to synthesize functional nucleic acids and various types of beneficial nucleic acids. Provided is the above method wherein at least one sprint oligo comprises a functional nucleic acid, or at least one sprint oligo encodes information other than information related to chemical library monomers. Provided is the above method further comprising the step of binding at least one DNA barcode module via click chemistry, the step of which does not use any sprint oligos.

[0066] In short, the present disclosure is a system for screening compounds, comprising: (a) a picowell array plate comprising a plurality of picowells, each picowell having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, and a wall existing between the upper hole and the floor; (b) at least one bead disposed within at least one picowell, comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds; and (c) at least one bead having DNA in the form of either a chained DNA barcode or an orthogonal DNA barcode. The system includes a barcode, and if the DNA barcode takes the form of a chained DNA barcode, the chained DNA barcode is prepared by (i) using click chemistry or (ii) using a repeating cycle of steps, wherein the step of the repeating cycle includes using a sprint oligo for annealing to a partially prepared DNA barcode, the annealed sprint oligo is used as a template for extending the partially prepared DNA barcode using DNA polymerase, and the sprint oligo contains bases complementary to the DNA barcode module to be polymerized to the partially prepared DNA barcode.

[0067] In another embodiment, the provided system comprises the DNA barcode, which includes (a) one or more DNA barcode modules, each of which codes information relating to the identity of a chemical library monomer; (b) optionally, one or more functional nucleic acids; and (c) optionally, one or more nucleic acids that code information of a type other than information relating to the identity of a chemical library monomer.

[0068] Furthermore, the system provided further includes a plurality of caps, each capable of fitting to a different picowell opening, each capable of minimizing or preventing evaporation of the fluid inside the picowell, and minimizing or preventing leakage of the fluid inside the picowell.

[0069] The system also includes a plurality of spherical caps, each capable of fitting into a hole in the picowell, the hole being circular, each capable of minimizing or preventing evaporation of the fluid inside the picowell, and each capable of minimizing or preventing leakage of the fluid inside the picowell.

[0070] Furthermore, the intended system is one in which at least one bead contains a DNA barcode in the form of a chained DNA barcode, wherein the chained DNA barcode comprises: (i) a sequencing primer binding site; (ii) a first DNA barcode module; (iii) a first annealing site that can hybridize with the first oligonucleotide sprint, which can be used by the first oligonucleotide sprint to induce the enzymatic synthesis of the second DNA barcode module; (iv) a second DNA barcode module; (v) a second annealing site that can hybridize with the second oligonucleotide sprint, which can be used by the second oligonucleotide sprint to induce the synthesis of a third DNA barcode; (vi) a third DNA barcode module; and (vii) a third annealing site that can hybridize with the third oligonucleotide sprint, which can be used by the third oligonucleotide sprint to synthesize a fourth DNA barcode.

[0071] In embodiments of the method, a method is provided for screening a compound library for compounds having desired properties, comprising: (a) providing a plurality of beads, each bead comprising a plurality of oligonucleotides attached to the bead surface and a plurality of substantially related compounds attached to the bead surface, wherein the sequence of oligonucleotides attached to the bead encodes the synthesis history of the plurality of substantially related compounds attached to the bead surface; (b) incorporating the plurality of beads into an assay for desired properties of compounds in a compound library; (c) capturing a signal from at least one bead, wherein the signal reflects the performance of a compound on the bead in the assay; (d) sequencing the plurality of oligonucleotides attached to at least one bead from which the assay signal has been further captured without removing the oligonucleotides from the beads; and (e) identifying at least one compound from the readout of the sequencing in step (d) and associating it with the corresponding assay performance captured by the signal in step (c).

[0072] More specifically, the above methods include the assay comprising a binding assay, or an activity assay, or an assay comprising a competitive binding assay or a competitive inhibition assay, or an assay comprising the interaction of an unbound compound with another assay reagent, wherein the unbound compound is a compound released from the surface of a bead, or a compound released by cleaving a cleavable linker that connects the compound to a bead, or the assay occurring in multiple limited volumes, typically with one bead dispersed per limited volume.

[0073] In another embodiment, further intended is that the limited volume contains aqueous droplets, or The above method involves aqueous droplets being suspended in an oily medium or a hydrophobic liquid medium, or a limited volume containing picowells, or picowells being organized into a regular array, or multiple limited volumes being organized into a regular array.

[0074] Furthermore, the method above includes a limited volume comprising a layer of aqueous medium adhered around the beads, wherein the beads are suspended in a hydrophobic medium, and the assay reagent is washed away before sequencing the oligonucleotide. The method above is performed in which the sequencing step (d) is carried out before the assay step (b). Further provided is the method above in which the oligonucleotide on the beads is removed after the sequencing step but before the assay step. Furthermore, the method above is intended to include the removal of the oligonucleotide comprising enzymatic digestion, chemical cleavage, thermal decomposition, or physical shear, and the binding assay comprising binding of an RNA molecule to the beads, and the signal from the beads comprising sequencing of the bound RNA molecule.

[0075] In yet another embodiment, the method described above is provided, wherein the binding assay comprises a fluorescently labeled binding assay, and the molecule that binds to the compound on the beads comprises a fluorophore, or the method described above comprises a nucleic acid labeled binding assay, and the molecule that binds to the compound on the beads comprises a nucleic acid tag, and the signal from the assay further comprises sequencing of the nucleic acid tag attached to the molecule that binds to the compound on the beads.

[0076] In yet another embodiment of the method relating to the property, provided is the above method, wherein the desired property comprises one or more of the following: (i) inhibiting or stimulating the catalytic activity of an enzyme; (ii) stimulating a Th1 immune response measurable by a cell line assay or in vivo assay; (iii) stimulating a Th2 immune response measurable by a cell line assay or in vivo assay; (iv) inhibiting a Th1 immune response measurable by a cell line assay or in vivo assay; (v) inhibiting a Th2 immune response measurable by a cell line assay or in vivo assay; or (vi) stimulating or inhibiting ubiquitin-mediated degradation of a protein measurable by a purified protein, a cell line assay, or an in vivo assay.

[0077] In an embodiment of the system, the system provided is for screening a compound library for compounds having desired activity, and includes: (a) a sample compartment for receiving beads to which multiple compounds are attached and which are encoded with oligonucleotides; (b) a plurality of encapsulation compartments within the sample compartment, each encapsulation compartment nominally containing a single bead dispersed in an assay medium, and further containing a reagent whose interaction with the compound on the bead is assayed and yields a measurable signal; (c) a detector for measuring the signal; (d) a sequencing platform; and (e) a user interface for receiving one or more commands from a user. Also provided is the above system, wherein the encapsulation compartment contains droplets. In another embodiment, the above system is provided, wherein the encapsulation compartment contains picowells, or further contains assay reagents, or the detector contains an optical detector, or the sequencer contains an optical detector.

[0078] In one embodiment, the Disclosure is characterized by a method of perturbing cells by (a) providing a nucleic acid-encoded perturbation and restricting cells with the nucleic acid-encoded perturbation; (b) contacting cells with the nucleic acid-encoded perturbation in a restricted volume, wherein the initiation and dose of the perturbation are controlled; (c) incubating cells with the nucleic acid-encoded perturbation for a specified period of time; and (d) transferring nucleic acids encoding the nucleic acid-encoded perturbation into the cells.

[0079] In some embodiments of this model, the nucleic acid-encoded perturbation is a nucleic acid-encoded compound or drug molecule. In some embodiments, the nucleic acid-encoded perturbation is a DNA-encoded library.

[0080] In some embodiments, the perturbation and the nucleic acid encoding the perturbation are free and not attached to the solution. In some embodiments, the perturbation and the nucleic acid encoding the perturbation are attached to each other. In some embodiments, the perturbation and the nucleic acid encoding the perturbation are attached to the same substrate but not to each other. In some embodiments, the attachment of the perturbation to the substrate and the attachment of the nucleic acid to the substrate are cleavable attachments. In certain embodiments, the cleavable attachments are selected from the group consisting of photocleavable attachments, temperature-cleavable attachments, pH-sensitive attachments, acid-cleavable attachments, base-cleavable attachments, sound-cleavable attachments, salt-cleavable attachments, redox-sensitive attachments, or physically cleavable attachments.

[0081] In some embodiments of this aspect of the present disclosure, restricting cells and perturbations includes droplet encapsulation, emulsion encapsulation, picowell encapsulation, macrowell encapsulation, physical adhesion, bubble encapsulation, or microfluidic restriction.

[0082] In some embodiments, control across perturbations includes controlling light exposure, temperature exposure, pH exposure, time exposure, sound exposure, salt exposure, chemical or physical redox potential, or mechanical agitation exposure.

[0083] In certain embodiments, incubation includes exposing cells to a perturbation after cleaving the perturbation from the substrate or after cleaving nucleic acids from the substrate. In some embodiments, incubation includes exposing cells to a perturbation without cleaving the perturbation from the substrate or without cleaving nucleic acids from the perturbation.

[0084] In some embodiments, transferring nucleic acids encoding perturbations into cells includes attaching the nucleic acids to the cell surface. In certain embodiments, attaching nucleic acids to the cell surface includes inserting the nucleic acids into the cell membrane. In certain embodiments, attaching nucleic acids to the cell surface includes attaching the nucleic acids to biomolecules on the cell surface. In certain embodiments, the biomolecules are proteins or carbohydrates. In other embodiments, attaching nucleic acids to the cell surface includes attaching them via any tags on the nucleic acids.

[0085] In another embodiment, the present disclosure features a method for perturbing cells with a perturbation and encoding cells with the identity of the perturbation. The method includes (a) providing a library encoded on beads-bound DNA, (b) restricting cells with the library encoded on beads-bound DNA, wherein the library encoded on beads-bound DNA comprises one or more copies of combinatorially synthesized compounds and one or more copies of encoding nucleic acid tags, the compounds and encoding nucleic acids are attached to beads, the encoding nucleic acids encode the identity of the compounds, and the library encoded on beads-bound DNA and cells are restricted to a restricting volume, (c) releasing the compounds from the beads and incubating the compounds with cells in a restricting volume, (d) optionally releasing the encoding nucleic acid tags from the beads, and (e) attaching the encoding nucleic acid tags to cells, thereby maintaining the identity of the compounds via the encoding nucleic acid tags attached to the cells.

[0086] In yet another embodiment, the Disclosure features a method for perturbing cells, encoding cells by the identity of the perturbation, and measuring the cell's response to the perturbation. The method comprises (a) contacting cells with a bead-bound DNA coding library in a first restricted volume, the bead-bound DNA coding library comprising one or more copies of combinatorially synthesized compounds and one or more copies of coding nucleic acid tags, wherein the compounds and coding nucleic acids are attached to beads and the coding nucleic acids encode the identity of the compounds; (b) releasing the compounds in the library from the beads and incubating the compounds in the library with cells in the first restricted volume; (c) optionally releasing the coding nucleic acid tags from the beads in the first restricted volume; (d) capturing the coding nucleic acid tags on the cell surface of the cells, thereby exposing the cells to the compounds in the library and capturing the identity of the exposed compounds on the cell surface; and (e) releasing the cells from the first restricted volume, wherein the coding nucleic acid tags are attached to the cells and the coding nucleic acid tags are exposed to the cells. (f) capturing previously perturbed and nucleic acid-tagged cells with response-detection beads in a second limited volume, wherein the cells are exposed to lysis conditions that expose the cellular contents of the cells to the response-capture beads, the response-capture beads comprising a capture probe for capturing the cellular contents and a nucleic acid tag encoding the perturbation in the previously perturbed and nucleic acid-tagged cells; (g) incubating the response-capture beads with the lysed cells in a second limited volume, thereby capturing both the cellular contents and the nucleic acid tag encoding the perturbation to the response-capture beads; (h) optionally converting the cellular response to the perturbation into a nucleic acid signal, wherein the cellular response to the perturbation is not a nucleic acid signal; and (i) sequencing the nucleic acid tag attached to the response-capture beads, thereby correlating the identity of the perturbation with the cellular response to the perturbation.

[0087] In yet another embodiment, the Disclosure provides (a) an array of picowells and a library of functionalized perturbation beads, wherein the picowells are capable of accommodating a single cell and a single functionalized perturbation bead, each functionalized perturbation bead comprising a plurality of substantially identical releaseable compounds and a plurality of nucleotide barcodes encoding the compounds, the nucleotide barcodes being functionalized barcodes capable of capturing the cellular contents of a cell, and the cellular contents of a cell comprising a cellular response to a perturbation contained in the functionalized perturbation bead; (b) capturing a single cell in each picowell of the picowell array; (c) capturing a single functionalized perturbation bead in a picowell containing a single cell; and (d) releasing a compound from the functionalized perturbation bead and incubating the cell with the released compound. The method is characterized by perturbing a cell and capturing the cell's response to a perturbation, comprising: (e) lysing the cell to release its cellular contents; (f) capturing one or more components of the cellular contents onto a functionalized oligonucleotide on a functionalized perturbation bead, wherein the capture includes hybridization and enzymatic extension for combining the nucleotide barcode with the nucleic acid elements of the cellular contents, thereby forming a hybrid of the nucleotide barcode and the nucleic acid elements of the cellular contents; and (g) releasing the hybrid, collecting the hybrid from a library of functionalized perturbation beads, sequencing the hybrid, thereby relating the perturbation to the cellular response to the perturbation. [Brief explanation of the drawing]

[0088] [Figure 1] Chain-type beads. In chain-type beads, the DNA barcode takes the form of a series of single-stranded DNA barcode modules linked together, along with any other nucleic acids that have functions such as primer annealing sites, spacers, or information about the manufacturing date. The numbers in this figure are not structural numbers. The numbers indicate the sequence of "DNA barcode modules" within the DNA barcode. [Figure 2] Orthogonal beads. In orthogonal beads, the DNA barcode takes the form of all DNA barcode modules, and instead of occurring together as a single strand, the DNA barcode modules occur individually linked at different positions on the bead. The numbers in this diagram are not structural numbers. The numbers indicate the sequence of "DNA barcode modules" within the DNA barcode. [Figure 3-1] Conditions for cleavable linkers, cleavage (UV light or chemical), and cleavage products. Information is from Yinliang Yang (2014) Design of Cleavable Linkers and Applications in Chemical Proteomics, Technische Universitat Munchen Lehrstuhl fur Chemie der Biopolymere. The alphabetical letters to the left of each linker are from this reference. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] Exemplary amino acid derivatives for compositions and methods of the present disclosure. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 5] Photographs showing increased degradation of fusion proteins in HeLa cells with increasing concentrations of added lenalidomide. Top: Expression of IKZF1 / GFP fusion protein. Bottom: Expression of mScarlett® control. Lenalidomide was added at concentrations of 0, 0.1, 1.0, or 10 micromoles. [Figure 6] Photographs showing increased degradation of the fusion protein with increasing concentrations of added lenalidomide in HeLa cells. Top: Expression of IKZF3 / GFP fusion protein. Bottom: Expression of mScarlett control. Lenalidomide was added at concentrations of 0, 0.1, 1.0, or 10 micromoles. [Figure 7]Methods and reagents for producing bead-bound DNA barcodes. The most accurate description of “DNA barcode” is the sum of all information contained in the sum of all DNA barcode modules. However, for convenience, the term “DNA barcode” is used herein to refer to the sum of all information in all DNA barcode modules, any additional nucleic acids that provide information such as the number of steps, or chemical monomers of the general formula constituting the bead-bound compound, and further, any additional nucleic acids that perform functions such as linkers, sequencing primer binding sites, hairpins with sequencing primer binding sites, or spacers. If the DNA barcode is produced at least partially by click chemistry, the DNA barcode may contain residual chemical groups from the click chemistry reaction. [Figure 8] Structure of Alexa Fluor® 488. The purpose of this diagram is to identify the compound without using a trade name. [Figure 9] A simplified diagram of a bead-bound release monitor. The release monitor provides the user with a measurement of the concentration of a soluble compound, followed by UV-induced release of the compound from the beads. In a preferred embodiment, one form of bead is dedicated solely to being the release monitor, i.e., this bead does not contain a bead-bound compound or a bead-bound DNA library. "PCL" is a photocleavable linker. [Figure 10] Detailed diagram of the bead release monitor. [Figure 11-1] Chemical synthesis of a bead release monitor. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 12] Amine-functionalized beads having a bifunctional linker containing a lysine residue. [Figure 13] Steps in the chemical synthesis of lenalidomide modified with type 1 carboxyl groups. [Figure 14] Steps in the chemical synthesis of lenalidomide modified with type II carboxyl groups. [Figure 15] Steps in the chemical synthesis of lenalidomide modified with a type 3 carboxyl group. [Figure 16] Lenalidomide analog. [Figure 17-1] A chemical synthesis step for deoxycytidine analogs suitable for click chemistry synthesis of DNA barcodes. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 17-4] Same as above. [Figure 18] A cap is placed to cover the top of the picowell and to seal the picowell. Figure 18A shows an active cap, to which a compound can be released via a cleavable linker. Figure 18B shows another type of active cap to which a reagent such as an antibody is bound. The bound reagent can be persistently linked, linked with a cleavable linker, or linked via hydrogen bonding, and can be released simply by exposure to the solution in the picowell and subsequently detaching from the active cap and diffusing into the solution. Figure 18C shows a passive cap, which can be used to absorb, adsorb, collect, or capture metabolites from the solution in the picowell, where the absorbed metabolites can then be analyzed. [Figure 19-1] Figure 19A shows a picowell plate without caps covering the picowells. Figure 19B shows a picowell plate with caps covering each picowell. Figure 19C shows a polyacrylamide solution poured over a picowell plate, with one cap securely and firmly fixed over each picowell. The polyacrylamide then penetrates the porous caps and solidifies, forming a stable adhesion to each cap. Figure 19D shows the next step: peeling off the solidified polyacrylamide "roof" from the picowell plate, revealing each cap. The metabolites absorbed by each cap can then be analyzed by moving them out of the picowell solution. Preferably, the solution poured over the picowell plate and over the beads is a hydrogel, and preferably, the beads are made from a hydrogel. [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0089] In an exclusive embodiment, the disclosure may exclude a system, a microtiter plate, a microtiter plate having microwells, nanowells, or picowells, and associated methods, wherein at least one well is capped, and a liquid polymer solution is poured over the plate and over the capped wells. Also excluded is the above, in which the liquid polymer polymerizes to form a solid polymer that adheres to each cap. Also excluded is a method in which the solid polymer is torn off and the adhering caps are removed, and the resulting composition. [Figure 20] Map of the circular plasmid used to integrate the IKZF1 gene into the cell genome. The plasmid is IKZF1 mNEON-p2a-mScarlet-w3-2FB (9081 base pairs). IKZF1 encodes the Ikarus protein. [Figure 21] Map of the circular plasmid used to integrate the IKZF3 gene into the cell genome. The plasmid is IKZF3 mNeon-p2a-mScarlet-w3-2FB(9051 bp). IKZF3 encodes the Aiolos protein. [Figure 22-1] Chemical monomers (compounds 1-6) and their DNA barcodes. [Figure 22-2] Same as above. [Figure 23] Chemical monomers (compounds 7-10) and their DNA barcodes. [Figure 24-1] Chemical monomers (compounds 11-16) and their DNA barcodes. [Figure 24-2] Same as above. [Figure 25-1] Chemical monomers (compounds 17-21) and their DNA barcodes. [Figure 25-2] Same as above. [Figure 26-1] Chemical monomers (compounds 22-16) and their DNA barcodes. [Figure 26-2] Same as above. [Figure 27-1] Chemical monomers (compounds 27-30) and their DNA barcodes. [Figure 27-2] Same as above. [Figure 28] Sequencing of a bead-bound DNA barcode. This figure shows the intensity of the fluorescence signal for each of the five consecutive bases, which are part of the bead-bound DNA barcode. [Figure 29] Picowell with steps. [Figure 30] Time course of fluorophore emission from beads. This shows the operation of the bead-bound emission monitor and the acquisition of emission data at t=0 sec, t=1 sec, t=11 sec, and t=71 sec. [Figure 31] Release data following catalytic action of aspartyl protease on quencher-fluorophore substrates. [Figure 32] A cross-sectional view of the picowell shows various steps. [Figure 33] Titration data showing how increasing UV dose increases cleavage of fluorophores from beads. In layman's terms, this shows how a stronger swing of the axe affects cleavage of fluorophores from beads (UV dose power is measured in joules / cm²). The notation "exposure" refers only to the parameter at the time of taking the photograph. This is simply the exposure time at the time of taking the photograph (this refers to the exposure time of the light that causes the cleavage, or the light that causes excitation). [Figure 34] TAMRA concentration versus luminous flux. Shown are the concentration of free TAMRA, followed by its emission after exposure to 365 nm UV light. [Figure 35-1] Hand-drawn diagrams are provided showing the quencher-fluorophore substrate and the cleavage of this substrate by the enzyme, resulting in the inhibition of the enzyme. The molecular structures of bead-bound pepstatin-A and bead-bound Fmoc-valine (negative control) are also shown. [Figure 35-2] Same as above. [Figure 36]The step involves preparing beads to ultimately capture mRNA from lysed cells, and then producing a cDNA library. This figure also arose in one of the provisional applications (Compositions and Methods for Screening Compound Libraries on Single Cells), for which priority is claimed in this application. [Figure 37] Tagging involves tagging cells with DNA barcodes via lipids embedded in the cell membrane. This figure also arises in one of the provisional applications (Composition and Method for Screening a Composite Library on a Single Cell), from which priority is claimed in this application. [Modes for carrying out the invention]

[0090] Where used herein, including in the attached claims, singular words such as “a,” “an,” and “the” include multiple corresponding references unless the context explicitly indicates otherwise. All references cited herein are incorporated by reference to the same extent as individual patents, published patent applications, figures, drawings, sequence listings, compact discs, etc., which are specifically and individually indicated to be incorporated by reference.

[0091] abbreviation Table 1 shows abbreviations and non-restrictive definitions. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0092] Reagents, kits, enzymes, buffers, live cells, and equipment are available. For example, see Sigma-Aldrich, St. Louis, MO; Oakwood Chemical, Estill, SC; Epicentre, Madison, WI; Invitrogen, Carlsbad, CA; ProMega, Madison, WI; Life Technologies, Carlsbad, CA; ThermoFisher Scientific, South San Francisco, CA; New England BioLabs, Ipswich, MA; American Type Culture Collection (ATCC), Manassas, VA; Becton Dickinson, Franklin Lakes, NJ; Illumina, San Diego, CA; 10X Genomics, Pleasanton, CA.

[0093] Barcoded gel beads, non-barcoded gel beads, and microfluidic chips are available from 1CellBio, Cambridge, MA. Guidance and instruments for flow cytometry are available (see, for example, FACSCalibur®, BD Biosciences, San Jose, CA, BD FACSAria II® User Guide, catalog number 643245, Rev. A, December 2007, page 344).

[0094] "Labeled" compositions can be detected directly or indirectly by spectroscopic, photochemical, fluorescence quantitative, biochemical, immunochemical, isotopic, or chemical methods, as well as by methods involving plasmon nanoparticles. For example, useful labels include: 32 P, 33 P, 35 S, 14 C, 3 H, 125I includes stable isotopes, epitope tags, fluorescent dyes, Raman tags, electron-dense reagents, substrates, or enzymes, and is used, for example, in enzyme-linked immunoassays or fluorettes (Rozinov and Nolan (1998) Chem. Biol. 5:713-728).

[0095] Table of Contents for Detailed Descriptions (I) Beads (II) One bead, one compound (OBOC) (III) Bind nucleic acids to beads (IV) DNA barcode (V) Attach the compound to the beads. (VI) To synthesize a compound, chemical monomers are bonded together. (VII) Split pool synthesis and parallel synthesis (VIII) Manufacture a picowell (IX) Deposit the beads into the picowell. (X) Sequencing of bead-bound nucleic acids in picowells (XI) Release the bead-bound compound from the beads. (XII compound biochemical assay) Cellular assay for compound (XIII) (XIV) Perturbation-response analysis in cells (I) Beads

[0096] The methods and compositions of this disclosure utilize beads such as mono-size TentaGel® M NH2 beads (diameters such as 10, 20, and 30 micrometers), standard TentaGel® amino resins (diameters such as 90 and 130 micrometers), and TentaGel Macrobeads® (diameters of 280 to 320 micrometers) (all of which are from Rapp Polymere, 72072 Tubingen, Germany). These have a polystyrene core derivatized with polyethylene glycol (Paulick et al (2006) J.Comb.Chem.8:417-426). TentaGel® resins are grafted copolymers consisting of a low-crosslinked polystyrene matrix grafted with polyethylene glycol (PEG). Accordingly, this disclosure provides modified beads or resins to contain one or both of the DNA barcode and / or compounds, while the unmodified beads take the form of grafted copolymers consisting of a low-crosslinked polystyrene matrix grafted with polyethylene glycol (PEG).

[0097] TentaGel® is characterized by the immobilization of PEG chains with a molecular weight of up to 20 kilodaltons on functionalized and crosslinked polystyrene. Graft copolymers having approximately 2000–3000 daltons of PEG chains have been proven optimal in terms of kinetic rate, mobility, swelling, and resin capacity (Rapp Polymere, Germany). Accordingly, this disclosure provides beads or resins in the form of graft copolymers having approximately 2000–3000 daltons of PEG chains. Regarding swelling, Comellas et al. provide guidance for measuring the swelling capacity of beads when immersed in, for example, DCM, DMF, methyl alcohol, water, or buffers used in enzyme assays (Comellas et al (2009) PLoS ONE.4:e6222 (p. 12)). The unit of swelling is milliliters per gram of beads.

[0098] In an embodiment of the alternative beads, the disclosure uses a resin in which the PEG spacer is attached to a polystyrene backbone via an alkyl chain, and the resin is microsphere and monosize (TentaGel® M resin).

[0099] Furthermore, in an embodiment of alternative beads, the disclosure uses a resin having PEG spacers attached to a polystyrene backbone via alkyl chains, and the resin type exists in two bifunctional species: the first surface-modified resin has reaction sites on the outer surface of the bead protected orthogonally to the reaction sites in the internal volume of the bead, and the second hybrid resin has cleavable and non-cleavable ligands present in this support and is developed for continuous cleavage (TentaGel® B resin).

[0100] Furthermore, in another embodiment, the disclosure uses a resin in which the PEG spacers are attached to the polystyrene backbone via alkyl chains, and the macrobead resin exhibits a very large particle size and high volume (TentaGel® MB resin). The disclosure also uses a resin in which the PEG spacers are attached to the polystyrene backbone via benzyl ether chains. This resin can be used for immunoassays or for the synthesis of PEG-modified derivatives (PEG-bound PEG-modified compounds) (TentaGel® PAP resin).

[0101] Furthermore, the beads may be made of HypoGel® 200 resin. These resins are composite materials of oligoethylene glycol (MW 200) grafted onto a low-crosslinked polystyrene matrix (Fluka Chemie GmbH, CH-9471 Buchs, Switzerland).

[0102] In some embodiments, amino-functionalized polystyrene beads without PEG linkers may be used, for example, mono-size polystyrene M NH2 microbeads (with diameters such as 5, 10, or 20 micrometers, from Rapp Polymere, 72072 Tubingen, Germany).

[0103] In some embodiments, the compound can be encapsulated within pores, chambers, or tunnels in the beads without covalent attachment to the beads. The compound can be diffused or forced into such pores in the beads by various means. In some embodiments, the compound can be loaded into the beads by diffusion. In some embodiments, high temperature can be used to swell the beads and load the compound into them. In some embodiments, high pressure can be used to force the compound into the beads. In some embodiments, a solvent that swells the beads can be used to load the compound into the beads. In some embodiments, vacuum or low pressure can be used to disperse the compound into the beads. In some embodiments, gentle or vigorous physical stirring can be used to load the compound into the beads.

[0104] In embodiments in which the compound is loaded onto beads without covalent adhesion, the compound can be unloaded from the beads by diffusion. In some embodiments, the compound can be removed from such beads in a non-limiting manner using temperature, pressure, solvent, pH, salt, buffer, or surfactant, or a combination of such conditions. In some embodiments, the compound contained within such beads can be released, for example, by using the physical integrity of the beads provided by non-crosslinked polymerized beads.

[0105] In an exclusionary embodiment, the disclosure may exclude any beads, and bead-compound complexes, or any method, including one of the beads described above.

[0106] The beads of this disclosure further include: Merrifield resin (chloromethyl polystyrene); PAM resin (4-hydroxymethylphenylacetamidomethyl polystyrene); MBHA resin (4-methylbenzhydrylamine); Brominated Wang resin (alpha-bromopriopiophenone); 4-nitrobenzophenone oxime (Kaiser) resin; Wang resin (4-hydroxymethylphenoxymethyl polystyrene); PHB resin (p-hydroxybenzyl alcohol); HMPA resin (4-hydroxymethylphenoxyacetic acid)); HMPB resin (4-hydroxymethyl-3-methoxyphenoxylbutanoic acid); 2-chlorotrityl resin; 4-carboxytrityl resin; Link acid resin (4-[(2,4-dimethoxypehenyl)hydro (Xyxymethyl)phenoxymethyl; Linkamide (RAM) resin "Knorr" resin (4-((2,4-dimethylphenyl)(Fmox-amino)methyl)phenoxyalkyl); PAL resin (5-[4-(Fmoc-amino)methyl-3,5-dimethoxyphenoxy]valeramidemethyl polystyrene); Severamide resin (9-Fmox-amino-xanthan-3-yl-oxymethyl); HMBA resin (hydroxymethylbenzoic acid); 4-sulfamoylbenzoyl resin "Kenner's safety device" resin (N-(4-sulfamoylbenzoyl)aminomethyl-polystyrene); FMP- resin (4-(4-formyl-3-methoxyphenoxy)-ethyl) (See ChemFiles Resins for Solid-Phase Peptide Synthesis Vol.3 (page 32) (Fluka Chemie GmbH, CH-9471 Buchs, Switzerland)).

[0107] The beads of this disclosure further include the above-mentioned beads used as passive encapsulating materials for compounds (passively holding compounds without covalent linkages to the compounds), and further include unfunctionalized polystyrene beads; silica beads; alumina beads; porous glass beads; polyacrylamide beads; titanium oxide beads; alginate beads; ceramic beads; PMMA (polymethyl methacrylate) beads; melamine beads; zeolite beads; polylactide beads; deblock copolymer micelles; dextran beads, and the like. Many of the beads listed in this paragraph can be purchased from vendors such as Microspheres-Nanospheres, Cold Spring, NY 10516, USA.

[0108] In addition to beads, vesicles or droplets may also be used as vehicles for delivering compounds for some embodiments of the present disclosure. Lipids, deblock copolymers, triblock copolymers, or other membrane-forming materials may be used to form internal volumes into which compounds can be loaded. Compounds may be released from these encapsulated volumes by adding detergents, mechanical agitation, temperature, salt, pH, or other means. Water-in-oil or oil-in-water emulsions are yet another means of passively encapsulating compounds that can be delivered to assay volumes.

[0109] In all embodiments in which passive encapsulation is used to deliver the compound, the DNA tag may be further passively loaded, or alternatively, the DNA tag may be covalently attached to beads, vesicles, or droplets.

[0110] In an exclusionary embodiment, the disclosure may exclude beads or resins made from any of the above chemicals, or from a derivative of any one of the above chemicals.

[0111] In embodiments, the beads may be spherical and may have diameters such as about 0.1 to 1 micrometer, about 1 to 5 micrometers, about 1 to 10, about 5 to 10, about 5 to 20, about 5 to 30, about 10 to 20, about 10 to 30, about 10 to 40, about 10 to 50, about 20 to 30, about 20 to 40, about 20 to 50, about 20 to 60, about 50 to 100, about 50 to 200, about 50 to 300, about 50 to 400, about 100 to 200, about 100 to 400, about 100 to 600, about 100 to 800, about 200 to 400, about 200 to 600, and about 200 to 800 micrometers.

[0112] Non-spherical beads that can be defined in the terms of the values ​​and ranges described above are also provided. For example, one of the axes, or one of the primary dimensions (e.g., side), or one of the secondary dimensions (e.g., diagonal) may include values ​​within the above ranges. In an exclusionary embodiment, this disclosure may exclude any reagent, composition, system, or method that includes spherical beads (or non-spherical beads) that fall within one or more of the above values ​​or ranges.

[0113] A chain of beads. In one embodiment, a plurality of bead dimers are provided, each bead dimer taking the form of two beads attached to each other, one bead containing a plurality of attached nucleic acid barcodes (either orthogonal nucleic acid modules or chain-like nucleic acid modules), and the other bead containing a plurality of attached compounds, all of which are substantially related to each other (or all of which are substantially identical to each other in chemical structure). The bead dimer can be synthesized by preparing a first bead with attached compounds and a second bead with attached nucleic acid barcodes separately, and then linking the two beads. In one embodiment, the beads are attached to each other by a reversible linker, and in another embodiment, the beads are attached to each other by an irreversible linker.

[0114] Bead permeability. In embodiments, the disclosure provides beads having a range or degree of permeability. Permeability can be measured as the percentage of the volume of beads reachable by a solvent, the unit of measurement being the percentage of the surface of the beads in the form of pores, or the unit of measurement being the percentage of the interior of the beads in the form of channels, networks, or chambers that are in fluid communication with the surface (and external medium) of the beads. The disclosure may or may not include porous beads.

[0115] Rothberg's U.S. Patent No. 9,062,304 discloses beads having external and internal regions. Various pore sizes, polymers such as poly(styrenesulfonic acid) and polystyrene are shown, including "internal surface (pore surface)," "preferred pores that would exclude larger molecules," and optionally "developed differential functionalization of the internal and external surfaces." Figure 1 of Rothberg's patent provides photographs of the bead surface and the pores of the beads. Bedre's U.S. Patent No. 9,745,438 provides transmission electron microscope images of porous beads. Smith's U.S. Patent No. 5,888,930 provides scanning electron microscope images of cross-sections of porous beads. Spherical beads with small pores on the surface and large pores internally are shown, and the beads are made from, for example, polystyrene, polyacrylonitrile, polycarbonate, cellulose, or polyurethane. Cooke's U.S. Patent No. 5,047,437 discloses the pore morphology of spherical poly(acrylonitrile) copolymers with a skinless surface (Figure 1) and beads with an external skin on the surface (Figure 5). Tsao's U.S. Patent No. 4,090,022 discloses the porous openings and internal spaces of cellulose beads.

[0116] Each of the above-mentioned patents, including all of the figures, is incorporated herein in whole, so that each of them may be individually incorporated by reference as a whole.

[0117] Without any limitation, the outer surface of a bead or microparticle can be determined by tightly encasing the entire bead or microparticle in an elastic film. Without any limitation, the outer surface of a bead is the part of the bead that is in physical contact with the wrapping, and the bead or microparticle can be wrapped via a thought experiment, or the wrapped bead can be depicted in a diagram or photograph, or the bead can actually be wrapped.

[0118] For example, the Disclosure provides beads having pores that account for at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, and at least 40% of the surface area. The Disclosure also provides beads in which the volume of internal channels or networks accounts for at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, and at least 80% of the total volume of the beads, and the internal channels or networks are in fluid communication with the outer surface (and external medium) of the beads.

[0119] Furthermore, the Disclosure provides beads having pores that account for less than 1%, less than 2%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, and less than 40% of the surface area. The Disclosure also provides beads in which the volume of internal channels or networks accounts for less than 1%, less than 2%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, and less than 80% of the total volume of the beads, and the internal channels or networks are in fluid communication with the outer surface (and external medium) of the beads.

[0120] Iron core beads. This disclosure encompasses iron core beads or magnetic beads. These beads are manipulated using magnets to move them from one reaction vessel to another, or from one vessel to another. By using these beads, robotic manipulation can be enhanced. Methods for manufacturing and using magnetic beads are available (Szymonifka and Chapman (1994) Tetrahedron Letters. 36: 1597-1600; Liu, Qian, Xiao (2011) ACS Comb. Sci. 13: 537-546; Alam, Maeda, Sasaki (2000) Bioorg. Med. Chem. 8: 465-473).

[0121] In an exclusionary embodiment, the disclosure may exclude any beads or any group of beads that satisfy either of the above values ​​or ranges.

[0122] Loading of compounds onto beads In many experiments, it is advantageous to load pre-synthesized compounds onto beads, which can then be used as vehicles for delivering the compounds to assays. Many standard techniques used for drug delivery to biological specimens can be adapted for delivering compounds to assays (see Wilczewska et al (2012) Nanoparticles as drug delivery systems. Pharmacological Reports. 64:1020-1037, Kohane DS (2007) Microparticles and nanoparticles for drug delivery. Biotechnol. Bioeng. 96:203-209, Singh et al (2010) Microencapsulation: A promising technique for controlled drug delivery. Res Pharm Sci. 5:65-77).

[0123] In embodiments where a pre-synthesized compound is loaded onto beads, the compound may be held in a conventional 96, 385, or 1536-well microtiter plate. Beads may be added to these plates and loaded with the compound by diffusion or other active loading methods. In preferred embodiments, the beads selected for impregnation have a pore size or permeability shape that prevents the compound from being immediately depleted upon removal from the mother liquor. Diffusion of the beads to the outside is enhanced, if necessary, by heat, pressure, additives, or other stimulants. In some embodiments, compound-loaded beads may be capped in a manner that prevents leakage of the internal contents until caused by an external shock. One way to cap the outside of porous beads is to add lipids or amphiphilic molecules to the bead compound solution so that the cavities exposed on the surface of the beads are sealed by a bilayer formed by the amphiphilic molecules. In some embodiments, pre-formed vesicles may be mixed with the drug-loaded beads, and as a result, upon agitation, the vesicles rupture, and a membrane is reformed on the surface of the drug-loaded beads, thereby sealing them. A method for sealing such beads is described (see Tanuj Sapra et al (2012), Nature Scientific Reports volume 2, Article No.: 848).Further experimental protocols for sealing silica beads are available in the report published by Ryan Davis et al. of Sandia Laboratories, Nanoporous Microbead Supported Bilayers: Stability, Physical Characterization, and Incorporation of Functional Transmembrane Proteins, SAND2007-1560, and the bSUM method is described by Hui Zheng et al. (bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles), J.Gen.Physiol.(2016)147:77-93.

[0124] In some embodiments utilizing pre-synthesized compounds, beads are generated from the compound by adding appropriate reagents, for example, lipids or diblock copolymers, followed by stirring, thereby forming vesicles containing the compound either internally or within a bilayer membrane. In some embodiments, the compound is extruded through a microfluidic T-junction to create droplets of the aqueous phase into the oil phase, with the compound contained within the aqueous phase or at the interface between the aqueous and oil phases. In some embodiments, the formed droplets can be further polymerized to produce hydrogels that are more robust and stable to handle than unpolymerized aqueous phase droplets. Droplet-based encapsulation and assays are disclosed in Oliver et al (2013) Droplet Based Microfluidics, SLAS Discovery Volume:19 issue:4, page(s):483-496. Sol-gel encapsulation processes may further be utilized to encapsulate compounds within beads. The formation of sol-gel beads is described in Sol-gel Encapsulation of Biomolecules and Cells for Medicinal Applications, Xiaolin Wang et al (2015), Current Topics in Medicinal Chemistry, 15:223.

[0125] One bead, one compound (OBOC) The method used to prepare combinatorial libraries involves three steps: (1) library preparation, (2) screening of compounds within the library, and (3) determining the structure of the compounds, for example, determining the structure of all compounds or only those compounds for which interesting results were obtained in the screening (see Lam et al (1997) The One-Bead-One-Compound Combinatorial Library Method. Chem. Rev. 97:411-448). The advantage of compound synthesis via bead-linked synthesis is that compounds can be rapidly prepared by the "split pool" method.

[0126] OBOCs combined with coding strategies. Another feature of OBOCs is that each bead contains not only a compound but also a coding strategy. When bead-bound nucleic acids are used to code the compound that is bound to the same bead, the term "coding" does not refer to the genetic code. Instead, the term "coding" means that the user possesses a legend, key, or code that associates each of thousands of short nucleic acid sequences with a single bead-bound compound.

[0127] The dramatic variations in the use of beads containing bead-bound compounds and bead-bound nucleic acids, where nucleic acids encode related compounds, are as follows: A dramatic variation is the fabrication of a library of complexes, where each member of the library takes the form of a complex of a small molecule and a DNA portion (where the DNA portion encodes the small molecule). This complex is soluble and not bead-bound. After screening with cells or purified proteins, the complex remains bound to the cells or purified proteins, thereby allowing the complex to be isolated and the compound to be finally identified by sequencing the complexed nucleic acid (see Satz et al (2015) Bioconjugate Chemistry. 26:1623-1632).

[0128] Here, as with much of this patent document, the term “encode” does not refer to a genetic code, but rather to the fact that researchers use a specific nucleic acid sequence to reveal a particular known structure of a compound attached to it.

[0129] Instead of using coding strategies such as DNA barcoding, the beads used to screen for positives (thus indicating the compounds that screen for positives) can be subjected to Edman digestion or mass spectrometry to identify the bead-bound compounds (see Shih et al (2017) Mol. Cancer Ther. 16:1212-1223). If the bead-bound compound is a peptide, the sequence of the positive screening peptide compound can be directly determined using MALDI mass spectrometry. Direct sequencing is possible because cleavage and ionization occur simultaneously under laser irradiation (Song, Lam (2003) J. Am. Chem. Soc. 125:6180-6188).

[0130] One subtle point when performing split-pool synthesis of combinatorial libraries is that it is possible to fabricate compounds so that all compounds share a common motif. This strategy is described as "generating a library of motifs, rather than a library of compounds" (see Sepetov et al (1995) Proc. Natl. Acad. Sci. 92:5426-5430; Lam et al, 418, above).

[0131] To provide a typical example of a large bead, the bead has a diameter of 0.1 mm and is about 10 times the size of the same compound. 13 Copies can be retained (Lam et al., see above). After preparing the library of bead-bound compounds, each bead can be used in an individual assay, which measures biochemical activity or, alternatively, binding activity. The assay can be a "bead-on" assay, or alternatively, the compound can be cleaved from the beads and used in a liquid-phase assay (Lam et al., see above).

[0132] Parameters for any type of bead include the tendency to swell in a given assay medium, whether the bead polymer is hydrophobic or hydrophilic, the identification of attachment sites on the beads for each compound to adhere to, the use of spacers such as polyethylene glycol to partially separate each compound from the bead surface, and providing internal volume of the beads.

[0133] While the need to attach compounds to beads is considered, the fact that they are far from the hydrophobic surface of the beads is a factor, as Lam et al. previously disclosed, that polyoxyethylene grafted styrene (TentaGel®) has the advantage that the functionalizable groups are located at the ends of the polyoxyethylene chain and therefore far from the hydrophobic polystyrene. Beads with water-soluble linkers include TentaGel and polydimethylacrylamide beads (PepSyn® gel, Cambridge Research Biochemicals, Northwitch, UK).

[0134] The internal volume parameter can offer advantages, as it is necessary to prevent interaction between the bead-bound DNA barcode and the target of the bead-bound compound. To leverage this advantage, beads can be manufactured so that the DNA barcode is located inside the bead, while the compound to be screened is attached to the surface of the bead (Lam et al., previously cited, 438-439). This advantage of internal volume may be irrelevant if the bead-bound compound is attached by a cleavable linker, and if the compound assay is performed only against the compound being cleaved and released.

[0135] Appell et al. provide a non-limiting example of the split-pool method for synthesizing a chemical library and subsequently screening it to detect active compounds (Appell et al (1996) J. Biomolecular Screening. 1:27-31). Library beads are placed one per well in an array of wells on a first microwell plate, nanowell plate, or picowell plate. The beads are exposed to light to cleave approximately 50% of the bead-bound compounds, releasing them into the solution in the wells. The released compounds are then transferred to a second microwell plate and an assay is performed to detect the wells containing the active compounds, thereby identifying which beads on the first plate contain the active bead-bound compounds. Then, "once the active [compound] is identified from a single bead, the bead is recovered and decoded, thus obtaining the synthesis history and structure of the active compound" (Appell et al, ibid.).

[0136] In a cell system screening assay for screening bead-binding compounds, Shih et al. provide a novel type of bead (Shih et al (2017) Mol. Cancer Ther. 16:1212-1223). This novel type of bead contains bead-binding compounds that are members of a library of "synthetic death ligands directed towards ovarian cancer." The beads are further decorated with biotin, and two additional chemicals are added to form a sandwich, which maintains adhesion between the cell and the bead. The sandwich contains streptavidin and a biotin-LXY30 complex, which links the bead to the LXY30 receptor, a well-known protein on the cell surface, namely an integrin. The aforementioned method by Shih et al. led to the discovery of a novel molecule ("LLS2") that can kill cancer cells. In the above method, bead-binding compounds are used, and the compounds bind to cells (even if the compound still binds via bead binding). Cho et al. prepared a similar single-bead, single-compound library, and the compounds screened were sufficient to bind to cells (without any need for the aforementioned sandwich) (Cho et al (2013) ACS Combinatorial Science. 15:393-400). The objective of Cho et al.'s report was to discover RGD-containing peptides that bind to integrins expressed by cancer cells. The reagents and methods disclosed above are useful for this disclosure.

[0137] Nucleic acids are attached to beads (orthogonal formula; chain-like formula). One way to understand the topic of chained barcodes and orthogonal barcodes is to note the advantages one has over the other. The advantages of orthogonal barcoding over chained barcoding are as follows: With the attachment of each monomer of the growing compound, DNA barcode modules are attached in parallel. In chained barcoding, if the attachment of a given module is incomplete (i.e., if not all attachment sites successfully bond with the required module), the sequence of the completed barcode will be inaccurate. The expression "inaccurate" means incomplete coupling, meaning that chunks may be missing from the small chunks that were assumed to be the completed, correct DNA barcode. Here, because the attachment of all modules failed, the completed barcode sequence will contain errors. In contrast, in orthogonal barcoding, each individual module covalently bonds to its own unique attachment site on the bead. Also, once a module has attached to a given site on the bead, no further modules will be connected to modules that are already attached.

[0138] This disclosure provides reagents and methods for reducing damage to bead-bound DNA barcodes and to partially synthesized bead-bound DNA barcodes. Each DNA barcode module can be in the form of double-stranded DNA (dsDNA) before attaching to a growing bead-bound DNA barcode, and this dsDNA is treated with a DNA crosslinking agent such as mitomycin C. After the synthesis of the DNA barcode in dsDNA form is complete, this dsDNA is converted to ssDNA. The conversion from dsDNA to ssDNA is effective when one of the DNA strands has a uracil (U) residue, and the cleavage of the DNA at the uracil residue is catalyzed by uracil-N-glycosidase (see Figure 5 of serial number 62 / 562,905 filed on 25 September 2017, which is incorporated herein by reference in its entirety). The above refers to the damage inflicted on the growing DNA barcode by the reagents used to create the bead-binding compound.

[0139] Another method to reduce damage to bead-bound DNA barcodes and to partially synthesized DNA barcodes is to synthesize the DNA barcodes in the form of double-stranded DNA, where each of the DNA barcode modules attached to each other takes the form of dsDNA, and each of the two strands is stabilized via a DNA headpiece. For final sequencing of the completed DNA barcode, one of the strands is cut and removed from the DNA headpiece. The above refers to the damage inflicted on the growing DNA barcode by the reagents used to construct the bead-bound compound (if this compound is a member of a chemical library).

[0140] Another method to reduce damage to bead-bound DNA barcodes is to synthesize the DNA barcodes via self-assembly to form hairpins, which self-assemble by the first projection of the hairpin annealing to a second projection of the hairpin.

[0141] When the synthesized DNA barcode takes the form of double-stranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can denature the DNA barcode. The above methods and reagents can prevent denaturation.

[0142] As stated above, the term "DNA barcode" can refer to a polynucleotide that identifies an entire compound, while, in contrast, a "DNA barcode module" can refer to only one of the monomers that make up the compound.

[0143] Another method to reduce damage to bead-bound DNA barcodes and to reduce damage to partially synthesized DNA barcodes is to use double-stranded DNA (dsDNA) and seal the ends of this dsDNA via 7-aza-dATP and dGTP.

[0144] In an alternative embodiment, the method may use an intermediate between "linked DNA barcoding" and "orthogonal DNA barcoding," which comprises blocks of DNA barcoding, i.e., each block containing two, three, four, or five DNA modules, and so on (but not all DNA modules that identify the full-length compound).

[0145] Figure 1 discloses an exemplary and non-exclusive diagram of a chain-structured bead. The bead contains multiple DNA barcodes (each made from a DNA barcode module) and multiple compounds (each made from a chemical library monomer). For simplicity, the term “DNA barcode” is used to refer to all nucleic acids that constitute the “DNA barcode module,” and the polymer containing all nucleic acids that provide some function. The function can be an annealing site for sequencing primers, or the function can be used to identify steps in the chemical synthesis of the bead-bound compound. Figure 1 further illustrates the bead-bound compounds, each compound being made from several chemical library members, each chemical library member being represented as a square, circle, or triangle. Figure 1 shows that each DNA barcode module is numbered sequentially from 1 to 8, with these numbers corresponding to the respective eight shapes (square, circle, triangle). For clarity, nucleic acids that perform a function (and do not represent or “code” a specific chemical unit) are not shown in the figure.

[0146] Figure 2 discloses exemplary and non-limiting embodiments of orthogonal structured beads. The beads contain multiple DNA barcodes (each made up of a DNA barcode module), but each DNA barcode module is attached to a separate binding site on the bead. The entire DNA barcode consists of eight DNA barcode modules, numbered 1 to 8 in the figure. If information from a particular DNA barcode is to be read and used to identify a compound bound to the same bead, DNA sequencing must be performed on each of the separately attached DNA barcode modules. In Figure 2, the beads contain multiple attached chemical compounds, each having eight units, as indicated by eight shapes (circle, square, triangle).

[0147] In Figure 2, for clarity, the functional nucleic acids attached to each DNA barcode module are not shown. Naturally, each DNA barcode module requires a nucleic acid to identify the position of the chemical library monomer in the completed, full-length compound. In the example shown in Figure 2, the position must be the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th position.

[0148] In one embodiment, chemical monomers are first attached, followed by the corresponding DNA barcode modules. In an alternative embodiment, DNA barcode modules are first attached, followed by the corresponding chemical monomers. Alternatively, the procedure may follow an organic synthesis method that uses either "one embodiment" or "an alternative embodiment." In yet another alternative embodiment, the method provides the addition of blocks of several chemical monomers attached to beads in parallel with the attachment of blocks of several DNA barcode modules.

[0149] In an exclusionary embodiment, those that can be excluded are reagents, compositions, and methods that use block-by-block additions of chemical monomers, DNA barcode modules, or both chemical monomers and DNA barcode modules to beads.

[0150] This relates to nucleic acids that may be present in bead-bound polynucleotides and include nucleic acids that help “code” or identify monomers in the bead-bound compound. In an exclusionary embodiment, the disclosure may exclude nucleic acids that code for “step-specific DNA sequencing primer sites.” In this scenario, for each chemical monomer present in the compound, there is a corresponding DNA barcode module. Each DNA barcode module is flanked by at least one corresponding primer binding site, i.e., a “step-specific DNA sequencing primer site.” Also excluded are nucleic acids that code for or specify a particular step in the chemical synthesis of the compound, such as step 1, step 2, step 3, or step 4.

[0151] Furthermore, the disclosure may include nucleic acids that function as spacers. For example, a spacer can create a distance along a polynucleotide chain between a first site, which is the annealing site of a sequencing primer, and a second site that is specific to a chemical monomer. The disclosure may also use nucleic acids that repeat or confirm information provided by another nucleic acid. The disclosure may also use nucleic acids that encode PCR primer binding sites. A polynucleotide having PCR primer binding sites has two PCR primer binding sites, and since both of these sites are designed to have the same melting point (the melting point when the PCR primer anneals to the PCR primer binding site), the PCR primer binding sites can be distinguished from the sequencing primer.

[0152] In an exclusionary embodiment, the Disclosure may exclude nucleic acids that function as spacers or as standalone spacers. The Disclosure may also exclude nucleic acids that repeat or confirm information provided by another nucleic acid. Furthermore, the Disclosure may exclude nucleic acids that function as PCR primer binding sites, and nucleic acids that function as binding sites for primers that are not PCR primers.

[0153] Furthermore, this disclosure may identify the date on which the chemical library was prepared, or the steps in the chemical synthesis of a particular compound, or exclude nucleic acids that function as primer annealing sequences.

[0154] Providing sequencing primers for specific DNA barcode modules. This disclosure provides DNA barcodes comprising DNA barcode modules and one or more sequencing primer annealing sites. Each DNA barcode module may have its own dedicated sequencing primer binding site. Alternatively, two, three, four, five, six, seven, eight, nine, ten, or more consecutive DNA barcode modules may be sequenced using one specific sequencing primer binding site, which may be present on a bead-bound DNA barcode.

[0155] The following describes how each DNA barcode module has its own dedicated sequencing primer binding site. This disclosure provides a bead-linked barcode including a primer-binding site capable of binding a DNA sequencing primer, the primer-binding site capable of directing sequencing of one or more of the following DNA barcode modules: a first DNA barcode module, a second DNA barcode module, a third DNA barcode module, a fourth DNA barcode module, a fifth DNA barcode module, and a sixth DNA barcode module, wherein the primer-binding site is located 3 primes to the first DNA barcode module with no other DNA barcode modules between it and the first DNA barcode module, 3 primes to the second DNA barcode module with no other DNA barcode modules between it and the second DNA barcode module, 3 primes to the third DNA barcode module with no other DNA barcode modules between it and the third DNA barcode module, 3 primes to the fourth DNA barcode module with no other DNA barcode modules between it and the fourth DNA barcode module, 3 primes to the fifth DNA barcode module with no other DNA barcode modules between it and the fourth DNA barcode module, 3 primes to the fifth DNA barcode module with no other DNA barcode modules between it and the sixth DNA barcode module with no other DNA barcode modules between it and the sixth DNA barcode module.

[0156] Code sequences and sequences complementary to code sequences. This disclosure may encompass any one, any combination, or all of the code sequences disclosed above or elsewhere in this document. In an exclusionary embodiment, any one, any combination, or all of the code sequences disclosed above or elsewhere in this document may be excluded. This further includes the exclusion of double-stranded nucleic acids encoding any one, any combination, or all of the code sequences described above or elsewhere in this document.

[0157] Orthogonal DNA barcode (each DNA barcode module is attached to a different position on the bead) Synthesis of orthogonal beads. In orthogonal synthesis, each DNA module is covalently attached to a separate site on the bead, resulting in the entire DNA barcode being provided by multiple DNA modules. When the DNA barcode has an orthogonal structure, the DNA barcode modules are not attached to each other; instead, each DNA barcode molecule has its own bead attachment site dedicated to that particular DNA barcode module.

[0158] A nucleic acid that identifies the number of synthesis steps for each DNA barcode module. In an embodiment, the orthogonal DNA barcode includes a short nucleic acid that identifies the first step of compound synthesis. In this embodiment, rather than the parallel attachment of the first chemical monomer and the first DNA barcode module, the first DNA barcode module actually takes the form of a complex of two nucleic acids, a short nucleic acid meaning "step 1", attached to the first DNA barcode module. All nucleotides in this complex are in-frame with each other and can be read by sequencing assay, although the first short nucleic acid may optionally be attached to the first DNA barcode module via a spacer nucleic acid.

[0159] The following is a continuation of the above description of orthogonal DNA barcodes, which include a short nucleic acid that identifies a second step in compound synthesis. In this embodiment, rather than the parallel attachment of a second chemical monomer and a second DNA barcode module, the second DNA barcode module actually takes the form of a complex of two nucleic acids, a short nucleic acid meaning "Step 2", attached to the second DNA barcode module. All nucleotides in this complex are in-frame with each other and can be read by sequencing assay, although the second short nucleic acid may optionally be attached to the second DNA barcode module via a spacer nucleic acid.

[0160] The method described above is repeated for any given beads up to the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and final DNA barcode modules and the final chemical monomer. The above method can be used when producing bead-bound DNA barcodes and compounds using split-pool synthesis.

[0161] Orthogonal structures offer the following advantages over chain structures: In chain synthesis (where all DNA barcode modules are attached to each other by a single continuous polymer), failure to achieve synthesis in any of the intermediate coupling steps can compromise the meaning of the ultimately completed chain DNA barcode. In contrast, in orthogonal synthesis (where each DNA barcode module is attached to a dedicated site on a bead), failure to attach any of the DNA barcode modules will simply result in an empty attachment site on the bead, without compromising the meaning of any of the other attached DNA barcode modules. In a preferred embodiment, each attached DNA barcode module contains an attached second nucleic acid, which identifies the step (a step in the parallel synthesis of the DNA barcode and the compound).

[0162] In orthogonal synthesis, it is permissible to use up all attachment sites on the bead (sites for attaching growing chemical library members). However, in orthogonal synthesis, the chemical reaction must be designed so that the entire population of attachment sites on the bead is only partially used due to the initial attachment of many DNA barcode modules. The following provides any limitations on site consumption during the chemical synthesis of orthogonal barcodes. For unmodified beads, the total number of sites available for attaching DNA barcode modules is 100%.

[0163] The extent to which the synthesis of orthogonal configuration beads (with respect to the first DNA barcode) consumes the attachment sites on a given bead. The following relates to the attachment of the first DNA barcode module. In embodiments, the attachment of the first DNA barcode module consumes about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the DNA barcode attachment sites on the bead. In other embodiments, less than about 2%, less than about 5%, less than about 10%, less than about 20%, less than about 30%, less than about 40%, or less than 50% of the DNA barcode attachment sites on the bead are consumed. In yet another embodiment, the attachment of the first DNA barcode module results in the consumption of 2-4%, 2-6%, 2-8%, 2-10%, 2-12%, 2-14%, 2-16%, 2-18%, 2-20%, 10-20%, 10-25%, 10-30%, 10-35%, and 10-40% of the DNA barcode attachment site.

[0164] Regarding limitations, when the last DNA barcode module constituting a particular DNA barcode is attached, less than 20% of the site is consumed, and less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 95%, or less than 98% of the site is consumed.

[0165] An exclusionary embodiment may exclude beads or methods that conform to any of the above values ​​or ranges. Alternatively, an exclusionary embodiment may exclude beads or methods that do not conform to any of the above values ​​or ranges.

[0166] The following relates to polymers comprising one or more nucleic acids, each of which is a DNA barcode, and polymers comprising two or more nucleic acids, where some nucleic acids have biochemical functions such as functioning as primer annealing sites or spacers, and other nucleic acids have informational functions and are DNA barcodes. In an exclusionary embodiment, the disclosure may exclude DNA barcodes comprising DNA crosslinking agents such as psoralens. Also excluded are DNA barcodes having a primer binding region with a higher (or lower) melting temperature than the DNA barcode module. This temperature may simply be "higher" or "lower," or may be at least 2 degrees higher, at least 4 degrees higher, at least 6 degrees higher, at least 8 degrees higher, or at least 2 degrees lower, at least 4 degrees lower, at least 6 degrees lower, at least 8 degrees lower.

[0167] Furthermore, methods for producing DNA barcodes using DNA ligase can be excluded. Also excluded are DNA barcodes and methods that constitute hairpins (where ssDNA is bent into a loop, and a portion of the ssDNA hybridizes with another portion of the same ssDNA). In addition, compositions containing nucleic acid hairpins, where the nucleic acid hairpins are covalently closed, for example, with a chemical linker, can be excluded. Furthermore, DNA barcodes that are covalently linked to a "headpiece" either directly or indirectly (indirectly via covalent bonds to one or more chemicals present between the DNA barcode and the headpiece) can be excluded.

[0168] In other exclusionary embodiments, the excluded DNA barcode is a bead-bound DNA barcode, the completed DNA barcode contains only single-stranded DNA (ssDNA) and no double-stranded DNA (dsDNA).

[0169] The extent to which attachment sites on a given bead are consumed by the synthesis of orthogonal-structured beads (with respect to the second DNA barcode). The following relates to the attachment of the second DNA barcode module. In embodiments, the attachment of the second DNA barcode module (in the case of the fabrication of orthogonal-structured beads) consumes approximately 5%, 10%, 20%, 30%, 40%, or 50% of the remaining free DNA barcode attachment sites on the bead. In other embodiments, less than approximately 5%, less than approximately 10%, less than approximately 20%, less than approximately 30%, less than approximately 40%, or less than 50% of the remaining free DNA barcode attachment sites on the bead are consumed. In yet another embodiment, the attachment of the first DNA barcode module consumes between 2-4%, 2-6%, 2-8%, 2-10%, 2-12%, 2-14%, 2-16%, 2-18%, 2-20%, 10-20%, 10-25%, 10-30%, 10-35%, and 10-40% of the remaining free DNA barcode attachment sites.

[0170] An exclusionary embodiment may exclude beads or methods that conform to any of the above values ​​or ranges. Alternatively, an exclusionary embodiment may exclude beads or methods that do not conform to any of the above values ​​or ranges.

[0171] The above embodiments, like the excluded embodiments described above, can also be applied to methods involving the attachment of a third DNA module barcode, a fourth DNA module barcode, or a fifth DNA barcode module.

[0172] Linked DNA barcodes (all DNA barcode modules reside on a single strand or polymer, and the entire strand or polymer is attached to a single position on a bead).

[0173] Synthesis of bead-linked chain-like DNA barcodes. This disclosure provides bead-linked chain-like DNA barcodes, where the beads comprise multiple chain-like DNA barcodes, and most or almost all of the multiple chain-like DNA barcodes have essentially the same structure. The chain-like DNA barcode may comprise one or more DNA barcode modules, and the order of these DNA barcode modules along the entire DNA barcode (from the bead-attached end to the distal end) is the same as the order in which the bead-linked chain-like DNA barcode is synthesized. Furthermore, the order of these DNA barcode modules along the entire DNA barcode is the same as the order in which the corresponding chemical library monomers are bound to the growing bead-linked compound.

[0174] A linked DNA barcode may include, in this order, a linker used to connect the entire linked DNA barcode to beads. It may also include, in this order, a first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, and a third annealing site.

[0175] A sequence of sites for hybridizing sequencing primers within a bead-bound DNA barcode. In an embodiment of the sites for hybridizing sequencing primers, a chain-like DNA barcode may include, in this sequence, a linker, a first DNA barcode module, a first annealing site, a first sequencing primer binding site, a second DNA barcode module, a second annealing site, a second sequencing primer binding site, a third DNA barcode module, a third annealing site, a third sequencing primer binding site, and so on.

[0176] An alternative sequence of sites that hybridize sequencing primers within a bead-bound DNA barcode. In an embodiment of the alternative sequence of sites that hybridize sequencing primers, the chain-like DNA barcode may include, in this order, a linker, a first DNA barcode module, a first sequencing primer binding site, a first annealing site, a second DNA barcode module, a second sequencing primer binding site, a second annealing site, a third DNA barcode module, a third sequencing primer binding site, a third annealing site, and so on.

[0177] The term "annealing site." The term "annealing site" refers to an annealing site that is part of a sprint oligonucleotide (sprint oligo), and is also used to refer to a corresponding bead-binding annealing site present on a growing bead-binding DNA barcode. Those skilled in the art will understand that an "annealing site" on a sprint oligo does not have the same DNA sequence as a corresponding "annealing site" on a growing bead-binding DNA barcode. In other words, those skilled in the art will understand that one sequence is complementary to another. Therefore, it is not important in this description that both annealing sites have the same name. In other words, it is not important that a second annealing site on a sprint oligo is disclosed as hybridizing to a second annealing site on a growing bead-binding DNA barcode.

[0178] Block Synthesis. In alternative embodiments, the sequences of growing compounds and growing DNA barcode modules can be synthesized in blocks. For example, a block consisting of two chemical library units can be attached to beads in parallel with the attachment of a block consisting of the corresponding two-DNA barcode modules. Similarly, a block consisting of three chemical library units can be attached to beads in parallel with the attachment of a block consisting of the corresponding three-DNA barcodes. Block synthesis involving four, five, six, seven, eight, nine, ten, and so on blocks is also provided. Each of these block transfer embodiments may also be excluded by this disclosure. Block-by-block transfers of DNA barcode monomers can be performed orthogonally, with each of the consecutive blocks of DNA barcode monomers having its own attachment point for receiving. Alternatively, block-by-block transfers of DNA barcode monomers can also be performed to generate concatemer structures (all DNA barcode modules arise only as a single consecutive linear polymer).

[0179] Furthermore, when bead-bound DNA barcodes and bead-bound compounds are synthesized in parallel using a split pool, block synthesis may occur. A block can take the form of two or more chemical library monomers, and a block can take the form of two or more DNA barcode modules.

[0180] Location of split-pool synthesis. Split-pool synthesis can be used for the parallel synthesis of bead-bound compounds and bead-bound chain-like DNA barcodes. It can also be used for the parallel synthesis of bead-bound compounds and bead-bound orthogonal DNA barcodes. Chain-like DNA barcodes can be prepared by the "sprint oligo" method. Alternatively, chain-like DNA barcodes can also be prepared by click chemistry. The "sprint oligo" method and click chemistry can also be used in combination. Split-pool synthesis can be performed in a 96-well plate, each well having a bed made of a 0.25 micrometer filter. Under normal gravity conditions, aqueous solutions do not pass through this filter. However, if, for example, it is necessary to replace a first aqueous solution with a second aqueous solution, suction can be applied to remove any aqueous solution from all 96 wells. This suction method is used when beads have been exposed to the first reagent set, or when it is necessary to wash away the first reagent set, or when it is necessary to replace the first reagent set with the second reagent set. A manifold was used to hold the 96-well plate (Resprep VM-96 manifold), and a pump was used to draw fluid from the bottom of all filters (BUCHI Vac V-500 pump). The 96-well plate with the filter bottom was AcroPrep Advance 96-well, 350uL, 0.45um, REF 8048 (Pall Corp., Multi-Well Plates, Ann Arbor, MI).

[0181] The distance from the primer annealing site to the DNA barcode module. For the purpose of sequencing a bead-bound DNA barcode, i.e., sequencing all DNA barcode modules that form the DNA barcode, the sequencing primer has a polynucleotide containing a first nucleic acid, which is the annealing site, and a second nucleic acid, which is the DNA barcode module. The first nucleic acid may be immediately upstream of the second nucleic acid. Alternatively, the first nucleic acid may be upstream of the second nucleic acid, and the first and second nucleic acids are separated from each other by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides, or by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides. The separation can use nucleic acids that simply function as spacers, or alternatively, the separation can use a third nucleic acid that codes for information such as the number of steps in a multi-step organic synthesis pathway, or the number of classes of compounds, or diseases that may be treatable with the bead-bound compound, or a date, or lot number.

[0182] Synthesis of bead-linked DNA barcodes using click chemistry Click chemistry can be used for the stepwise synthesis of DNA barcodes. Here, the first DNA barcode module can be directly bound to a bead, or to a bead-binding linker.

[0183] Furthermore, the binding site is a polynucleotide in the form of the first nucleic acid, which is the first DNA barcode module, attached to the second nucleic acid, which is the first sequencing primer binding site. This sequencing primer binding site allows the operator to determine the sequence of the first DNA barcode module.

[0184] To provide another example, the binding is possible to a second DNA barcode module directly bound to a first DNA barcode module. Alternatively, the binding is possible to a polynucleotide in the form of the first nucleic acid, which is the second DNA barcode module, attached to a second nucleic acid, which is the second sequencing primer binding site. This sequencing primer binding site allows the operator to determine the sequence of the second DNA barcode module. If there is a read-through to the first DNA barcode module, the sequences of both of these DNA barcode modules can be determined.

[0185] To provide yet another example, what can be bound is a polynucleotide containing a first nucleic acid, which is a first DNA barcode module, and a second nucleic acid, which identifies the steps in the multi-step parallel synthesis of the DNA barcode and the compound. Furthermore, or alternatively, the second nucleic acid can identify a general class of compounds produced by split-pool synthesis. Furthermore, or alternatively, the second nucleic acid can identify the disease treated by the compound being screened. Furthermore, the second nucleic acid can identify a date or the name of a chemist, etc.

[0186] A preferred method for synthesizing DNA barcodes is described below, using the same reaction cycle to gradually attach each DNA barcode module.

[0187] Step 1. Provide beads to which TCO groups are attached. In practice, the beads will have hundreds or thousands of similarly attached TCO groups, each TCO group attached to a different site on the bead. In practice, the split pool method is used to simultaneously modify a large number of beads by click chemistry.

[0188] Step 2. Add [Tetrazine]-[First DNA barcode module]-[Azide] to the beads and condense the TCO group with the tetrazine group. The result is the following construct: BEAD-TCO-Tetrazine-First DNA barcode module-Azide. In fact, this construct does not contain any TCO or tetrazine, but it does contain the condensation product produced when TCO condenses with tetrazine.

[0189] Step 3. Any cleaning.

[0190] Step 4. Cap the azide and add DBCO-TCO to create a TCO terminus. The resulting structure is as follows.

[0191] BEAD-TCO-Tetrazine-First DNA Barcode Module-Azide-DBCO-TCO

[0192] Step 5. Optional cleaning.

[0193] Step 6. Add the following reagents to attach the second DNA barcode module. Attachment is at the distal end of the growing DNA barcode. The reagents are:

[0194] The structure for the beads is [tetrazine]-[second DNA barcode module]-[azide], and the TCO group is condensed with the tetrazine group. The result is the following construct: BEAD-TCO-Tetrazine-First DNA barcode module-Azide-DBCO-TCO-[Tetrazine]-[Second DNA barcode module]-[Azide] The above scheme involves a series of steps for progressively adding more DNA barcode modules, with these additions running parallel to the addition of more chemical monomers. As mentioned elsewhere, this “parallel” synthesis can involve attaching a chemical monomer and then attaching a DNA barcode module that identifies that monomer, or attaching a DNA barcode module and then attaching chemical monomers identified by that particular chemical monomer.

[0195] Compounds for click chemistry synthesis of DNA barcodes Figure 17 discloses the chemical synthesis of a compound suitable for linking deoxycytidine residues (dC) during the synthesis of DNA barcode modules, and ultimately the entire DNA barcode. The starting material is N4-acetyl-2'-deoxy-5'-O-DMTcytidine. The abbreviation "DMT" stands for 4,4-dimethoxytrityl. The final product of this multi-step organic synthesis pathway has a cytosine moiety, a triphosphate group, and a propargyl group attached at the 3' position of the ribose group. The propargyl group is used in click chemistry and condenses with the azide group to produce a covalent bond. The result of the condensation is that the remaining chemicals (which never naturally exist in nucleic acids) emerge as "traces" from the click chemistry being performed. Available is a DNA polymerase that can be used for sequencing-by-synthesis of DNA barcodes produced by click chemistry, and the DNA polymerase can move across the traces, which do not cause sequencing errors. TBAI is tetrabutylammonium iodide.

[0196] Synthesis of linkage-shaped DNA barcodes In the following description, DNA barcode modules are assembled in a single line to produce DNA barcodes. However, in the diagrams shown in the text below, the term "DNA barcode" is used instead of "DNA barcode module" to fit the diagrams on the page. Figure 7 shows the same steps as shown here, but with added details such as a diagram of beads. A series of repeated reactions can be used to add each additional DNA barcode module.

[0197] An option to create DNA barcodes containing terminal nucleic acids that encode DNA hairpins. This involves a DNA barcode containing a 3' end, a nucleic acid with an annealing site for a sequencing primer, a bend that takes the form of approximately 4 non-base-paired bases, and a sequencing primer that has an annealing site for a sequencing primer and can be bent to form a base pair. Again, the sequencing primer anneals to the annealing site of the sequencing primer, and the actual sequencing reaction starts from the 3' end of the annealed sequencing primer.

[0198] When performing the final step of synthesizing the DNA barcode and when binding the final DNA barcode module to the growing bead-bound DNA barcode, the “sprint oligo” may include a sequence encompassing a DNA hairpin (the DNA hairpin, in this order, includes the annealing site for the sequencing primer, several nucleotides that are not base-paired with each other or near any base, and the sequencing primer). After annealing the “sprint oligo”, DNA polymerase and dNTPs are then added, and polymerization occurs at the 3' end of the growing DNA barcode, with the sprint oligo being used as a template for polymerization, in the following order: (1) the annealing site for the sequencing primer, (2) the bend in the hairpin taking the form of four or five deoxyribonucleotides that do not form base-paired with teaching to others, and (3) the sequencing primer.

[0199] A reversible terminator group at the 3' end of a hairpin sequencing primer. This disclosure provides reagents, compositions, and methods for attaching a pre-formed nucleotide / reversible terminator group complex to the 3' end of an annealed sequencing primer. The reversible terminator group is an optional component of the hairpin sequencing primer and is part of a bead-bound DNA barcode.

[0200] Step 1. First, beads are placed in picowells, each bead having a bound polynucleotide, the 5' end of which is optionally bound to the bead using a linker. Figure 7 shows that the bead-bound polynucleotide contains a first DNA barcode and a first annealing site. The linker can be made from nucleic acid or it can be made chemically by several other means. Preferably, the linker is hydrophobic, and preferably the linker separates the bead-bound DNA barcode from hydrophobic polystyrene beads, e.g., TentaGel® beads.

[0201] For convenience, the first annealing site, which is part of the bead-bound DNA barcode, and the first annealing site, which is part of the soluble "sprint oligo," do not have the same base sequence, but both are referred to as the "first annealing site." (Instead, the base sequences are complementary to each other, and as a result, the sprint oligo can hybridize to the first annealing site of the bead-bound DNA barcode, and thus function as a template for DNA polymerase, extending the bead-bound DNA barcode by copying what is on the sprint oligo.)

[0202] Furthermore, for convenience, the second annealing site, which is part of the bead-bound DNA barcode, and the second annealing site, which is part of the soluble "sprint oligo," are both referred to as "second annealing sites," even though they do not have the same sequence (but instead have complementary bases).

[0203] Bead-bound DNA barcodes from the 5' end to the 3' end may contain nucleic acids in the following order: Beads / First DNA barcode / First annealing site / Alternatively, a bead-attached growth DNA barcode from the 5' end to the 3' end may contain nucleic acids that code for the step number, and the bead-attached growth DNA barcode may have nucleic acids in the following order: Beads / First DNA barcode / Nucleic acid encoding the step number / First annealing site / Alternatively, bead-bound growth DNA barcodes can include nucleic acids that are functional nucleic acids (sequencing primer annealing sites), as shown below: Beads / First DNA barcode / Sequencing primer annealing site / First annealing site / Not shown in the diagrams in these texts are any linkers that mediate the coupling of DNA barcodes to beads. Linkers can take the form of nucleic acids or can be made from some other organic chemicals.

[0204] Step 2. Add a soluble sprint oligonucleotide (sprint oligo), which consists of a first annealing site, a second DNA barcode module, and a second annealing site.

[0205] Figure 7 also shows the step in which the hybridized sprint oligo is used as a template, and DNA polymerase catalyzes the attachment of the second DNA barcode module and the second annealing site to the bead-bound growing DNA barcode. Figure 7 shows DNA polymerase using the sprint oligo as a template, resulting in the bead-bound DNA barcode growing slightly longer (growth due to covalent attachment of the second DNA barcode and the second annealing site). Shown immediately below the text is the complex of the sprint oligo hybridized to the bead-bound growing DNA barcode.

[0206] Beads / First DNA barcode / First annealing site / First annealing site / Second DNA barcode / Second annealing site To reiterate some of the information shown in Figure 7, what is shown immediately below is the sprint oligo:

[0207] "First annealing site / Second DNA barcode / Second annealing site" Step 3. Add DNA polymerase and dNTPs to extend the bead-bound DNA barcode. Below is the bead-bound DNA barcode, and the bead-bound grown barcode is longer than before because the sprint oligo is still hybridized and has nucleic acids attached, which are the "second DNA barcode module" and the "second annealing site". Figure 7 further illustrates this step. The sprint oligo is shown below the bead-bound barcode: Beads / First DNA barcode / First annealing site / Second DNA barcode / Second annealing site

[0208] First annealing site / Second DNA barcode / Second annealing site Step 4. Wash the sprint oligos. The sprint oligos can be encouraged to dissociate from the bead-bonded growth barcodes by heating, i.e., by heating the entire picowell plate to, for example, about 60°, 65°, 70°, 75°, and 80° for about 10 minutes, or alternatively, by adding diluted NaOH to the picowell array to neutralize it.

[0209] Step 5. Add the second sprint oligo, which can be used as a template to mediate the DNA polymerase-catalyzed attachment of the third DNA barcode and third annealing site after hybridization to the bead-bound growth sprint oligo. The second sprint oligo is a soluble reagent and is shown below (but not shown in Figure 7).

[0210] Second annealing site / Third DNA barcode / Third annealing site / Step 6. Enable this oligonucleotide to anneal to the corresponding bead-bound "second annealing site" and allow DNA polymerase to extend the bead-bound oligonucleotide, thereby including the following supplement: "third DNA barcode / third annealing site /

[0211] Step 7. Wash the second sprint oligo.

[0212] Step 4. Add the following sprint oligo (this specific addition is not shown in Figure 7).

[0213] Third annealing site / Fourth DNA barcode / Fourth annealing site / This soluble oligonucleotide has nucleic acid that can anneal to the "third annealing site" of the bead-bound oligonucleotide. Upon annealing, a DNA polymerase with four dNTPs is utilized to extend the bead-bound oligonucleotide and encode yet another DNA barcode module (a fourth DNA barcode). The cycle of the above steps is repeated throughout the split-pool procedure, in which a library of compounds and associated DNA barcodes are prepared in parallel, with each DNA barcode being associated with a given compound (each DNA barcode indicates the history of the chemical synthesis of the associated compound). If the chemical synthesis of the compound library is complete, the cycle of the above steps is stopped. With the completed bead-bound DNA barcode chemical library controlled, the beads can then be distributed into the picowells of a picowell array.

[0214] The DNA barcode on each bead further constitutes a DNA barcode associated with each picowell. The DNA barcode can identify the bead-bound compound. The sequence sequencing method of this disclosure is performed in the picowell while the beads are still in the picowell. In an exclusionary embodiment, this disclosure may exclude any sequencing method and any reagents used for sequencing, where sequencing is not performed on a bead-bound DNA template, or where sequencing is not performed on a bead-bound DNA template immobilized in the picowell.

[0215] Annealing sites of sequencing primers. In one embodiment, each DNA barcode module within the completed DNA barcode is functionally linked to an annealing site of its own sequencing primer, thus providing the operator with the ability to perform separate sequencing procedures on each DNA barcode module. (In this embodiment, it is preferable that each DNA barcode module is also functionally linked to its own nucleic acid that identifies (encodes) the steps of synthesis for the entire DNA barcode.)

[0216] In another embodiment, each DNA barcode has only one sequencing primer annealing site, which can be located at or near the 3' end of the bead-bound DNA barcode, and the sequencing primer itself is soluble and can be added to the picowell and then hybridized to the sequencing primer annealing site. Alternatively, if the sequencing primer is part of a DNA hairpin, this DNA hairpin is added via a “sprint oligo” in the final step of constructing the bead-bound DNA barcode. Figure 7 does not show the sequencing primer annealing site.

[0217] Nucleic acids bound to beads via the 3' end of the nucleic acid Various embodiments disclosed in this invention relate to coupling DNA to beads via the 5' end of the DNA, while in other embodiments, DNA such as DNA barcodes or DNA tags may be attached to beads via their 3' ends. The 3' hydroxyl group of DNA may react under certain chemical synthesis conditions (e.g., Mitsunobu transformation), rendering the 3' end damaged and unable to participate in extension, ligation, or other steps. Thus, DNA tags can adhere to beads via their 3' ends, preventing unwanted chemical reactions and thus preventing damage to DNA barcodes.

[0218] Exclusionary embodiments relating to the bead-linked DNA barcodes of the present disclosure. Exclusionary are any beads, particulate matter, fine particles, resin, or polymer composition, wherein the chained DNA barcodes are linked to the beads via photocleavable or cleavable linkers.

[0219] The substances that can be excluded are beads, fine particles, granules, resins, or polymer compositions that do not include (1) chained DNA barcodes attached to a first position on a bead, or (2) compounds attached to a second position on a bead where the first position is not the same as the second position. In a preferred embodiment, this “compound” is made up of a plurality of chemical library monomers.

[0220] Excluded are materials such as beads, fine particles, granules, resins, or polymer compositions that do not have an outer surface (or outer surface) and an inner surface (or inner surface, or internal region), and the beads do not contain at least 10,000 substantially identical linked DNA barcodes attached to the beads, and at least 90% of the at least 10,000 substantially identical linked DNA barcodes are attached to the outer surface. In other words, excluded are any beads in which at least 90% of the attached linked DNA barcodes are not attached to the outer surface.

[0221] Excluded are materials in the form of beads, fine particles, granules, resins, or polymer compositions that are substantially made of polyacrylamide or contain any polyacrylamide.

[0222] The substances that can be excluded are beads, fine particles, granules, hydrogels, resins, or polymer compositions that contain a promoter such as a T7 promoter, or contain a poly-A region, or contain both a promoter and a poly-A region.

[0223] A method using only 1 cycle of annealing / polymerization to generate a bead-bound DNA barcode having two DNA barcode modules. This disclosure encompasses systems, reagents, and methods where the bead-bound DNA barcode includes only 1 annealing / polymerization step. This embodiment is represented by the following figures, where the first figure shows the annealing of the split oligo, and the second figure shows the filling using DNA polymerase. The final result is a bead-bound DNA barcode that includes two DNA barcode modules. In this particular procedure, the bead-bound starting material can optionally include a linker (but preferably not any cleavable linker), optionally a nucleic acid encoding information other than the identity of the compound, and optionally a functional nucleic acid such as a sequencing primer or a DNA hairpin. Two figures are shown within the text (see immediately below):

[0224] Bead / First DNA barcode / First annealing site / First annealing site / Second DNA barcode / Second annealing site Bead / First DNA barcode / First annealing site / Second DNA barcode / Second annealing site

[0225] First annealing site / Second DNA barcode / Second annealing site A method using 2 cycles of annealing / polymerization to generate a bead-bound DNA barcode having three DNA barcode modules. This disclosure encompasses bead-bound compositions, systems, and methods where two different split oligos are used (a first split oligo; a second split oligo). In this context, the first split oligo consists of the structure of a first annealing site / second DNA barcode / second annealing site, and the second split oligo consists of the structure of a second annealing site / third DNA barcode / third annealing site.

[0226] A method using 3 cycles of annealing / polymerization to generate bead-bound DNA barcodes having 4 DNA barcode modules. The present disclosure encompasses bead-bound compositions, systems, and methods, in which 3 different split oligos are used (first split oligo; second split oligo; third split oligo). In this context, the first split oligo consists of the structure of a first annealing site / a second DNA barcode / a second annealing site, the second split oligo consists of the structure of a second annealing site / a third DNA barcode / a third annealing site, and the third split oligo consists of the structure of a third annealing site / a fourth DNA barcode / a fourth annealing site.

[0227] A method using 4 cycles of annealing / polymerization to generate bead-bound DNA barcodes having 5 DNA barcode modules. The present disclosure encompasses bead-bound compositions, systems, and methods, in which 4 different split oligos are used (first split oligo; second split oligo; third split oligo; fourth split oligo). In this context, the first split oligo consists of the structure of a first annealing site / a second DNA barcode / a second annealing site, the second split oligo consists of the structure of a second annealing site / a third DNA barcode / a third annealing site, the third split oligo consists of the structure of a third annealing site / a fourth DNA barcode / a fourth annealing site, and the fourth split oligo consists of the structure of a fourth annealing site / a fifth DNA barcode / a fifth annealing site.

[0228] Embodiments using multiple-step annealing / polymerization to generate bead-bound DNA barcodes having multiple DNA barcode modules. The disclosure relates to chained barcodes and includes bead-bound compositions, systems, and methods such as using only one sprint oligo (to produce a 2-module DNA barcode), using only two sprint oligos (to produce a 3-module DNA barcode), using only three sprint oligos (to produce a 4-module DNA barcode), using only four sprint oligos (to produce a 5-module DNA barcode), using only five sprint oligos (to produce a 6-module DNA barcode), and using only six sprint oligos (to produce a 7-module DNA barcode).

[0229] Included are bead-binding compositions, systems, and methods using at least one sprint oligo, at least two sprint oligos, at least three sprint oligos, at least four sprint oligos, at least five sprint oligos, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least four, at least twenty sprint oligos, or fewer than 20, fewer than 15, fewer than 10, fewer than eight, fewer than six, fewer than four, fewer than three, or fewer than two sprint oligos. These numbers represent the number of sprint oligos themselves, as well as the number of steps to add the sprint oligos and the number of DNA modules added to the growing bead-binding DNA barcode.

[0230] Reduce damage to DNA barcodes. Reduce damage using orthogonal DNA barcoding (instead of linkage DNA barcoding). One way to understand the topic of linkage DNA barcoding and orthogonal DNA barcoding is to note the advantages one has over the other. The advantages of orthogonal barcoding over linkage barcoding are as follows: With the attachment of each monomer of the growing compound, the compound library monomers for constructing the chemical library and the DNA barcoding module for constructing the completed full-length DNA barcode are attached in parallel.

[0231] In chain-based barcoding, if any given module is not properly attached (i.e., if all attachment sites do not successfully bind to the required module), the resulting barcode sequence will be inaccurate. The term "inaccurate" means that incomplete coupling causes missing pieces, and the user assumed the resulting product was a complete and correct DNA barcode. Here, because all DNA modules failed to attach, the resulting DNA barcode sequence will contain errors. In contrast, orthogonal barcoding covalently binds each individual DNA module to its own unique attachment site on the bead. Furthermore, once a DNA module is attached to a given site on the bead, there is no need to bind additional DNA modules to those already bound to the bead.

[0232] Damage is reduced by using a crosslinking agent. This disclosure provides reagents and methods for reducing damage to bead-bound DNA barcodes and to partially synthesized bead-bound DNA barcodes. Each DNA barcode module can be in the form of double-stranded DNA (dsDNA) before attaching to a growing bead-bound DNA barcode, and this dsDNA is treated with a DNA crosslinking agent such as mitomycin C. After the synthesis of the DNA barcode in dsDNA form is complete, this dsDNA is converted to ssDNA. The conversion from dsDNA to ssDNA is effective when one of the DNA strands has a uracil (U) residue, and the cleavage of the DNA at the uracil residue is catalyzed by uracil-N-glycosidase (see Figure 5 of serial number 62 / 562,905 filed on 25 September 2017, which is incorporated herein by reference in its entirety). The above refers to the damage inflicted on the growing DNA barcode by the reagents used to create the bead-binding compound.

[0233] Damage is reduced when using double-stranded DNA (dsDNA) to construct DNA barcodes. Another method to reduce damage to bead-bound DNA barcodes and to partially synthesized DNA barcodes is to synthesize the DNA barcodes in the form of double-stranded DNA, where each of the DNA barcode modules attached to each other takes the form of dsDNA, and each of the two strands is stabilized via a DNA headpiece. For final sequencing of the completed DNA barcode, one of the strands is cut and removed from the DNA headpiece. The above refers to the damage inflicted on the growing DNA barcode by the reagents used to construct the bead-bound compound (if this compound is a member of a chemical library).

[0234] Damage is reduced by including hairpins. Yet another method to reduce damage to bead-bound DNA barcodes is to synthesize the DNA barcodes via self-assembly to form hairpins, which self-assemble by the first projection of the hairpin annealing to a second projection of the hairpin.

[0235] When the synthesized DNA barcode takes the form of double-stranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can denature the DNA barcode. The above methods and reagents can prevent denaturation.

[0236] Damage is reduced by using sealed ends of dsDNA. Another method to reduce damage to bead-bound DNA barcodes and to reduce damage to partially synthesized DNA barcodes is to use double-stranded DNA (dsDNA) and seal the ends of this dsDNA via 7-aza-dATP and dGTP.

[0237] Damage is reduced by avoiding proteinaceous solvents, strong acids and bases, and strong reducing and oxidizing agents. The types of chemicals are compatible with the presence of deoxyribonucleic acid (DNA), and bead-bound or non-bead-bound DNA may require the absence of proteinaceous solvents, avoidance of strongly acidic conditions, avoidance of strong bases such as t-butyllithium, avoidance of strong reducing agents such as lithium aluminum hydride, avoidance of reagents that react with DNA bases such as some alkyl halides, and avoidance of some oxidizing agents (see Luk and Sats (2014) DNA-Compatible Chemistry (Chapter 4), A Handbook for DNA-Encoded Chemistry, 1st ed. John Wiley and Sons, Inc.).

[0238] As mentioned elsewhere, the term "DNA barcode" can refer to a polynucleotide that identifies an entire compound, while, in contrast, a "DNA barcode module" can refer to only one of the monomers that make up the compound.

[0239] Damage to nucleic acids is reduced by using DNA-compatible chemicals. Satz et al. disclose a variety of chemicals compatible with bead-bound nucleic acids (Satz et al (2015) Bioconjugate Chemistry. 26:1623-1632; corrected in Satz et al (2016) Bioconjugate Chemistry. 27:2580-2580). While the aforementioned description by Satz et al. concerns chemical reactions performed on DNA / chemical library member complexes, the types of DNA-compatible chemicals described are also relevant, as organic chemistry is performed on beads containing bead-bound compounds and bead-bound DNA.

[0240] DNA-compatible reactions for the formation of benzimidazole compounds, imidazolidinone compounds, quinazolinone compounds, isoindolinone compounds, thiazole compounds, and imidazopyridine compounds are disclosed (see Satz et al., Table 1, entries 1-6).

[0241] Furthermore, DNA-compatible protecting groups are disclosed to include alloc deprotection, BOC deprotection, t-butyl ester hydrolysis, methyl / ethyl ester hydrolysis, and nitro reduction with hydrazine and Raney nickel (see Satz et al., Table 1, entries 7-11).

[0242] Furthermore, methods for coupling reagents to DNA are disclosed, where binding occurs at functional groups already attached to the DNA. These methods include the Suzuki coupling, an optimized procedure for Sonogashira coupling between alkynes and aryl halides; the conversion of aldehydes to alkynes using dimethyl-1-diazo-2-oxopropylphosphonate; a novel method for directly adding triazoles from purified alkynes; an improved reaction using isocyanate reagents in pH 9.4 buffer; and an improved method for the reaction of isocyanate components with amine-functionalized DNA (see Satz et al., Table 1, entries 12-15).

[0243] Additional methods for coupling reagents to DNA are disclosed, where the coupling occurs at functional groups already attached to the DNA. These include methods for binding primary amines to DNA, optimized procedures for forming DNA-complexed thioureas, methods for alkylating secondary amines and bisalkylating aliphatic primary amines, monoalkylating primary amine DNA complexes using hetalyl halides as components that can react with amine-functionalized DNA complexes, and methods for the Wittig reaction (see Satz et al., Table 1, entries 16-20).

[0244] Reduce damaged DNA via DNA repair enzymes. Various proteins, including enzymes, DNA damage-binding proteins, and helicases, are available for the repair of DNA damage. Commercially available are DNA repair proteins that can repair damage in the form of oxidation-induced damage, radiation-induced damage, ultraviolet-induced damage, damage from formaldehyde adducts, and damage in the form of alkyl group adducts. Glycosylase enzymes (which do not cleave ssDNA or dsDNA) that remove damaged bases are available for the repair of 5-formyluracil, deoxyuridine, and 5-hydroxymethyluracil. T4PDG is available for the repair of pyrimidine dimers. hNEIL1 and Fpg are available for the repair of oxidized pyrimidines, oxidized purines, apurinic sites, and pyrimidine sites. EndoVIII is available for the repair of oxidized pyrimidines and pyrimidine sites. EndoV is available for the repair of mismatches. HaaG is a glycosylase available for the repair of alkylated purines. When DNA repair enzymes leave gaps, the double-stranded DNA has gaps and one or more consecutive deoxyribonucleotides are missing from one of the strands, and various DNA polymerases are available to fill the gaps (see Catalog(2018)New England BioLabs, Ipswich, MA).

[0245] Various DNA repair enzymes and DNA repair systems have been isolated from mammals, yeast, and bacteria. These include those that mediate nucleotide excision repair (NER), direct repair, base excision repair, transcription-coupled DNA repair, and recombination repair. Interstrand DNA crosslinks can be repaired by using a combination of NER and homologous recombination. Direct repair includes the repair of cyclobutane pyrimidine dimers and 6-4 products via photolyase enzymes. Direct repair includes the repair of O 6- methylguanine from O 6-It also includes the removal of methyl groups. See Sancar et al (2004) Ann.Rev.Biochem. 73:39-85; Hu, Sancar (2017) J.Biol.Chem. 292:15588-15597.

[0246] The present disclosure provides systems, reagents, and methods for repairing damage to bead-bound DNA barcodes by treatment with DNA repair enzymes or by DNA repair protein complexes, etc.

[0247] Damage is reduced by coupling DNA to beads via their 3' ends. Certain chemical conversions can damage the exposed 3'-hydroxyl group of nucleic acids. For example, the Mitsunobu reaction enables the conversion of primary and secondary alcohols to esters, phenyl ethers, thioethers, and various other compounds, thereby rendering the exposed 3' end non-reactive to subsequent processing steps or the currently modified 3' end being potentially involved in further chemical reactions. In some embodiments, DNA tags can be attached to beads via their 3' ends, such that only the 5' ends are exposed in solution.

[0248] The reagents, systems, and methods of this disclosure involve bead-binding nucleic acids, such as bead-binding DNA or bead-binding DNA tags, where coupling to the beads involves the 3' end (terminus) (or 3' end (end)) of the DNA. If the ssDNA constituting the DNA barcode is bound via the 3' end of the ssDNA, sequencing can be initiated by hybridizing only one sequencing primer, which hybridizes upstream of the entire DNA barcode, and this hybridization is at or near the bead-binding end of the bound ssDNA. Instead of using only one sequencing primer, multiple sequencing primers can be used, each sequencing primer hybridizing upstream to a specific DNA barcode module. For example, if a given DNA barcode contains five DNA barcode modules, the DNA may be bound to the beads via the 3' end, and the DNA barcode may contain five different primer annealing sites, each primer annealing site located directly upstream or just upstream of a given DNA barcode module.

[0249] Embodiments of double-stranded DNA (dsDNA) coupling. In other embodiments, dsDNA is bound to the bead, and only one 3' end of the strand in the dsDNA is bound to the bead. In embodiments of 5' coupling with dsDNA, only dsDNA can be bound, and only one 5' end of the strand of the dsDNA is bound to the bead.

[0250] (V) Coupling of compound and beads This disclosure provides (1) linkers for attaching chemical library members to substrates such as beads, (2) linkers for attaching nucleic acid barcodes to substrates such as beads, (3) cleavable linkers, for example, cleavable by UV light or by enzymes such as proteases, (4) non-cleavable linkers, (5) bifunctional linkers, (6) multifunctional linkers, and (7) multiple beads used for chaining. For example, available linkers include 4-hydroxymethylbenzoic acid (HMBA) linkers and 4-hydroxymethylphenyl acetate linkers (see Camperi, Marani, Cascone (2005) Tetrahedron Letters. 46:1561-1564).

[0251] An "uncleavable linker" may be characterized as a linker that is not detectably cleaved by any reagent, condition, or environment used during a given step in an organic chemistry procedure. Alternatively, an "uncleavable linker" may be characterized as a linker that cannot be cleaved except by a reagent, condition, or environment that is unacceptably destructive to other reactants, products, or reagents in the given organic chemistry procedure.

[0252] A bifunctional linker, or other multifunctional linker, can take the form of a fork (a fork used by humans to consume food), where the fork's handle is attached to a bead, and each prism of the fork is linked to one of various chemicals. For example, one prism can be linked to a chemical library member; another prism can be linked to a DNA barcode; yet another prism of the fork can be linked to a metal ion.

[0253] With regard to the use of multiple beads, the disclosure provides embodiments of multiple beads, such as (1) a first bead comprising an attached nucleic acid barcode linked to a second bead, the second bead comprising an attached chemical library member, and (2) a first bead comprising an attached nucleic acid barcode linked to a second bead, the second bead comprising an attached chemical library member, and a third bead comprising a reagent to which a reagent is covalently attached (to the first bead and / or the second bead). The attached reagent may be an enzyme, which is used to assay the activity of the attached chemical library member.

[0254] (VI) Bond monomers to create a compound. Exemplary chemical monomers. Suitable amino acid derivatives for use as chemical monomers for the compositions and methods of the present disclosure are shown in Figure 4. The figure shows, for example, suppliers of chemicals such as AnaSpec EGT Group, Fremont, CA; Sigman-Aldrich, St. Louis, MO; Acros Organics (part of ThermoFisher Scientific); or Combi-Blocks, San Diego, CA.

[0255] Additional chemical monomers are shown in Figures 22–27. Each of Figures 22–27 provides the structure, chemical name, and associated DNA module barcode. As disclosed in Figures 1–6 (Figure 22), the respective barcodes are ACGT, ACTC, AGAC, AGCG, AGTA, and ATAT. For compounds 7–10 (Figure 23), the respective barcodes are ATGA, CACG, CAGC, and CATA. For compounds 11–16 (Figure 24), the respective barcodes are CGAG, CGCT, CGTC, CTAC, CTGT, and GACT. For compounds 17–21 (Figure 25), the respective barcodes are GAGA, GCAC, GCTG, GTAG, and GTCA. For compounds 22–26 (Figure 26), the respective barcodes are GTGC, TAGT, TATC, TCAG, and TCGC. Furthermore, for compounds 27-30 (Figure 27), the barcodes are TCTA, TGAT, TGCA, and TGTG, respectively. These barcodes are merely illustrative. For any given library of compounds, different collections of DNA barcodes can be used to identify each of the chemical monomers used to construct the compounds in that library.

[0256] Coupling reaction. The following describes the coupling of chemical monomers to beads and to each other, i.e., the first step is to directly couple a first chemical monomer to beads via a cleavable linker, and then subsequent chemical monomers are coupled one by one to each other. The conditions shown below are DNA compatible.

[0257] This paper describes a method for preparing three amino acid compounds on Tentagel® beads. Fmoc-protected resin (1 mg, Rapp Polymere GmbH, 10 μm, TentaGel M-NH2, 0.23 mmol / g) modified with Fmoc-Photo-Linker, 4-{4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butanoic acid), or another suitable linker with Fmoc protection, was suspended in each well of a reaction plate (Merck Millipore Ltd, 0.45 μm hydrophobic PTFE) in DMA (150 μL). The bottom of the plate was vacuumed and the solvent removed using a Resprep VM-96 vacuum manifold. The Fmoc protecting group was removed by suspending the resin in 150 μL of a mixture of 5% piperazine and 2% DBU in DMF. The plate was sealed with Excel Scientific Alumna Seal and shaken at 40°C for 15 minutes. Vacuum was applied to remove the solvent, and the deprotection procedure was repeated for 5 minutes. After filtration, each well was washed with 150 μL each of 2XDMA, 3XDCM, and 1XDMA, with vacuum applied between each wash to remove the solvent. Next, 150 μL of a pre-activation mixture of 60 mM Fmoc-amino acids, 80 mM Oxyma, 200 mM DIC, and 80 mM 2,4,6-trimethylpyridine, which had been left at room temperature for 2 minutes, was added to acylate each well of the resin with the appropriate amino acid. The plate was resealed and shaken at 40°C for 1 hour. After filtration, each well was washed with 150 μL each of 2XDMA and 3XDCM. The beads in each well were resuspended in 150 μL of DCM, and the contents of each well were combined into one container by pipetting. The combined beads were thoroughly mixed and redistributed to the plate by pipetting equal volumes into the appropriate wells (1 mg / well). By removing the solvent through vacuum, each well is prepared for the next appropriate step. For each additional amino acid coupling, the Fmoc deprotection step is repeated first, followed by the coupling step with the desired amino acid. If a split pool is required, the coupling and redistribution method is repeated.

[0258] A method for producing trimer amino acids on beads by the split-pool method is described. Fmoc-Photo-Linker, 4-{4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butanoic acid-modified Fmoc-protecting resin (1 mg, Rapp Polymere GmbH, 10 μm, TentaGel M-NH2, 0.23 mmol / g), or any other suitable linker was suspended in each well of a reaction plate (Merck Millipore Ltd, 0.45 μm hydrophobic PTFE) in DMA (150 μL). The bottom of the plate was vacuumed and the solvent removed using a Resprep® VM-96 vacuum manifold. The Fmoc protecting group was removed by suspending the resin in 150 μL of a mixture of 5% piperazine and 2% DBU in DMF. The plate was sealed with Excel Scientific Alumna Seal and shaken at 40°C for 15 minutes. Vacuum was applied to remove the solvent, and the deprotection procedure was repeated for 5 minutes. After filtration, each well was washed with 150 μL each of 2XDMA, 3XDCM, and 1XDMA, with vacuum applied between washes to remove the solvent. Next, 150 μL of a pre-activated mixture of 60 mM Fmoc-amino acid, 80 mM Oxyma, 200 mM DIC, and 80 mM 2,4,6-trimethylpyridine, which had been left at room temperature for 2 minutes, was added to acylate each well of the resin with the appropriate AA. The plate was resealed and shaken at 40°C for 1 hour. After filtration, each well was washed with 150 μL each of 2XDMA, 3XDCM, and 1XDMA. For each additional AA coupling, the Fmoc deprotection step was repeated first, followed by the coupling step with the AA of interest. To analyze each successive coupling, a 1 mg portion of the beads was suspended in 100 μL of DMSO and exposed to full power of a 365 nm LED for 2 hours. The resin was filtered, and the filtrate was injected into an Agilent 1100 series LCMS equipped with an Agilent Poroshell SB-C-18, 3.0 x 50 mm, 2.7 μm column. A gradient from 5% CH3CN in 0.1% TFA in water to 100% CH3CN in 0.1% TFA was measured at a flow rate of 1.2 mL / min over 4 minutes, and monitored at 220 nm.

[0259] Experiment to prepare non-amino acid pendants with lenalidomide (Revlimid®) attached. This attaches to the final amino acid after deprotection. This was also performed in spin mode. Each well of resin was acylated with 150 μL of a mixture pre-treated for 5 minutes with 40 mM chloroacetic acid, 40 mM Oxyma, 80 mM DIC, and 40 mM TMP in DMA (after Fmoc deprotection). The plate was sealed and shaken at 40°C for 1 hour. Each well was washed with 150 μL each of 3X DMA, 3X DCM, and 2X DMA. The resin was then resuspended in a suspension of 100 mM K2CO3 and 100 mM Rev in DMA. The plate was sealed and shaken at room temperature for 3 hours. The resin was washed with 150 μL each of 2X50 / 50 DMA / water, 3X DMA, 3X DCM, and 2X DMA.

[0260] This defines the degree of fidelity in the synthesis of the compound attached to a given bead. This relates to the finished compound, which is a member of a chemical library. Each chemical compound can be made partially or completely from chemical monomers. The following are the characteristics of the compound attached to a given bead. This given bead may be the product of a split-pool system synthesis of a library of compounds, and each bead has a unique compound.

[0261] Members of the chemical library can be synthesized on solid supports, such as beads, via solid-phase synthesis. Solid-phase synthesis of chemicals containing peptide bonds is characterized by the use of one of two chemical groups. The first chemical group is N-alpha-9-fluorenyl-methyloxycarbonyl (Fmoc, basic unstable). The second chemical group is tert-butyloxycarbonyl (tBoc, acid unstable) (see Vagner, Barany, Lam (1996) Proc. Natl. Acad. Sci. 93:8194-8199). Fmoc and tBoc are protecting groups that can be used to protect peptide substrates, and either the Fmoc group or the tBoc group is attached to an alpha-amino group (Sigler, Fuller, Verlander (1983) Biopolymers. 22:2157-2162).

[0262] Preferably, at least 99.5%, at least 99.0%, at least 95%, at least 90%, at least 85%, or at least 80% of the members of the chemical library bonded to the given beads have exactly the same chemical structure after the synthesis is complete. Incomplete coupling may occur, which may occur in one or more steps in the multi-step synthesis of the chemical library members. For this reason, the compositions of this disclosure may be characterized and limited by one of the following limitations or scopes.

[0263] Further provided by this disclosure are methods and reagents in which at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% of the members of a chemical library bound to a given bead have exactly the same chemical structure after complete synthesis (these figures reflect and take into account errors that may occur during solid-phase synthesis, such as one growing compound failing to receive one of the chemical monomers; and these figures also reflect and take into account any chemical damage to any monomer that may occur during solid-phase synthesis).

[0264] In an exclusionary embodiment, the disclosure may exclude any method or reagent that does not satisfy one of the above cutoff values ​​for “exactly the same structure.”

[0265] In alternative embodiments, 2 beads, 3 beads, 4 beads, 5 beads, about 5-10 beads, about 10-20 beads, about 20-40 beads, and about 40-80 beads contain similar and equal compounds in the bead population (without considering any errors in the incorporation of chemical monomers during solid-phase synthesis and without considering chemical damage to chemical monomers during organic synthesis).

[0266] Introduction to Click Chemistry. Jewett et al. defined click reactions as being selective, high-yielding, and having good reaction rates. A subclass of click reactions in which the components are inert to the surrounding biological environment is called biorthogonal (Jewett and Bertozzi (2010) Chem. Soc. Rev. 39:1272-1279). "Click chemistry" can be used to link small units together with heteroatom linkages such as carbon-X-carbon. Click chemistry can be used alone or in combination with other types of chemical reactions for the synthesis of drugs or drug candidates. Click chemistry works well in procedures used in combinatorial chemistry. Click chemistry reactions are characterized by high yield, irreversibility, and inactivity with oxygen or water. The class of chemical reactions used in "click chemistry" includes (1) cyclic addition reactions, particularly the 1,3-bipolar family and hetero-Diels-Alder reactions; (2) nucleophilic ring-opening reactions as well as strained heterocyclic molecules such as epoxides, aziridines, and cyclic sulfates; (4) non-aldol type carbonyl chemistry; and (5) addition to carbon-carbon multiple bonds, as well as oxidation reactions and some Michael addition reactions. Click chemistry reactions are distinguished by high thermodynamic driving forces, usually exceeding 20 kcal / mol, whereas non-click chemistry reactions involve bond formation with only moderate thermodynamic driving forces (Kolb and Sharpless (2003) Drug Discovery Today. 8:1128-1137, Kolb, Finn, Sharpless (2001) Angew. Chem. Int. Ed. 40:2004-2021).

[0267] Tetrazine and trans-cyclooctene (TCO). Tetrazines such as 1,2,4,5-tetrazine can react with trans-cyclooctene (TCO) via Diels-Alder cycloaddition (Devaraj, Haun, Weissleder (2009) Angew. Chem. Intl. 48:7013-7016).

[0268] Buchwald-Hartwig amination. The Buchwald-Hartwig amination reaction can be used in the solid-phase synthesis of pharmaceuticals. This amination reaction is used to synthesize a carbon-nitrogen bond, and the reaction involves palladium catalysis of an aryl halide and an amine (R1-NH-R2), in which the amine is replaced by an aryl product, and the nitrogen of the amino group is directly attached to the aromatic ring. The final result is a product with a carbon (of the aryl group) and nitrogen (of the amino group) bond. In other words, this reaction converts an aryl halide to the corresponding aniline. Buchwald-Hartwig amination is compatible with a variety of amines and is suitable for combinatorial chemistry (Zimmermann and Brase (2007) J.Comb.Chem.9:1114-1137).

[0269] Huesgene cycloaddition. The 1,3-dipolar cycloaddition reaction of Huesgene involves alkynes and organic azides. Alkynes have an RC=CH structure. Azides have an RN structure. + =N=N - It has the following structure. Copper catalysts accelerate the rate of the Huesgen cycloaddition reaction. The Huesgen reaction functions via "click chemistry" or "click reaction". When the Huesgen reaction is catalyzed with copper, it can produce 1,2,3-triazole nuclei suitable for the preparation of small molecule agents. The Huesgen reaction is compatible with the presence of amino acid side chains, at least in a protected form. Molecules prepared from 1,2,3-triazoles may have bonds similar to the amide bonds of polypeptides, and therefore these molecules can substitute for peptide bonds (Angell and Burgess (2007) Chem. Soc. Rev. 36: 1674-1689).

[0270] Peptide nucleic acids (PNAs). This disclosure provides split-pool, combinatorial chemistry, or solid-phase chemistry methods for synthesizing peptide nucleic acids. Peptide nucleic acids are analogs of oligonucleotides. They are resistant to hydrolysis by nucleases. They can strongly bind to their target RNA sequences. The uptake of peptide nucleic acids into cells can be enhanced by "cell-permeable peptides" (Turner, Ivanova, Gait (2005) Nucleic Acids Res. 33:6837-6849; Koppelhus (2008) Bioconjug. Chem. 19:1526-1534). Peptide nucleic acids can be prepared by solid-phase synthesis and combinatorial synthesis (see Quijano, Bahal, Glazer (2017) Yale J. Biology Medicine. 90:583-598; Domling (2006) Nucleosides Nucleotides. 17:1667-1670).

[0271] This disclosure encompasses bead-bound compounds, each compound existing in the form of a single monomer. For example, this bead-bound compound may exist in the form of lenalidomide, or in the form of lenalidomide with a carboxylic acid group attached, or in the form of lenalidomide with an amino group modified by a small chemical moiety having a carboxylic acid group, or the compound may be a lenalidomide analog which is a stereoisomer or enantiomer of lenalidomide.

[0272] (VII) Split pool synthesis and parallel synthesis This relates to the use of the “split pool” method for synthesizing a library of compounds, and to the method of using the “split pool” method for the simultaneous synthesis of bead-bound compounds and bead-bound DNA barcodes. It also describes splitting and pooling for preparing mixed sets of compounds. Disclosed below at some point later are non-amino acid coupling and the preparation of polyethylene glycol (PEG) modified beads.

[0273] This disclosure provides a split-pool synthesis for generating a chemical library. In one embodiment, the method involves the steps of (a) dividing beads into different containers, and (b) adding different components to each container. For example, if three containers are used, seed A is added to the first container, seed B to the second container, and seed C to the third container and reacted so that the seeds covalently bind to the attachment sites of the beads in the containers, (c) all the beads are pooled into one container, (d) the beads are divided into three containers, and (e) different components are added to each container, with seed A added to the first container, seed B added to the second container, and seed C added to the third container, so that the seeds covalently bind to the first seed that was previously attached (see Stockwell (2000) Trends Biotechnol. 18:449-455).

[0274] The split pool synthesis of this disclosure includes a DNA barcode coupling step either before or after each chemical coupling step (to construct chemical library members), wherein the DNA barcode identifies the chemicals coupled in that step.

[0275] In an exclusionary embodiment, the disclosure may exclude, for a given step in parallel synthesis, a method and reagents to which the barcode is applied before the chemical is applied. Conversely, the disclosure may exclude, for a given step in parallel synthesis, a method and reagents to which the chemical is applied before the barcode is applied.

[0276] One characteristic of bead-bonded chemical libraries prepared by the split-pool method is that only one compound is attached to each bead. If the coupling is incomplete, for example, if only 4,000 out of 5,000 attachment sites are successfully coupled with the desired chemical species in a given split-pool step, some heterogeneity will occur.

[0277] Parallel Synthesis. In preferred embodiments of this disclosure, parallel synthesis can be used for the organic synthesis of compounds and associated DNA barcodes. In practice, the modification of beads with one or more chemical monomers and the modification of the same beads with one or more DNA barcode modules are not strictly parallel. In practice, a bead receives one or more chemical units (chemical monomers), and subsequently receives DNA barcode modules encoding those particular chemical units. The term “parallel” refers to the fact that as the polymer of the chemical library monomers grows, the polymer of the DNA barcode modules also grows. If all DNA barcode modules are attached to the bead and form a chain-like or orthogonal structure, the full-length DNA barcode is referred to as a “DNA barcode” (not just the DNA barcode modules).

[0278] The ratio of the number of externally attached DNA barcodes to the total number of attached chemical library members.

[0279] This relates to the outer and inner surfaces of the beads. For a given bead having DNA barcodes attached to the outside (without considering the number of DNA barcodes attached to the inside) and attached chemical library members (attached to both the outer and inner surfaces), the ratio of the number of DNA barcodes attached to the outside to the total number of attached chemical library members may be, for example, about 0.1:100, about 0.2:100, about 0.5:100, about 1.0:100, about 2:100, about 5:100, about 10:100, about 20:100, about 30:100, about 40:100, about 50:100, about 60:100, about 70:100, about 80:100, about 90:100, about 1:1, about 100:150, about 100:200, about 100:400, about 100:600, etc. In an exclusionary embodiment, the disclosure may exclude any beads or any group of beads that conform to one of the above values.

[0280] Uniformity of DNA barcodes in typical beads; uniformity of chemical library members in typical beads. This disclosure provides "homogeneity of a chemical library" such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99.5% for any given beads (or any group of beads).

[0281] In less precise embodiments, the disclosure provides "homogeneity of a chemical library" of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of any given beads, or alternatively, any given group of beads.

[0282] Similarly, this disclosure provides the above-mentioned cutoff values ​​for evaluating the uniformity of barcodes, such as DNA barcodes.

[0283] The uniformity of DNA barcodes and chemical library members can be planned in the methods section of the laboratory manual or notes and defined as the proportion of the total population that matches the exact sequence as desired.

[0284] In an exclusionary embodiment, the disclosure may exclude any reagent, composition, or method that does not meet one or more of the above cutoff values.

[0285] When evaluating the uniformity of a group of beads, if uniformity is desired throughout the entire group, it is necessary to consider the combined uniformity of beads #1, #2, #3, #4, #5, #6, #7, etc.

[0286] In an exclusionary embodiment, the disclosure may exclude any beads or any group of beads such that the uniformity of the DNA barcodes is not at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99.5%, etc. Also, in an exclusionary embodiment, the disclosure may exclude any beads or any group of beads such that the uniformity of the chemical library members is not at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99.5%, etc.

[0287] Ratio of DNA barcodes attached internally to DNA barcodes attached externally In some embodiments of this disclosure, it may be desirable to manufacture and use beads in which the DNA barcode is attached primarily to the outer surface. One reason for not manufacturing and using beads with internal DNA barcodes is the low penetration of DNA oligomers into the internal space and the low penetration of DNA ligase (ligase for connecting DNA modules to each other in order to create a completed DNA barcode) into the internal space. For sequencing purposes, another reason for not manufacturing and using internal DNA barcodes is the low penetration of enzymes required to amplify the DNA necessary for the final sequencing of the barcode. Yet another reason for not manufacturing and using beads with internal DNA barcodes is the cost of the internal space for attaching members of a chemical library.

[0288] This disclosure provides beads associated with DNA barcodes, where the ratio of internally attached DNA barcodes to externally attached DNA barcodes is approximately 0.1:100, approximately 0.2:100, approximately 0.4:100, approximately 0.8:100, approximately 1:100, approximately 2:100, approximately 4:100, approximately 8:100, approximately 10:100, approximately 20:100, approximately 40:100, approximately 50:100, approximately 60:100, approximately 70:100, approximately 80:100, approximately 90:100, approximately 1:1, etc.

[0289] Furthermore, this disclosure provides beads related to DNA barcodes, where the ratio of internally attached DNA barcodes to externally attached DNA barcodes is less than 0.1:100, less than 0.2:100, less than 0.4:100, less than 0.8:100, less than 1:100, less than 2:100, less than 4:100, less than 8:100, less than 10:100, less than 20:100, less than 40:100, less than 50:100, less than 60:100, less than 70:100, less than 80:100, less than 90:100, less than 1:1, and so on.

[0290] A collection of beads in an aqueous suspension can be brought into contact with a substrate such as a picowell array, resulting in the beads entering and occupying the picowells. The desired occupancy rate can be achieved by adjusting the ratio of the number of beads in the suspension to the number of picowells in the substrate. For example, if the suspension contains only one bead, all picowells containing beads will contain only one bead, and the remaining picowells will contain no beads at all. If the suspension contains 20,000 beads and the substrate contains 200,000 picowells, at least 180,000 picowells will be completely empty of beads, and most of the picowells containing beads will contain only one bead. A small fraction of the occupied picowells will contain two beads.

[0291] In a valuable embodiment, the ratio of beads to picowells in the suspension may be approximately 0.2:100, 0.4:100, 0.6:100, 0.8:100, 1:100, 2:100, 4:100, 6:100, 8:100, 10:100, 20:100, 30:100, 40:100, 50:100, 60:100, 80:100, 100:100 (same as 1:1), 2:1, 4:1, 6:1, 8:1, 10:1, etc.

[0292] In an exclusionary embodiment, the disclosure may exclude any method or system that falls under one of the above values ​​or ranges.

[0293] In the embodiments within this range, the ratio of beads to picowells in the suspension is approximately 0.2:100 to 0.4:100, 0.4:100 to 0.6:100, 0.6:100 to 0.8:100, 0.6:100 to 1:100, 1:100 to 2:100, 2:100 to 4:100, 4:100 to 6:100, 0.6:100 to 8:100, 8:100 to 10:100, and 10:100. The ratios can be approximately 20:100, 20:100 to 30:100, 30:100 to 40:100, 40:100 to 50:100, 50:100 to 60:100, 60:100 to 80:100, 80:100 to 100:100 (same as 1:1), 100:100 (same as 1:1) to 2:1, 2:1 to 4:1, 4:1 to 6:1, 6:1 to 8:1, 8:1 to 10:1, etc.

[0294] In an exclusionary embodiment, the disclosure may exclude any method or system that falls under one of the above values ​​or ranges.

[0295] (VIII) Manufacture a picowell A combination of UV light, photomask, and photoresist for fabricating a picowell array plate. A plate containing many microwells or picowells can be fabricated for use in this disclosure as follows: Briefly, a three-layer sandwich is assembled. The top layer is photoresist. The middle layer is a glass wafer. The bottom layer is a photomask. The picowells will be separated from the photoresist by UV light. After the picowells are separated from the flat sheet of photoresist, the photoresist resembles a typical metal dish containing cups for baking muffins, with the cups of the dish used to hold the muffin batter having slanted sides. The UV light acts as a "non-crosslinking agent" because it breaks down the polymer of the photoresist. After UV treatment, a solvent is added to wash away the UV-treated photoresist, leaving behind clean-looking picowells.

[0296] The inclined walls are created by tilting and rotating. Picowells with inclined walls are fabricated as follows: The photomask has many holes, each corresponding to the desired bottom dimension of the picowell. The bottom dimension can include circumference, diameter, and shape, i.e., circular shape. The top dimension of the well is created by directing inclined UV light onto the holes in the photomask, either by rotating the light source or by rotating the stage that holds the sandwich (photomask / glass wafer / photoresist sandwich). In rotation, the light source is tilted slightly from the 90-degree position to cut the inclined wall of each picowell, rather than at a 90-degree angle to the photomask / wafer / photoresist sandwich. The resulting picowell array plate containing many picowells can be used as is. Alternatively, this picowell array plate can be used as a mold to inexpensively fabricate many picowell array plates.

[0297] Han et al. describe the equipment and reagents for manufacturing microwell plates with tilted walls (see Han et al (2002) J. Semiconductor Technology and Science. 2:268-272). The equipment described includes a UV light source, a contact stage, a tilting stage, and SU-8 photoresist. Manufacturing begins with a single-sided polished silicon wafer. SU-8 photoresist is coated onto the wafer to a thickness of approximately 0.10–0.15 mm. The photoresist is then gently baked on a 65-degree hot plate for 10 minutes, and then gently baked on a 95-degree hot plate for 30 minutes. The resulting photoresist / wafer sandwich is then brought into contact with a UV mask using a contact stage. The term "tilting and rotating UV lithography" refers to a method for manufacturing microwell array plates or picowell array plates, where each well has a tilted wall. Here, the floor of the well has a small diameter, and the top of the well (the part where the upper edge of the well contacts the flat surface of the plate) has a wider diameter. A turntable is used to expose the well to UV light, and the UV light is tilted (Han et al., previously mentioned). The mask contacts the photoresist, each of which is circular. Figure 8 by Han et al., mentioned above, provides a photograph of the direction of the UV light, the UV mask, the photoresist structure, the wafer substrate, and the turntable. Han et al. describe a method for manufacturing a frustum of a cone. A soft material such as PDMS (polydimethylsiloxane) is poured over a conical array and cured, causing the PDMS layer to peel off and form a conical well.

[0298] A mold is created for mass production of picowell array plates. If the picowell array plate is being manufactured, epoxy is poured over the plate, filling all the picowells, and all the filled picowells can be brought into contact with the epoxy platform. Once the epoxy has hardened, the solid platform with the array of picowell protrusions attached is removed (the picowell protrusions are the inverse of the desired picowells). The solid platform with picowell protrusions is a reusable mold that can be used to manufacture many picowell array plates.

[0299] The procedure for creating replicas from an epoxy mold (or a conical array mold made of any rigid material) is referred to as "hot embossing." Briefly, a substrate material is heated to its glass transition temperature or softening temperature, at which point a mold with pico-protrusions is uniformly pressed into the thermosoftened material. After the pico-protrusions are transferred to the substrate material as pico-indentations, the mold can be separated from the substrate. This disclosure preferably discloses pico-cones and pico-wells as patterns for the mold and substrate, respectively.

[0300] This section describes hot embossing, epoxy masters, and photoresists such as SU-8 photoresist (see Bohl et al (2005) J. Micromechanics and Microengineering. 15:1125-1130, Jeon et al (2011) Biomed Microdevices. 13:325-333, Liu, Song, Zong (2014) J. Micromechanics and Microengineering. 24: Article ID: 035009, and del Campo and Greiner (2007) J. Micromechanics and Microengineering. 17: R81-R95).

[0301] Other embodiments of microwell plates. Plastic microwell arrays can be manufactured via thermoforming using a silicon mold, the silicon mold having an array of microwells, e.g., an array of 800,000 microwells. Tapered shapes, smooth sidewalls, and high levels of control resulting in submicron tolerances can be fabricated using non-pulsed dry etching processes. In contrast, methods using pulsed dry etching processes, such as the Bosch process, may result in rougher sidewalls and a lack of control over lateral dimensions during etching.

[0302] Plastic arrays are manufactured by thermoforming plastic onto a silicon master fabricated by a non-pulsed isotropic dry etching process using a chromium mask, employing a non-pulsed dry etching process. This process uses three gases: Ar, SF6, and C4F8. The process is carried out with 1200–2000 watts of RF power and a 150 watt bias. By varying the gas flow between the three gases, the taper of the silicon mold can be fine-tuned to produce smooth sidewalls. The ratio of SF6 to C4F8 is changed, and the result of changing the ratio is a tapered wall of the mold (silicon pillar) that exists at inclinations of, for example, 18 degrees (very inclined wall), 9 degrees (slightly inclined wall), or 2 degrees (wall almost perpendicular to the substrate) (Perry, Henley, and Ramsey (Oct. 26–30, 2014) Development of Plastic Microwell Arrays for Improved Replication Fidelity.18). th International Conference on Miniaturized Systems for Chemistry and Life Sciences. San Antonio, TX (See pages 1700-1703).

[0303] In embodiments, the disclosure provides substrates, arrays, grids, microfluidic devices, etc., including arrays of microwells. In one embodiment, all microwells have essentially the same volume. This volume can be about 1 femtoliter, about 2, about 4, about 6, about 8, about 10, about 20, about 40, about 60, about 80, about 100, about 200, about 400, about 600, about 800, or about 1,000 femtoliters.

[0304] Furthermore, the volume can take the form of a range between either of the two adjacent values ​​above, such as between approximately 40 femtoliters and approximately 60 femtoliters. Alternatively, the volume can take the form of a range between two of the values ​​above that are not directly adjacent to each other in the list above.

[0305] Furthermore, the volume can be approximately 1 picoliter, approximately 2, approximately 4, approximately 6, approximately 8, approximately 10, approximately 20, approximately 40, approximately 60, approximately 80, approximately 100, approximately 200, approximately 400, approximately 600, approximately 800, or approximately 1,000, approximately 2,000, approximately 5,000, approximately 10,000, approximately 20,000, approximately 50,000, approximately 100,000, approximately 200,000, approximately 500,000, or approximately 1,000,000 picoliters. Also, the volume can take the form of a range between two of the above values ​​that are not directly adjacent to each other within the list above.

[0306] In an exclusionary embodiment, the disclosure may exclude any substrate containing microwells or any array containing microwells, wherein the volume of each microwell may be defined by one of the above values, or by a range of either of the two above values ​​adjacent to each other, or by a range of either of the two above values ​​not adjacent to each other in the list.

[0307] Spherical plugs on picowells (also known as capped beads). This disclosure provides spherical plugs, or alternatively, porous spherical plugs for each well or substantially all wells of a picowell array. The purpose of the plugs is to retain drugs, candidate drugs, cell contents, or metabolites within the wells. The plugs also help to isolate the contents of the picowells from each other. The spherical plugs do not need to be perfectly spherical, as long as the purpose of covering the top (or opening, or mouth) of the picowell is fulfilled. The wells may have a top diameter and a bottom diameter. The diameters of the spherical plugs before capping the wells are approximately 10 micrometers, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 70, approximately 90, approximately 120, or approximately 200 micrometers. The plugs can be added to cover the picowells by simply flowing them over those of the picowell array. Beads can be packed into the top (or mouth or hole) of a picowell and tightly sealed using centrifugation, pressure, stirring, or other methods. In some embodiments, a solvent can be used to swell and / or change the size of the capped beads. In some embodiments, the capped beads are loaded with a solvent that shrinks the beads, and when replaced with assay buffer or a different solvent, the capped beads return to their original size or swell, thereby tightly sealing the picowell. In some embodiments, temperature can be used to swell or shred the capped beads to obtain a better seal at the mouth of the picowell. If necessary, the capped beads can be held in place and prevented from falling further into the picowell by one of the steps in a stepped picowell array.

[0308] Capped beads may be the same type of bead that carries the compounds of this disclosure, or they may be different types of beads. In some embodiments, capped beads may actually be compounds having beads themselves. Capped beads may function as passive caps that prevent or delay the diffusion of molecules from picowells, or the beads may be active beads that can capture reagents from picowells using a functional portion attached to the capped beads. In some embodiments, porous capped beads may passively capture metabolites released from cell line assays performed in picowells. In some embodiments, capped beads may nonspecifically capture cellular material such as lipids, proteins, carbohydrates, and nucleic acids. In some embodiments, capped beads may be functionalized with antibodies to specifically capture proteins released from healthy, diseased, lysed, or fixed cells. In some embodiments, capped beads may be functionalized with DNA or RNA oligonucleotides that specifically capture cellular nucleic acids. In some embodiments, DNA or RNA-functionalized capped beads may be used to capture microRNAs released from cells in capped picowells. In some embodiments, the picowell comprises two beads, compound-containing beads inside the picowell, and a capped bead covering the mouth of the picowell. In some embodiments, the capped bead is also a compound-containing bead. In some embodiments, the capped bead captures material released from the composite bead. In some embodiments, the capped bead captures a sample of the compound released from the composite bead. In some embodiments, the capped bead captures a DNA barcode released from the composite bead. In some embodiments, the capped beads capture different types of analytes released from within the picowell they cap.

[0309] The relative hardness of the cap and the picowells. A preferred device is a microtiter plate, where each microtiter contains thousands of picowells on its bottom surface. The ability of the cap to properly position, or to seal each picowell, may be a function of the hardness of the plastic constituting the holes and inner walls of the picowells relative to the hardness of the cap.

[0310] The hardness of plastics can be defined in terms of "durometer" values. Hardness is defined and tested as the material's resistance to press-fitting. The hardness of a spherical stopper and the hardness of a picowell wall can be defined in terms of their "durometer" values. Hardness can be, for example, approximately 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100. When attributing any of these durometer values ​​to a plastic or other material, the scale used must also be specified. For example, the scale can be the ASTM D2240 Type A scale used for softer materials, or the ASTM D2240 Type D scale used for harder materials (Silicon Design Manual, 6). th (See ed., Albright Technologies, Inc., Leominster, MA).

[0311] Picowell Shape. In some embodiments, the picowell may be cylindrical, where the diameter of the cylinder is approximately the same at the top and bottom of the picowell. In some embodiments, the picowell may have a slight taper, with the top of the picowell being slightly larger than the bottom. In some embodiments, the picowell may be conical, with an angle deviating from the normal somewhere between 1 and 30 degrees. In some embodiments, the picowell is stepped, having a discontinuous step from the top diameter to the bottom diameter (in contrast to a conical picowell where the diameter changes smoothly from top to bottom). In some embodiments, the stepped picowell has a wider cylinder near the opening of the picowell and a narrower cylinder near the bottom of the picowell. In some embodiments, the stepped picowell may have multiple discontinuous steps from top to bottom. In some embodiments of the multi-stepped picowell, the diameter of each step may be greater than the diameter of the step below it. In some embodiments, small beads may be deposited at the bottom of the stepped picowell, and capped beads may be deposited at the top opening of the stepped picowell. In some embodiments, the picowell may contain more than two beads.

[0312] A method for constructing a stepped picowell. Figure 29 discloses a stepped picowell. The embodiment shown has three compartments and two steps. The upper compartment is the widest, and in the case where the picowell is capped, the majority of the upper compartment is occupied by the cap and is configured to receive the cap. The middle compartment is configured to be occupied mainly by a reagent, or by a reagent alone. The reagent may include a buffer, an enzyme substrate, one or more salts, and a preservative or stabilizer such as dithiothreitol, an RNAse inhibitor, glycerol, or DMSO. The bottom compartment is configured to be occupied by a bead, i.e., a bead to which both a DNA library and a releaseable compound are bound. In addition to having a DNA barcode and a releaseable compound, the same bead may also have a "response capture element" that holds the capped bead in place and prevents it from falling further into the picowell by one of the steps in the stepped picowell. In Figure 29, structure 1 is the cap, structure 2 is the bead, and structure 3 is the upper region located just above the first step. Structure 4 is the central region, which can be used to place assay reagents. The central region is just above the second step. Assay reagents in the central region can diffuse to the lowest region. Structure 5 is the lowest region, which can be used to place beads and one or more cells.

[0313] For the space of the lowest compartment occupied by the bead (assuming only one bead exists in the picowell), the diameter of the bead may be approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the diameter of the lowest compartment (assuming the picowell is a circular well). If the picowell is not a circular well, the above values ​​may refer to the widest dimension of the well. In an exclusionary embodiment, this disclosure may exclude systems or beads that do not satisfy any of the above parameters.

[0314] Furthermore, regarding the space occupied by the beads (assuming only one bead exists in the picowell), approximately 50% of the beads are in the lowest compartment, and approximately 50% of the same beads are in the central compartment. These parameters can also be approximately 55% in the lowest and 65% in the central, 60% in the lowest and 40% in the central, 65% in the lowest and 45% in the central, 70% in the lowest and 30% in the central, 75% in the lowest and 25% in the central, 80% in the lowest and 20% in the central, 85% in the lowest and 15% in the central, 90% in the lowest and 10% in the central, 95% in the lowest and 5% in the central, and approximately 100% in the lowest compartment. To perform these calculations, the space occupied by the beads is assumed (hypothetically) to be that the beads are not porous. In an exclusionary embodiment, this disclosure may exclude systems or beads that do not satisfy any of the above parameters.

[0315] Similar to conical and cylindrical picowells, the use of a molding system is one preferred embodiment for fabricating stepped picowells. For this purpose, a mold containing a multilayered columnar array is desired and, when pressed into a thermoplastic or other curable polymer substrate, can form the trace of a stepped picowell. A layered columnar array with multiple steps, each step of a different diameter (getting smaller towards the top), can be formed by a multilayer lithography process. Briefly, to crosslink the first layer of the micropillar array, a first layer of a first photoresist is exposed through a first mask. A second layer of photoresist may be deposited directly on the first layer (previously exposed), and a second pattern of the second photoresist may be crosslinked later using a second photomask. At the end of multilayer patterning, the resist lamination is developed to wash away uncrosslinked areas, leaving the multilayered columnar array. A detailed protocol for fabricating multilayered columnar arrays is described in Francisco Perdigones et al., (January 8) th(2011). Microsystem Technologies for Biomedical Applications, Biomedical Engineering, Trends in Electronics, Anthony N. Laskovski, can be found in IntechOpen. Once the multilayer column array is fabricated, a standard process can be used to imprint a stepped picowell array using a mold.

[0316] Removing the capped beads. In many embodiments, it is advantageous to collect the capped beads to study the reaction, analyte, or cellular response to chemical perturbations within the picowells. In some embodiments, the capped beads can be removed from the mouths of the picowells by inverting the picowell array and using mechanical agitation. In some embodiments, they can be easily removed from the mouths of the picowells by using a solvent to shrink the picowells. In some embodiments, a liquid with a density higher than the capped beads can be added to the top of the picowell array, causing the capped beads to rise by buoyancy and float on top of the high-density medium.

[0317] In some embodiments, the capped beads can be crosslinked with each other to transform them into a capped sheet that can be peeled off the top of the picowell array. In some embodiments, a crosslinked gel can be poured onto the capped picowells, and the crosslinked gel crosslinks the capped beads and themselves so that the capped beads are embedded in a peelable crosslinked sheet.

[0318] The relative positions of the picowells are maintained in the form of exfoliated layers. In embodiments in which the capped beads interlock with a gel layer that can be exfoliated, it should be understood that the relative positions of the capped beads to each other and to the picowells are maintained in the exfoliated layers. This allows for a direct connection between the picowells, the assay in the picowells, the beads within the picowells, and any material captured by the capped beads.

[0319] In some embodiments, alignment markers can be used to orient the relative features of the picowell array to the capped beads of the exfoliated layer.

[0320] Reference markers enable the alignment and repositioning of picowells. Arranging picowells in an irregular array allows for easy identification of shifts and drifts during imaging of the picowell array. In some embodiments, the picowells are arranged in an irregular order to facilitate the detection of optical and mechanical drifts during imaging. In some embodiments, the picowell array includes reference markers to help identify shifts and drifts during imaging. In some embodiments, the reference markers are easily identifiable shapes, patterns, or features scattered among the picowells of the picowell array. In some embodiments, a small number of picowells are arranged in an easily identifiable pattern, allowing for easy alignment in case of optical or mechanical drifts during imaging. In some embodiments, external markers, such as fluorescent beads, can be atomized onto the picowell array to provide a reference pattern.

[0321] Embodiments of a cap-free mat. Embodiments of a cap-free mat can take the form of a “capless film” in at least some forms or examples. Instead of sealing the opening at the top of the picowell, the mat can be sealed, for example, to prevent evaporation of any cell culture medium or enzyme assay medium that may be in the picowell. Preferably, the mat is sized to cover all picowells in a given picowell array. Alternatively, the mat can be sized to cover a given section of picowells in the array. The mat can be fixed to the top of a picowell plate to cover the picowells and also cover the substantially flat top surface of the picowell plate between the picowells. Secure contact can be achieved by (i) maintaining constant pressure, for example, by a rigid rubber platen placed on top of the mat and acting as a weight on top of the mat; (ii) using a mat connected to a weight such as a rigid rubber platen; or (iii) one or more reversible chemical adhesives that can be applied to the entire mat (if the mat is not an absorbent mat). If the mat is an absorbent mat, it includes circular absorbent pads surrounded by a reversible chemical adhesive. Here, the mat is aligned in contact with the picowell array, and the circular absorbent pads cover only the openings of each picowell, preventing any "spillout" from the openings and contact with the plane of the picowell plate.

[0322] Membrane films are available for use as mats for contacting the substantial plane of picowell plates and for use in capless sealing of picowells. Flat sheet films such as Dow Film Tex, GE Osmonics, Microdyn Nadir, Toray, TriSep, Synder, Novamem, Evonik, and Aquaporin flat sheet films are available from Sterlitech Corp. in Kent, Washington. These include films made from polyamide-TFCs, cellulose acetate, polyamide-urea-TFCs, cellulose acetate blends, polypiperazine-amide-TFCs, PES, composite polyamide-TFCs, PES, PAN, PVDF, PSUH, RC, PESH, polyetheretherketones, and polyimides. Pore ​​sizes in terms of molecular weight cutoff include 150 Da, 200 Da, 300 Da, 500 Da, 900 Da, 600 Da, 1,000 Da, 2,000 Da, 3,000 Da, 5,000 Da, 10,000 Da, 50,000 Da, 20,000 Da, 30,000 Da, 70,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 400,000 Da, 500,000 Da, 800,000 Da, 3500 Da, 0.005 micrometers, 0.030 micrometers, 0.05 micrometers, 0.10 micrometers, 0.20 micrometers, and so on. With respect to the systems, compositions, reagents, and methods of this disclosure, these cutoff values ​​can allow for the selective collection of a particular class of compounds by excluding other classes of compounds. For example, some of the above membranes can exclude proteins and other macromolecules while allowing small molecule metabolites to pass through and be absorbed by the absorbent mat. Flat sheet membranes that are impermeable to all molecules, including water, metal ions, salts, metabolites, proteins, and nucleic acids, are also available for use in the systems, compositions, and methods of this disclosure.

[0323] Reversible adhesion is mediated by "molecular Velcro," for example, metalloporphyrin-containing polymers and pyridine-containing polymers (Sievers, Namyslo, Lederle, Huber (2018) eXPRESS Polymer Letters. 12:556-568). Other molecular Velcro adhesives involve copolymers containing L-3,4-dihydroxyphenylalanine, complementary strands of ssDNA (one type of ssDNA covalently attached to the flat upper surface of the picowell plate, and another type of ssDNA covalently attached to the mat), and catechol side chains (see Sievers et al. above). Furthermore, reversible bonding can be mediated by gallium adhesive, and the degree of bonding can be controlled by slight changes in temperature (Metin Sitti (May 18, 2016) Switch and Stick. The chemical element gallium could be used as a new reversible adhesive that allows its adhesive effect to be switched on and off with ease. Max-planck-Gesellschaft). Yet another reversible adhesive is available from DSM-Niaga Technology in Zwol, Netherlands.

[0324] Absorbent materials (non-specific absorbents; specific absorbents). Absorbent materials that can be incorporated into a mat to provide absorption properties include "molecular sieve" beads such as Sepharose®, Sephadex®, and Agarose®, and ion-exchange beads made from DEAE cellulose, carboxymethylcellulose, phosphocellulose, or any combination of the above, all of which can be combined into a single absorbent mat. Absorbent ligands include those used in high-pressure liquid chromatography (HPLC) (see BioRad catalog, Hercules, CA). Specific absorbents include response capture elements such as poly(dT) that can capture mRNA by hybridizing with a polyA tail. Response capture elements also include exon-targeted RNA probes, antibodies, and aptamers. Any one or any combination of these can be covalently attached to a mat to create an absorbent mat, which, when in contact with the top surface of a picowell, can capture aqueous assay medium or aqueous cell culture medium that may be inside the picowell.

[0325] (IX) Deposit beads into picowells. Picowell plates can take the form of 96-well plates, each of which contains thousands of picowells. Alternatively, picowell plates can take the form of 24-well plates, each of which contains thousands of picowells. For 96-well plates, each well can be filled with 0.1–0.2 mL of a bead suspension in water or aqueous solution. For 24-well plates, each well can be filled with 0.5 mL of a bead suspension in water or aqueous solution. The suspension can be added using a standard pipette with a disposable tip. The number of beads in the suspension can result in approximately one-third of picowells containing only one bead, approximately one-third containing two beads, and approximately one-third containing no beads or two or more beads. Furthermore, the number of beads in the suspension can depend on the circumstances, with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the wells containing one or more beads, or at least 98% of those wells containing only one bead.

[0326] After the beads have settled, any excess liquid can be removed by touching the pipette tip to the wall of each well in a 96-well plate or to the wall of each well in a 24-well plate to remove the excess liquid.

[0327] Regarding assay reagents, when picowells are used to perform a reaction, for example, in the case of DNA sequencing, biochemical assays, or cultured cell assays, the assay reagent can be added to the picowells that already contain settled beads. As mentioned above for the initial addition of the bead suspension, the addition of assay reagent is done using a pipette. After the assay reagent has been equilibrated with the solution already present in each picowell, any excess solution in each of the 96 wells of a 96-well plate, or in each of the 24 wells of a 24-well plate, can be removed with a pipette tip that touches the wall of each of the 96 wells of a 96-well plate, or the wall of each of the 24 wells of a 24-well plate.

[0328] An embodiment of a flow cell for a picowell array. The picowell array may be part of a flow cell, with a fluid chamber having an inlet and an outlet mounted on top of the picowell array. In such an embodiment, the beads, cells, and other assay materials of the present disclosure may flow in through the inlet and out through the outlet. Gravity or centrifugal force is used to retain the beads in the picowells as they flow through the flow cell.

[0329] (X) Sequencing of bead-bound nucleic acids in picowells Bead-bound nucleic acids can be sequenced while still attached to the beads. Alternatively, or additionally, bead-bound nucleic acids can be sequenced after the DNA barcode has been cleaved from the beads.

[0330] The DNA barcode is cleaved from the beads before sequencing. In some embodiments, the disclosure may encompass a method for bead-bound DNA barcodes being cleaved from the beads, thereby releasing the DNA barcode in a soluble form before amplification, or before sequencing, or before any type of sequence identification technique, such as hybridization with a nucleic acid probe.

[0331] Exclusionary Embodiments. In embodiments, the Disclosure may exclude any method, associated reagents, systems, compositions, or beads in which the bead-bound DNA barcode is cleaved before amplification, or before sequencing, or before any type of sequence identification technique, such as hybridization with a nucleic acid probe. The Disclosure may also exclude any method in which a polynucleotide containing the DNA barcode is cleaved, or a nucleic acid containing only a portion of the DNA barcode is cleaved before amplification, before sequencing, or before any type of sequence identification technique, such as hybridization with a nucleic acid probe.

[0332] Polymerase chain reaction (PCR); quantitative PCR (qPCR). Both PCR and qPCR methods rely on a three-step process: (1) denaturing the DNA template at high temperature, annealing the primers at a reduced temperature, and finally extending the primers via DNA synthesis catalyzed by DNA polymerase (Gadkar and Filion (2014) Curr. Issues Mol. Biol. 16:1-6). qPCR is also called "real-time PCR" (Kralik and Ricchi (2017) Frontiers Microbiology. 8 (p. 9)).

[0333] Recent modifications or improvements to PCR and qPCR methods include the use of helicase-dependent (HDA) amplification, the use of internal amplification standards, the use of locked nucleic acids (LNAs), and the use of inhibitor-binding additives (Gadkar and Filion (2014) Curr. Issues Mol. Biol. 16:1-6). Locked nucleic acids offer the advantage of recognizing and binding to their targets with extreme precision.

[0334] qPCR allows for the simultaneous amplification and quantification of target DNA molecules. The qPCR method compares the number of amplification cycles required for the response curve to reach a specific fluorescence threshold (Pabinger, Rodiger, Kriegner (2014) Biomolecular Detection Quantification. 1:23-33). Refsland et al. provide a concise description of clearly typical conditions for performing qPCR (Refsland, Stenglein, Harris (2010) Nucleic Acids Res. 38:4274-4284).

[0335] Guidance is available on the design and validation of PCR primers, as well as on variables such as annealing temperature (Ta), melting temperature (Tm), extension step temperature, and buffer type (Bustin and Huggett (2017) Biomolecular Detection Quantification. 14:19-28).

[0336] Rolling Circle Amplification (RCA). DNA can be amplified while still attached to beads. The amplified form of DNA is simpler than the sequence of unamplified DNA. In the rolling circle amplification method, a DNA tag (DNA barcode) is created as a single strand. Once single-stranded, sprint oligos are added to crosslink the ends of the tag DNA, followed by sprint oligo extension and ligation. Using DNA polymerase (minus 5'→3' exonuclease activity), the DNA ensures a ligable junction after catalyzing the extension of the sprint oligos. The circularized DNA can then be subjected to rolling circle amplification by adding a strand-displacing DNA polymerase such as phi29 DNA polymerase. The ability to perform rolling circle amplification (RCA) with DNA barcode tags allows for thermal amplification to a sufficient amount to easily sequence any surviving DNA molecule, thus eliminating the need for synthetic chemicals that can damage DNA. DNA can be made into single strands by exonuclease digestion, nicking, and melting at high temperatures, or by treatment with sodium hydroxide.

[0337] Details of Rolling Circle Amplification (RCA) are revealed in the following steps that can be used to implement RCA.

[0338] Step 1: Begin with bead-bound ssDNA. If the bead-bound DNA is initially double-stranded from (dsDNA), the strand not used for RCA can be modified so that thymine (T) residues are replaced with uracil (U) residues at or near the bead-bound end. When dsDNA is prepared in this way, the uracil residues can be cleaved using uracil N-glycosidase, thereby leaving an unstable sugar phosphate (as part of the DNA backbone), which can then be cleaved by nuclease treatment (Ostrander et al (1992) Proc. Natl. Acad. Sci. 89:3419-3423).

[0339] Step 2: Add the "sprint oligo" to the bead-bound ssDNA. The sprint oligo is designed to hybridize approximately 10-20 base pairs at the end (5' end) of the ssDNA covalently bound to the bead, and also hybridize approximately 10-20 base pairs at the free end (3' end) of the bead-bound ssDNA. The sprint oligo does not need to bring the bead-bound end of the ssDNA close to the free end of the bead-bound ssDNA. All that is needed is to splinter the far ends of the bead-bound ssDNA sequence to form a large loop.

[0340] Step 3: Add sulfolobus DNA polymerase IV, which uses the large loop of ssDNA as a template to create a complementary large loop that is covalently attached to the sprint oligo at one end.

[0341] Step 4: Using DNA ligase, the complementary large loop is covalently closed, generating a circular ssDNA. It is this closed ssDNA loop that performs the "rolling" between the RCAs.

[0342] Step 5: Add a DNA polymerase with strand displacement activity and add dNTPs. The added DNA polymerase covalently attaches the dNTPs to the bead-bound ssDNA, and the distal end of the bead-bound ssDNA is extended to create a complementary copy of the one on the "rolling circle," then further extended to create yet another complementary copy of the one on the "rolling circle," and further extended to create yet another complementary copy of the one on the "rolling circle." During this process of potentially infinite amplification, the strand displacement activity of the DNA polymerase allows for the continuous activity of the DNA polymerase.

[0343] Optionally, the methods of this disclosure include real-time monitoring of rolling circle amplification (RCA) via a fluorescent molecular beacon (Nilsson, Gullberg, Raap (2002) Nucleic Acids Res. 30:e66 (p. 7)). Reagents for RCA are available from Sigma-Aldrich (St. Louis, MO), Sygnis TruePrime Technology (TruePrime® RCA kit), Heidelberg, Germany, and GE Healthcare (TempliPhi 500® amplification kit). Fluorophores and quenchers are available from ThermoFisher Scientific (Carlsbad, CA), Molecular Probes (Eugene, OR), Cayman Chemical (Ann Arbor, MI), and Sigma-Aldrich (St. Louis, MO).

[0344] Step 6. The ssDNA amplified by RCA is used as a template for PCR amplification, primers are added, and thermostable DNA polymerase is added. The PCR product is then sequenced by next-generation sequencing.

[0345] In one aspect of this disclosure, the RCA-amplified ssDNA is cleaved from the beads before the PCR amplification that produces the PCR product. In another aspect of this disclosure, the PCR amplification that produces the PCR product can be performed without cleaving the RCA-amplified ssDNA from the beads.

[0346] As Baner et al. described, "the RCA reaction can generate a chain representing many tandem copies of complement to a cyclic molecule" (Baner, Nilsson, Landegren (1998) Nucleic Acids Res. 26:5073-5078). Bacillus subtilis phi29 DNA polymerase is a suitable enzyme due to its strand displacement activity and high processing capacity. RCA was similarly characterized by Li et al., "In RCA, the cyclic template is amplified isothermally by the DNA polymerase phi29, which has strand displacement properties. The long single-stranded DNA product contains thousands of sequence repeats" (Li and Zhong (2007) Anal. Chem. 79:9030-9038).

[0347] The sequencing of the DNA barcodes in this disclosure may be performed using the method of Vander Horn's U.S. Patent No. 8,632,975, which is incorporated herein by reference in its entirety. The DNA barcodes in this disclosure may also be sequenced by methods using synthetic sequencing, such as Sanger sequencing, or by methods using "next-generation sequencing."

[0348] The Illumina method for DNA sequencing. The Illumina method for DNA sequencing is as follows: DNA can be fragmented into sizes ranging from 100 to 400 base pairs (bp) by sonication (Hughes, Magrini, Demeter (2014) PLoS Genet. 10:e1004462). In the Illumina method, a DNA library is prepared and DNA fragments from cells or from cells are modified by DNA adapters (attached to the ends of the fragments). The reaction product takes the form of a sandwich, with the DNA to be sequenced in the center of the sandwich. The reaction product takes the form of (first adapter)-(DNA to be sequenced)-(second adapter). The adapter-DNA-adapter complex is further associated with another adapter, which is covalently attached to a solid surface. The solid surface may be a flat plate. The solid surface has a lawn of many adapters protruding from the plane. The adapters have DNA sequences complementary to one of the adapters in the sandwich. In fact, the turf contains two types of adapters, one of which hybridizes to one of the adapters in the complex, non-covalently anchoring the complex to the plate. These can be referred to as the "first turf-binding adapter" and the "second turf-binding adapter." The first task of DNA polymerase is to use the anchored (but non-covalently bound) DNA as a template to create the daughter strand, and as DNA polymerization occurs, the daughter strand becomes covalently attached to the "first turf-binding adapter." This covalent linkage is generated by the catalytic action of DNA polymerase. After the daughter strand is fully synthesized, its distal end (the end that protrudes into the medium) contains a DNA sequence complementary to the second adapter in the sandwich described above. This complementary DNA sequence allows the distal end of the newly synthesized daughter DNA to bend and hybridize to the "second turf-binding adapter." The above describes how both adapters in the sandwich are used, as well as how both the "first grass bonding adapter" and the "second grass bonding adapter" are used.

[0349] Subsequently, the reaction cycle is repeated many times, resulting in a cluster of amplified versions of the original dsDNA. In fact, the cluster takes the form of covalently attached (anchored) ssDNA molecules, each of which corresponds to only one strand of the original dsDNA (dsDNA isolated from a living cell or tissue). This cluster of anchored ssDNA molecules is called a "polony." Polony generation is achieved by a technique called "bridge amplification." Finally, after bridge amplification and pollony creation, the reverse strand covalently attached to the solid surface is cleaved from its anchorage, washed away, and discarded, leaving only the forward strand.

[0350] Information regarding the Illumina® trademark is available from Goodwin, McPherson, McCombie (2016) Nature Rev. Genetics. 17:333-351, Gierahn, Wadsworth, Hughes (2017) Nature Methods. 14:395-398, Shendure and Hanlee (2008) Nature Biotechnology. 26:1135-1145, Reuter, Spacek, Snyder (2015) Molecular Cell. 58:586-597, and Illumina Sequencing by Synthesis (a 5-minute video on YouTube).

[0351] Sequencing by oligonucleotide ligation and detection (SOLiD sequencing). SOLiD determines the sequence of DNA fragments by measuring the fluorescence intensity from dye-labeled molecules. A library of DNA fragments is prepared from the sample to be sequenced and used to prepare a population of clone beads (only one type of fragment on the surface of each magnetic bead). The fragments attached to the beads are given an attached universal P1 adapter sequence such that the start sequences of all fragments are both known and identical. PCR is performed, and the PCR products attached to the beads are covalently bound to the slide.

[0352] Next, the primers hybridize to the P1 adapter sequence in the library template. A set of four fluorescently labeled dinucleotide probes compete for ligation to the sequencing primers. The specificity of the dinucleotide probes is achieved by examining all first and second bases in each ligation reaction. Multiple cycles of ligation, detection, and cleavage are performed with a number of cycles that determines the length of the final readout. Following a series of ligation cycles, the extension product is removed, and the template is reset with a primer complementary to the n-1 position of the second ligation cycle (see Wu et al (2010) Nature Methods. 7:336-337).

[0353] pH-based DNA sequencing. pH-based DNA sequencing is a system and method in which base incorporation is determined by measuring hydrogen ions produced as a byproduct of polymerase-catalyzed extension reactions. DNA templates, each having operably bound primers and polymerases, are loaded into a reaction chamber or microwell, followed by repeated cycles of adding deoxynucleoside triphosphates (dNTPs) and washing. The DNA templates are then attached to a solid support as a clonal population. Each such incorporation releases hydrogen ions, and the hydrogen-releasing population of templates assembles, causing a detectable change in the local pH of the reaction chamber (see Pourmand (2006) Proc. Nat'l. Acad. Sci. 103:6466-6470). This disclosure may exclude pH-based DNA sequencing.

[0354] For linked DNA barcodes, the entire linked DNA barcode can be sequenced in a single run (only one sequencing primer is required for sequencing the entire linked DNA barcode). Alternatively, some or all of the DNA barcode modules that make up a linked DNA barcode can be sequenced separately (each separately sequenced DNA barcode module will obtain its own sequencing primer). For orthogonal DNA barcodes, each DNA barcode module that makes up an orthogonal DNA barcode is attached to its own site on the bead, so each DNA barcode module that makes up an orthogonal DNA barcode requires its own dedicated sequencing primer.

[0355] Exclusionary Embodiments. In embodiments, the Disclosure may exclude any systems, devices, combinations of devices, and methods involving a microfluidic, an aqueous droplet present in an oil medium, and an aqueous droplet produced by a first channel containing an aqueous reagent being connected to a second channel containing oil, and generating an aqueous droplet moving through a third channel beginning from the connecting region through the oil medium. Microfluidics devices and reagents are described (see, for example, Brouzes, Medkova, Savenelli (2009) Proc. Natl. Acad. Sci. 106:14195-14200, Guo, Rotem, Hayman (2012) Lab Chip. 12:2146-2155, Debs, Utharala, Balyasnikova (2012) Proc. Natl. Acad. Sci. 109:11570-11575, and Sciambi and Abate (2015) Lab Chip. 15:47-51).

[0356] In other exclusionary embodiments, what can be excluded are any reagents, compositions, nucleic acids, or beads that constitute the "DNA headpiece," or reagents, compositions, nucleic acids, or beads that are covalently attached to the "DNA headpiece." MacConnell, Price, Paegel (2017) ACS Combinatorial Science. 19:181-192 provides an example of a DNA headpiece in which beads are functionalized with an azide DNA headpiece.

[0357] Additional exclusionary embodiments relating to sequencing methods and sequencing reagents. In embodiments, the disclosure may exclude reagents, systems, or methods that do not involve the use of a “reversible terminator” in DNA sequencing. Also excluded are any reagents, systems, or methods that do not contain a methoxy protecting group. Furthermore, any reagents, systems, or methods that involve DNA sequencing but in which the DNA being sequenced is not covalently attached to beads when information regarding the order of polynucleotides is detected and collected may be excluded. Furthermore, any reagents, systems, or methods that amplify the DNA template before performing the sequencing reaction, for example by PCR or rolling circle techniques, may be excluded. In embodiments, any method of barcoding may be excluded, for example, nucleic acid barcoding of linked (all information regarding the synthesis of members of a chemical library present on a single nucleic acid). In another embodiment, any method of barcoding may be excluded, for example, nucleic acid barcoding of orthogonal (information regarding the synthesis of a given monomer of a compound library dispersed at multiple attachment positions on beads). In an exclusionary embodiment relating to DNA ligases, this disclosure may exclude any reagents, systems, or methods that use DNA ligases to connect modules of nucleic acid barcodes.

[0358] Fluorophores, Quenchers, and FRET System Assays. This disclosure provides fluorophores and quenchers for screening members of a chemical library or for characterizing isolated members of a chemical library. FRET stands for Förster Resonance Energy Transfer.

[0359] The assay can be performed on a bead-bound chemical library. Alternatively, the assay can be performed on a free chemical library member immediately after cleavage from the beads, i.e., in the same microwell as the beads or in the vicinity of the same hydrogel matrix as the beads. Furthermore, the assay can be performed on a soluble chemical library member that is neither attached to any beads nor has been cleaved from the beads and subsequently purified.

[0360] Suitable fluorophores for reagents in this disclosure include Alexa 350, Alexa 568, Alexa 594, Alexa 633, A647, Alexa 680, fluorescein, Pacific Blue, coumarin, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 660, ATTO655, ATTO647n, Setau-665 (SETA Biochemicals, Urbana, IL), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, tetramethylrhodamine (TMR), Texas Red, tetrachlorofluorescein (TET), hexachlorofluorescein (HEX), and jaw dye (4'-5'-dichloro-2',7'-dimethoxy-6-carboxyfluorescein), SYBR Green I (absorbs at 497 nm, 520 nm) This includes 6-carboxyfluorescein (6-FAM) (absorbs at 492 nm, emits at 518 nm), 5-carboxyfluorescein (5-FAM) (absorbs at 492 nm, emits at 518 nm), FITC, and rhodamine. Quenchers include TAMRA quencher, black hole quencher-1 (BHQ1), black hole quencher-2 (BHQ2), and DABCYL quencher. Note that TAMRA can be a fluorophore or a quencher, as disclosed elsewhere in this patent document.

[0361] Guidance is available for reagents used in FRET assays, which include fluorophores and quenchers (see Johansson (2006) Choosing reporter-quencher pairs for efficient quenching. Methods Mol. Biol. 335:17-29). An example of a FRET assay involves measuring the activity of signal peptidase (SpsB) using the substrate "SceD peptide". The FRET pair attached to the peptide was 4-(4-dimethylaminophenylazo)5-((2-aminoethyl)amino)-1-nephthalenesulfonic acid (see Rao et al (2009) FEBSJ. 276:3222-3234). Another example comes from an assay of HIV-1 protease using the peptide substrate KVSLNFPIL. The donor / acceptor FRET pair was EDANS (donor) and DABCYL (acceptor). EDANS fluorescence can be quenched by DABCYL via resonance energy transfer to non-fluorescent DABCYL (see Meng et al (2015) J. Biomolecular Screening. 20:606-615). Yet another example comes from the assay of botulinum toxin. The activity of SNAP-25 can be measured using the substrate of BoNT-A. In the FRET assay, the substrate has fluorescein isothiocyanate (FITC) linked at the N-terminus, and the quencher linked at the C-terminus is 4-(4-dimethylaminophenyl)diazenylbenzoic acid (DABSYL). The peptide substrate corresponds to amino acids 190-201 of SNAP-25 (see Rasooly and Do (2008) Appl. Environ. Microbiol. 74:4309-4313).

[0362] This disclosure provides reagents, compositions, and methods for screening a library of compounds to discover and identify enzyme inhibitors and enzyme activators, and to discover compounds that can enhance the rate of in vivo degradation of a given protein. These reagents, compositions, and methods can be used with FRET assays, or alternatively, with non-FRET assays.

[0363] Molecular beacons are described (see Baruch, Jefferey, Bogyo (2004) Trends Cell Biology. 14:29-35). A molecular beacon is a reagent in which a fluorophore is bound to a quencher via a linker. The linker may be cleavable by a nuclease, and thus nuclease activity can be measured. This disclosure provides a method for screening a chemical library to identify nuclease inhibitors, or alternatively, to identify nuclease activators. Feng et al. described the use of molecular beacons and FRET assays to measure the activity of various nucleases (Feng, Duan, Liu (2009) Angew Chem. Int. Ed. Engl. 48:5316-5321). Feng et al. demonstrated the use of FRET assays to measure the activity of various restriction enzymes.

[0364] (XI) Releases bead-bound compounds Cleavable linkers. Provided are non-cleavable linkers. Also provided are cleavable linkers (see Holmes and Jones ((1995) J. Org. Chem. 60:2318-2319, Whitehouse et al (1997) Tetrahedron Lett. 38:7851-7852, and Yoo and Greenberg ((1995) J. Org. Chem. 60:3358-3364, cited by Gordon et al (1999) J. Chem. Technology Biotechnology. 74:835-851). Cleavable linkers also include acyl sulfonamide linkers present in alkaline hydrolysis, as well as activated N-alkyl derivatives that are cleaved under mild conditions, and traceless linkers based on aryl-silicon bonds, and traceless linkers based on silyl ether linkages (Gordon et al (1999) J. Chemical Technology (Described on pages 839 and 842 of Biotechnology. 74:835-851). Furthermore, the linker provided is tartaric acid-based, which produces a C-terminal aldehyde upon cleavage, and the cleavage is by periodic acid oxidation (see Paulick et al (2006) J. Comb. Chem. 8:417-426).

[0365] Figure 3 discloses various cleavable linkers suitable for the compositions and methods of this disclosure. Figure 3 is reproduced from Table 1 of Yinliang Yang (2014) Design of Cleavable Linkers and Applications in Chemical Proteomics. Technische Universitat Munchen Lehrstuhl fur Chemie der Biopolymere. From Figure 3, preferred cleavable linkers for this disclosure are linkers a, c, d, p, q, r, and t. Linker p was used in the experimental results disclosed herein. These cleavage conditions are DTT (linker a), Na2SO4 (linker c), Na2SO4 (linker d), UV light (linker p), UV light (linker q), UV light (linker r), and TEV protease (linker t). These particular cleavage conditions are mild and do not damage the beads, the bead-binding compounds, or the chemical library members (units) of the bead-binding compounds.

[0366] Chemically cleavable linkers compatible with click chemistry. Qian et al. (2013) describe many cleavable linkers compatible with click chemistry (Qian, Martell, Pace (2013) ChemBioChem. 14:1410-1414). These include linkers with an azo bond, which is cleavable with dithionite. The structure of this linker is as follows: R1-benzene1-N=N-benzene2-R2. The first benzene ring has a para hydroxyl group relative to R1, and the second benzene ring has a carbonyl group linked to R2, which is para relative to the azo moiety.

[0367] Photocleavable Linkers This disclosure includes photocleavable linkers having an o-nitrobenzyl group. This group can be cleaved by irradiation at 330–370 nm (see Saran and Burke (2007) Bioconjugate Chem. 18:275–279, Mikkelsen, Grier, Mortensen (2018) ACS Combinatorial Science. DOI:10.1021). A linker with a shorter photocleavage time than the o-nitrobenzyl linker is the 2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC) linker. A variation of the o-nitrobenzyl linker is the o-nitrobenzylamino linker. When attached to a peptide chain and subsequently cleaved, this linker releases an amide. Linkers having an o-nitroveratril group are available, which have shorter photocleavage times and higher release yields than the unsubstituted o-nitrobenzyl linker. Phenacyl linkers, benzoin linkers, and pivaloyl linkers are also available (see Mikkelsen et al (2018) ACS Combinatorial Science. DOI:10.1021).

[0368] Linkers with photocleavable ether bonds are available. These photocleavable linkers can be used when the linker is attached to the beads and the cleavable group is an "R group," and after cleavage, the released group takes the form of ROH (see Glathar and Giese (2000) Organic Letters. 2:2315-2317). Linkers with photocleavable ester bonds are also available (see Rich et al (1975) 97:1575, Renil and Pillai (1994) Tetrahedron Lett. 35:3809-3812, Holmes (1997) J. Org. Chem. 62:2370-2380, cited by Glathar and Giese above). The ether bond of the linker can be cleaved by acid, base, oxidation, reduction, and fluoride-sensitive silyl-oxygen bonds, as well as photolysis (Glatthar and Giese, previously mentioned).

[0369] Another photocleavable linker used to link the peptide (R1) and nucleic acid (R2) is as follows: R1 is directly attached to the methylene moiety of the benzyl group. The para linkage to the methylene group is a ring-attached nitro group. The meta linkage to the methylene moiety is a ring-attached ethyl group. One carbon of the ethyl group has a phosphoric acid molecule. The R2 group is attached to the oxygen atom of this phosphoric acid molecule (Olejnik et al (1999) Nucleic Acids Res. 27:4626-4631).

[0370] Akerblom et al. disclose alpha-methyl 2-nitrobenzyl type photounstable linkers containing amino, hydroxyl, bromo, and methylamino groups, as well as 4-nitrophenoxycarbonyl activated hydroxyl and amino groups (see Akerblom and Nyren (1997) Molecular Diversity. 3:137-148). Cathepsin B can cleave linkers having the target sequence "valine-citrulline" (Dal Corso, Cazzamalli, Neri (2017) Bioconjugate Chemistry. 28:1826-1833).

[0371] Enzyme-cleavable linkers. Linkers that can be cleaved by enzymes such as proteases are available (see Leriche, Chisholm, Wagner (2012) Bioorganic Medicinal Chem. 20:571-582). The hydroxymethylphenoxy linker can be cleaved with chymotrypsin (see Maltman, Bejugam, Flitsch (2005) Organic Biomolecular Chem. 3:2505-2507). Linkers that can be cleaved with tobacco etch virus protease are available (see Weerapana, Speers, Cravatt (2007) Nature Protocols. 2:1414-1425, Dieterich, Link, Graumann (2006) Proc. Nat'l. Acad. Sci. 103:9482-9487). The linker sequences of LVPRG and LVPRGS can be cleaved by thrombin (Jenny, Mann, Lundblad (2003) Protein Expression Purification. 31:1-11). Plasmin-cleavable linkers are available (Devy, Blacher, Noel (2004) FASEBJ. 18:565-567).

[0372] Bead-bound release monitor. This disclosure provides a novel and unique release monitor capable of evaluating the release of bead-bound compounds. The release monitor takes the form of a bead-bound complex of a fluorophore and a quencher, where the fluorophore is connected to the bead via a cleavable linker. Preferably, the cleavable linker is a photocleavable linker. Preferably, the bead-bound release monitor is placed in a dedicated picowell, which does not contain other types of beads. When the photocleavable linker is cleaved, the fluorophore is released from the bead and diffuses into the medium in the picowell, achieving a certain distance from the bead-bound quencher, resulting in an enhancement of fluorescence proportional to the amount released. The enhancement of fluorescence makes it possible to calculate the concentration of free fluorophore in the picowell, and more importantly, to calculate the amount of compound released from other beads in other wells.

[0373] In summary, the bead-binding release monitor is located in its own dedicated well, while the other wells contain the bead-binding compound, which is the drug candidate.

[0374] Figure 8 discloses a simplified version of a preferred non-limiting example of a bead-bound release monitor. The release monitor takes the form of a quencher held near the fluorophore, resulting in the quenching of the fluorophore. In embodiments, the quenching is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, at least 99.95%, and so on. In the picowell, one bead is dedicated to the release monitor, while another or more beads are used for compound attachment and DNA library attachment. Exposure of all beads in the picowell to UV light simultaneously cleaves the fluorophore and compound. QSY7 is a preferred quencher. The structure and CAS number of QSY7 are as follows (see below):

[0375] CAS name / number: Xanthilium, 9-[2-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-1-piperidinyl]sulfonyl]phenyl]-3,6-bis(methylphenylamino)-,chloride 304014-12-8

[0376] The increase in fluorescence resulting from the separation of the fluorophore from the quencher can be used to estimate the concentration of the simultaneously released compound in the picowell. The increase in fluorescence resulting from the separation of the fluorophore from the quencher can also be used to estimate the number of molecules present in the picowell in free form (molecules that take the form of compounds that were previously bead-bound compounds). In a more preferred embodiment, the release monitor includes the quencher and the fluorophore, and cleavage results in the release of the fluorophore (without the release of the quencher). This embodiment provides lower background noise than the less preferred embodiment described below. In the less preferred embodiment, cleavage releases the quencher, and readout takes the form of increased fluorescence from the bead-bound fluorophore.

[0377] The release monitor provides the user with a measurement of the concentration of a soluble compound, followed by UV-induced release of the compound from the beads. In a preferred embodiment, one form of bead is dedicated to being the release monitor. "Dedicated" means that this bead does not contain a bead-bound compound or a bead-bound DNA library.

[0378] As a general principle, the mere fact that a compound is released from a bead by the cleavage of a photosensitive linker should not be assumed to mean that the compound has become soluble. First, note that just because a compound is considered "hydrophobic" or "water-insoluble" does not mean that the molecule cannot move freely in the solvent. For example, even cholesterol has measurable solubility in water (see Saad and Higuchi (1965) Water Solubility of Cholesterol. J. Pharmaceutical Sciences. 54:1205-1206). Furthermore, the biochemical efficacy of bead-bound water-insoluble compounds can be increased by additives such as surfactants, detergents, DMSO, or carriers such as human serum albumin. Therefore, a release monitor can be used to assess the overall concentration of compounds whose water solubility is limited or who are not water-soluble under conditions where one of the above agents is present in the picowell, or alternatively, water-insoluble compounds are released near the plasma membrane of living cells cultured inside the picowell.

[0379] Figure 9 discloses a simplified version of a preferred embodiment of the bead-coupled release monitor, and Figure 10 discloses the complete and detailed structure of this preferred embodiment of the bead-coupled release monitor.

[0380] Figure 30 provides data illustrating the use of a bead release monitor, with the beads in a picowell. The bead-bound fluorophore, bound using a photocleavable linker, is TAMRA (excitation wavelength 530 nm, emission wavelength 570 nm). The figure shows the time course of fluorophore release from the beads. This shows the operation of the bead-bound release monitor and acquisition of fluorescence data at t=0 sec, t=1 sec, t=11 sec, and t=71 sec. Figure 30 also includes insets showing enlargements of two of the four smaller figures. Figure 30 was obtained from incubation of the aspartyl protease cathepsin-D with "peptide Q-fluorosubstrate" and beads. The reagents were placed in the wells at 4°C. Using 365 nm ultraviolet light, the fluorophore was cleaved from the beads, thereby releasing the fluorophore and separating it from the quencher. The purpose of this assay was to evaluate the time course of release performed in a separate well, which contained a different type of bead. Different types of beads possessed the same photocleavable linker, but this linker was attached to pepstatin-A. The release of pepstatin-A can be inhibited by binding to aspartyl protease present in the same assay medium. This setup, using bead-bound pepstatin-A and aspartyl protease, serves as a positive control.

[0381] UV light was applied through a 20x objective lens. Images were acquired with Gain=5 and exposure time was 400ms. TAMRA was excited at 530nm. TAMRA emits light at 570nm.

[0382] Figure 35 discloses further details regarding the enzyme assay, in which bead-bound pepstatin-A is released, and the released pepstatin-A results in enzyme inhibition. 10 μm TentaGel beads displaying photocleavable pepstatin-A (positive control) and covalently bound Cy5 label were mixed with 10 μm TentaGel beads displaying photocleavable Fmoc-valine (negative control) in PBST buffer. This bead population was introduced into picowells, and the buffer was subsequently used to detect cathepsin D protease and peptide Q-fluorescent substrate (λ). ex =480nm, λ em The slides were replaced with a protease inhibition assay including UV (365 nm, 77 J / cm²). The wells were encapsulated with air, and the entire slide was replaced with UV (365 nm, 77 J / cm²). 2 The mixture was exposed to ) to cleave the photounstable linker and release the compound to reach approximately 13 μM. The flow cell was incubated (30 minutes, 37°C). Wells containing positive control beads should inhibit cathepsin-D peptide proteolysis and exhibit a low fluorescence signal. Wells containing negative control beads should not show any cathepsin-D inhibition and should exhibit fluorescence intensity similar to that of empty wells.

[0383] The terms quencher and fluorophore can vary depending on other chemicals occurring in the immediate vicinity of a given chemical. While TAMRA is a fluorophore in the context of laboratory data from bead-coupled release monitors, under other circumstances, TAMRA can be a quencher. TAMRA functions as a quencher in TaqMan® probes, including FAM and TAMRA.

[0384] Further description of experimental setup and laboratory data. This disclosure provides data on controlled concentrations of 5(6)-carboxytetramethylrhodamine (TAMRA) in phosphate buffer (10 mM phosphate, 154 mM sodium, pH 8.0) in air-separated, filled picowells. Well regions quantified by captured fluorescence images (10 ms, 2 ms exposures) and average pixel intensity (n≧100) generate a calibration curve of concentration versus fluorescence intensity. The above data takes the form of a standard curve showing fluorescence at various predetermined concentrations of free TAMRA (2, 10, 30, 60, 100 mM TAMRA). This standard curve was adjusted under two different conditions, i.e., photographic images were taken with 2 ms exposure or 10 ms exposure. The experiments used to adjust the standard curve were performed in picowells, but no beads were used in these experiments (a known amount of TAMRA). Since the data simply takes the form of a standard curve, which may also be referred to as a calibration curve, photographic images are not shown in this patent document.

[0385] The experimental setup includes the following: In scheme X), a TentaGel-Lys(PCL1-Tamra)-QSY7 bead structure. QSY7 (gray) quenches the Tamra fluorophore (orange) but is covalently attached to the bead via a photocleavable linker (purple). Irradiation from UV (365 nm) provides quantitative release of the compound at its original location.

[0386] Figure 31 discloses emission data after the catalytic action of aspartyl protease on a quencher-fluorophore substrate. Higher fluorescence indicates that the enzyme is more catalytically active. Lower fluorescence indicates that the enzyme is less catalytically active, i.e., the enzyme is more inhibited by the release inhibitor, the inhibitor is released from the beads, and the release is obtained by cleavage of a photocleavable linker. Images were captured after UV emission and incubation of the cathepsin-D assay (λ). ex =480nm, λ em (λ = 525 nm). Wells containing positive control beads can be spectrally identified by the Cy5 fluorophore (λ).ex =645nm, λ em (=665nm, orange false color). Sections were analyzed by line plotting across the open well volumes. Wells containing negative control beads did not induce cathepsin-D inhibition. Assay volumes in wells containing positive control beads were dark, indicating strong inhibition. Assay volumes in empty wells were comparable to those in wells containing negative control beads.

[0387] Figure 32 shows the procedure. Furthermore, for scheme X), picowell substrate (46 pL per well) is sealed in a flow cell, the wells are moistened under vacuum, a suspension of TentaGel-Lys(PCL1-TAMRA)-QSY7 beads is introduced, air is drawn across the flow cell, and each well is compartmentalized (top). Before taking fluorescence microscope images to quantify the released compound (TAMRA) concentration (bottom), the flow cell is illuminated with a controlled beam of UV LED (λ 平均値 Irradiated with 365 nm and equilibrated (20 minutes) (Figure 32). In detail, Figure 32 shows a cross-section of the picowells and illustrates the steps of wetting the picowells in the flow cell, introducing beads in suspension to cover the picowells, resulting in one bead per picowell, drawing air into the entire flow cell to reduce excess dispersion and allow the meniscus to fall below the flat top surface of the picowell plate, controlled UV exposure (365 nm), resulting in the emission of some TAMRA, and detecting the fluorescence signal with a fluorescence microscope to induce emission from TAMRA (excited at 531 / 40 nm) (emitted at 594 / 40 nm). The notation "slash 40" refers to the bandwidth, meaning that the cutoff filter limited the light to the range of 531 nm plus 20 nm and minus 20 nm, and 594 nm plus 20 nm and minus 20 nm (this slash notation can also be used for the excitation and emission wavelengths).

[0388] The inventors obtained a photograph showing the following data (see Figure 33): Fluorescence emission of fluorophore (TAMRA) (λ ex 531 / 40nm, λem 593 / 40) was released from 10 μm TentaGel-Lys(PCL1-TAMRA)-QSY7 beads after exposure to UV LED (365 nm) in a picowell flow cell. A) Due to the FRET quenching effect of QSY7, (0 J / cm³) was released before UV exposure. 2 There is no significant emission above the background. TAMRA emission is (B) 25 J / cm². 2 (C)257J / cm 2 (D) 489 J / cm 2 (E)721J / cm 2 (F)953J / cm 2 Following UV exposure, equilibrium (20 minutes) was reached, and then imaging was performed using appropriate exposure times. To measure TAMRA concentration, fluorescence emission was measured within the volume surrounding each bead (Figure 33). The notation "40" with a slash refers to the bandwidth, meaning that the cutoff filter limited the light to the range of 531 nm plus 20 nm and minus 20 nm (this slash notation can also be used for the excitation and emission wavelengths).

[0389] The following are some interpretations of fluorescence data from concentration tests of bead-released TAMRA in picowells (45 pL) after UV exposure (365 nm) of a bead-bound release monitor, conducted by the inventors (see Figure 34). The image analysis used was the average pixel intensity of the solution surrounding the bead-filled wells (n≧14), normalized with respect to the image exposure time, and correlated with a standard curve of known TAMRA concentrations in picowells. Error bars represent 1σ calculated from RSD%. The UV-released compound concentrations were 1.1 μM (8.9 RSD%), 54.3 μM (5.2 RSD%), 142 μM (4.2 RSD%), 174 μM (7.7 RSD%), and 197.3 μM (10.1 RSD%) (Figure 34).

[0390] (XII) Biochemical assay of compounds (non-cell-based assay) The beads in the picowells can be used to perform a variety of biochemical assays. Non-exclusive examples include binding assays, enzyme assays, catalytic assays, fluorescence assays, luminescence assays, and scattering assays. Examples are given below.

[0391] A biochemical assay sensitive to protease and peptidase inhibitors. The objective is to detect and develop drugs that inhibit proteases, to screen assays that can use a mixture of specific proteases or peptidases, suitable cleavable substrates, and to screen color or fluorescence assays sensitive to the degree of inhibition by candidate drug compounds. For example, one reagent may be a bead-conjugated compound, the compound not yet tested for activity. Another reagent may take the form of a bead-conjugated pepstatin (an established inhibitor of HIV-1 protease) (Hilton and Wolkowicz (2010) PLoS ONE.5:e10940 (p. 7)). Yet another reagent may be a cleavable substrate of HIV-1 protease, where cleavage by HIV-1 protease results in a color or fluorescence change. Positive cleaning drug candidates that produce a color difference (or fluorescence difference) in a specific assay (in a given microwell) are identified. The cleavable substrate covalently binds to the quencher and the fluorescent agent, taking the form of an adjacent sensitive peptide. Before cleavage, the fluorophore does not fluoresce because of the proximity of the quencher, but fluorescence is generated after cleavage (see Lood et al (2017) PLoS ONE.12:e0173919 (p. 11), Ekici et al (2009) Biochemistry.48:5753-5759, Carmona et al (2006) Nature Protocols.1:1971-1976). The reagents and methods of this disclosure encompass the techniques disclosed above.

[0392] The enzyme-based screening assay for compounds that inhibit ubiquitin ligases involves reagents containing MDM2 (enzyme) and p53 (substrate). The applicant conducted practical tests based on the following technology: MDM2 controls the amount of p53 in cells. MDM2 is overexpressed in several cancers. MDM2 is an enzyme, and "in vivo studies have shown that purified MDM2 is sufficient for p53 ubiquitination" (Leslie et al (2015) J. Biol. Chem. 290:12941-12950). The applicant's objective is to discover inhibitors of MDM2, which are expected to reduce p53 ubiquitination and thereby reduce subsequent p53 degradation. Given the expected increase in p53 in cells, inhibitors with the above properties are expected to be useful in the treatment of cancer.

[0393] The applicants used the following enzymatic assay to evaluate the effect of lenalidomide on the ubiquitination of p53 mediated by MDM2 / HDM2. The applicants used reagents from the following kit: MDM2 / HDM2 Ubiquitin Ligase Kit - p53 Substrate (Boston Biochem, Cambridge, MA). One of the reagents used in the assay was a bead containing a covalently bound antibody. The bead was TentaGel® M NH2 (catalog number M30102, Rapp Polymere GmbH, Germany), and the antibody was a mouse-biosynthesized anti-human p53 monoclonal antibody. MDM2 is an E3 ligase that can use p53 as a substrate, and MDM2 catalyzes the ubiquitination of p53.

[0394] The objective is to activate p53 to reduce cancer. The relationship between MDM2, the transcription factor called "p53," and anticancer therapy is proposed based on the following description: "MDM2 is an E3 ubiquitin ligase that ubiquitinates p53 and targets it for proteasomal degradation" (Ortiz, Lozano (2018) Oncogene. 37:332-340). p53 has tumor suppressor activity. p53 activity is inhibited by MDM2. According to Wu et al., MDM2 is a "p53-binding protein" (see Wu, Buckley, Chernov (2015) Cell Death Disease. 6:e 2035). For example, by blocking the interaction between MDM2 and p53, the compound is expected to prevent p53 ubiquitination and function as an anticancer agent.

[0395] Objective of the Screening Assay. The objective of the screening assay is to discover compounds that affect p53 ubiquitination, e.g., compounds that stimulate p53 ubiquitination and compounds that inhibit p53 ubiquitination. More specifically, the objective is to discover compounds that inhibit or activate p53, and whose effects are mediated by MDM-2 and either E1, E2, or E3 ligases. MDM2 stands for “mouse double microchromosome.” MDM2 is referred to as “E3 ubiquitin ligase.” When MDM2 occurs intracellularly, evidence suggests that the activity to catalyze p53 ubiquitination requires many other proteins, such as CUL4A, DDB1, and RoC1 (see Banks, Gavrilova (2006) Cell Cycle. 5:1719-1729, Nag et al (2004) Cancer Res. 64:8152-8155). Banks et al. described physical interactions involving p53 and MDM2, stating, "We found that L2DTL, PCNA, and the DDB1 / CUL4A complex physically interact with the p53 tumor suppressor and its regulator MDM2 / HDM2" (Banks, Gavrilova (2006) Cell Cycle. 5:1719-1729). Nag et al. further described physical interactions involving p53 and MDM2, stating, "Cul4A functions as an E3 ligase and is involved in the proteolysis of several regulatory proteins via the ubiquitin-proteasome pathway. Here, we show that Cul4A is associated with MDM2 and p53" (Nag et al (2004) Cancer Res. 64:8152-8155).

[0396] Desired readout from a bead assay of p53 ubiquitination modifiers. Screening a compound and obtaining a positive screening hit, i.e., increased AF488 fluorescence, means that an activator has been found. Screening a compound and obtaining a positive cleaning hit, i.e., decreased fluorescence, means that an inhibitor has been found. Compounds that inhibit p53 ubiquitination suggest that the compound can be used to treat cancer. Furthermore, if a compound specifically inhibits p53 ubiquitination, i.e., the compound does not inhibit the ubiquitination of other proteins, or the compound inhibits the ubiquitination of other proteins but less severely than p53, it also suggests that the compound can be used to treat cancer.

[0397] Materials. The materials include the E3 ligase kit K-200B from Boston Biochem. The Boston Biochem catalog lists kits such as the Mdm2 / HDM2 ubiquitin ligase kit - p53 substrate. The following pertains to Mdm2, which is part of this kit. This kit does not contain cereblon. Lenalidomide and similar compounds can bind to either cereblon or Mdm2, with the final result being activation of the ubiquitin ligase. The materials further include Diamond White Glass microscope slides, 25mm x 75mm (Globe Scientific, Paramus, NJ). Corning stirrer / hot plate (0-10 settings) 698 watts, model PC-420. N-hydroxysuccinimide (NHS). Methyltetrazine (mTET). AlexaFluor488 (AF488) (ThermoFisher Scientific). TentaGel beads M NH2 (catalog number M30102) (Rapp Polymere GmbH). Parafilm (Sigma-Aldrich, St. Louis, MO). Figure 8 shows the structure of Alexa Fluor® 488. The structure of Alexa Fluor 488 (AF488) is shown in the product information for AlexaFluor488-Nanogold-Streptavidin (Nanoprobes, Inc., Yaphank, NY).

[0398] Cellular assay for compound (XIII) Cell line assays performed in picowells can use human cells, non-human cells, human cancer cells, non-human cancer cells, bacterial cells, and parasitic cells such as malaria parasite cells. Furthermore, cell line assays can be performed using "killed but metabolically active" human or non-human cells, i.e., their genomes are cross-linked to enable metabolism but prevent cell division (Dubensky U.S. Patent Publication No. 2007 / 0207170, incorporated herein by reference in its entirety). Additionally, cell line assays can be performed with apoptotic, necrotic, or dead cells. Cell line assays using bacterial cells can be used for antibiotic screening. Virus-infected human cells can be used for antiviral screening. Cell combinations are provided for cell line assays. For example, combinations of dendritic cells and T cells are provided for screening and identifying compounds that stimulate antigen presentation or, alternatively, impair antigen presentation.

[0399] Cell line assays can be based on primary cultures of cells obtained, for example, from biopsies of normal tissue, solid tumors, or hematological malignancies, or from circulating solid tumor cells. Alternatively, cell line assays can be based on cells that have been passaged one or more times.

[0400] Cell line assays performed in picowells can use cultures containing only one cell, or two, three, four, or five cells, or approximately two, three, four, or five cells, or more than three, four, or five cells.

[0401] The applicant conducted practical tests based on the following technology, which describes a cell line assay for screening compounds of exemplary embodiments in which lenalidomide (test compound) inhibits ubiquitin-mediated proteolysis of transcription factors. The transcription factors include Icarus and Aeolus.

[0402] This disclosure provides a cell line assay for screening compounds on bead-bound compounds, the screening of which is performed in a plate having many picowells. The components of the cell line assay include picowells for holding a bead-bound chemical library, each bead to which substantially only one homogeneous type of compound is attached. The compounds are released via a cleavable linker. Mammalian cells are cultured in the picowells. The picowells further contain culture medium. The non-limiting example of lenalidomide disclosed here is an example of proof of principle which can be used to screen a chemical library for discovering other compounds that control the ubiquitination of a given target protein.

[0403] A brief description of the cell line assay: Recombinant cells are used as reagents to detect and screen compounds that induce the proteolysis of green fluorescent protein (GFP). The readout to identify a positive screening compound is a situation in which green cells become colorless or cells with reduced green color. Regarding the mechanism of this cell line assay, the mechanism of action of lenalidomide, which causes green cells to become colorless or cells with reduced green color, is that lenalidomide binds to a protein called "cereblon." In cells, cereblon is part of a protein complex called "E3 ubiquitin ligase." Cereblon is a direct target of the anticancer drugs lenalidomide, thalidomide, and pomalidomide. The normal constitutive activity of E3 ubiquitin ligase and its relationship with cereblon is described as follows: "Cereblon promotes proteasomal degradation [of target proteins] by binding to E3 ubiquitin ligase" (see Akaffo et al (2018) J. Biol. Chem. 293:6187-6200). In contrast to the normal activity of E3 ubiquitin ligase, when drugs such as lenalidomide, thalidomide, or pomalidomide are added, the result is that "lenalidomide, thalidomide, and pomalidomide promote the ubiquitination and degradation of substrates by E3 ubiquitin ligase, and each of these drugs induces the degradation of transcription factors IKZF1 and IKZF3" (Kronke et al (2015) Nature. 523:183-188).

[0404] Regarding terminology, cereblon is described as part of a protein complex referred to as "E3 ligase" and also as "E3 ubiquitin ligase." Generally, cereblon itself is not referred to as "E3 ligase." The following excerpt illustrates the usage of the word "cereblon." According to Akuffo et al (2018) J. Biol. Chem. 293:6187-6200, "When thalidomide binds to the E3 ligase substrate receptor cereblon, proteasomal disruption [of the substrate] is promoted by the involvement of the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase." Consistently, Yang et al (2018) J. Biol. Chem. 293:10141-10157 discloses that "cereblon functions as a substrate receptor for cullin-4 RING E3 ligase to mediate protein [substrate] ubiquitination." Zhu et al (2014) Blood. 124:536-545 states that "thalidomide binds to CRBN [cereblon] and alters the function of the E3 ubiquitin ligase complex composed of CRBN, DDB1, and CUL4." Lopez-Girona et al (2012) Leukemia. 26:2326-2335 states that "studies have identified the E3 ligase protein cereblon (CRBN) as a direct molecular target of thalidomide, and that CRBN and DDB1 form a functional E3 ligase complex with Cul4A and Roc1."

[0405] To see the overall picture of the cell line assay devised and used by the applicant, the first step is to add lenalidomide to the cells. The final step is the deactivation of IKZF1 and IKZF3. If IKZF1 arises as a fusion protein with GFP, the final step is the degradation of the entire fusion protein by the proteasome. Similarly, if IKZF3 arises as a fusion protein with GFP, the final step is the degradation of the entire fusion protein by the proteasome. As a result of GFP degradation, cells that previously emitted green fluorescence are converted into cells that do not emit fluorescence.

[0406] This is a lengthy description of a cell line assay. It relates to the name of the E3 ubiquitin ligase (protein complex), the name of the protein that binds to this complex, and the name of the protein that is the target of this complex. The published literature is inconsistent regarding these names. Sometimes proteins are referred to by their protein name, and sometimes they are referred to by the name of the gene that codes for them. For this reason, in the following reports, protein names and gene names are used together, such as "Cereblon" (protein name) and "CRBN" (gene name). Also, "Ikaros" is the name of the protein, and the gene name is IKZF1. Similarly, "Aeolus" is the name of the protein, and IKZF3 is the name of the gene. "Cullin-ring finger ligase-4" is the name of the protein, and the gene name is CRL4. "Cullin-1 regulator" is the name of the protein, and the gene name is ROC1. ROC1 is also known as RBX1 (Jia and Sun (2009) Cell Division. 4:16. DOI: 10.1186). "Cullin-4A" is the name of the protein, while the gene name is CUL4A. See Schafer, Ye, Chopra (2018) Ann. Rheum. Dis. DOI: 10.1136, Chen, Peng, Hu (2015) Scientific Reports. 5: 10667, Matyskiela et al (2016) Nature. 535: 252-257, and Akuffo et al (2018) J. Biol. Chem. 293: 6187-6200).

[0407] E3 ubiquitin ligases catalyze the transfer of ubiquitin residues to target proteins, which are then transported to the proteasome for degradation. E3 ligases catalyze the binding of ubiquitin to one or more lysine residues of the target protein. Humans express approximately 617 different E3 ubiquitin ligase enzymes (see Shearer et al (2015) Molecular Cancer Res. 13:1523-1532). E3 ubiquitin ligases are complexes of proteins including Cullin-4 (CUL4A or CUL4B), the regulator of Cullins-1 (RoC1), and the RING Box domain protein (RBX1). As noted above, RoC1 is the same protein as RBX1 (see Jia and Sun (2009) Cell Division. 4:16. DOI:10.1186). When cereblon (CRBN) is linked to the E3 ubiquitin ligase complex, a larger complex is formed, CRL4 CRBN It is referred to as (Matyskiela et al (2016) Nature. 535:252-257). The term "CRL4" means "Cullin-4 RING ligase" (Gandhi et al (2013) Brit. J. Haematol. 164:233-244, Chamberlain et al (2014) Nature Struct. Mol. Biol. 21:803-809). When reading literature on cereblon, it is necessary to take into account the above inconsistencies in nomenclature.

[0408] The following is a longer version of the short excerpt disclosed above. Below is yet another form of nomenclature, namely, "CRL4 CRBNThe term is "E3 ubiquitin ligase." Longer reports more completely integrate various names and cellular events. "The relationship between cereblon (CRBN) and the E3 ubiquitin ligase complex is described as "promoting proteasomal degradation [of target proteins] by utilizing the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase" (Akuffo et al (2018) J. Biol. Chem. 293:6187-6200). Regarding anticancer drugs, "Lenalidamide, thalidomide, and pomalidomide promote substrate ubiquitination and degradation by E3 ubiquitin ligase. These compounds bind to CRBN and CRL4 CRBN These are substrate adapters for E3 ubiquitin ligases, and each of these drugs induces the degradation of transcription factors IKZF1 and IKZF3 (Kronke et al (2015) Nature. 523:183-188).

[0409] This relates to a cell line assay in which any given microwell, nanowell, or picowell contains beads, the beads having covalently linked compounds attached via a cleavable linker, and the well contains one or more cultured mammalian cells. The response to the compounds and drug candidates of this disclosure can be evaluated by one or more biomarkers.

[0410] Biomarkers include diagnostic biomarkers, biomarkers that predict whether a given patient will respond (improve) to a given drug, and biomarkers that predict whether a given patient will experience unacceptable toxicity to a given drug (Brody, T. (2016) Clinical Trials: Study Design, Endpoints and Biomarkers, Drug Safety, and FDA and ICH Guidelines, 2 nd(ed., Elsevier, San Diego, CA). This disclosure also describes the use of yet another type of biomarker, namely, biomarkers that monitor a patient's response to a given drug after drug therapy has been initiated. For example, the following concerns the biomarker "peroxiredoxin 6 (PRDX6)" and lung cancer. According to Hughes et al., "PRDX6 levels in cell culture media from cell lines increased after gefitinib treatment versus vehicle PRDX6 accumulation over time and were positively correlated with gefitinib sensitivity. Serum PRDX6 levels increased significantly in the first 24 hours of therapy change in serum PRDX6 during the course of gefitinib therapy and were superior to imaging strategies for monitoring the response to anti-EGFR agents." Note that biomarkers are superior to the use of "imaging" to detect a more direct measurement of the effectiveness of the response, i.e., a reduction in tumor size and number (Hughes et al (2018) Cancer Biomarkers. 22:333-344). Other biomarkers used to monitor the response to anticancer drugs include CA125 for monitoring the response to platinum therapy in ovarian cancer, and serum HSPB1 for monitoring the response to chemotherapy in ovarian cancer (see Rohr et al (2016) Anticancer Res. 36:1015-1022 and Stope et al (2016) Anticancer Res. 36:3321-3327).

[0411] Cytokine expression. The response can be evaluated by measuring expressed cytokines such as IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha. These specific cytokines can be measured simultaneously using gold nanostructures with antibodies that specifically recognize one of these cytokines, and detection is accompanied by plasmon resonance (Spackova, Wrobel, Homola (2016) Proceedings of the IEEE. 104:2380-2408, Oh et al (2014) ACS Nano. 8:2667-2676). Cytokines expressed by single cells, such as single T cells, can be measured via fluorescent antibodies in a device containing microwells (Zhu, Stybayeva (2009) Anal. Chem. 81:8150-8156). The above methods are useful reagents and methods for the present disclosure.

[0412] In some embodiments, antibodies against cytokines can be attached to the walls of picowells, and cytokines released from cells as a function of drug exposure, or differentially released, can be captured by antibodies bound to the picowell walls. Captured cytokines can be identified by a second set of labeled antibodies. In some embodiments, antibodies against cytokines can be attached to capped beads. The capped beads can then be detached and embedded in a cross-linked hydrogel sheet, which can be subjected to further analysis by, for example, ELISA, mass spectrometry, or other analytical techniques.

[0413] Apoptosis. Real-time data on apoptosis, and initial events in single-cell apoptosis, can be measured using surface-enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) (see Stojanovic, Schasfoort (2016) Sensing Bio-Sensing Res. 7:48-54, Loo, Lau, Kong (2017) Micromachines. 8:338. DOI:10.3390). Stojanovic, mentioned above, detects the release of cytochrome C, EpCam, and CD49e from cells. Loo et al., also mentioned above, measured the release of cytochrome C from cells, and the detection is accompanied by a DNA aptamer (this DNA aptamer functions like an antibody). Zhou et al. used SERS to detect early apoptosis in single cells, measuring phosphatidylserine on the cell membrane (see Zhou, Wang, Yuan (2016) Analyst. 141:4293-4298). In addition to collecting data on apoptosis, SERS can be used to evaluate drug activity by collecting data on mitotic stages, metabolite release, and expression of biomolecules bound to the plasma membrane (see Cialla-May et al (2017) Chem. Soc. Rev. 46:3945-3961). Plasmon resonance can measure protein denaturation and DNA fragmentation that occur in apoptosis (see Kang, Austin, El-Sayed (2014) ACS Nano. 8:4883-4892). Plasmon resonance (SERS) can distinguish between cancer cells and normal cells by measuring the proportion of mitotic proteins in alpha-helix and beta-sheet morphologies (Panikkanvalappil, Hira, El-Sayed (2014) J.Am.Chem.Soc.136:159-15968). The above method is suitable as a reagent and method for the present disclosure.

[0414] Apoptosis can also be measured in cultured cells using a method that does not use plasmon resonance, but instead uses immunocytochemistry with an anti-cleavage caspase 3 antibody (Shih et al (2017) Mol. Cancer Ther. 16:1212-1223).

[0415] General information regarding cell line assays. The cell line assays described herein may be used to test responses from human cancer cells, cells from solid tumors, cells from hematological malignancies, human stem cells, human hepatocytes, pathogens, infectious bacteria, bacterial-infected human cells, virus-infected human cells, and others. The assays can detect morphological responses of cells, such as migration, as well as genetic and biochemical responses.

[0416] The assays of the present disclosure may be designed to detect the response of cells located inside the picowells, or to detect the response of cells located outside the picowells, such as in a nutrient medium located as the top layer of the picowell array. The assays of the present disclosure may also be designed to detect the response of cells, where the cells and beads are located in the medium, where the cells are located in the medium and the beads are located in the top or bottom of the medium, where the cells are located in the top of the medium and the beads are located in the top, middle or bottom of the medium.

[0417] This disclosure provides a population of cells in a picowell array. In embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the cell population is located inside the picowells (not in any region located above the picowells). In embodiments, this is the percentage of cells located inside the wells, with the remainder located in the layer of nutrient medium above the array of wells, and may be in a range defined by two of these numbers, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or in the range of "about 60% to about 90%".

[0418] Cell matrix. In assays of the biological activity of cells, cells are exposed to compounds released from beads or to compounds bound to beads. Suitable matrices include those containing one or more of the following: poly-D-lysine (PDL), poly-L-lysine (PLL), poly-L-ornithine (PLO), vitronectin, osteopontin, collagen, peptides containing the RGD configuration, polypeptides containing the RGD configuration, laminin, laminin / fibronectin complexes, laminin / entactin complexes, etc. Suitable matrices also include products available from Corning, Inc., such as PuraMatrix® Peptide Hydrogel®, Cell-Tak® Cell and Tissue Adhesive, and Matrigel®. See Corning Life Sciences (2015) Corning Cell Culture Surfaces, Tewksbury, MA (page 20), and De Castro, Orive, Pedraz (2005) J. Microencapsul. 22:303-315. In an exclusionary embodiment, this disclosure may exclude any composition or method comprising one of the above matrices or one of the above polymers.

[0419] In embodiments, the Disclosure provides an array in which each picowell comprises either a bead, one or more cells, and a solution (without any matrix), or a matrix, or a combination of solution and matrix. The matrix may be a hydrogel, polylysine, vitronectin, MatriGel®, etc.

[0420] The activity of bead-binding or bead-releasing compounds can be evaluated. Assays for evaluating activity include activation or inhibition of enzymes, activation or inhibition of cell signaling cascades or individual cell signaling proteins, binding to antibodies (or antibody complementarity-determining regions (CDRs), antibody variable regions), and inhibition of binding of ligands or substrates to enzymes (or antibodies, or antibody variable regions).

[0421] For the above assays, readout can be determined, for example, by a fluorescence assay containing a chain of fluorophores in the quencher (FQ). The linker can be designed to be cleavable by endoproteases, DNAse, RNAse, or phospholipases (see Stefflova, Zheng (2007) Frontiers Bioscience. 12:4709-4721). The term "molecular beacon" refers to this type of FQ molecule, while "molecular probe" is also used to refer to constructs in which the separation of F and Q is induced by hybridization, such as in the TaqMan® assay (Tyagi and Kramer (1996) Nature Biotechnol. 14:303-308, Tsourkas, Behlke, Bao (2003) Nucleic Acids Res. 15:1319-1330).

[0422] Transcriptional profiling in response to drug exposure. The DNA barcodes of this disclosure may be modified to include a response capture element, which captures the cell's response to a perturbation encoded by the encoding portion of the barcode. In some embodiments, the DNA barcode may terminate with a poly-T moiety (multiple repeats of thymidine nucleotides), and the poly-T sequence may be used to capture poly-A terminated mRNA molecules released from lysed cells. In some embodiments, the response capture sequence may be complementary to the gene of interest, thereby capturing the expression profile of the desired gene via hybridization to the beads of this embodiment. In some embodiments, the picowells may include single-cell picowells in which the transcriptional profile is captured on the beads. In some other embodiments, multiple cells may be included within the picowells in which the transcriptional profile is captured.

[0423] In one exemplary workflow, the following steps may track a cell's transcriptional response to a drug: (a) Provide picowells designed to capture a single cell per well. (b) Introduce compound-loaded DNA barcode beads into the picowells so that one bead is present in each picowell. (c) Release the compound from the beads in each picowell by an appropriate method (e.g., a UV treatment agent for the compound attached via a UV-cleavable linker suitable for the beads of the embodiment, or diffusion by immersion of the beads in an acid-cleavable, base-cleavable, or temperature-cleavable compound). (d) The picowells may be isolated from each other via capped beads that retain their contents within the picowells, or by other means such as an air or oil barrier at the top of the picowells. (e) Incubate the cells in the picowells for a certain period of time in the presence of the compound released from the beads. (f) After a suitable time, for example, 1 hour, 2 hours, 5 hours, 9 hours, 12 hours, 15 hours, 18 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, or another suitable time based on the assay, the cells are lysed by a lysis method. The lysis method may involve the addition of a washing agent, repeated freeze-thaw cycles, heating, addition of a membrane-breaking peptide, mechanical stirring, or other suitable means. (g) Upon lysis, the contents of the cells are exposed to the beads in the picowells, at which point the response capture elements on the beads in the picowells enable the capture of their intended response. In some embodiments, the response capture is a poly-T sequence that captures the complete mRNA profile of the cell(s) (or more) in each picowell. In some embodiments, the response capture element is designed to capture a specific DNA or RNA sequence from the cell(s). In some embodiments, the transcriptional response of the cell(s) may be captured as a function of the dose(s) (or concentration) of the compound.

[0424] (XIV) Perturbation-response analysis in cells The methods described herein may include a perturbation library and a cell library. In some embodiments, the perturbation and cells are incubated in a restricted environment. During or after incubation, a barcode identifying the perturbation ("perturbation barcode") may be transferred to the cells being incubated. The method may further include releasing the cells from the perturbation (i.e., separating or removing them) and subjecting the cells to a second restriction where the cell contents are captured along with the perturbation barcode. In some embodiments, the second restriction may include a cell-specific barcode which may then be used to study the cell contents and perturbation barcode, thereby relating the perturbation to a cellular response.

[0425] In some embodiments, the methods described herein may comprise two responses: a perturbation response encoded by a perturbation barcode and a measurement response. In the perturbation response, a cell may be subjected to a perturbation. In the measurement response, the cellular response as a result of the perturbation may be measured. In some embodiments, the measurement response may comprise a measurement barcode. In other embodiments, the method may comprise associating the perturbation with the cellular response by passing the perturbation barcode to the measurement response and capturing both the measurement barcode and the perturbation barcode contained in the measurement response.

[0426] The methods described herein may include perturbation responses encoded by perturbation barcodes. A perturbation response involves subjecting cells to a perturbation and subsequently measuring the cells' cellular response to the applied perturbation. The perturbation barcode is decoded either before or after measuring the cellular response, and thus the identity of the perturbation can be associated with the measured cellular response.

[0427] In some embodiments, the method involves providing a DNA-encoded, bead-bound compound library, the compounds of which may be released from beads, and contacting the DNA-encoded, bead-bound compound library with a library of cells, the contact may be performed by confining the beads with one or more cells in a first restricted volume, releasing the compounds from the beads, and incubating the compounds with the cells in the first restricted volume. In some embodiments, DNA barcodes identifying the compounds may be released from the beads and attached to cells, either simultaneously or after incubation. Cells with attached DNA barcodes are released from the first restricted volume and confined again in a second restricted volume, the second restricted volume having a reagent for lysing the cells, as well as a mechanism for capturing cell contents and bead-specific barcodes carried by the cells. In some embodiments, the mechanism used to capture cell contents and barcodes may involve the use of capture barcodes and may function to uniquely identify single cells or small clusters of cells. In some embodiments, capture barcodes and bead-specific barcodes may be concatenated. In some embodiments, all of the limited volumes containing individually barcoded materials can be merged to create a pool of barcoded cellular contents and barcodes that are specific to cells and beads. The pool of barcoded materials can be analyzed by sequencing to study individual cellular responses and link them to the perturbations that caused those responses.

[0428] In some embodiments, the method involves capturing individual cells within a droplet. The cells may contain nucleic acid barcodes on their cell membranes to uniquely identify the perturbations the cells experience. The cells may be lysed within the droplet, and the cellular mRNA and cell membrane-bound nucleic acid barcodes may be captured with a set of barcoded capture oligonucleotides ("capture barcodes"). Each droplet may contain different unique barcoded capture oligonucleotides, and the barcodes within a single droplet may have substantially identical sequence segments. The mRNA and cell membrane-bound barcodes may be copied to the droplet-barcoded oligonucleotides using reverse transcriptase. The nucleic acid material may be pooled together from the droplet by disrupting the droplet. All nucleic acid material may be sequenced to study the transcriptional profiles of individual cells and relate them to perturbations associated with those transcriptional profiles.

[0429] The methods described herein may involve subjecting a library of cells to two barcoded restrictions: perturbation restriction and lysis restriction. Cells may be restricted individually or as small clusters. The barcodes may include barcodes introduced into cells while perturbing the cells, and barcodes introduced while lysing the cells. In some embodiments, the perturbation barcodes may be transported by the cells to the lysis step. In some embodiments, the lysis barcodes may be applied to the cell contents and / or perturbation barcodes, resulting in the establishment of barcoded cell contents that associate the cell contents with the perturbations experienced by the cells. In some embodiments, the perturbation beads may further include response capture probes. In this case, a single picowell compartmentalization step may suffice instead of two compartmentalization steps. In such embodiments, the composite barcodes may be functionalized to capture cellular responses. In some embodiments, the perturbation barcodes terminate with a poly(T) segment into which the poly(A) tail of the mRNA molecule can hybridize.

[0430] In some embodiments, the workflow for single-cell perturbation response analysis is as follows: (1) Provide functionalized perturbation beads, the perturbation barcodes ending in a capture sequence, the capture sequence may include a set of poly(T)nucleotides for mRNA capture or other suitable capture probes for capturing other cellular responses; (2) Capture a library of cells in a picowell array; (3) Capture a library of functionalized perturbations in the same picowells, where in some embodiments, single cells and single functionalized beads are captured per well, and in other embodiments, clusters of cells may be captured in the picowells; (4) Optionally, cover the picowells with an oil medium to prevent cross-contamination of reagents between wells; (5) Extract compounds from the perturbation beads. (6) release the perturbation bead and incubate the cells in each well with the compound released from the perturbation bead; (7) lyse the cells in the picowell by covering the picowell and flowing lysis buffer over them; (8) capture mRNA or other cellular responses directly at the tip of the perturbation barcode; (9) copy the cellular response to the perturbation barcode using polymerase or reverse transcriptase; (10) release the beads from the picowell by sonication, then cleave the perturbation barcode extended from the released beads, or simply cleave the perturbation barcode from the beads while the beads are still inside the picowell; and (11) subject the cleaved nucleotides (extended perturbation barcodes) to a suitable library preparation method and sequence the prepared nucleotides. In some embodiments, the sequenced nucleotides include two regions: a perturbation barcode that identifies the perturbation / compound to which the cell has been subjected, and a response region corresponding to the mRNA expression of the cell to which the perturbation / compound identified by the perturbation barcode has been subjected. This workflow is shown in Figure 36 and optionally includes an imaging step for process QC. Reverse transcriptase can function to extend the captured RNA onto the bead-attached DNA, thereby transferring the cell contents information to the functionalized beads. The beads can then be pooled, extracted, and analyzed with a sequencer. In some embodiments, DNA from a single cell may be further captured on specific primers on the functionalized beads.In such embodiments, polymerase may be replaced with reverse transcriptase.

[0431] In some embodiments, the capture of perturbations and cellular responses may occur in two different constraints, as shown in Figure 37. Perturbation barcodes may be transferred to the cell surface before being subjected to cellular response capture constraints. Cellular response capture also involves capturing perturbation barcodes transported on the cell surface, thereby directly relating the cellular response to the perturbation to which the cell is exposed. In some embodiments, the capture of cellular responses can be achieved by the Drop-seq method. In some embodiments, the capture of cellular responses may occur in any commercially available single-cell analyzer, such as the 10X Genomics single-cell instrument, Raindance single-cell analysis protocol, BioRad single-cell isolation instrument, Mission Bio single-cell analysis protocol, GigaGen instrument and protocol, and / or any other commercially available single-cell analyzer or service.

[0432] In some embodiments, cell suspensions can be used as a starting point for cells restricted by perturbation beads. Methods for suspending cells in aqueous medium or culturing cells in suspensions are well known to those skilled in the art. Methods for suspending cells and culturing cells in suspensions are also described in the art. For example, in some embodiments, globular cell cultures can be used as a starting point for perturbation because they capture more intercellular interaction signatures than single cells in isolation (see, for example, Edmondson et al., Assay Drug Dev Technol. 12:207-218, 2014; Fennema et al., Trends Biotechnol. 31:108-115, 2013; Han et al., Sci Reports 5:11891, 2015; Zanoni et al., Sci Reports 6:19103, 2016, all of which are incorporated herein by reference in their entirety). In some embodiments, organoids may be used instead of single cells to undergo high-throughput perturbations (see, for example, Foley, Nat Methods 14:559-562, 2017; Liu et al., Front Pharmacol. 7:334, 2016; Neugebauer et al., BioRxiv April 2017; Skardal et al., Drug Discov Today 21:1399-1411, 2016; Boehnke et al., J Biomol Screen 21:931-941, 2016, all of which are incorporated herein by reference in their entirety).

[0433] In some embodiments, cells are obtained from disease models. The methods described herein enable large-scale, high-throughput screening of compounds across disease model cells to determine whether a therapeutic response is obtained by exposure to one or more compounds in a perturbation / compound library. In other embodiments, cells are healthy cells of various strains. The methods described herein enable large-scale, high-throughput mapping of cellular responses to various compounds. In some embodiments, data collected by unbiased screening of combinatorial compound libraries on cells enables novel drug prediction based on known maps of drug-cell interactions.

[0434] In some embodiments, the limitations used in the methods described herein include droplet limitations. In some embodiments, the droplets comprise aqueous droplets in an oil matrix. In some embodiments, the droplets are produced in a microfluidic junction comprising a mixture of aqueous and oil phases. In some embodiments, the microfluidic junction comprises cells, perturbed beads, and an oil phase. One embodiment of a microfluidic construction for producing droplets using cells and beads is shown by the “Drop-Seq” method (see, for example, Macosko et al., Cell 161:1202-1214, 2015).

[0435] In some embodiments, the method includes hydrogel restriction. In some embodiments, the restriction used in the method described herein includes hydrogel restriction, and the cells and beads are embedded in a hydrogel matrix that ...

Claims

1. A system for screening compounds, (a) A picowell array plate comprising a plurality of picowells, wherein each picowell has an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor by a wall, and the wall being located between the upper hole and the floor, (b) Beads placed within picowells, comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds, (c) The beads include bead-attached DNA barcodes that take the form of either a chained DNA barcode or an orthogonal DNA barcode, and if the DNA barcode takes the form of a chained DNA barcode, the chained DNA barcode is (i) Using click chemistry, or (ii) A method of production using one or both of the following: the repeating cycle of step, wherein the repeating cycle of step comprises using a partially produced bead-bound DNA barcode and a hybridizable sprint oligonucleotide (sprint oligo), wherein the hybridization is mediated by an annealing site on the sprint oligo and a corresponding complementary annealing site on the partially produced bead-bound DNA barcode. The annealed sprint oligo is used as a template for extending the partially constructed DNA barcode using DNA polymerase, the sprint oligo contains bases complementary to the DNA barcode module polymerized onto the partially constructed bead-bound DNA barcode, and the sprint oligo also contains bases complementary to the annealing site polymerized onto the partially constructed bead-bound DNA barcode. (d) A system comprising: each of the plurality of substantially identical bead-bound compounds comprising one or more chemical library monomers, each bead-bound DNA barcode module identifying the corresponding chemical library monomer, the term “compound” used to refer to a finished product comprising one or more chemical library members, and the finished DNA barcode identifying the compound.

2. The system according to claim 1, further comprising oligonucleotide sequencing primers capable of inducing sequencing of one or more DNA barcode modules contained in a bead-bound DNA barcode, and optionally, the system comprising a DNA sequencing instrument, wherein the DNA sequencing instrument is not a luminescent sequencer and is not a pH-based DNA sequencing instrument.

3. The system according to claim 1, further comprising a plurality of spherical caps, each cap capable of fitting into the hole of the picowell, the hole being circular, each cap capable of minimizing or preventing evaporation of the fluid inside the picowell, and each cap capable of minimizing or preventing leakage of the fluid inside the picowell.

4. The system according to claim 1, wherein the at least one bead disposed in the at least one picowell includes at least one response capture element coupled with the at least one bead.

5. At least one of the beads placed in the picowell is It includes at least one response capture element coupled to the at least one bead, and the at least one response capture element is (a) Poly(dT), (b) Exon-targeting RNA probes, (c) Antibody, or (d) The system according to claim 1, comprising an aptamer.

6. The DNA barcode is either a chained DNA barcode or an orthogonal DNA barcode, and the DNA barcode includes one or more DNA barcode modules, each of which codes information identifying a chemical library monomer, and the chained DNA barcode or the orthogonal DNA barcode is (a) one or more functional nucleic acids, and (b) The system according to claim 1, further comprising one or both of one or more nucleic acids that encode information of a type other than the identity of chemical library monomers.

7. The aforementioned bead-linked DNA barcode, (i) The first DNA barcode module, or (i) a first DNA barcode module, a first annealing site, and a second DNA barcode module, or (ii) A first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, and a third DNA barcode module, or (iii) A first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, a third annealing site, and a fourth DNA barcode module, or (iv) First DNA barcode module, first annealing site, second DNA barcode module, second annealing site, third DNA barcode module, third annealing site, fourth DNA barcode module, fourth annealing site, and fifth DNA barcode module, or (v) The system according to claim 1, comprising a first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, a third annealing site, a fourth DNA barcode module, a fourth annealing site, a fifth DNA barcode module, a fifth annealing site, and a sixth DNA barcode module.

8. The bead includes a DNA barcode which is an orthogonal DNA barcode, the bead includes an outer surface, and the orthogonal DNA barcode is (a) A first nucleic acid comprising an annealing site for a first DNA barcode module and sequencing primer, the first nucleic acid which binds to the beads at a first position, (b) A second nucleic acid comprising an annealing site for a second DNA barcode module and sequencing primer, the second nucleic acid which binds to the beads at a second position, (c) A third nucleic acid comprising an annealing site for a third DNA barcode module and sequencing primer, wherein the second nucleic acid binds to the beads at a third position, The system according to claim 1, wherein the first, second, and third positions on the bead are each located at different positions on the outer surface of the bead.

9. The aforementioned linked DNA barcode, (i) both click chemistry and the repeated cycle of the step using the sprint oligo, (ii) Both click chemistry and non-click chemistry chemical methods, (iii) Click Chemistry only, or (iv) The system according to claim 1, manufactured by using only the repeated cycle of the step using the sprint oligo.

10. The system according to claim 1, wherein each of the plurality of substantially identical bead-binding compounds is bonded to the beads via a cleavable linker, or via a cleavable linker which is a photocleavable linker, or via an incleavable linker.

11. The system according to claim 1, wherein at least one of the beads comprises a grafted copolymer consisting of a low-crosslinked polystyrene matrix grafted with polyethylene glycol (PEG).

12. At least one picowell contains at least one cell, The plurality of substantially identical bead-bound compounds are bound to at least one bead via a cleavable linker, and cleaving the cleavable linker releases the bead-bound compound from the bead, generating the released compound. The released compound is capable of coming into contact with the at least one cell, and the at least one cell, (i) Mammalian cells that are not cancer cells, (ii) Mammalian cancer cells, (iii) Dead mammalian cells, (iv) Apoptotic mammalian cells, (v) Necrotic mammalian cells, (vi) bacterial cells; (vii) Malaria parasite cells, (vii) Cells that are metabolically active but have a cross-linked genome and are incapable of cell division, or The system according to claim 1, wherein the (ix) cells are mammalian cells infected with the virus.

13. Each picowell has an upper hole defining an opening at the top of the picowell, a bottom defined by the floor, the upper hole being separated from the floor, and a wall existing between the upper hole and the floor. The system according to claim 1, wherein the hole is circular, the floor is circular, the wall is in the shape of a truncated cone, the hole has a first diameter, the floor has a second diameter, and the first diameter is greater than the second diameter.

14. Each picowell has an upper hole defining an opening at the top of the picowell, a bottom defined by the floor, the upper hole being separated from the floor, and a wall existing between the upper hole and the floor. The hole is circular, the floor is circular, the wall is in the shape of a truncated cone, the hole has a first diameter, the floor has a second diameter, and the first diameter is larger than the second diameter. The apparatus further includes a cap that fits snugly into the hole, wherein the hole is made of a polymer having a larger durometer (harder), and the cap is made of a polymer having a smaller durometer (softer), and the relative durometers of the cap and the hole allow the cap to fit reversibly and snugly into the hole. The aforementioned cap, (i) A cap intended solely to stop the picowell and prevent leakage. (ii) A passive cap that can absorb metabolites released by cells while cells in a cell culture medium are being cultured in the picowell, (iii) An active cap, which takes the form of beads containing multiple essentially identical compounds, Each of the aforementioned plurality of essentially identical compounds is bonded to the beads by a cleavable linker, a cap, (iv) The system according to claim 1, an active cap, which takes the form of beads containing a plurality of identical reagents, each of which is bonded to the beads by a cleavable linker.

15. The system according to claim 14, further comprising at least one spherical cap.

16. The system according to claim 14, further comprising at least one non-spherical cap.

17. The aforementioned DNA barcode does not code for any chemical monomer, but instead (a) Class of compounds that are cleavably attached to the beads, (b) A step in a multi-step organic synthesis pathway in which a bead-bound nucleic acid corresponds to a predetermined chemical monomer used to produce a bead-bound compound, and the bead-bound nucleic acid corresponding to the predetermined chemical monomer identifies that chemical monomer. (c) The date on which the bead-bonded compound was synthesized, (d) The bead-bound compound is intended to treat a disease, (e) The bead-bound compound stimulates or inhibits cellular events, or (f) The system according to claim 1, comprising one or more nucleic acids that specify one or more reaction conditions used to bind a predetermined chemical library monomer to the beads.

18. The system according to claim 1, wherein there is no headpiece for linking any of the bead-binding compounds to any of the bead-binding DNA barcodes.

19. The linked DNA barcode comprises at least one nucleic acid which is a DNA barcode module, and (a) Can be used as an annealing site for sequencing primers, (b) A hairpin structure can be formed, wherein the hairpin structure includes a sequencing primer, an annealing portion of the sequencing primer, and beads, wherein the beads are 5-prime with respect to the sequencing primer and 3-prime with respect to the annealing portion of the sequencing primer, or (c) The system according to claim 1, comprising at least one functional nucleic acid which is a spacer nucleic acid.

20. The orthogonal DNA barcode comprises a plurality of DNA barcode modules, each of which is linked to a different site on the bead by direct or linker, and each of the plurality of DNA barcode modules is (a) Suitable for use as an annealing site for sequencing primers, (b) A hairpin structure capable of forming a hairpin structure, wherein the hairpin structure includes a sequencing primer, an annealing portion of the sequencing primer, and beads, wherein the beads are 5-prime with respect to the sequencing primer and 3-prime with respect to the annealing portion of the sequencing primer, or (c) The system according to claim 1, comprising at least one functional nucleic acid which is a spacer nucleic acid.

21. A method for controlling the concentration of a compound in a solution present in a picowell, wherein the method is applied to a bead-bound compound in the picowell, the picowell contains a solution, the bead-bound compound is bound to the beads via a cleavable linker, and the method is (a) A step of exposing the bead-bonded compound to conditions that result in the cleavage of the cleavable linker, wherein the conditions include light capable of cleaving the cleavable linker. (b) a step of allowing the release of the bead-bound compound from the beads and generating the released compound, wherein, following the release, a substantially uniform concentration of the compound in the solution is obtained by diffusion or dispersion of the released compound in the solution. (c) A method comprising the step of adjusting the conditions to produce a determined concentration of substantially uniform concentration, wherein the determined concentration is calculated taking into account the concentration of released fluorophores released from a bead-bonded release monitor.

22. The above conditions are adjusted by adjusting one or more of the wavelength of light and the intensity of light, and by the duration of exposure, optionally (i) The concentration of the released fluorophore released from the bead-bound release monitor is determined at the same time as the release of the bead-bound compound from the beads and the generation of the released compound, or (ii) The method according to claim 21, wherein the concentration of the released fluorophore released from the bead-bound release monitor is determined substantially prior to the release of the bead-bound compound from the beads and the generation of the released compound.

23. A cap combined with a picowell plate containing multiple picowells, The cap can be used together with the picowell plate. Each of the plurality of picowells is defined by a hole, a floor, and a wall, the wall being defined by the upper part of the hole and the bottom part of the floor, the hole being circular, the floor being circular, and the wall being in the form of a truncated cone surface. The hole has a first diameter, and the floor has a second diameter, and the first diameter is larger than the second diameter. The cap is a spherical cap capable of fitting snugly into the hole, the hole is made of a polymer having a larger durometer (harder), and the cap is made of a polymer having a smaller durometer (softer). The relative durometer of the cap and the hole allows the spherical cap to fit reversibly and snugly into the hole, and the cap (i) The picowell can be plugged to prevent leakage, (ii) A passive cap that can absorb metabolites released from cells while cells in a cell culture medium are being cultured in the picowell, (iii) Active caps, taking the form of beads containing multiple essentially identical compounds, A cap in which each of the plurality of essentially identical compounds is bound to a bead by a cleavable linker, and at least one of the plurality of picowells contains an aqueous medium, wherein cleavage of the cleavable linker releases at least some of the plurality of essentially identical compounds from the beads into the aqueous medium.

24. A picowell array plate having a generally flat top and a plurality of picowells, each picowell having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor by a wall, and the wall being located between the upper hole and the floor, A system comprising, optionally, a bead placed in at least one of the plurality of picowells, the bead comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds, The picowell array plate further includes a mat capable of reliably covering, or substantially reliably covering, the openings located above at least one or all of the plurality of picowells, wherein the reliably covering is reversible, and the mat is optionally (a) an absorbent surface that, when positioned in contact with the general upper plane of the picowell array plate, is capable of absorbing any metabolites, biochemicals, or proteins that may be contained in one or more of the picowells, (b) A system comprising one or all of the bonding surfaces capable of maintaining reversible adhesion to the general plane on the upper part of the picowell array plate.

25. A method for determining the signal from an assay and the readout of sequencing on beads, thereby identifying one or more target compounds from the assay, (a) Providing a plurality of beads, each bead comprising a plurality of compounds attached to the bead and a plurality of oligonucleotides which are substantially related to each other, wherein the plurality of oligonucleotides attached to each bead identify the plurality of compounds attached to the same bead; (b) The step of performing the assay with the plurality of compounds attached to the beads, (c) A step of determining at least one signal that reflects the performance of the compound in the assay of step b, (d) A step of sequencing the plurality of oligonucleotides attached to the beads without removing the oligonucleotides from the beads, thereby determining the readout of the sequence for each bead, A method comprising: (e) identifying the compound attached to the beads by reading out the sequencing in step d and associating it with the assay performance contained in the determined signal in step c, wherein the beads having signals from the assay and the reading out of the sequencing identify and associate the compound of interest.

26. A method for screening a compound library for compounds having desired properties, (a) To provide a plurality of beads, each bead comprising a plurality of oligonucleotides attached to the surface of the bead and a plurality of substantially related compounds attached to the surface of the bead, wherein the sequence of the oligonucleotides attached to the bead codes for the identity of the plurality of substantially related compounds attached to the surface of the bead, (b) Incorporating the plurality of beads into an assay for desired properties of compounds in the compound library, (c) capturing a signal from at least one bead, wherein the signal reflects the performance of the compound on the bead in the assay. (d) Sequencing the plurality of oligonucleotides attached to at least one bead from which the assay signal has been further captured, without removing the oligonucleotides from the beads. (e) A method comprising identifying at least one compound from the sequence readout of step (d) and relating it to the corresponding assay performance captured in the signal of step (c).

27. The method according to claim 26, wherein each bead comprises a plurality of different oligonucleotides and a plurality of substantially related compounds.

28. The method according to any one of claims 25 to 27, wherein the plurality of oligonucleotides are a plurality of DNA oligonucleotides.

29. The method according to claim 25, wherein the plurality of compounds are attached to the surface of the beads by linking the plurality of compound components in tandem, and all of the compound components together constitute the compound.

30. The method according to claim 29, wherein each DNA module and each compound component is assembled over time and alternatively.

31. The method according to any one of claims 25 to 30, wherein each compound in a plurality of identical compounds is attached to the bead surface via a cleavable linker.

32. The method according to claim 31, wherein the cleavable linker is a light-cleavable linker, a protease-cleavable linker, or an acid-cleavable linker.

33. The method according to any one of claims 25 to 32, wherein the compound is cut from the bead surface after step (a) and before step (d).

34. The method according to any one of claims 25 to 33, wherein the signal reflecting the desired properties of the compound is a fluorescence signal.

35. The method according to any one of claims 25 to 34, wherein the size of each bead is 1 μm to 100 μm.

36. The method according to claim 35, wherein the size of each bead is 1 μm to 10 μm.

37. The method according to claim 36, wherein the size of each bead is approximately 3 μm.

38. The method according to any one of claims 25 to 37, further comprising identifying a candidate target among a plurality of potential targets, wherein the compound having the desired properties binds to the candidate target.

39. The method according to claim 38, wherein step (b) comprises incubating the plurality of beads in the plurality of potential targets.

40. The method according to claim 37 or 38, wherein the potential target is a protein or nucleic acid.

41. The method according to any one of claims 25 to 40, wherein the sequencing is performed by single-molecule real-time sequencing, ion semiconductor sequencing, pyrosequencing, synthesis sequencing, bridge amplification sequencing, ligation sequencing, nanopore sequencing, chain termination sequencing, ultra-parallel signature sequencing, Polony sequencing, heliscope single-molecule sequencing, shotgun sequencing, SOLiD sequencing, Illumina sequencing, tunnel current DNA sequencing, hybridization sequencing, sequencing using mass spectrometry, microfluidic Sanger sequencing, and oligonucleotide extension sequencing.

42. A method for screening a compound library for compounds having desired properties, (a) To provide a plurality of beads, each bead comprising a plurality of oligonucleotides attached to the surface of the bead and a plurality of substantially related compounds attached to the surface of the bead, wherein the sequence of the oligonucleotides attached to the bead encodes the synthesis history of the plurality of substantially related compounds attached to the surface of the bead, (b) Incorporating the plurality of beads into an assay for desired properties of compounds in the compound library, (c) capturing a signal from at least one bead, wherein the signal reflects the performance of the compound on the bead in the assay. (d) Sequencing the plurality of oligonucleotides attached to at least one bead from which the assay signal has been further captured, without removing the oligonucleotides from the beads. A method comprising (e) identifying at least one compound from the sequence readout of step (d) and relating it to the corresponding assay performance captured in the signal of step (c).

43. The method according to claim 42, wherein the assay includes a binding assay.

44. The method according to claim 42, wherein the assay includes an activity assay.

45. The method according to claim 42, 43, or 44, wherein the assay comprises a competitive binding assay or a competitive inhibition assay.

46. The method according to claim 42, wherein the assay comprises an interaction between a non-conjugated compound and other assay reagents, and the non-conjugated compound is a compound released from the surface of the beads.

47. The method according to claim 45, wherein the compound is released by cutting a severable linker connecting the compound to the beads.

48. The method according to claims 42 to 47, wherein the assay occurs within a plurality of restricted volumes, and a nominal single bead is dispersed within each restricted volume.

49. The method according to claim 48, wherein the limited volume includes aqueous droplets.

50. The method according to claim 49, wherein the aqueous droplets are suspended in an oil medium or a hydrophobic liquid medium.

51. The method according to claim 48, wherein the limited volume includes picowells.

52. The method according to claim 50, wherein the picowells are organized into a regular array.

53. The method according to claim 51, wherein the plurality of limited volumes are organized into a regular array.

54. The method according to claim 48, wherein the limited volume includes a layer of aqueous medium adhered around the beads, and the beads are suspended in a hydrophobic medium.

55. The method according to claim 42, wherein the assay reagent is washed away before sequencing the oligonucleotide.

56. The method according to claim 42, wherein the sequencing step (d) is performed before the assay step (b).

57. The method according to claim 56, wherein the oligonucleotide on the beads is removed after the sequencing step but before the assay step.

58. The method according to claim 57, wherein the removal of the oligonucleotide includes enzymatic digestion, chemical cleavage, thermal decomposition, or physical shearing.

59. The method according to claim 43, wherein the binding assay includes binding of RNA molecules to the beads.

60. The method according to claim 43, wherein the signal from the beads includes sequencing of a bound RNA molecule.

61. The method according to claim 42, wherein the binding assay comprises a fluorescently labeled binding assay, and the molecule that binds to the compound on the beads comprises a fluorophore.

62. The method according to claim 42, wherein the binding assay comprises a nucleic acid-labeled binding assay, the molecule that binds to the compound on the beads comprises a nucleic acid tag, and the signal from the assay further comprises sequencing of the nucleic acid tag attached to the molecule that binds to the compound on the beads.

63. The aforementioned desired characteristics are (i) Inhibiting or stimulating the catalytic activity of an enzyme, (ii) Stimulating a Th1 immune response that can be measured by a cell line assay or in vivo assay, (iii) Stimulating a Th2 immune response that can be measured by a cell line assay or in vivo assay. (iv) Inhibiting the Th1 type immune response measurable by cell line assays or in vivo assays, (v) Inhibiting the Th2 type immune response measurable by cell line assays or in vivo assays, (vi) The method according to claim 42, comprising one or more of stimulating or inhibiting ubiquitin-mediated degradation of a protein, which can be measured by purified protein, cell line assay, or in vivo assay.

64. A system for screening a compound library for compounds having desired activity, (a) A sample compartment for receiving beads to which multiple compounds are attached and which are encoded with oligonucleotides, (b) A plurality of encapsulation compartments within the sample compartment, each encapsulation compartment nominally containing a single bead dispersed in an assay medium, and further comprising a reagent whose interaction with the compound on the bead is assayed and yields a measurable signal, (c) A detector for measuring the signal, (d) Sequencing platform and (e) A system including a user interface for receiving one or more commands from a user.

65. The system according to claim 64, wherein the encapsulated compartment contains a liquid droplet.

66. The system according to claim 64, wherein the encapsulated compartment includes a picowell.

67. The system according to claim 64, wherein the encapsulated compartment further contains an assay reagent.

68. The system according to claim 64, wherein the detector includes an optical detector.

69. The system according to claim 64, wherein the sequencer includes an optical detector.

70. A method of perturbing cells, (a) providing a perturbation encoded by a nucleic acid and restricting a cell with the perturbation encoded by the nucleic acid, (b) Contacting the cells with a perturbation encoded by the nucleic acid within a limited volume, wherein the onset and dose of the perturbation are controlled, (c) Incubating the cells with a perturbation encoding the nucleic acid for a specific period of time, (d) A method comprising transferring the nucleic acid encoding a perturbation into the cell.

71. The method according to claim 70, wherein the perturbation encoded in the nucleic acid is a compound or drug molecule encoded in the nucleic acid.

72. The method according to claim 70 or 71, wherein the perturbation encoded in the nucleic acid is a DNA-encoded library.

73. The method according to any one of claims 70 to 72, wherein the perturbation and the nucleic acid encoding the perturbation are not attached to the solution but are free.

74. The method according to any one of claims 70 to 73, wherein the perturbation and the nucleic acid encoding the perturbation are attached to each other.

75. The method according to any one of claims 70 to 72, wherein the perturbation and the nucleic acid encoding the perturbation are attached to the same substrate but are not attached to each other.

76. The method according to claim 75, wherein the attachment of the perturbation to the substrate and the attachment of the nucleic acid to the substrate are cleavable attachments.

77. The method according to claim 7, wherein the cleavable adhesion is selected from the group consisting of light-cleavable adhesion, temperature-cleavable adhesion, pH-sensitive adhesion, acid-cleavable adhesion, base-cleavable adhesion, sound-cleavable adhesion, salt-cleavable adhesion, redox-sensitive adhesion, or physically cleavable adhesion.

78. The method according to any one of claims 70 to 77, wherein restricting the cells and the perturbation includes droplet encapsulation, emulsion encapsulation, picowell encapsulation, macrowell encapsulation, physical adhesion, bubble encapsulation, or microfluidic restriction.

79. The method according to any one of claims 70 to 78, wherein the control over the perturbation includes controlling light exposure, temperature exposure, pH exposure, time exposure, sound exposure, salt exposure, chemical or physical redox potential, or mechanical stirring exposure.

80. The method according to any one of claims 75 to 79, wherein the incubation includes exposing the cells to the perturbation after the perturbation has been cleaved from the substrate, or after the nucleic acid has been cleaved from the substrate.

81. The method according to any one of claims 75 to 79, wherein the incubation includes exposing the cells to the perturbation without cleaving the perturbation from the substrate or without cleaving the nucleic acid from the perturbation.

82. The method according to any one of claims 70 to 81, wherein transferring the nucleic acid encoding the perturbation described in the nucleic acid to the cell comprises attaching the nucleic acid to the cell surface of the cell.

83. The method according to claim 82, wherein attaching the nucleic acid to the cell surface of the cell includes inserting the nucleic acid into the cell membrane.

84. The method according to claim 82, wherein attaching the nucleic acid to the cell surface of the cell includes attaching the nucleic acid to biomolecules on the cell surface.

85. The method according to claim 84, wherein the biomolecule is a protein or a carbohydrate.

86. The method according to any one of claims 82 to 85, wherein attaching the nucleic acid to the cell surface of the cell includes attaching it via an arbitrary tag on the nucleic acid.

87. A method for perturbing cells with a perturbation and encoding the cells with the identity of the perturbation, (a) To provide a library encoded in bead-bound DNA, (b) Restricting cells with a library encoded on the bead-bound DNA, wherein the library encoded on the bead-bound DNA comprises one or more copies of combinatorially synthesized compounds and one or more copies of encoded nucleic acid tags, the compounds and the encoded nucleic acids are attached to beads, the encoded nucleic acids encode the identity of the compounds, and the library encoded on the bead-bound DNA and the cells are restricted to a restricting volume. (c) Releasing the compound from the beads and incubating the compound with the cells in the restricted volume, (d) Optionally, releasing the coded nucleic acid tag from the beads, (e) A method comprising attaching the coded nucleic acid tag to the cell, thereby maintaining the identity of the compound via the coded nucleic acid tag attached to the cell.

88. A method for perturbing a cell, encoding the cell by the identity of the perturbation, and measuring the cell's response to the perturbation, (a) Contacting cells with a bead-bound DNA coding library in a first limited volume, wherein the bead-bound DNA coding library comprises one or more copies of combinatorially synthesized compounds and one or more copies of coding nucleic acid tags, the compounds and the coding nucleic acids are attached to beads, and the coding nucleic acids encode the identity of the compounds, (b) releasing the compound from the library from the beads and incubating the compound from the library with the cells in the first limited volume, (c) Optionally, releasing the encoded nucleic acid tag from the beads within the first limited volume, (d) The encoded nucleic acid tag is captured on the cell surface of the cell, thereby exposing the cell to the compound in the library, and the identity of the exposed compound is captured on the cell surface. (e) Releasing the cells from the first limited volume, wherein the encoding nucleic acid tag is attached to the cells and the encoding nucleic acid tag codes for the identity of the compound to which the cells are exposed. (f) Capture a previously perturbed and nucleic acid-tagged cell with a second limited volume of response-detection beads, wherein the cell is exposed to lysis conditions that expose the cell contents to the response-capture beads, and the response-capture beads include a capture probe for capturing the cell contents and a nucleic acid tag encoding the perturbation in the previously perturbed and nucleic acid-tagged cell. (g) Incubating the response capture beads with lysed cells in the second limited volume, thereby capturing both the cell contents and the nucleic acid tag encoding the perturbation to the response capture beads, (h) Optionally, converting the cell's response to the perturbation into a nucleic acid signal, wherein the cell's response to the perturbation is not a nucleic acid signal. (i) A method comprising sequencing the nucleic acid tag attached to the response capture bead, thereby correlating the identity of the perturbation with the cell's response to the perturbation.

89. A method for perturbing cells and capturing the cell's response to the perturbation, (a) To provide a picowell array and a library of functionalized perturbation beads, wherein the picowell is capable of accommodating a single cell and a single functionalized perturbation bead, and each functionalized perturbation bead comprises a plurality of different substantially identical releaseable compounds and a plurality of nucleotide barcodes encoding the compounds, the nucleotide barcodes being functionalized barcodes capable of capturing the cellular contents of the cell, and the cellular contents of the cell comprising a cellular response to the perturbation contained in the functionalized perturbation bead, (b) capturing a single cell in each picowell of the picowell array, (c) capturing a single functionalized perturbation bead in the picowell containing a single cell, (d) Releasing the compound from the functionalized perturbation beads and incubating the cells with the released compound, wherein the compound between the picowells has minimal diffusion during incubation. (e) Lysizing the cells and releasing the cellular contents, (f) capturing one or more components of the cell contents onto functionalized oligonucleotides on the functionalized perturbed beads, wherein the capture includes hybridization and enzymatic extension for combining the nucleotide barcode with the nucleic acid elements of the cell contents, thereby forming a hybrid of the nucleotide barcode and the nucleic acid elements of the cell contents. (g) A method comprising releasing the hybrid, collecting the hybrid from the library of functionalized perturbation beads, sequencing the hybrid, and thereby relating the perturbation to the cellular response to the perturbation.

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