Chemical libraries encoded by oligonucleotides

Abstract

The present application is entitled Oligonucleotide-encoded chemical libraries. The present application provides a bead having a covalently attached compound and a covalently attached DNA barcode and methods of using such beads. The bead has a plurality of substantially identical copies of the compound and a plurality of substantially identical copies of the DNA barcode. The compound is comprised of one or more chemical monomers, wherein the DNA barcode is in the form of a barcode module, wherein each module corresponds to a corresponding chemical monomer and allows identification of the corresponding chemical monomer. The nucleic acid barcode can have a cascade structure or an orthogonal structure. A method for sequencing the bead-bound nucleic acid barcode, for cleaving the compound from the bead, and for assessing the biological activity of the released compound is provided.

Description

[0001] This application is a divisional application of the original application with the filing date of September 24, 2018, application number 2018800720827, and the title of "Oligonucleotide-encoded chemical libraries". TECHNICAL FIELD

[0002] The present disclosure relates to high-throughput screening using a library of compounds, wherein the compounds are bound to or contained in beads, each bead containing multiple copies of one compound, wherein further the beads also contain a DNA tag encoding the identity or synthesis history of the compound contained within or on the bead. The present disclosure also relates to high-throughput assays performed in picowells, wherein the picowells contain beads loaded with compounds and assay material. The present disclosure further relates to releasing the bead-bound compounds and screening them for biological activity. Broadly speaking, the present disclosure envisions assays in which the beads are used as delivery vehicles for the compounds, as well as methods for producing such beads loaded with compounds.

[0003] The present disclosure relates to bead-bound compounds, wherein each compound is made from one or more monomers belonging to a chemical library. The present disclosure also relates to bead-bound DNA barcodes, i.e. to nucleic acids, wherein the sequence of each nucleic acid is a code (unrelated to the genetic code) referring to a specific chemical library monomer. The present disclosure further relates to releasing the bead-bound compounds and then screening the released compounds for biological activity.

[0004] The present disclosure also generally relates to methods for perturbing a cell or several cells with dose-controlled compounds and analyzing the change in cell state by RNA and / or protein analysis. The methods disclosed herein can be applied on a single cell level or on multiple cells for the purpose of high-throughput screening, target discovery or diagnostics and other similar applications.

[0005] Cross Reference to Related Applications

[0006] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 62 / 562,905, filed September 25, 2017, and also to U.S. Provisional Patent Application Serial No. 62 / 562,912, also filed September 25, 2017, the contents of which are incorporated by reference in their entirety. BACKGROUND

[0007] For example, combinatorial chemistry involving split and pool chemistry can be used to synthesize large numbers of compounds. Compounds made in this way are used in the field of pharmaceutical chemistry, where the compounds can be screened for various biochemical activities. These activities include binding to one or more proteins, where the protein is known at the time the screening test is performed. Alternatively, the protein bound by the compound being tested is identified only after a binding event is detected. Compounds can also be screened for inhibiting or activating the activity of a known protein (this is not just screening for "binding" activity). Alternatively, compounds can be screened for inhibiting or activating a cellular function, and where the molecular target is unknown to the researcher at the time of screening.

[0008] Screening of compounds, such as those belonging to a large chemical library made by split and pool methods, can be facilitated by screening with arrays of thousands of micro-wells, nano-wells, or pico-wells. Furthermore, screening can be facilitated by providing different compounds to each pico-well in the form of a bead, and where each bead contains hundreds of copies of the same compound, and where the same bead also contains hundreds of copies of a "DNA barcode" that can be used to identify the compound attached to the same bead. Furthermore, screening of compounds is further facilitated using a cleavable linker, where the cleavable linker allows for the controlled release of the compound from the bead, and where the released compound is then used in a biochemical assay or cell-based assay in the same pico-well.

[0009] For example, screening of compounds in very small confined volumes, such as droplets, pico-wells, or microfluidic environments, has wide advantages due to the small amount of assay reagents required, thus not necessarily limiting to combinatorially generated compounds. Any method that allows for the loading of compounds onto beads, also allows for the elution of the compounds from the beads at a later time, can be used to deliver the bead-bound compounds to an assay in a small confined volume. The addition of nucleic acid barcodes to the beads enables the identity of the compound present in the bead to be carried into the assay volume. In this way, very high throughput assays can be performed without the need for spatial indexing of the compounds within a robot or microtiter plate. Millions to billions of compounds can be saved in one vial, with the identity of the compounds marked on the same bead (with DNA) containing each individual compound.

[0010] A common approach to drug discovery involves selecting a target of interest and monitoring the interaction of the target protein or enzyme with a large library of compounds. In many cases, a large number of initial hits are found to be toxic to the body or cross-react with other proteins in the body, making target-based selection an inefficient method for drug screening. The need for a pre-selected target is also an inherent limitation, as it requires that the biological basis of the disease be known and understood. Screening compounds against an entire organism is a difficult, expensive, and extremely low throughput task.

[0011] Conventional phenotypic screening of cells involves establishing a model of a diseased state cell, contacting the cell with various drug libraries, and monitoring whether the disease phenotype is corrected by a measurable assay. Such screening methods are referred to as phenotypic screening because the underlying biological mechanism is not necessarily known at the outset, but measurable phenotypic changes indicative of a therapeutic response are taken as relevant indicators. Today, there are a large number of cell lines and disease models that reflect various baseline and diseased cell states. There are also a large number of compound libraries and candidate biologic drugs that can be used. The apparent screening activity of combining different cell models with different candidate drugs to find phenotypic responses is fraught with technical limitations because the assays are limited to microtiter plate format and imaging modalities, both of which are severely limited in throughput.

[0012] One approach to overcome flux limitations is to employ high flux single cell screening methods for drug discovery (see, e.g., Heath et al., Nat Rev Drug Discov. 15:204-216, 2016). In these methods, single cells are isolated and partitioned into compartments where individual assays can be performed on each cell. Genomic analysis by mRNA sequencing of single cells, for example using droplet encapsulation, is a popular approach that reveals 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, the disclosures of which are incorporated by reference herein in their entireties). The most advanced single cell analysis platforms currently available have enabled quantification of mRNA transcripts with single cell resolution, allowing cells to be characterized and fingerprinted based on their transcriptional state. This approach allows for comparison of differences between tissue samples extracted from subjects or prepared in experiments and examination of single cell transcription, and thus the expression state of proteins. Measuring single cell mRNA by transcriptome sequencing and profiling is an important approach not only to study molecular mechanisms of cell lineage phenotypes during disease progression, but also to study molecular mechanisms of drug efficacy, resistance, and discovery of therapeutic targets (see, e.g., Chu et al., Cell Biol and Toxicol 33:83-97, 2017, Wang, Cell Biol and Toxicol 32:359-361, 2016, and Wang et al., Cell Biol and Toxicol 33:423-427, 2017). The application of single cell RNA sequencing has been used to define intercellular heterogeneity, evidenced by transcriptomic intercellular variation, which is highly relevant to drug efficacy and specificity, transcriptional stochasticity, transcriptomic plasticity, and genomic evolution. Encapsulation in picowells has also been demonstrated (see, e.g., Gierahn et al., Nat Methods 14:395-398, 2017). Single cell protein measurements can also be performed using similar isolation methods (Butnik et al., BioRxiv, January 2017, Su et al., Proteomics 17:3-4, 2017).

[0013] While high-throughput single-cell RNA sequencing (RNA-seq) methods, including commercialized versions of automated platforms such as Fluidigm C1, 10X Genomics or 1CellBiO systems, are rapidly emerging, the lack of methods to effectively partition different drugs to different cells limits the application of single-cell RNA profiling in targeted-agnostic high-throughput drug screening and target discovery. The number of drugs that can be detected is limited by the plate capacity when cells or tissues are incubated with different perturbations in a well plate and then compared between single-cell analysis and transcriptional profiling. Further, the requirement to prepare barcoded mRNA from each isolated sample and then perform a comprehensive RNA profile of each sample also creates a major bottleneck. SUMMARY

[0014] Briefly, the disclosure provides a system for screening compounds, the system comprising: (a) a picowell array plate comprising a plurality of picowells, wherein each picowell has a top aperture defining an opening at a top of the picowell, a bottom defined by a floor, wherein the top aperture is spaced apart from the floor by a wall, and wherein the wall is between the top aperture and the floor; (b) a bead disposed in a picowell, wherein the bead comprises a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds; (c) wherein the bead comprises bead-bound DNA barcodes in the form of a concatenated DNA barcode or an orthogonal DNA barcode, and wherein if the DNA barcode is in the form of a concatenated DNA barcode, the concatenated DNA barcode is prepared by a method using one or both of: (i) using click chemistry, or (ii) using a repeat step cycle, wherein the repeat step cycle comprises using a splint oligonucleotide that is hybridizable to a partially made bead-bound DNA barcode, and wherein the hybridization is mediated by an annealing site on the splint oligonucleotide and a corresponding complementary annealing site in the partially made bead-bound DNA barcode, wherein the annealed splint oligonucleotide serves as a template for extending the partially made DNA barcode using a DNA polymerase, and wherein the splint oligonucleotide contains bases that are complementary to a DNA barcode module to be polymerized with the partially made DNA barcode, (d) wherein each compound of the plurality of substantially identical bead-bound compounds comprises one or more chemical library monomers, and wherein each bead-bound DNA barcode module identifies a corresponding chemical library monomer, wherein the term “compound” is used to refer to a complete product that comprises one or more chemical library members, and wherein a complete DNA barcode identifies the compound.

[0015] The floor of the microwell, nanowell, or picowell need not be flat. The floor can be curved like the bottom of a glass test tube or a metal centrifuge tube. In addition, the floor can be tapered like a tapered centrifuge tube. The floor can be flat but have notches, for example, that facilitate movement of assay solution or cell culture fluid near the bottom of any bead located in the picowell. In embodiments of a flat floor, the present system and method can require a flat floor. The cascade DNA barcode can be made entirely by organic chemistry methods, for example, by click chemistry. In addition, the orthogonal DNA barcode can be made entirely by organic chemistry methods, for example, including click chemistry.

[0016] Also provided is the above system further comprising a plurality of lids, each lid being capable of fitting into the opening of a different picowell, and each lid being capable of minimizing or preventing evaporation of fluid inside the picowell, and each being capable of minimizing or preventing leakage of fluid inside the picowell.

[0017] Further included is the above system wherein the cascade DNA barcode is made by a method that uses both: (i) click chemistry and the repeating step cycle using the splint oligonucleotide; (ii) click chemistry and a chemical method that is not a click chemistry method; (iii) click chemistry only; or (iv) the repeating step cycle using the splint oligonucleotide only. For this particular implementation, the "cascade DNA barcode" in question does not include any chemical coupling agent used to couple a nucleic acid directly to a bead.

[0018] In embodiments of a spherical lid, the above system is provided further comprising a plurality of spherical lids, wherein each lid is capable of fitting into the orifice of a picowell, wherein the orifice is circular, and each lid is capable of minimizing or preventing evaporation of fluid inside the picowell, and each lid is capable of minimizing or preventing leakage of fluid inside the picowell.

[0019] In embodiments of a response element, the above system is provided wherein the at least one bead disposed in at least one picowell comprises at least one response capture element coupled to the at least one bead. In addition, the above system is contemplated wherein the at least one bead disposed in at least one picowell comprises at least one response capture element coupled to the at least one bead, wherein the at least one response capture element comprises: (a) poly(dT) or (b) an exon-targeting RNA probe.

[0020] Also contemplated are the above systems, wherein the DNA barcode is a concatenated DNA barcode or an orthogonal DNA barcode, and wherein the DNA barcode comprises one or more DNA barcode modules, wherein each DNA barcode module of the one or more DNA barcode modules encodes information identifying a chemical library monomer, and wherein the concatenated DNA barcode or the orthogonal DNA barcode further comprises one or both of: (a) one or more functional nucleic acids; (b) one or more nucleic acids encoding information of a type other than the identity of a chemical library monomer.

[0021] Disclosed below are “consisting only of’ embodiments and “comprising” embodiments as they apply to the number of bead-bound DNA barcode modules that make up a DNA barcode. Embodiments are provided in which a DNA barcode consists only of one DNA barcode module or only two DNA barcode modules, or contains only three DNA barcode modules or only four DNA barcode modules, and so on, or in which a DNA barcode comprises at least one DNA barcode module, or comprises at least two DNA barcode modules, or comprises at least three DNA barcode modules, or comprises at least four DNA barcode modules, and so on,

[0022] Contemplated is a system, wherein the bead-bound concatenated DNA barcode comprises: (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 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 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.

[0023] Further, contemplated is the above system, further comprising a primer binding site capable of binding a DNA sequencing primer, wherein the primer binding site is capable of directing sequencing of one or more of the first DNA barcode module, the second DNA barcode module, the third DNA barcode module, the fourth DNA barcode module, the fifth DNA barcode module, or the sixth DNA barcode module, and wherein the primer binding site is located 3' of the first DNA barcode module, 3' of the second DNA barcode module, 3' of the third DNA barcode module, 3' of the fourth DNA barcode module, 3' of the fifth DNA barcode module, or 3' of the sixth DNA barcode module, or wherein the primer binding site is located between the first DNA barcode module and the second DNA barcode module, or between the second DNA barcode module and the third DNA barcode module, or between the third DNA barcode module and the fourth DNA barcode module, or between the fourth DNA barcode module and the fifth DNA barcode module, or between the fifth DNA barcode module and the sixth DNA barcode module.

[0024] Additionally, provided is the above system, wherein the primer binding site is located between the first DNA barcode module and the second DNA barcode module, or between the second DNA barcode module and the third DNA barcode module, or between the third DNA barcode module and the fourth DNA barcode module, or between the fourth DNA barcode module and the fifth DNA barcode module, or between the fifth DNA barcode module and the sixth DNA barcode module. In embodiments regarding the location of the primer binding site relative to upstream DNA barcode modules and relative to downstream DNA barcode modules, provided is the above system, wherein a primer binding site is located between each and every pair of consecutive DNA barcode modules.

[0025] Further, provided is the above system, wherein the bead comprises a DNA barcode that is itself an orthogonal DNA barcode, wherein the bead comprises an outer surface, and wherein the orthogonal DNA barcode comprises: (a) a first nucleic acid comprising a first DNA barcode module and an annealing site for a sequencing primer, wherein the first nucleic acid is coupled to the bead at a first location, (b) a second nucleic acid comprising a second DNA barcode module and an annealing site for a sequencing primer, wherein the second nucleic acid is coupled to the bead at a second location, and (c) a third nucleic acid comprising a third DNA barcode module and an annealing site for a sequencing primer, wherein the second nucleic acid is coupled to the bead at a third location, and wherein the first location, the second location, and the third location on the bead are each located at different locations on the outer surface of the bead.

[0026] In an encoding embodiment, the above system is provided, wherein the DNA barcode comprises one or more nucleic acids that do not identify any chemical library monomer, but instead identify: (a) a class of compounds that are cleavably linked to the bead; (b) a step in a multi-step organic synthesis pathway; (c) a date of synthesis of the compound bound to the bead; (d) a disease that the compound bound to the bead is intended to treat; (e) a cellular event that the compound bound to the bead is intended to stimulate or inhibit; or (f) a reaction condition used to couple a given chemical library monomer to the bead.

[0027] In a linker embodiment, the above system is provided, wherein each compound in the plurality of substantially identical bead-bound compounds is coupled to the bead by a cleavable linker. Also provided is the above system, wherein each compound in the plurality of substantially identical bead-bound compounds is coupled to the bead by a photocleavable linker. Also provided is the above system, wherein each compound in the plurality of substantially identical bead-bound compounds is coupled to the bead by a non-cleavable linker.

[0028] In a release monitor embodiment, the above system is provided, wherein the at least one picowell contains a release monitor bead, and does not contain any other type of bead, In an embodiment, the above system is provided, wherein the at least one bead comprises a graft copolymer consisting of a low cross-linked polystyrene matrix grafted with polyethylene glycol (PEG).

[0029] In a release monitor embodiment, the above system is provided, wherein the at least one picowell contains a release monitor bead, and does not contain any other type of bead,

[0030] wherein the release monitor bead comprises a bead-bound quencher and a bead-bound fluorophore, wherein the bead-bound quencher is positioned in close proximity to the bead-bound fluorophore in a quenching relationship and is capable of quenching fluorescence of 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 bead-bound fluorophore, and wherein the bead-bound fluorophore is bound by a first photocleavable linker, wherein the picowell containing the release monitor bead is a first picowell, wherein the first picowell contains a first solution, wherein exposure of the first picowell to cleavage conditions is capable of cleaving the photocleavable linker and releasing the fluorophore into the first solution of the first picowell, wherein exposure causes the fluorophore to diffuse throughout the first solution in the first picowell, and wherein a fluorescence signal obtained by illuminating light on the first picowell (containing the first solution including the dispersed fluorophore) allows a user to use the fluorescence signal to calculate a percentage of release of the bead-bound fluorophore from the release monitor bead, resulting in a calculated value of the percentage of release, and wherein a second picowell contains a bead-bound compound coupled to a photocleavable linker of the same type as the first photocleavable linker, wherein the second picowell contains a second solution,

[0031] and wherein the calculated value of the percentage of release of the release monitor bead from the first picowell allows calculation of the concentration of the released compound in the second solution of the second picowell.

[0032] In embodiments relating to the identity of all compounds bound to a given bead or relating to the identity of all DNA barcodes bound to a given bead, the above-described system is provided, wherein the at least one bead comprises a plurality of substantially identical bead-bound DNA barcodes, wherein the plurality is 10 million to 1 billion copies of the substantially identical bead-bound DNA barcodes. Also provided is the above-described system, wherein the at least one bead comprises a plurality of substantially identical bead-bound compounds, wherein the plurality is 10 million to 1 billion copies of the substantially identical bead-bound compounds.

[0033] In embodiments relating to cells (e.g., mammalian cells, cancer cells, bacterial cells), the above-described system is provided, wherein the at least one picowell comprises at least one cell, wherein the plurality of substantially identical bead-bound compounds are bound to the at least one bead by a cleavable linker, and wherein cleaving the cleavable linker releases the bead-bound compound from the bead to produce a released compound, and wherein the released compound is capable of contacting the at least one cell. In other cell embodiments, the above-described system is provided, wherein the at least one picowell comprises at least one cell, wherein the plurality of substantially identical bead-bound compounds are bound to the at least one bead by a cleavable linker, and wherein cleaving the cleavable linker releases the bead-bound compound from the bead to produce a released compound, and wherein the released compound is capable of contacting the at least one cell, and wherein the at least one cell is: (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 Plasmodium cell, (vii) a cell that has metabolic activity but has a cross-linked genome and is unable to undergo cell division, or (ix) a mammalian cell infected with a virus.

[0034] In device embodiments, the above-described system is provided, wherein each picowell has a top aperture defining an opening at a top of the picowell, a bottom defined by a floor, wherein the top aperture is spaced apart from the floor, and wherein there is a wall between the top aperture and the floor, and wherein the aperture is circular, wherein the floor is circular, and wherein the wall takes the form of a truncated cone, and the aperture has a first diameter, the floor has a second diameter, and wherein the first diameter is greater than the second diameter.

[0035] In other device-related embodiments, the above system is provided, wherein each picowell has a top aperture defining an opening at a top of the picowell, a bottom defined by a floor, wherein the top aperture is spaced apart from the floor, and wherein there is a wall between the top aperture and the floor, and wherein the aperture is circular, wherein the floor is circular, and wherein the wall takes the form of a truncated cone, and the aperture has a first diameter, the floor has a second diameter, and wherein the first diameter is greater than the second diameter, further comprising further comprising a cap that fits tightly into the aperture, wherein the aperture is composed of a polymer that is greater (harder) on the durometer scale, and wherein the cap is made of a polymer that is less (softer) on the durometer scale, and wherein the relative durometer of the cap and the aperture allows the cap to reversibly and tightly fit into the aperture, and wherein the cap is: (i) a cap that is intended only to plug the picowell and prevent leakage, (ii) a cap that is passive in itself and is capable of absorbing metabolites released by cells in the case of cells in cell culture medium being cultured in the picowell, (iii) a cap that is active in itself and takes the form of a bead comprising a plurality of substantially identical compounds, and wherein each compound of the plurality of substantially identical compounds is coupled to the bead by a cleavable linker; (iv) a cap that is active in itself and takes the form of a bead comprising a plurality of identical reagents, and wherein each reagent of the plurality of substantially identical reagents is coupled to the bead by a cleavable linker. The above system is also provided, wherein the cap is spherical, or wherein the cap is non-spherical.

[0036] In a cushion embodiment, the above system comprises a picowell array plate comprising a generally planar upper surface, a plurality of picowells, wherein each picowell has a top aperture defining an opening at a top of the picowell, a bottom defined by a floor, wherein the top aperture is spaced apart from the floor by a wall, and wherein the wall is between the top aperture and the floor, and optionally, a bead disposed in at least one of the plurality of picowells, wherein the bead comprises a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds, wherein the picowell array plate further comprises a cushion capable of securely covering, or indeed covering, the opening at the top of at least one or all of the plurality of picowells, wherein the securely covering is reversible, wherein the cushion optionally comprises one or all of: (a) an absorbent surface capable of absorbing any metabolites, biochemicals, or proteins that can be included by one or more of the plurality of picowells when the absorbent surface is positioned in contact with the generally planar upper surface of the picowell array plate, (b) an adhesive surface capable of reversible adhesion to the generally planar top surface of the picowell array plate.

[0037] In a biochemical assay embodiment, contemplated is the above system comprising at least one picowell, wherein the at least one picowell comprises a bead comprising a plurality of substantially identical compounds and a plurality of substantially identical barcodes, wherein the at least one picowell comprises an assay medium, the medium comprising a cereblon E3 ubiquitin ligase, a substrate of the cereblon E3 ubiquitin ligase (such as Ikaros or Aiolos), and wherein the system is capable of screening for a compound that activates the cereblon E3 ubiquitin ligase activity, thereby capable of decreasing the concentration of Ikaros or Aiolos within a cell.

[0038] In another biochemical assay embodiment, contemplated is the above system comprising at least one picowell, wherein the at least one picowell comprises a bead comprising a plurality of substantially identical compounds and a plurality of substantially identical barcodes, wherein the at least one picowell comprises an assay medium, the medium comprising a MDM2 E3 ubiquitin ligase, a substrate of the MDM2 E3 ubiquitin ligase (such as p53), and wherein the system is capable of screening for a compound that activates the MDM2 E3 ubiquitin ligase activity, thereby capable of increasing the concentration of p53 within a cell.

[0039] In more barcode embodiments, the above system is provided, wherein the DNA barcode comprises one or more nucleic acids that do not encode any chemical library monomer, but rather identify one or more of: (a) a class of compounds that are cleavably linked to the bead; (b) a step in a multi-step organic synthesis pathway, wherein the bead-bound nucleic acid corresponds to a given chemical monomer used to make the bead-bound compound, and wherein the bead-bound nucleic acid corresponding to the given chemical monomer identifies the chemical monomer; (c) a date of synthesis of the bead-bound compound; (d) a disease that the bead-bound compound is intended to treat; (e) a cellular event that the bead-bound compound is intended to stimulate or inhibit.

[0040] In embodiments lacking any headpiece, the above system is provided, wherein the at least one bead comprises a plurality of substantially identical bead-bound compounds, and further comprises a plurality of substantially identical bead-bound DNA barcodes, and wherein there is no headpiece linking any of the bead-bound compounds to any of the bead-bound DNA barcodes.

[0041] Further, contemplated is the above system, wherein at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the substantially identical bead-bound DNA barcodes have the same structure. Additionally, contemplated is the above system, wherein at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the substantially identical bead-bound compounds have the same structure.

[0042] Further, provided is the above system, wherein the concatenated DNA barcode comprises at least one nucleic acid that is itself a DNA barcode module, or the above system, wherein the concatenated DNA barcode comprises only one nucleic acid that is itself a DNA barcode module.

[0043] In embodiments of a sequencing primer annealing site, the above system is provided, wherein the concatenated DNA barcode comprises at least one nucleic acid that is itself a DNA barcode module and at least one functional nucleic acid that: (a) is capable of serving as an annealing site for a sequencing primer, (b) is capable of forming a hairpin structure, and wherein the hairpin structure comprises a sequencing primer, an annealing site for the sequencing primer, and a bend in the hairpin structure, wherein the bend is 5' of the sequencing primer and 3' of the annealing site for the sequencing primer, or (c) is a spacer nucleic acid.

[0044] In other sequencing primer embodiments, the above system is provided, wherein the orthogonal DNA barcode contains a plurality of DNA barcode modules, wherein each of the DNA barcode modules is directly coupled to or coupled to a different site on the bead via a linker, and wherein each of the plurality of DNA barcode modules contains at least one functional nucleic acid that: (a) is capable of being used as an annealing site for a sequencing primer, (b) is capable of forming a hairpin structure, and wherein the hairpin structure includes a sequencing primer, an annealing site for the sequencing primer, and a kink in the hairpin structure, wherein the kink is 5' of the sequencing primer and 3' of the annealing site for the sequencing primer, or (c) is a spacer nucleic acid.

[0045] In embodiments reciting functionalization language in relation to splint oligos, a bead comprising a concatenated DNA barcode is provided, wherein the concatenated DNA barcode comprises: (a) a first DNA barcode module and a first annealing site of a first splint oligo, wherein the splint oligo comprises three nucleic acids, wherein the three nucleic acids are: a nucleic acid that is itself a hybridization complement of a first annealing site, a nucleic acid that is itself a hybridization complement of a second DNA barcode module, and a nucleic acid that is itself a second annealing site, and (b) a second DNA barcode module and a second annealing site of a second splint oligo, wherein the second splint oligo comprises three nucleic acids, wherein the three nucleic acids are: a nucleic acid that is itself a hybridization complement of a second annealing site, a nucleic acid that is itself a third DNA barcode module, and a nucleic acid that is itself a third annealing site.

[0046] In another embodiment containing functionalization language in relation to splint oligos, the above bead is provided, further comprising: a third DNA barcode module and a third annealing site of a third splint oligo, wherein the third splint oligo comprises three nucleic acids, wherein the three nucleic acids are: a nucleic acid that is itself a hybridization complement of a third annealing site, a nucleic acid that is itself a fourth DNA barcode module, and a nucleic acid that is itself a fourth annealing site.

[0047] Further, in yet another embodiment containing functionalization language in relation to splint oligos, the above bead is provided, further comprising one or more of: (i) a fourth DNA barcode module and a fourth annealing site of a fourth splint oligo, wherein the fourth splint oligo comprises three nucleic acids, wherein the three nucleic acids are: a nucleic acid that is itself a hybridization complement of a fourth annealing site, a nucleic acid that is itself a fifth DNA barcode module, and a nucleic acid that is itself a fifth annealing site, (ii) a response capture element, (iii) a release monitor.

[0048] In a linker embodiment, included are the above-described beads, wherein the concatenated DNA barcodes are coupled to the bead, but: (i) are not coupled to the bead via any photocleavable linker, (ii) are not coupled to the bead via any enzymatically cleavable linker; or (iii) are not coupled to the bead via any type of cleavable linker.

[0049] In an embodiment involving different coupling locations, provided are the above-described beads, wherein the concatenated DNA barcodes are coupled to a first location on the bead, wherein the bead further comprises a compound coupled to a second location on the bead, and wherein the first location is different from the second location.

[0050] In a surface embodiment (both inner and outer surfaces), provided are the above-described beads, wherein the bead comprises an outer surface and an inner surface, wherein the bead comprises at least 10,000 substantially identical concatenated DNA barcodes coupled to the bead, and wherein at least 90% of the at least 10,000 substantially identical concatenated DNA barcodes are coupled to the outer surface.

[0051] In an exclusive embodiment that can distinguish the present disclosure from other embodiments, provided are the above-described beads, which do not comprise any polyacrylamide, and wherein the concatenated DNA barcodes: (i) do not comprise any nucleic acid that is a promoter in its own right; (ii) do not comprise any nucleic acid that is a polyA in its own right; or (iii) do not comprise any nucleic acid that is a promoter in its own right and do not comprise any nucleic acid that is a polyA in its own right.

[0052] In a release monitor bead embodiment, the present disclosure provides a release monitor bead capable of functioning in an aqueous medium, wherein the release monitor bead comprises a bead-bound quencher and a bead-bound fluorophore, wherein the bead-bound quencher is positioned proximal to the bead-bound fluorophore in a quenching relationship and is capable of quenching at least 50% of the fluorescence of the bead-bound fluorophore, and wherein the bead-bound fluorophore is bound by a first photocleavable linker, wherein the picowell containing the release monitor bead is a first picowell, wherein the first picowell contains a first solution, wherein exposure of the first picowell to a cleavage condition is capable of cleaving the photocleavable linker and releasing the fluorophore into the first solution of the first picowell, wherein exposure results in diffusion of the fluorophore throughout the first solution in the first picowell, and wherein a fluorescence signal obtained by illuminating the first picowell (containing the first solution including the dispersed fluorophore) with light allows a user to calculate a percent release of the bead-bound fluorophore from the release monitor bead using the fluorescence signal, resulting in a calculated percent release value, and wherein a second picowell contains a bead-bound compound coupled to a photocleavable linker of the same type as the first photocleavable linker, and wherein the second picowell contains a second solution, and wherein the calculated percent release value from the release monitor bead in the first picowell allows calculation of a concentration of the released compound in the second solution of the second picowell. In other release monitor embodiments, release monitor beads are provided (wherein the fluorophore is TAMRA and the quencher is QSY7), release monitor beads having the structure shown Figure 9 release monitor beads having the structure shown Figure 10 release monitor beads and release monitor beads wherein the quenching ability is at least 90%, at least 98%, at least 99%, or at least 99.9%.

[0053] In an embodiment of the method of manufacture, included is a method for synthesizing a release monitor bead, wherein the release monitor bead comprises a bead, a quencher, a fluorophore, and a photocleavable linker coupling the fluorophore to the bead, the method comprising the following 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 the quencher using a reagent that is itself the quencher, N-hydroxysuccinimide (quencher-NHS), as a source of the quencher, (v) removing the 4-methyltrityl protecting group using a reagent comprising trifluoroacetic acid, (vi) coupling the photocleavable linker to the epsilon amino group of the lysine, wherein the photocleavable linker is provided by a reagent, i.e., Fmoc-photocleavable linker-OH, (vii) coupling the fluorophore. Also provided are the above embodiments, but independent of the order of the steps. In other method embodiments, the above method is provided, wherein the fluorophore is TAMRA, and wherein the quencher is QSY7.

[0054] In a method related to the use of a release monitor bead, provided is a method for controlling the concentration of a compound in a solution present in a picowell, wherein the method applies to a bead-bound compound in a picowell, wherein the picowell contains a solution, and wherein the bead-bound compound is coupled to the bead by a cleavable linker, the method comprising: (a) exposing the bead-bound compound to conditions that effect cleavage of the cleavable linker, and releasing the bead-bound compound from the bead to generate a released compound, wherein after release, the released compound diffuses or disperses in the solution, thereby producing a substantially uniform concentration of the compound in the solution, (b) wherein the conditions comprise light capable of cleaving the cleavable linker, (c) adjusting the conditions to produce a determined concentration of the substantially uniform concentration, and (d) wherein the determined concentration is determined with respect to the concentration of a released fluorophore released from a bead-bound release monitor. Also provided are the above methods, wherein the conditions are adjusted by adjusting one or more of the wavelength of the light, the intensity of the light, and the duration of the exposure, and the above methods, wherein the concentration of the released fluorophore released from the bead-bound release monitor is determined at the same time that the bead-bound compound is released from the bead to generate the released compound, and the above methods, wherein the concentration of the released fluorophore released from the bead-bound release monitor is determined at a time point long before the bead-bound compound is released from the bead to generate the released compound.

[0055] The term "determined" can mean a concentration that is predetermined and decided upon prior to exposing the beads to light at a desired concentration. Additionally, the term "determined" can mean a concentration that is decided "on the fly," i.e., a concentration that is decided at the same time as exposing the beads to light.

[0056] In a cap embodiment, what is included is a cap in combination with a pico-well plate comprising a plurality of pico-wells, wherein the cap is usable with a pico-well plate comprising a plurality of pico-wells, wherein each pico-well of the plurality of pico-wells is definable by an orifice, a floor, and a wall, wherein the wall is defined by the orifice on a top and the floor on a bottom, and wherein the orifice is circular, wherein the floor is circular, and wherein the wall takes the form of a truncated cone surface, and wherein the orifice has a first diameter, the floor has a second diameter, and wherein the first diameter is greater than the second diameter,

[0057] wherein the cap is a spherical cap that is fittable into the orifice tightly, wherein the orifice is composed of a polymer that is greater (harder) on a durometer scale, and the cap is made of a polymer that is smaller (softer) on a durometer scale, and wherein the relative durometer of the cap and the orifice allows the spherical cap to be reversibly and tightly fitted into the orifice, and wherein the cap: (i) is able to plug the pico-well and prevent leakage, (ii) is a passive cap and is able to absorb metabolites released by cells in a cell culture medium if cells are cultured in the pico-well, (iii) is an active cap that takes the form of a bead comprising a plurality of substantially identical compounds, and wherein each compound of the plurality of substantially identical compounds is coupled to the bead by a cleavable linker, and wherein cleavage of the cleavable linker releases at least some of the plurality of compounds from the bead, (iv) is an active cap that takes the form of a bead comprising a plurality of substantially identical reagents, and wherein each reagent of the plurality of substantially identical reagents is coupled to the bead by a cleavable linker, and wherein cleavage of the cleavable linker releases at least some of the plurality of reagents from the bead.

[0058] In a porous cover embodiment, a plurality of porous covers are provided in combination with a pico-well plate and a solid polymer coating, wherein each of the plurality of porous covers comprises an upper surface and a lower surface, wherein the pico-well plate comprises a plurality of pico-wells, wherein at least one porous cover is in contact with and reversibly tightly fitted in a pico-well, wherein the pico-well plate and each upper surface of the plurality of porous covers are covered with a solid polymer coating, wherein the solid polymer coating contacts and is connected to at least some of the upper surface of each cover, and wherein (i) each of the plurality of pico-wells is capable of containing an aqueous solution, wherein a product of a reaction is produced in the solution, and wherein at least some of the product is absorbed by the lower surface of each of the plurality of porous covers, (ii) wherein a solution of a polymerizable reagent capable of polymerization is poured over the plurality of porous covers along with the pico-well plate, and wherein the polymerizable reagent is polymerized to form a substantially flat surface that substantially covers all of the top surface of the pico-well plate, thereby affixing the polymerized reagent to each of the plurality of porous covers, and (iii) all of the plurality of porous covers are removable by an action of peeling from the plurality of pico-wells, wherein the adhesion between the plurality of porous covers and the polymerized reagent is maintained, thereby resulting in a series of adhered covers, partially, the upper surface of each cover is partially embedded in the polymerized reagent, and the lower surface of each cover is available for analysis of any absorbed reaction product.

[0059] This provides manufacturing method embodiments for using splint oligos to direct the enzymatic synthesis of DNA barcodes. Methods are provided for preparing a bead-bound concatenated DNA barcode, wherein the bead-bound concatenated DNA barcode comprises a plurality of DNA barcode modules, and optionally one or more functional nucleic acids, and optionally one or more identity-encoding nucleic acids that encode an identity other than the identity of a chemical library monomer, the method comprising: (a) the step of providing a bead with a conjugated polynucleotide comprising a first DNA barcode module and a first annealing site, wherein the first annealing site is capable of hybridizing to a first splint oligo, the first splint oligo is capable of being used as a template for a DNA polymerase to catalyze the polymerization of nucleotides complementary to the hybridized first splint oligo to the conjugated polynucleotide, wherein the polymerized nucleotides after polymerization (complementary to the hybridized first splint oligo) comprise a second DNA barcode module and a second annealing site conjugated to the bead; (b) the step of providing the bead with the conjugated polynucleotide with the first oligo and allowing the first splint oligo to hybridize to the conjugated polynucleotide; (c) the step of adding a DNA polymerase and deoxynucleotide triphosphates (dNTPs) and allowing the DNA polymerase to catalyze the polymerization of the dNTPs to the conjugated polynucleotide, wherein the conjugated polynucleotide has a free 3' end, and wherein the polymerization is at the free 3' end, (d) the step of washing away the first splint oligo. Also contemplated are the above methods, wherein the first splint oligo comprises the first annealing site, the second DNA barcode module, and the second annealing site.

[0060] In further manufacturing method embodiments, the above methods are provided, wherein the first splint oligo comprises the first annealing site, the second DNA barcode module, the second annealing site, and a nucleic acid that encodes a first sequencing primer annealing site, wherein the first sequencing primer annealing site is capable of hybridizing to a sequencing primer, resulting in a hybridized sequencing primer, and wherein the hybridized sequencing primer is capable of directing sequencing of the second DNA barcode module and the first DNA barcode module.

[0061] Further, contemplated are the above methods, wherein the first splint oligo, the DNA polymerase, and the dNTPs are all added simultaneously, or wherein the first splint oligo, the DNA polymerase, and the dNTPs are each added separately.

[0062] With respect to internal versus external positions on a bead, the above methods are provided, wherein the bead comprises an external position and an internal position, and wherein the bead-bound concatenated DNA barcode is conjugated to the bead at a position that is substantially external to the bead and rarely at an internal position of the bead, and wherein the bead further comprises a plurality of conjugated compounds, wherein all of the plurality of conjugated compounds have substantially the same structure when compared to one another, and wherein the bead is substantially composed of a hydrophobic polymer.

[0063] In a further method embodiment, the above-described method is provided, further comprising: (a) the step of providing the bead with a conjugated first longer polynucleotide comprising a first DNA barcode module, a first annealing site, a second DNA barcode, and a second annealing site, wherein the second annealing site is capable of hybridizing to a second splint oligonucleotide, the second splint oligonucleotide is capable of being used as a template for a DNA polymerase to catalyze the polymerization of nucleotides complementary to the hybridized second splint oligonucleotide to the conjugated first longer polynucleotide, wherein the polymerized nucleotides after polymerization (complementary to the hybridized second splint oligonucleotide) comprise a third DNA barcode module and a third annealing site bound to the bead; (b) the step of providing the bead with a conjugated polynucleotide having the second oligonucleotide and allowing the second oligonucleotide to hybridize to the conjugated first longer polynucleotide; (c) the step of adding a DNA polymerase and deoxynucleotide triphosphates (dNTPs) and allowing the DNA polymerase to catalyze the polymerization of the dNTPs to the conjugated longer polynucleotide, wherein the conjugated longer polynucleotide has a free 3’ end, and wherein the polymerization is at the free 3’ end, (d) the step of washing away the second splint oligonucleotide.

[0064] This involves the use of a first DNA barcode module, a second DNA barcode module, a third DNA barcode module, etc. for the manufacture of a serially numbered entire DNA barcode. This also involves the use of a cycle of repeating method steps over and over again in the manufacture of the entire DNA barcode. The above-described method is provided, wherein each of the plurality of DNA barcode modules is identified or named by a number, the method further comprising repeating the recited steps, wherein for a first repetition, the name of the DNA barcode module is increased by adding one number to the existing name, the name of the annealing site is increased by adding one number to the existing name, the name of the splint oligonucleotide is increased by adding one number to the existing name of the distal DNA barcode module, and the name of the “first longer polynucleotide” is changed by adding one number to the existing name, wherein the repeating the recited steps is one repetition, or two repetitions, or three repetitions, or four repetitions, or five repetitions, or more than five repetitions, or more than ten repetitions.

[0065] Also contemplated is the above-described method, comprising a plurality of splint oligonucleotides, wherein each splint oligonucleotide comprises a sequencing primer annealing site, wherein the sequencing primer annealing site is capable of hybridizing to a sequencing primer, thereby producing a hybridized sequencing primer, and wherein the hybridized sequencing primer is capable of directing sequencing of at least one bead-bound DNA barcode module and at least one bead-bound DNA barcode module.

[0066] This relates to embodiments involving splint oligos that direct DNA polymerase to synthesize functional nucleic acids and various types of informative nucleic acids. Provided are the above methods, wherein at least one splint oligo comprises a functional nucleic acid, or wherein at least one splint oligo encodes information other than information on the identity of the chemical library monomer. Provided are the above methods, further comprising a step of coupling at least one DNA barcode module by click chemistry, wherein the step does not use any splint oligo.

[0067] In brief, the present disclosure provides a system for screening a compound, the system comprising: (a) a picowell array plate comprising a plurality of picowells, wherein each picowell has a top aperture defining an opening at a top of the picowell, a bottom defined by a floor, wherein the top aperture is spaced apart from the floor, and wherein a wall is located between the top aperture and the floor; (b) at least one bead disposed in at least one picowell, wherein the at least one bead comprises a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds; (c) wherein the at least one bead comprises a DNA barcode that is in the form of a concatenated DNA barcode or an orthogonal DNA barcode, and wherein, if the DNA barcode is in the form of a concatenated DNA barcode, the concatenated DNA barcode is made using a method that uses one or both of: (i) using click chemistry, or (ii) using a repeating step cycle, wherein a step in the repeating cycle comprises annealing a splint oligo to a partially made DNA barcode, wherein the annealed splint oligo is used as a template to extend the partially made DNA barcode using a DNA polymerase, and wherein the splint oligo contains bases that are complementary to a DNA barcode module that is to be polymerized to the partially made DNA barcode.

[0068] In another aspect, provided is the above system, wherein the DNA barcode comprises: (a) one or more DNA barcode modules, wherein each DNA barcode module of the one or more DNA barcode modules encodes information about the identity of a chemical library monomer, and (b) optionally one or more functional nucleic acids, and (c) optionally one or more nucleic acids that encode information of a type other than the identity of the chemical library monomer.

[0069] Further, provided is the above system, further comprising a plurality of covers, each cover being capable of fitting into the opening of a different picowell, and each cover being capable of minimizing or preventing evaporation of fluid inside the picowell, and each being capable of minimizing or preventing leakage of fluid inside the picowell.

[0070] Also contemplated is the above system, further comprising a plurality of spherical caps, wherein each cap is capable of fitting into the orifice of a picowell, wherein the orifice is circular, and each cap is capable of minimizing or preventing evaporation of a fluid inside the picowell, and each cap is capable of minimizing or preventing leakage of a fluid inside the picowell.

[0071] Also contemplated is the above system, wherein if the at least one bead comprises a DNA barcode in the form of a concatenated DNA barcode, the concatenated DNA barcode comprises: (i) a sequencing primer binding site, (ii) a first DNA barcode module, (iii) a first annealing site capable of hybridizing to a first oligonucleotide splint, wherein the first oligonucleotide splint is capable of being used to direct the enzymatic synthesis of a second DNA barcode module, (iv) a second DNA barcode module, (v) a second annealing site capable of hybridizing to a second oligonucleotide splint, wherein the second oligonucleotide splint is capable of being used to direct the synthesis of a third DNA barcode, (vi) a third DNA barcode module, (vii) a third annealing site capable of hybridizing to a third oligonucleotide splint, wherein the third oligonucleotide splint is capable of being used to synthesize a fourth DNA barcode.

[0072] In a method embodiment, a method for screening a library of compounds for a compound having a desired property is provided, the method comprising: (a) providing a plurality of beads, wherein each bead comprises a plurality of oligonucleotides attached to a surface of a bead and a plurality of substantially related compounds attached to the surface of the bead, and wherein the sequence of the oligonucleotides attached to the bead encodes a synthetic history of the plurality of substantially related compounds attached to the surface of the bead; (b) binding the plurality of beads in an assay for a desired property of a compound in the library of compounds; (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 the at least one bead for which assay signal was also captured without removing the oligonucleotides from the bead; and (e) identifying at least one compound from the sequencing reads of step (d) and correlating the compound to its corresponding assay performance captured in the signal of step (c).

[0073] In further detail, contemplated are the above methods, wherein the assay comprises a binding assay, or wherein the assay comprises an activity assay, or wherein the assay comprises a competitive binding assay or a competition inhibition assay, or wherein the assay comprises the interaction of an un-tethered compound with other assay reagents, wherein the un-tethered compound is a compound released from the surface of the bead, or wherein the compound is released by cleavage of a cleavable linker that links the compound to the bead, or wherein the assay occurs in a plurality of finite volumes, wherein nominally one bead is dispersed in each finite volume.

[0074] In another aspect, further contemplated are the above methods, wherein the finite volume comprises an aqueous droplet, or

[0075] wherein the aqueous droplet is suspended in an oil medium or a hydrophobic liquid medium, or wherein the finite volume comprises a picowell, or wherein the picowell is organized in a regular array, or wherein the plurality of finite volumes is organized in a regular array.

[0076] Further, contemplated are the above methods, wherein the confined volume comprises a layer of adherent aqueous medium surrounding the bead, wherein the bead is suspended in a hydrophobic medium, and the above methods, wherein the assay reagents are washed away prior to sequencing the oligonucleotide. And the above methods, wherein the sequencing step (d) is performed prior to the assaying step (b). Also provided are the above methods, wherein the oligonucleotide on the bead is removed after the sequencing step but prior to the assaying step. Further, contemplated are the above methods, wherein the removal of the oligonucleotide comprises enzymatic digestion, chemical cleavage, thermal degradation, or physical shearing, and the above methods, wherein the binding assay comprises the binding of an RNA molecule to the bead, and the above methods, wherein the signal from the assay comprises sequencing the bound RNA molecule.

[0077] In yet another aspect, provided are the above methods, wherein the binding assay comprises a fluorescently labeled binding assay, wherein the molecule bound to the compound on the bead comprises a fluorophore, or the above methods, wherein the binding assay comprises a nucleic acid labeled binding assay, wherein the molecule bound to the compound on the bead comprises a nucleic acid tag, wherein further the signal from the assay comprises sequencing the nucleic acid tag linked to the molecule bound to the compound on the bead.

[0078] In yet another method embodiment related to a property, the above method is provided, wherein the desired property comprises one or more of: (i) inhibiting or stimulating catalytic activity of an enzyme, (ii) stimulating a Thl-type immune response, as measurable by an in vitro or in vivo assay, (iii) stimulating a Th2-type immune response, as measurable by an in vitro or in vivo assay, (iv) inhibiting a Thl-type immune response, as measurable by an in vitro or in vivo assay, (v) inhibiting a Th2-type immune response, as measurable by an in vitro or in vivo assay, (vi) stimulating or inhibiting ubiquitin-mediated protein degradation, as measurable by a purified protein, by a cell-based assay, or by an in vivo assay.

[0079] In a system embodiment, a system for screening a library of compounds for a compound having a desired activity is provided, the system comprising: (a) a sample chamber for receiving a plurality of compound-linked oligonucleotide-encoded beads; (b) a plurality of enclosed chambers within the sample chamber, each enclosed chamber nominally comprising a single bead dispersed in an assay medium, wherein further the assay medium comprises a reagent whose interaction with a compound on the bead is assayed, thereby generating 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 enclosed chambers comprise droplets. In another aspect, the above system is provided, wherein the enclosed chambers comprise picowells, or wherein further the enclosed chambers comprise assay reagents, or wherein the detector comprises an optical detector, or wherein the sequencer comprises an optical detector.

[0080] In an aspect, the disclosure features a method for perturbing a cell by: (a) providing a nucleic acid-encoded perturbation and confining a cell with the nucleic acid-encoded perturbation; (b) contacting the cell with the nucleic acid-encoded perturbation in a limited volume, wherein perturbation initiation and dosage are controlled; (c) incubating the cell with the nucleic acid-encoded perturbation for a specified period of time; (d) transferring the nucleic acid encoding the nucleic acid-encoded perturbation to the cell.

[0081] In some embodiments of this aspect, 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.

[0082] In some embodiments, the perturbation and the nucleic acid encoding the perturbation are unlinked and free in solution. In some embodiments, the perturbation and the nucleic acid encoding the perturbation are linked to one another. In some embodiments, the perturbation and the nucleic acid encoding the perturbation are linked to the same substrate but are not linked to one another. In some embodiments, the linkage of the perturbation to the substrate and the linkage of the nucleic acid to the substrate are cleavable linkages. In particular embodiments, the cleavable linkages are selected from the group consisting of: photocleavable linkages, temperature-cleavable linkages, pH-sensitive linkages, acid-cleavable linkages, base-cleavable linkages, sound-cleavable linkages, salt-cleavable linkages, oxidation-reduction sensitive linkages, or physically cleavable linkages.

[0083] In some embodiments of this aspect of the disclosure, confining the cell and the perturbation comprises droplet encapsulation, emulsion encapsulation, picowell encapsulation, macrowell encapsulation, physical linkage, bubble encapsulation, or microfluidic confinement.

[0084] In some embodiments, controlling the perturbation comprises controlling light exposure, controlling temperature exposure, controlling pH exposure, controlling time exposure, controlling sound exposure, controlling salt exposure, controlling chemical or physical oxidation-reduction potential, or controlling mechanical agitation exposure.

[0085] In particular embodiments, the incubating comprises exposing the cell to the perturbation after cleaving the perturbation from the substrate or after cleaving the nucleic acid from the perturbation. In some embodiments, the incubating comprises exposing the cell to the perturbation without cleaving the perturbation from the substrate or cleaving the nucleic acid from the perturbation.

[0086] In some embodiments, transferring the nucleic acid encoding the perturbation encoded by the nucleic acid to the cell comprises linking the nucleic acid to a cell surface of the cell. In particular embodiments, linking the nucleic acid to the cell surface of the cell comprises inserting the nucleic acid into a cell membrane. In particular embodiments, linking the nucleic acid to the cell surface of the cell comprises linking the nucleic acid to a biomolecule on the cell surface. In particular embodiments, the biomolecule is a protein or a carbohydrate. In other embodiments, linking the nucleic acid to the cell surface of the cell comprises linking via an optional tag on the nucleic acid.

[0087] In another aspect, a method for perturbing a cell by perturbation and encoding the cell with the identity of the perturbation. The method comprises: (a) providing a bead-bound DNA-encoded library; (b) restricting a cell with the bead-bound DNA-encoded library, wherein the bead-bound DNA-encoded library comprises one or more copies of a combinatorially synthesized compound and one or more copies of an encoding nucleic acid tag, wherein the compound and encoding nucleic acid are linked to a bead, wherein the encoding nucleic acid encodes the identity of the compound, and wherein the bead-bound DNA-encoded library and the cell are restricted in a finite volume; (c) releasing the compound from the bead and incubating the compound with the cell within the finite volume; (d) optionally releasing the encoding nucleic acid tag from the bead; and (e) linking the encoding nucleic acid tag to the cell, whereby the identity of the compound is retained by the encoding nucleic acid tag linked to the cell.

[0088] In yet another aspect, the disclosure features a method for perturbing a cell, encoding the cell with an identity of the perturbation, and measuring a response of the cell to the perturbation. The method includes: (a) contacting a cell with a bead-bound DNA-encoded library in a first finite volume, wherein the bead-bound DNA-encoded library comprises one or more copies of a combinatorially synthesized compound and one or more copies of an encoding nucleic acid tag, wherein the compound and encoding nucleic acid are linked to a bead, and wherein the encoding nucleic acid encodes an identity of the compound; (b) releasing the compound in the library from the bead and incubating the compound in the library with the cell within the first finite volume; (c) optionally releasing the encoding nucleic acid tag from the bead within the first finite volume; (d) capturing the encoding nucleic acid tag to a cell surface of the cell, whereby the cell is exposed to the compound in the library, and the identity of the exposed compound is captured onto the cell surface; (e) releasing the cell from the first finite volume, wherein the encoding nucleic acid tag is linked to the cell, and the encoding nucleic acid tag encodes the identity of the compound to which the cell was exposed; (f) capturing a previously perturbed and nucleic acid-labeled cell with a response detection bead in a second finite volume, wherein the cell is exposed to a lysis condition that exposes a cell content of the cell to a response capture bead, wherein the response capture bead comprises a capture probe that captures the cell content and a nucleic acid tag encoding a perturbation in the previously perturbed and nucleic acid-labeled cell; (g) incubating the response capture bead with the lysed cell in the second finite volume, whereby both cell content and the nucleic acid tag encoding the perturbation are captured onto the response capture bead; (h) optionally transducing a response of the cell to the perturbation into a nucleic acid signal, wherein the response of the cell to the perturbation is not a nucleic acid signal; and (i) sequencing the nucleic acid tag linked to the response capture bead, whereby the identity of the perturbation is correlated to the response of the cell to the perturbation.

[0089] In still another aspect, a method for perturbing a cell and capturing a response of the cell to the perturbation by: (a) providing a picowell array and a library of functionalized perturbation beads, wherein a picowell is capable of housing a single cell and a single functionalized perturbation bead, wherein each functionalized perturbation bead comprises a different plurality of substantially identical releasable compounds and a plurality of nucleotide barcodes encoding the compounds, wherein the nucleotide barcodes are functionalized barcodes capable of capturing cellular contents of the cell, wherein the cellular contents of a cell comprise a cellular response to a perturbation contained in the functionalized perturbation bead; (b) capturing a single cell into each picowell of the picowell array; (c) capturing a single functionalized perturbation bead into the picowell containing a single cell; (d) releasing the compounds from the functionalized perturbation bead and incubating the cell with the released compounds, wherein the compounds have minimal diffusion between picowells; (e) lysing the cell to release the cellular contents; (f) capturing one or more components of the cellular contents onto a functionalized oligonucleotide on the functionalized perturbation bead, wherein the capturing comprises hybridization and enzymatic extension to combine a nucleotide barcode with a nucleic acid element of the cellular contents, thereby forming a hybrid of the nucleotide barcode and the nucleic acid element of the cellular contents; and (g) releasing the hybrid, collecting the hybrid from the library of functionalized perturbation beads, and sequencing the hybrid, thereby correlating the perturbation with a cellular response to the perturbation. BRIEF DESCRIPTION OF DRAWINGS

[0090] Figure 1 . Cascade bead. In a cascade bead, the DNA barcode takes the form of all DNA barcode modules connected to each other in a single strand, and with any other functional nucleic acid (such as a primer annealing site) used as a spacer or information about the date of production. The numbers on this figure are not structural numbers. These numbers refer to the order of "DNA barcode modules" in the DNA barcode.

[0091] Figure 2 . Orthogonal bead. In an orthogonal bead, the DNA barcode takes the form of all DNA barcode modules, where the DNA barcode modules do not appear together in a single strand, but instead appear separately at different locations on the bead. The numbers on this figure are not structural numbers. These numbers refer to the order of "DNA barcode modules" in the DNA barcode.

[0092] Figure 3. Cleavable linkers, cleavage conditions (UV light or chemicals), and cleavage products. Information from Yinliang Yang (2014) Design of Cleavable Linkers and Applications in Chemical Proteomics. Biochemistry, Technical University of Munich. The letter to the left of each linker comes from this reference.

[0093] Figure 4 . Exemplary amino acid derivatives for use in the compositions and methods of the disclosure.

[0094] Figure 5 . This photograph discloses that degradation of the fusion protein inside HeLa cells increases as the concentration of added lenalidomide increases. Top: expression of IKZF1 / GFP fusion protein. Bottom: expression of mScarlett control. Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar. Expression of control. Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.

[0095] Figure 6 . This photograph discloses that degradation of the fusion protein inside HeLa cells increases as the concentration of added lenalidomide increases. Top: expression of IKZF3 / GFP fusion protein. Bottom: expression of mScarlett control. Lenalidomide was added at zero, 0.1, 1.0, or 10 micromolar.

[0096] Figure 7 . Methods and reagents for creating bead-bound DNA barcodes. The most accurate description of a “DNA barcode” is the sum of all information contained in the sum of all DNA barcode modules. But for convenience, the term “DNA barcode” is used herein to refer to the sum of all information of all DNA barcode modules plus any additional nucleic acids that provide information such as step number or general type of chemical monomer (constituent of bead-bound compound) and plus any additional nucleic acids that provide functionality such as linkers, sequencing primer binding sites, hairpins with sequencing primer binding sites, or spacers. In the case of DNA barcodes made at least in part by way of click chemistry, the DNA barcode can contain residual chemical groups from the click chemistry reaction.

[0097] Figure 8 . Structure of Alexa 488. The goal of this figure is to identify the compound without using the trademark name.

[0098] Figure 9. Simplified diagram of bead-bound release monitors. After UV-induced release of compound from the beads, the release monitors provide the user with a measure of the concentration of soluble compound. In a preferred embodiment, one type of bead is dedicated to serve as release monitors, i.e., the beads do not also contain bead-bound compound and do not also contain bead-bound DNA library. "PCL" is a photo-cleavable linker.

[0099] Figure 10 . Detailed diagram of bead release monitors.

[0100] Figure 11 . Chemical synthesis of bead release monitors.

[0101] Figure 12 . Amine-functionalized beads with bifunctional linker, where the linker contains a lysine residue.

[0102] Figure 13 . Steps in chemical synthesis of lenalidomide modified with first type of carboxyl group.

[0103] Figure 14 . Steps in chemical synthesis of lenalidomide modified with second type of carboxyl group.

[0104] Figure 15 . Steps in chemical synthesis of lenalidomide modified with third type of carboxyl group.

[0105] Figure 16A 、 Figure 16B 、 Figure 16C . Lenalidomide analogs.

[0106] Figure 17 . Steps in chemical synthesis of deoxycytidine analogs suitable for DNA barcode click chemistry synthesis.

[0107] Figure 18A 、 18B , 18C. Cap for placement on top of picowell and sealing of picowell. Figure 18A Active cap is shown, where compound is releasable through a cleavable linker. Figure 18B Another type of active cap is shown, where a reagent such as an antibody is bound. The bound reagent can be permanently linked, it can be linked through a cleavable linker, or it can be bound through hydrogen bonds and be releasable only by exposure to solution in the picowell, then diffusing from the active cap into that solution. Figure 18C Passive cap is shown, which can be used to absorb, adsorb, collect, or capture metabolites from solution in the picowell. The absorbed metabolites can be subsequently analyzed.

[0108] Figure 19A 、 19B , 19C. Figure 19AA picowell plate is shown with no lid on the picowells. Figure 19B A picowell plate is shown with a lid on each picowell. Figure 19C A solution of polyacrylamide is poured over a picowell plate with a lid firmly affixed to each picowell. The polyacrylamide then penetrates the porous lid, solidifies and forms a stable bond with each lid. Figure 19D The solidified polyacrylamide "topping" is then peeled off the picowell plate, taking each lid with it. The metabolites transferred from the picowell solution and absorbed into each lid can then be analyzed. Preferably, the solution poured over the picowell plate and over the beads becomes a hydrogel, and preferably the beads are made of hydrogel.

[0109] In exclusive embodiments, the present disclosure can exclude systems, microtiter plates, microtiter plates with microwells, nanowells or picowells and related methods, wherein at least one well is capped and a liquid polymer solution is poured over the plate and over the capped well. Additionally, excluded can be the above, wherein the liquid polymer has polymerized to form a solid polymer adhered to each cap. Additionally, excluded can be methods and resulting compositions, wherein the solid polymer is torn and the adhered caps are removed.

[0110] Figure 20 . Map of circular plasmid used to integrate IKZF1 gene into the cell genome. Plasmid is: IKZF1 mNEON-p2a-mScarlet-w3-2FB (9081 base pairs). IKZF1 decodes the Ikarus protein.

[0111] Figure 21 . Map of circular plasmid used to integrate IKZF3 gene into the cell genome. Plasmid is: IKZF3 mNeon-p2a-mScarlet-w3-2FB (9051 base pairs). IKZF3 decodes the Aiolos protein.

[0112] Figure 22 . Chemical monomers (compounds 1-6) and their DNA barcodes.

[0113] Figure 23 . Chemical monomers (compounds 7-10) and their DNA barcodes.

[0114] Figure 24 . Chemical monomers (compounds 11-16) and their DNA barcodes.

[0115] Figure 25 . Chemical monomers (compounds 17-21) and their DNA barcodes.

[0116] Figure 26. Chemical monomers (compounds 22-26) and their DNA barcodes.

[0117] Figure 27 Chemical monomers (compounds 27-30) and their DNA barcodes are shown.

[0118] Figure 28 . Sequencing bead-bound DNA barcodes. The plot discloses the fluorescent signal intensity of each base in a five consecutive base stretch that is part of a bead-bound DNA barcode.

[0119] Figure 29 . Staircase picowells.

[0120] Figure 30 . Time course of fluorophore release from a bead. This shows the operation of a bead-bound release monitor, with fluorescence data taken at t=0 seconds, t=1 second, t=11 seconds, and t=71 seconds.

[0121] Figure 31A and B. Figure 31A Photo showing catalysis of aspartyl protease on quencher fluorophore substrate. Figure 31B Emission data produced after showing catalysis of aspartyl protease on quencher fluorophore substrate.

[0122] Figure 32 . Cross-sectional view of picowell, illustrating each step.

[0123] Figure 33 . Titration data showing how increasing UV dose leads to greater cleavage of fluorophore from bead. In layman's terms, this shows how a more powerful swing of an axe affects the cleaving of a fluorophore from a bead (the power of the UV dose is measured in Joules / square centimeter). The notation "exposure" refers only to the parameter at the time of the photo. It is only the exposure time at the time the photo was taken (not the exposure time of the light that does the cleaving or the light that does the excitation).

[0124] Figure 34 . TAMRA concentration vs. light fluence. This shows the concentration of free TAMRA released after exposure to 365 nm UV light.

[0125] Figure 35 Hand-drawn diagram of quencher fluorophore substrate and cleavage of this substrate by enzyme, thus inhibiting the enzyme. Also shown are the molecular structures of bead-bound gastrin-A and bead-bound Fmoc-valine (negative control).

[0126] Figure 36. Preparation of beads that are ultimately used to capture mRNA from a cut cell and subsequently make a cDNA library. This figure also appears in a provisional application (Compositions and Method for Screening Compound Libraries on Single Cells) from which priority is claimed for the present application.

[0127] Figure 37 . Labeling of cells with DNA barcodes, where the labeling is through lipids that are embedded in the cell membrane. This figure also appears in a provisional application (Compositions and Method for Screening Compound Libraries on Single Cells) from which priority is claimed for the present application. DETAILED DESCRIPTION

[0128] As used herein, including the appended claims, the singular forms "a," "an," and "the" include their plural referents unless the context clearly dictates otherwise. All references cited herein are incorporated by reference, as if each individual publication, patent, published patent application, and document was specifically and individually indicated to be incorporated by reference.

[0129] Abbreviations

[0130] Table 1 provides abbreviations and non-limiting definitions.

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] Reagents, kits, enzymes, buffers, live cells, instruments, etc. can be obtained. See, e.g., 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.

[0137] Barcoded gel beads, non-barcode gel beads, and microfluidic chips are available from 1CellBio, Cambridge, MA. Guidance and instruments for flow cytometry are available (see, e.g., BD Biosciences, San Jose, CA, FACSDiva® User Guide, Part No. 643245, Rev. A, December 2007, 344 pages). BD FACSAria User Guide, Part No. 643245, Rev. A, December 2007, 344 pages).

[0138] “Labeled” compositions can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical, isotopic, or chemical methods, and by methods that involve the use of a nanoparticle of plasmonic nanoparticles. For example, useful labels include 32 P, 33 P, 35 S, 14 C, 3 H, 125 I, stable isotopes, epitope tags, fluorescent dyes, Raman tags, electron-dense reagents, substrates or enzymes, e.g., for use in enzyme-linked immunoassays, or fluorettes (Rozinov and Nolan (1998) Chem. Biol. 5:713-728).

[0139] Table of Contents

[0140] (I) Beads

[0141] (II) One bead one compound (OBOC)

[0142] (III) Coupling nucleic acids to beads

[0143] (IV) DNA barcodes

[0144] (V) Coupling compounds to beads

[0145] (VI) Coupling chemical monomers to each other to make compounds

[0146] (VII) Split and pool synthesis and parallel synthesis

[0147] (VIII) Fabricating picowells

[0148] (IX) Placing beads into picowells

[0149] (X) Sequencing nucleic acids bound to beads in picowells

[0150] (XI) Releasing bead-bound compounds from beads

[0151] (XII) Biochemical assays of compounds

[0152] (XIII) Cell-based assays of compounds

[0153] (XIV) Perturbation response analysis of cells

[0154] (I) Beads

[0155] The methods and compositions of the present disclosure use beads, such as single-sized M NH2beads (10, 20, 30, etc. microns in diameter), standard Amino Resins (90, 130, etc. microns in diameter), TentaGel (280-320 microns in diameter) (all of the above from Rapp Polymere, 72072 Tubingen, Germany). These beads have a polystyrene core derivatized with polyethylene glycol (Paulick et al. (2006) J. Comb. Chem. 8:417-426). The resins are graft copolymers consisting of a low-crosslinked polystyrene matrix grafted with polyethylene glycol (PEG). Accordingly, the present disclosure provides beads or resins modified to contain one or both of a DNA barcode and a compound, wherein the unmodified beads take the form of a graft copolymer consisting of a low-crosslinked polystyrene matrix grafted with polyethylene glycol (PEG).

[0156] characterized as "PEG chains up to 20 kilodaltons are immobilized on functionalized cross-linked polystyrene. Graft copolymers with PEG chains of about 2000-3000 daltons proved to be optimal in terms of kinetic rate, mobility, swelling and resin capacity". (Lapko Polymer GmbH, Germany). Thus, the present disclosure provides a bead or resin that takes the form of a graft copolymer with PEG chains of about 2000-3000 daltons. Regarding swelling, Comellas et al. provide guidelines for measuring the swelling capacity of beads, for example when immersed in DCM, DMF, methanol, water or buffers used in enzyme assays (Comellas et al. (2009) PLoS ONE 4: e6222 (12 pages)). The unit of swelling is milliliters per gram of bead.

[0157] In an alternative bead embodiment, the present disclosure uses a resin with PEG spacers (linked to the polystyrene backbone by alkyl bonds) and wherein the resin is microspherical and monosized M resin).

[0158] In yet another alternative bead embodiment, the present disclosure uses a resin with PEG spacers (linked to the polystyrene backbone by alkyl bonds) wherein the resin type exists in two bifunctional species: a first, surface-modified resin: orthogonally protecting the reactive sites on the outer surface of the bead from the reactive sites in the internal volume of the bead and a second, mixed resin: cleavable and non-cleavable ligands exist in this support species - developed for sequential cleavage B resin).

[0159] Furthermore, in another embodiment, the present disclosure uses a resin wherein the PEG spacer is linked to the polystyrene backbone by alkyl bonds and wherein the large bead resin shows very large particle diameter and high capacity MB resin). Additionally, the present disclosure uses a resin wherein the PEG spacer is linked to the polystyrene backbone by a benzyl ether bond. This resin can be used for immuno-processes or for the synthesis of PEG-modified derivatives (PEG-linked PEG-modified compounds) PAP resin).

[0160] Furthermore, the beads can be 200 resin. These resins are complexes of oligoethylene glycol (MW 200) grafted onto a low cross-linked polystyrene matrix (Fluka Chemie GmbH, CH-9471 Buchs, Switzerland).

[0161] In some embodiments, amino-functionalized polystyrene beads (without PEG linkers) can be used, e.g., single-size polystyrene M NH2beads (5, 10, 20, etc. microns in diameter, also from Rapp Polymers, 72072 Tubingen, Germany).

[0162] In some embodiments, compounds can be encapsulated within pores or chambers or channels within the beads without being covalently attached to the beads. Compounds can be diffused into or forced into such pores of the beads by various means. In some embodiments, compounds can be loaded within the beads by diffusion. In some embodiments, high temperature can be used to swell the beads and load the compounds within the beads. In some embodiments, high pressure can be used to force the compounds into the beads. In some embodiments, solvents that swell the beads can be used to load the compounds within the beads. In some embodiments, vacuum or low pressure can be used to partition the compounds into the beads. In some embodiments, gentle or vigorous physical agitation can be used to load the compounds into the beads.

[0163] In such embodiments, where compounds are loaded on the beads without being covalently attached, the compounds can be unloaded from the beads by diffusion. In some embodiments, in a non-limiting manner, temperature, pressure, solvent, pH, salt, buffer, or detergent, or combinations of such conditions can be used to unload the compounds from such beads. In some embodiments, the physical integrity of the beads, e.g., by uncrosslinked polymeric beads, can be used to release the compounds contained in such beads.

[0164] In exclusive embodiments, the present disclosure can exclude any beads and bead-compound complexes or any methods involving one of the above-described beads.

[0165] The beads of the present disclosure further comprise the following. Merrifield resin (chloromethyl polystyrene); PAM resin (4-hydroxymethyl phenylacetylamidomethyl polystyrene); MBHA resin (4-methylbenzhydrylamine); Brominated Wang resin (a-bromo-p- acetophenone); 4-nitrobenzophenone oxime (Kaiser) resin; Wang resin (4 hydroxymethyl phenoxymethyl polystyrene); PHB resin (p-hydroxybenzyl alcohol); HMPA resin (4-hydroxymethyl phenoxyl acetic acid); HMPB resin (4-hydroxymethyl-3-methoxy phenoxyl butyric acid); 2-chlorotrityl resin; 4-carboxytrityl resin; Rink acid resin (4-[(2,4-dimethoxyphenyl)hydroxymethyl]phenoxymethyl); Rink amide (RAM) resin "Knorr" resin (4-[((2,4-dimethylphenyl)(Fmox-amino)methyl)phenoxylalkyl); PAL resin (5-[4-(Fmoc-amino)methyl-3,5-dimethoxyphenoxyl]pentanoylamidomethyl polystyrene); Sieber amide resin (9-Fmox-amino-xan-thr-3-yl-oxymethyl); HMBA resin (hydroxymethyl benzoic acid); 4-sulfamoylbenzoyl resin "Kenner's safety catch" resin (N-(4-sulfamoylbenzoyl)aminomethyl-polystyrene); FMP-resin (4-(4-formyl-3-methoxyphenoxyl)-ethyl) (see ChemFiles Resins for Solid Phase Peptide Synthesis, Vol. 3, (page 32) (Fluka Chemie AG, CH-9471 Fluka, Switzerland).

[0166] The beads of the present disclosure further comprise the above beads that serve as passive encapsulants of compounds (passively holding the compound without covalent attachment to the compound), and further include the following: non-functionalized polystyrene beads; silica beads; alumina beads; porous glass beads; polyacrylamide beads; titania beads; alginate beads; ceramic beads; PMMA (polymethyl methacrylate) beads; melamine beads; zeolite beds; polylactic acid beads; de-blocked copolymer micelles; dextran beads, and the like. Many of the beads listed in this paragraph can be purchased from suppliers such as Microspheres-Nanospheres, Inc., Cold Spring Harbor, NY 10516, USA.

[0167] In addition to beads, for some embodiments of the present disclosure, vesicles or droplets can be used as vehicles for delivery of compounds. Lipids, de-blocked copolymers, tri-block copolymers, or other film forming materials can be used to form an internal volume into which a compound can be loaded. The compound can be released from these encapsulated volumes by the addition of a detergent, mechanical agitation, temperature, salt, pH, or other means. Water-in-oil droplet emulsions or oil-in-water droplet emulsions are other ways of passively encapsulating a compound that can be delivered to an assay volume.

[0168] In all embodiments where passive encapsulation is used to deliver a compound, the DNA tag can also be passively loaded, or alternatively, the DNA tag can be covalently attached to the bead, vesicle, or droplet.

[0169] In exclusive embodiments, the present disclosure can exclude any bead or resin made from any of the above chemicals or made from a derivative of any of the above chemicals.

[0170] In embodiments, the bead can be spherical and have a diameter of about 0.1-1 micron, about 1-5 microns, about 1-10, about 5-10, about 5-20, about 5-30, about 10-20, about 10-30, about 10-40, about 10-50, about 20-30, about 20-40, about 20-50, about 20-60, about 50-100, about 50-200, about 50-300, about 50-400, about 100-200, about 100-400, about 100-600, about 100-800, about 200-400, about 200-600, about 200-800 microns, etc.

[0171] Non-spherical beads that can be defined according to the above values and ranges are also provided. For example, one of the axes, one of the major dimensions (e.g., sides), or one of the minor dimensions (e.g., diagonals) can comprise a value within the above ranges. In exclusive embodiments, the present disclosure can exclude any agent, composition, system, or method that encompasses a spherical bead (or non-spherical bead) that falls within one or more of the above values or ranges.

[0172] Bead chains. In one embodiment, a plurality of bead dimers is provided, wherein the bead-dimers take the form of two beads connected to one another, and wherein one bead contains a plurality of linked nucleic acid barcodes (orthogonal nucleic acid modules or concatenated nucleic acid modules) and the other bead contains a plurality of linked compounds, wherein all of the compounds are substantially related to one another (or the chemical structures of all of the compounds are substantially identical to one another). The bead dimers can be synthesized by preparing a first bead with linked compounds, separately preparing a second bead with linked nucleic acid barcodes, and then connecting the two beads together. In one aspect, the beads are connected to one another by a reversible linker, and in another aspect, the beads are connected to one another by an irreversible linker.

[0173] Bead permeability. In embodiments, this disclosure provides beads having various ranges or permeabilities. Permeability can be measured as a percentage of the bead volume accessible to the solvent, wherein the unit of measurement is a percentage of the bead surface, which takes the form of pores, or a percentage of the bead interior, which takes the form of channels, networks, or chambers in fluid communication with the bead surface (and the external medium). This disclosure may cover porous beads, or alternatively, porous beads may be excluded.

[0174] Rothberg's U.S. Patent No. 9,062,304 discloses a bead having external and internal regions. What is shown is the "internal surface (pore surface)," and the options of "suitable pores will...exclude larger molecules" and "utilizing different functionalizations of the inner and outer surfaces," along with various pore sizes and polymers (such as poly(styrene sulfonate) and polystyrene). Rothberg's... Figure 1 Images of the bead surface and the pores of the beads are provided. Bedre's U.S. Patent No. 9,745,438 provides transmission electron micrographs of porous beads. Smith's U.S. Patent No. 5,888,930 provides scanning electron micrographs of cross-sections of porous beads. Shown are spherical beads with small pores on the surface and large pores internally, wherein the beads are made of, for example, polystyrene, polyacrylonitrile, polycarbonate, cellulose, or polyurethane. Cooke's U.S. Patent No. 5,047,437 discloses beads with a skinless surface (…). Figure 1 The porous structure of spherical poly(acrylonitrile) copolymers and the beads with an outer skin on the surface Figure 5 Tsao's U.S. Patent No. 4,090,022 discloses the porous openings and internal void spaces of cellulose beads.

[0175] Each of the aforementioned identified patents (including all figures) is incorporated herein in its entirety, as if each patent were incorporated herein by reference in its entirety.

[0176] Without implying any limitations, the outer surface of a bead or particle can be determined by tightly wrapping the entire bead or particle with an elastic membrane. This wrapping can be imagined through thought experiments, depicted in drawings or photographs, or physically performed. Without implying any limitations, the outer surface of the bead is the portion of the bead that is in physical contact with the wrapping.

[0177] For example, the present disclosure provides a bead having pores that comprise at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40% of the surface area. Additionally, the present disclosure provides a bead wherein the volume of the internal channels or network comprises 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%, at least 80% of the total volume of the bead, and wherein the internal channels or network are in fluid communication with the outer surface of the bead (and the external medium).

[0178] Additionally, the present disclosure provides a bead having pores that comprise less than 1%, less than 2%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40% of the surface area. Additionally, the present disclosure provides a bead wherein the volume of the internal channels or network comprises 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%, less than 80% of the total volume of the bead, and wherein the internal channels or network are in fluid communication with the outer surface of the bead (and the external medium).

[0179] Iron core beads. The present disclosure encompasses iron core beads or magnetic beads. These beads can be manipulated with a magnet to move them from one reaction vessel to another reaction vessel, or from one vessel to another vessel. The use of these magnetic beads can enhance the operation of a robot. Methods of making 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).

[0180] In exclusive embodiments, the present disclosure can exclude any bead or any population of beads wherein the bead or population meets one of the above values or ranges.

[0181] Compounds loaded into the beads

[0182] In many experiments, it is advantageous to load pre-synthesized compounds into beads, which can be used as a vehicle to deliver the compounds to the assay. Many standard techniques for drug delivery to biological samples can be adapted for delivery of compounds to the assay (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).

[0183] In such embodiments, where pre-synthesized compounds are loaded into the beads, the compounds can be held in traditional 96, 385, or 1536 well microtiter plates. Beads can be added to these plates and the compounds loaded into them by diffusion or by other active loading methods. In preferred embodiments, the beads chosen for impregnation have a pore size or a percolation geometry that prevents the compound from draining immediately upon removal from the mother liquor. If desired, diffusion from the beads can be enhanced by heat, pressure, additives, or other stimulants. In some embodiments, the beads loaded with compounds can be capped in a manner that prevents the internal contents from leaking until triggered by an external impetus. One method of capping the exterior of the porous beads involves adding a lipid or amphiphilic molecule to the bead-compound solution such that the cavities exposed to the bead surface are sealed by bilayers formed from the amphiphilic molecules. In some embodiments, preformed vesicles can be mixed with the drug-loaded beads such that upon agitation, the vesicles rupture and the membranes reform on the surface of the drug-loaded beads, thereby sealing them. Methods of performing such bead sealing are described (see Tanuj Sapra et al. (2012) Nature Scientific Reports, vol. 2, article number: 848). Further protocols for sealing silica beads can be found in the report 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 method described by Hui Zheng et al., bSUM (bSUM: Bead-Supported Monolayer Membrane System Facilitates Unidirectional Insertion of Membrane Proteins into Giant Vesicles), J. Gen. Physiol (2016) 147:77-93.

[0184] In some embodiments utilizing pre-synthesized compounds, vesicles containing the compound inside or in the bilayer membrane are formed by adding the appropriate reagents, such as by adding a lipid or diblock copolymer, followed by agitation, from the compound. In some embodiments, the compound can be pushed through a microfluidic T-junction to produce water phase droplets in an oil phase, with the compound contained within the water phase or at the interface between the water and oil phases. In some embodiments, the formed droplets can be further polymerized, resulting in hydrogels that are more robust and stable to handling than un-polymerized water phase droplets. Droplet-based encapsulation and assays are disclosed by Oliver et al. (2013) “Droplet Based Microfluidics,” SLAS Discovery, Vol. 19, No. 4, pp. 483-496. Sol-gel encapsulation methods can also be used 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.

[0185] One-bead-one-compound (OBOC)

[0186] Methods for manufacturing combinatorial libraries involve three steps: (1) preparing the library; (2) screening the compounds in the library, and (3) determining the structure of the compounds, e.g., all of the compounds or only those that provided interesting results by screening (see Lam et al. (1997) “The One-Bead-One-Compound Combinatorial Library Method” Chem. Rev. 97:411-448). The advantage of synthesizing compounds by the way of bead-bound synthesis is that the compounds can be rapidly prepared by the “split and pool” method.

[0187] OBOC is combined with a coding strategy. Another feature of OBOC is that each bead can contain not only a compound but also a coding strategy. When the bead-bound nucleic acid encodes for the compound (bound to the uniform bead), the term "coding" does not refer to the genetic code. Instead, the term "coding" means that the user has a legend, key, or code that relates each of the thousands of short nucleic acid sequences to the individual bead-bound compound.

[0188] The following is a dramatic change in the use of beads with bead-bound compounds and bead-bound nucleic acids, where the nucleic acids encode for the associated compound. The dramatic change is to make a library of conjugates, where each member of the library takes the form of a conjugate of a small molecule plus a DNA moiety, where the DNA moiety encodes for the small molecule. The conjugate is soluble and not bead-bound. After screening with cells or purified proteins, the conjugate remains bound to the cells or purified proteins, enabling isolation of the conjugate and ultimately identification of the compound by sequencing the nucleic acid of the conjugate (see Satz et al. (2015) Bioconjugate Chemistry 26: 1623-1632).

[0189] In this document, as in much of this patent document, the term "coding" does not refer to the genetic code, but rather to the fact that the researcher uses a particular nucleic acid sequence to represent a particular known structure of a compound (linked to it).

[0190] As an alternative to using a coding strategy, such as using DNA barcodes, the bead that screens positive (thus indicating the compound that screens positive) can be subjected to Edman degradation or mass spectrometry to identify the bead-bound compound (see Shih et al. (2017) Mol. Cancer Ther. 16: 1212-1223). If the bead-bound compound is a peptide, then MALDI mass spectrometry can be used to directly determine the sequence of the positive screening peptide compound. Direct sequencing is possible because cleavage and ionization occur simultaneously under laser irradiation (Song, Lam (2003) J. Am. Chem. Soc. 125: 6180-6188).

[0191] One advantage of performing split-and-pool synthesis of combinatorial libraries is that the compounds can be made so that all of the compounds have the same motif. This strategy has been 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., supra, at page 418).

[0192] To provide a typical example of large beads, the beads can have a diameter of 0.1 mm and can hold approximately 10 of the same compound. 13 Each bead can be used as a copy (Lam et al., ibid.). After preparing a library of bead-bound compounds, each bead can be used in an individual assay, wherein the assay measures biochemical activity or, alternatively, binding activity. The assay can be an "on-bead" assay, or alternatively, the compound can be cleaved from the bead and used in a solution-phase assay (Lam et al., ibid.).

[0193] The parameters of any type of bead include its tendency to swell in a given measurement medium, whether the polymer of the bead is hydrophobic or hydrophilic, the identity of the connection sites on the bead used to connect each compound, whether spacers (such as polyethylene glycol (PEG)) are used for some kind of separation of each compound from the bead surface, and the internal volume of the bead.

[0194] Regarding the issue of needing to attach compounds to beads, but away from the hydrophobic surface of the beads, Lam et al. (ibid.) disclosed polyoxyethylene-grafted styrene. It has the following advantages: the functionalizable groups are located at the ends of the polyoxyethylene chain, thus away from hydrophobic polystyrene. Beads with water-soluble linkers include TentaGel and polydimethylacrylamide beads (…). Gel, Cambridge Research Biochemicals, Novage, UK.

[0195] Internal volume parameters can provide an advantage in situations where it is necessary to prevent interactions between the DNA barcode bound to the bead and the target of the bead-bound compound. To utilize this advantage, beads can be fabricated with the DNA barcode located inside the bead, and conversely, the selected compound linked to the surface of the bead (Lam et al., ibid., pp. 438-439). This advantage of internal volume may be irrelevant in cases where the bead-bound compound is linked by a cleavable linker, and only in this case are the compounds determined by cleavage and release.

[0196] Appell et al. provide the following non-limiting example of a split and pool method for synthesizing a chemical library, which is then screened to detect active compounds (Appell et al. (1996) J. Biomolecular Screening 1 :27-31). In a series of wells in a first microplate, nanowell plate, or picowell plate, each well is placed with a library bead. The beads are exposed to light to cleave about 50% of the bead-bound compounds, which are then released into the well. The released compounds are then transferred to a second microplate and assayed to detect wells containing active compounds, thereby determining which beads in the first plate contain active bead-bound compounds. Then, “once an active [compound] is identified from a single bead, the bead is recovered and decoded to yield the synthetic history and... the structure of the active compound” (Appell et al., supra).

[0197] For a cell-based screening assay to screen bead-bound compounds, Shih et al. provide a novel bead (Shih et al. (2017) Mol. Cancer Ther. 16:1212-1223). This novel bead contains a bead-bound compound that is a member of a “synthetic death ligand” library against ovarian cancer. The bead is also decorated with biotin, has more than two chemicals added to it to form a sandwich, and wherein the sandwich maintains the cell’s attachment force to the bead. The sandwich comprises a streptavidin plus biotin-LXY30 complex. The sandwich links the bead to the receptor for LXY30, which happens to be a well-known protein on the surface of cells, namely integrin. The method of Shih et al. (supra) led to the discovery of a new molecule that can kill cancer cells (“LLS2”). The above method uses a bead-bound compound, wherein the compound is bound to the cell (even though the compound is still bead-bound). Cho et al. created a similar bead-compound library, wherein the screened compounds are sufficient to bind to cells (without the sandwich described above) (Cho et al. (2013) ACS Combinatorial Science 15:393-400). The purpose of the report by Cho et al. was to discover RGD-containing peptides that bind to integrin expressed by cancer cells. The above-disclosed reagents and methods can be used in the present disclosure.

[0198] Coupling nucleic acids to beads (orthogonal; concatenated)

[0199] One approach to the subject of concatenated barcodes and orthogonal barcodes is to note the advantages one has over the other. The advantages of orthogonal barcodes over concatenated barcodes are as follows. With each ligation of a monomer of a growing compound, ligated in parallel is a DNA barcode module. With concatenated barcodes, if the ligation of any given module is not perfect (meaning not all ligation sites have successfully coupled with the desired module), then the order of the complete barcode will be incorrect. The statement "incorrect" means that imperfect coupling means that a blob of possibly missing mass is considered to be a complete, correct DNA barcode. In this context, the complete barcode sequence will contain errors due to the failure of all modules to ligate. In contrast, with orthogonal barcodes, each individual module is covalently bound to its own unique ligation site on the bead. And, once a module is ligated to a given site on the bead, no other module can be ligated to the ligated module.

[0200] The present disclosure provides reagents and methods for reducing damage to bead-bound DNA barcodes, and for reducing damage to partially synthesized bead-bound DNA barcodes. Each DNA barcode module, prior to ligation to a growing bead-bound DNA barcode, can take the form of double-stranded DNA (dsDNA), where this dsDNA is treated with a DNA cross-linking agent such as mitomycin-C. After synthesis of the DNA barcode in the dsDNA form is complete, this dsDNA is converted to ssDNA. Conversion of dsDNA to ssDNA can be achieved in the context where one of the DNA strands has uracil (U) residues and where a uracil-N-glycosylase catalyzes cleavage of the DNA at the uracil residue positions (see U.S. Serial No. 62 / 562,905, filed September 25, 2017, which is incorporated by reference in its entirety herein). The above refers to damage caused by reagents used to make bead-bound compounds to growing DNA barcodes. Figure 5 The above refers to damage caused by reagents used to make bead-bound compounds to growing DNA barcodes (where the compound is a member of a chemical library).

[0201] Another approach to reducing damage to bead-bound DNA barcodes and to reducing damage to partially synthesized DNA barcodes is by synthesizing the DNA barcode in the form of double-stranded DNA, where each of the DNA barcode modules that are ligated to one another take the form of dsDNA, and where each of the two strands is stabilized by a DNA padlock. To perform final sequencing of the complete DNA barcode, one of the strands is cleaved from the DNA padlock and removed. The above refers to damage caused by reagents used to make bead-bound compounds to growing DNA barcodes (where the compound is a member of a chemical library).

[0202] Yet another method to reduce damage to bead-bound DNA barcodes is to synthesize the DNA barcode in a manner that self-assembles into a hairpin, and wherein the DNA barcode self-assembly anneals the first prong of the hairpin to the second prong of the hairpin.

[0203] If the DNA barcode being synthesized is in the form of double-stranded DNA (dsDNA), solvents such as DCM, DMF, and DMA denature the DNA barcode. The methods and reagents described above can prevent denaturation.

[0204] As described above, the term "DNA barcode" can refer to a polynucleotide that collectively identifies a compound, whereas a "DNA barcode module" can refer to only one of the monomers that make up the compound.

[0205] 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 to seal the ends of this dsDNA with 7-aza-dATP and dGTP.

[0206] In alternative embodiments, the method can use intermediates between "cascading DNA barcodes" and "orthogonal DNA barcodes," wherein the intermediates involve blocks of DNA barcodes, i.e., wherein each block contains two DNA modules, or contains three DNA modules, or contains four DNA modules, or contains five DNA modules, etc. (but does not contain all of the DNA modules that identify the full-length compound).

[0207] Figure 1 Exemplary and non-limiting figures of cascading structured beads are disclosed. The beads contain multiple DNA barcodes (each made of DNA barcode modules) and multiple compounds (each made of chemical library monomers). For ease of speaking, the term "DNA barcode" can be used to refer to the polymer that contains all of the nucleic acids that are "DNA barcode modules" as well as all of the nucleic acids that provide certain functions. The functions can be annealing sites for sequencing primers, or the functions can be used to identify chemical synthesis steps of the bead-bound compounds. Figure 1 Also shown are bead-bound compounds, wherein each compound is composed of several chemical library members, each chemical library member represented by a square, circle, or triangle. Figure 1 Each DNA barcode module is shown numbered consecutively from 1 to 8, wherein these numbers correspond to the eight shapes (squares, circles, triangles), respectively. For clarity, the nucleic acids that provide functions (do not represent or "encode" any particular chemical unit) are not shown in the figure.

[0208] Figure 2Exemplary and non-limiting embodiments of orthogonally structured beads are disclosed. The beads contain multiple DNA barcodes (each made of a DNA barcode module), but each DNA barcode module is attached to a separate attachment site on the bead. The entire DNA barcode is made of eight DNA barcode modules, numbered 1-8 in the figure. When reading information from a particular DNA barcode, which is then used to identify the compound bound to the same bead, DNA sequencing must be performed on each of the individually attached DNA barcode modules. In Figure 2 the beads also contain multiple attached compounds, each with eight units, as shown by the eight shapes (circles, squares, triangles).

[0209] In Figure 2 for clarity, the functional nucleic acids attached to each DNA barcode module are not shown. Of course, each DNA barcode module needs to have a nucleic acid that identifies the position of the chemical library monomer in the complete full-length compound. For the example shown, the position must be first, second, third, fourth, fifth, sixth, seventh, or eighth. Figure 2

[0210] In one embodiment, the chemical monomers are attached first, and then the corresponding DNA barcode modules are attached. In an alternative embodiment, the DNA barcode modules are attached first, and then the corresponding chemical monomers are attached. Additionally, the flow of organic synthesis can be followed, sometimes using “one embodiment” and sometimes using “alternative embodiment.” In yet another alternative embodiment, the present method provides for segmented addition of blocks of several chemical monomers (attached to the bead) in parallel with attachment of blocks of several DNA barcode modules.

[0211] In exclusive embodiments, the use of reagents, compositions, and methods for segmented addition of chemical monomers, DNA barcode modules, or both chemical monomers and DNA barcode modules can be excluded.

[0212] This relates to the nucleic acids that can be present in the bead-bound polynucleotides, including nucleic acids that “code” or are used to identify the monomers of the bead-bound compound. In exclusive embodiments, the present disclosure can exclude nucleic acids that code for “step-specific DNA sequencing primer sites.” In this case, for each chemical monomer present in the compound, there is a corresponding DNA barcode module, where the flanks of each DNA barcode module are at least one corresponding primer binding site, i.e., “step-specific DNA sequencing primer site.” Additionally, excluded can be 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.

[0213] ​Additionally, the present disclosure can include nucleic acids that are spacers. For example, a spacer can create a distance along a polynucleotide strand between a first site that is a sequencing primer annealing site and a second site that identifies a chemical monomer. Additionally, the present disclosure can use nucleic acids that repeat or corroborate information provided by another nucleic acid. Additionally, the present disclosure can use nucleic acids that encode PCR primer binding sites. PCR primer binding sites can be distinguished from sequencing primer because a polynucleotide with a PCR primer binding site has two PCR primer binding sites, and because the two sites are designed to have the same melting point (the site at which the melting point of a PCR primer anneals to a PCR primer binding site).

[0214] In exclusive embodiments, the present disclosure can exclude nucleic acids that are spacers or only spacers. Additionally, the present disclosure can exclude nucleic acids that repeat or corroborate information provided by another nucleic acid. Furthermore, the present disclosure can exclude nucleic acids that function as PCR primer binding sites, and can exclude nucleic acids that function as binding sites for primers that are not PCR primers.

[0215] Additionally, the present disclosure can exclude nucleic acids that identify the date of manufacture of a chemical library or identify a chemical synthesis step of a particular compound or function as a primer annealing sequence.

[0216] Specialized sequencing primers for specific DNA barcode modules. The present disclosure provides a DNA barcode that contains DNA barcode modules and one or more sequencing primer annealing sites. Each DNA barcode module can have its own specialized sequencing primer binding site. Alternatively, one particular sequencing primer binding site can be used to sequence two, three, four, five, 6, 7, 8, 9, 10 or more consecutive DNA barcode modules, which can exist on a DNA barcode bound to a bead.

[0217] Each DNA barcode module has its own dedicated sequencing primer binding site. The present disclosure provides a bead-bound concatenated barcode comprising a primer binding site capable of binding a sequencing primer for DNA, wherein the primer binding site is capable of directing sequencing of one or more of 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, and wherein the primer binding site is located 3' of the first DNA barcode module (no other DNA barcode module between the first DNA barcode module and the primer binding site), 3' of the second DNA barcode module (no other DNA barcode module between), 3' of the third DNA barcode module (no other DNA barcode module between), 3' of the fourth DNA barcode module (no other DNA barcode module between), 3' of the fifth DNA barcode module (no other DNA barcode module between), or 3' of the sixth DNA barcode module (no other DNA barcode module between).

[0218] Coding sequences and sequences complementary to coding sequences. The present disclosure can encompass any one of, any combination of, or all of the coding sequences disclosed above or elsewhere in this document. Excluded in exclusive embodiments can be any one of, any combination of, or all of the coding sequences disclosed above or elsewhere in this document. Also encompassed or excluded can be double-stranded nucleic acids encoding any one of, any combination of, or all of the coding sequences disclosed above or elsewhere in this document.

[0219] Orthogonal DNA barcodes (each DNA barcode module attached to a separate location on a bead)

[0220] Synthesis of orthogonal beads. By orthogonal synthesis, each DNA module is covalently attached to a separate site on a bead, and wherein the result is that the entire DNA barcode is contributed by multiple DNA modules. In the case of a DNA barcode having an orthogonal structure, no DNA barcode module is connected to another, rather each DNA barcode molecule has its own bead binding site specific to that particular DNA barcode module.

[0221] The nucleic acid identifies the step number of synthesis of each DNA barcode module. In an embodiment, the orthogonal DNA barcode comprises a short nucleic acid that identifies the first step of compound synthesis. For this embodiment, where a first chemical monomer and a first DNA barcode module are connected in parallel, the first DNA barcode module is in fact in the form of this complex of two nucleic acids: [the short nucleic acid that means "step one"] connected to [the first DNA barcode module]. All of the nucleotides of this complex are in frame and can be read in a sequencing analysis, but the first short nucleic acid can optionally be connected to the first DNA barcode module by a spacer nucleic acid.

[0222] The above description of orthogonal DNA barcodes continues below. The orthogonal DNA barcode comprises a short nucleic acid that identifies the second step of compound synthesis. For this embodiment, where a second chemical monomer and a second DNA barcode module are connected in parallel, the second DNA barcode module is in fact in the form of this complex of two nucleic acids: [the short nucleic acid that means "step two"] connected to [the second DNA barcode module]. All of the nucleotides of this complex are in frame and can be read in a sequencing analysis, but the second short nucleic acid can optionally be connected to the second DNA barcode module by a spacer nucleic acid.

[0223] The above method is repeated for a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and until the last DNA barcode module and the last chemical monomer for any given bead. When split and pool synthesis is used, the above method can be followed to create DNA barcoded and bead-bound compounds.

[0224] The orthogonal structure has the following advantages over the concatenated structure. When using concatenated synthesis (all DNA barcode modules connected to each other in a continuous polymer), any intermediate coupling step that fails the synthesis can destroy the meaning of the final completed concatenated DNA barcode. In contrast, by orthogonal synthesis (each DNA barcode module connected to a dedicated site on the bead), not connecting any DNA barcode module will only result in the connected site on the bead being empty, without destroying the meaning of any other connected DNA barcode module. In preferred embodiments, each connected DNA barcode module comprises a connected second nucleic acid, wherein the second nucleic acid identifies the step (step during parallel synthesis of DNA barcode and compound).

[0225] For orthogonal synthesis, it is acceptable for all ligation sites (sites for ligation of growing chemical library members) on a bead to be exhausted. However, for orthogonal synthesis, it is desirable to design chemical reactions such that all ligation sites on a bead are only partially exhausted in the case of ligation of the first DNA barcode module among many DNA barcode modules. The following provides optional limits on exhaustion of sites during orthogonal barcode chemical synthesis. For unmodified beads, the total number of sites available for ligation of DNA barcode modules is 100%.

[0226] The extent to which ligation sites on a given bead are exhausted is through synthesis of the bead in an orthogonal configuration (with respect to the first DNA barcode). The following relates to ligation of the first DNA barcode module. In embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of DNA barcode ligation sites on a bead are exhausted in the case of ligation of the first DNA barcode module. 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 about 50% of DNA barcode ligation sites on a bead are exhausted. In still other embodiments, between 2-4%, between 2-6%, between 2-8%, between 2-10%, between 2-12%, between 2-14%, between 2-16%, between 2-18%, between 2-20%, between 10-20%, between 10-25%, between 10-30%, between 10-35%, between 10-40% of DNA barcode ligation sites are exhausted in the case of ligation of the first DNA barcode module.

[0227] With respect to limits, less than 20% of sites are exhausted in the case of ligation of the last DNA barcode module making up a particular DNA barcode, 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 sites are exhausted.

[0228] Exclusive embodiments can exclude beads or methods that match any of the above values or ranges. Additionally, exclusive embodiments can exclude beads or methods that do not match any of the above values or ranges.

[0229] The following relate to polymers comprising one or more nucleic acids (each a DNA barcode), and to polymers comprising two or more nucleic acids, where some of the nucleic acids have a biochemical function, such as serving as a primer annealing site or as a spacer, and where other nucleic acids have an informational function and are DNA barcodes. In exclusive embodiments, the present disclosure can exclude DNA barcodes that contain a DNA crosslinker such as psoralen. Also excluded can be DNA barcodes that have a primer binding region that has a higher melting temperature (or a lower melting temperature) than the DNA barcode module. The temperature can simply be "higher" or "lower," or can be at least 2 degrees Celsius higher, at least 4 degrees Celsius higher, at least 6 degrees Celsius higher, at least 8 degrees Celsius higher, or at least 2 degrees Celsius lower, at least 4 degrees Celsius lower, at least 6 degrees Celsius lower, at least 8 degrees Celsius lower.

[0230] Also excluded can be methods for making DNA barcodes that use DNA ligase. Also excluded can be DNA barcodes and methods of making them that include a hairpin (ssDNA bent into a loop, such that a portion of the ssDNA is hybridized to another portion of the same ssDNA). Also excluded can be compositions having a nucleic acid hairpin that is covalently closed, for example, by a chemical linker. Further, excluded can be DNA barcodes that are covalently linked (directly or indirectly) to a "headpiece" (indirectly by way of being covalently bound to one or more chemicals that reside between the DNA barcode and the headpiece).

[0231] In other exclusive embodiments, excluded can be bead-bound DNA barcodes, where the intact DNA barcode does not include any double-stranded DNA (dsDNA), but only single-stranded DNA (ssDNA).

[0232] The extent to which the ligation sites on a given bead are exhausted is through the synthesis of orthogonally configured beads (with respect to the second DNA barcode). The following relates to ligation of a second DNA barcode module. In embodiments, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the remaining free DNA barcode ligation sites on a bead are exhausted in the ligation of a second DNA barcode module (for creating orthogonally configured beads). In other embodiments, less than about 5%, less than about 10%, less than about 20%, less than about 30%, less than about 40%, or less than about 50% of the remaining free DNA barcode ligation sites on a bead are exhausted. In still other embodiments, between 2-4%, between 2-6%, between 2-8%, between 2-10%, between 2-12%, between 2-14%, between 2-16%, between 2-18%, between 2-20%, between 10-20%, between 10-25%, between 10-30%, between 10-35%, between 10-40% of the remaining free DNA barcode ligation sites are exhausted in the ligation of a first DNA barcode module.

[0233] Exclusive embodiments can exclude beads or methods that match any of the above values or ranges. Additionally, exclusive embodiments can exclude beads or methods that do not match any of the above values or ranges.

[0234] The above embodiments, as well as the above exclusive embodiments, can also apply to methods of ligating a third DNA module barcode, or ligating a fourth DNA module barcode, or ligating a fifth DNA barcode module, etc.

[0235] Cascaded DNA barcodes (all DNA barcode modules reside in one strand or one polymer, where the entire strand or polymer is ligated to one location on a bead).

[0236] Synthesis of bead-bound cascaded DNA barcodes. The present disclosure provides a bead-bound cascaded DNA barcode, where the bead contains a plurality of cascaded DNA barcodes, and where most or nearly all of the plurality of cascaded DNA barcodes have substantially the same structure. The cascaded DNA barcode can contain one or more DNA barcode modules, where the order of these DNA barcode modules along the entire DNA barcode (from the bead ligation end to the distal end) is the same as the order when synthesizing the bead-bound cascaded DNA barcode. Additionally, the order of these DNA barcode modules along the entire DNA barcode is the same as the order when the corresponding chemical library is singly coupled to the growing bead-bound compound.

[0237] A concatenated DNA barcode can include, in this order, a linker for coupling the entire concatenated DNA barcode to a bead. In addition, it can 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, a third annealing site.

[0238] One order of sequencing primer hybridization sites in a bead-bound DNA barcode. In an embodiment of a sequencing primer hybridization site, a concatenated DNA barcode can include, in this order, 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, and a third sequencing primer binding site, etc.

[0239] Another order of sequencing primer hybridization sites when occurring in a bead-bound DNA barcode. In another embodiment of a sequencing primer hybridization site, a concatenated DNA barcode can 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, and a third annealing site, etc.

[0240] The term "annealing site". The term "annealing site" is used to refer to an annealing site that is part of a splint oligonucleotide and also to refer to a corresponding bead-bound annealing site that resides on a growing bead-bound DNA barcode. The skilled artisan understands that the "annealing site" on the splint oligonucleotide does not have the same DNA sequence as the corresponding "annealing site" on the growing bead-bound DNA barcode. In other words, the skilled artisan understands that one sequence is complementary to another. Thus, for the description herein, two annealing sites having the same name are not related. In other words, the following are not related: the second annealing site on the splint oligonucleotide is disclosed to be the annealing site that hybridizes to the second annealing site on the growing bead-bound DNA barcode.

[0241] Block synthesis. In alternative embodiments, the growing sequence of growing compounds and growing DNA barcode modules can be synthesized in blocks. For example, a block consisting of 2 chemical library units can be attached to a bead in parallel with a block consisting of a corresponding 2 DNA barcode modules. Similarly, a block consisting of 3 chemical library units can be attached to a bead in parallel with a block consisting of a corresponding 3 DNA barcode modules. Block synthesis involving four blocks, five blocks, six blocks, seven blocks, eight blocks, nine blocks, ten blocks, etc. are also provided. Each of these block transfer embodiments can also be excluded by the present disclosure. Block transfer of DNA barcode monomers can be performed orthogonally, with unique attachment points for receiving each successive block of DNA barcode monomers. Alternatively, block transfer of DNA barcode monomers can be performed to create a concatemer structure (all DNA barcode modules present only in one continuous linear polymer).

[0242] Additionally, during parallel split-and-pool synthesis of bead-bound DNA barcodes and bead-bound compounds, synthesis can be performed in blocks. The blocks can take the form of two or more than two chemical library monomers, and the blocks can take the form of two or more than two DNA barcode modules.

[0243] Location of split-and-pool synthesis. Split-and-pool synthesis can be used for parallel synthesis of bead-bound compounds and bead-bound concatenated DNA barcodes. Additionally, split-and-pool synthesis can be used for parallel synthesis of bead-bound compounds and bead-bound orthogonal DNA barcodes. Concatenated DNA barcodes can be made by a “clamper oligo” approach. Alternatively, concatenated DNA barcodes can be made by a click chemistry approach. Additionally, a combination of “clamper oligo” approach and click chemistry can be used. Split-and-pool synthesis can be performed in a 96-well plate, where the floor of each well is made of a 0.25 micron filter. Under normal gravity conditions, an aqueous solution will not flow through this filter. However, in cases where a first aqueous solution needs to be replaced with a second aqueous solution, a suction can be applied to remove any aqueous solution from all 96 wells. This suction method is used when the beads are exposed to a first set of reagents, or when the first set of reagents needs to be washed away, or when the first set of reagents needs to be replaced with a second set of reagents. A manifold is used to hold the 96-well plate (Resprep VM-96 manifold), and a pump can be used to draw fluid out of the bottom of each filter (BUCHI Vac V-500 pump). The 96-well plate with filter bottoms is AcroPrep Advance 96-well, 350uL, 0.45um, REF 8048 (Pall Corp., Ann Arbor, MI).

[0244] Distance from primer annealing site to DNA barcode module. For the purpose of sequencing the bead-bound DNA barcode, i.e. for the purpose of sequencing all DNA barcode modules forming the DNA barcode, the polynucleotide comprises a first nucleic acid, which is the annealing site for a sequencing primer, and a second nucleic acid, which is the DNA barcode module. The first nucleic acid can be directly upstream of the second nucleic acid. Alternatively, the first nucleic acid can be upstream of the second nucleic acid, wherein the first nucleic acid and the second nucleic acid are separated from each other by one, two, three, four, five, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides, or about one, about two, about three, about four, about five, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15 nucleotides. The separation can be performed using a nucleic acid that is only a spacer, or alternatively, the separation can be performed using a third nucleic acid that encodes information, such as the number of steps in an organic synthesis, or the number of a class of compounds, or the disease that can be treated by the bead-bound compound, or the date or batch, etc.

[0245] Synthesis of bead-bound cascade DNA barcodes by click chemistry

[0246] Click chemistry can be used for the step-by-step synthesis of DNA barcodes. In this context, the first DNA barcode module can be coupled directly to the bead, or the first DNA barcode module can be coupled to a bead-bound linker.

[0247] Alternatively, what can be coupled is a polynucleotide in the form of a first nucleic acid, which is itself the first DNA barcode module, linked to a second nucleic acid, which is itself a first sequencing primer binding site. This sequencing primer binding site allows the operator to determine the sequence of the first DNA barcode module.

[0248] Providing a further example, a second DNA barcode module can be directly linked to the bead-bound first DNA barcode module. Alternatively, what can be coupled is a polynucleotide in the form of a first nucleic acid, which is itself the second DNA barcode module, linked to a second nucleic acid, which is itself a second sequencing primer binding site. This sequencing primer binding site allows the operator to determine the sequence of the second DNA barcode module. If the first DNA barcode module is read through, the sequences of both DNA barcode modules can be determined.

[0249] Providing yet another example, what can be coupled is a polynucleotide comprising a first nucleic acid (itself a first DNA barcode module) and a second nucleic acid (identifying a step in the multi-step parallel synthesis of a DNA barcode and a compound). Additionally, or alternatively, the second nucleic acid can identify a general class of compounds made by a split and pool synthesis. Additionally, or alternatively, the second nucleic acid can identify a disease to be treated by the compound to be screened. Additionally, the second nucleic acid can identify a date or a chemist's name, among others.

[0250] One preferred method for synthesizing DNA barcodes is shown below, where the same reaction cycle is used and each DNA barcode module is attached stepwise.

[0251] Step 1. Provide the bead with a linked TCO group. In practical practice, the bead will have hundreds or thousands of the same linked TCO groups, with each TCO group linked to a different site on the bead. Additionally, in practical practice, a large number of beads will be modified simultaneously using the click split and pool method and the click chemistry method.

[0252] Step 2. Add [tetrazine]-[first DNA barcode module]-[azide] to the bead and allow the TCO group to condense with the tetrazine group. The result is the following structure: Bead-TCO-tetrazine-first DNA barcode module-azide. In practical practice, this construct does not contain any TCO or tetrazine, but rather has the condensation product that results when the TCO condenses with the tetrazine.

[0253] Step 3. Optional wash.

[0254] Step 4. Add DBCO-TCO to cap the azide and create a TCO end. The result is the following structure:

[0255] Bead-TCO-tetrazine-first DNA barcode module-azide-DBCO-TCO.

[0256] Step 5. Optional wash.

[0257] Step 6. Add reagents that link a second DNA barcode module. The linkage is at the distal end of the growing DNA barcode. The reagents are:

[0258] to the bead [tetrazine]-[second DNA barcode module]-[azide] and allow the TCO group to condense with the tetrazine group. The result is the following structure:

[0259] Bead-TCO-tetrazine-first DNA barcode module-azide-DBCO-TCO-[tetrazine]-[second DNA barcode module]-[azide]

[0260] The above protocol contains a cycle of steps for the stepwise addition of more and more DNA barcode modules, where these additions are in parallel with the addition of more and more chemical monomers. As described elsewhere, this "parallel" synthesis can involve the ligation of a chemical monomer, followed by the ligation of a DNA barcode module that identifies that monomer, or alternatively, the ligation of a DNA barcode module, followed by the ligation of a chemical monomer that is identified by that particular chemical monomer.

[0261] Compounds for click chemistry synthesis of DNA barcodes

[0262] Figure 17 Chemical synthesis of compounds suitable for ligation of deoxycytidine residues (dC) during synthesis of DNA barcode modules and ultimately the entire DNA barcode is disclosed. The starting material is N4-acetyl-2'-deoxy-5'-O-DMT cytidine. The abbreviation "DMT" stands for 4,4-dimethoxytrityl. The end product of this multi-step pathway of organic synthesis carries a cytosine moiety, a triphosphate group and an alkyne group attached to the 3' position of the ribose group. The alkyne group is used in click chemistry, where it condenses with an azide group to yield a covalent bond. After condensation, the result is that a chemical that is never naturally occurring in nucleic acids remains as a "scar" from the performed click chemistry. Available DNA polymerases can be used for sequencing-by-synthesis of DNA barcodes made by click chemistry, and where the DNA polymerase can pass through the scar, and where the scar does not cause sequencing errors. TBAI is tetrabutylammonium iodide.

[0263] Synthesis of cascade configuration DNA barcodes

[0264] In the following description, DNA barcode modules are lined up to create a DNA barcode. However, in the in-text figures shown below, the term "DNA barcode" is used in place of "DNA barcode modules" in order to fit the in-text figures on the page. Figure 7 The same steps are shown as here, but with more detail, such as the beads. The repeated reaction sequence can be used to add each additional DNA barcode module.

[0265] An option to create a DNA barcode that contains an end nucleic acid that encodes a DNA hairpin. This involves a DNA barcode that has a nucleic acid at the 3' end that has a sequencing primer annealing site, a bend in the form of about four base pairs (not base paired), and a sequencing primer that is able to bend and forms base pairs with the sequencing primer annealing site. For repetition, the sequencing primer anneals to the sequencing primer annealing site, where the actual sequencing reaction starts at the 3' end of the annealed sequencing primer.

[0266] When the final step of synthesizing the DNA barcode needs to be performed, and the final DNA barcode module is to be coupled to the bead-bound DNA barcode that is growing, the "clamp oligo" can comprise a sequence that encompasses a DNA hairpin (the DNA hairpin comprises, in this order, an annealing site for a sequencing primer, a number of nucleotides that do not base pair with each other or with adjacent base sequences, and a sequencing primer). After the "clamp oligo" is annealed, then a DNA polymerase and dNTPs are added, where polymerization occurs at the 3' end of the growing DNA barcode, where the order of the polymerization using the clamp oligo as a template is: (1) the annealing site for a sequencing primer; (2) a kink in the hairpin that takes the form of four or five deoxyribonucleotides that do not base pair with each other; (3) the sequencing primer.

[0267] Reversible terminator groups at the 3' end of a hairpin sequencing primer. The present disclosure provides reagents, compositions, and methods for linking a preformed nucleotide / reversible terminator group complex to the 3' end of an annealed sequencing primer. The reversible terminator group is an optional component of a hairpin sequencing primer that is part of a bead-bound DNA barcode.

[0268] Step 1. At the start, we have a bead in a picowell, where the bead has a coupled polynucleotide, and where the 5' end of the polynucleotide is optionally coupled to the bead through a linker. Figure 7 The coupled polynucleotide is shown to include a first DNA barcode and a first annealing site. The linker can be made of nucleic acid, or can be made of some other chemical. Preferably, the linker is hydrophobic, and preferably the linker separates the bead-bound DNA barcode from a hydrophobic polystyrene bead (e.g., The bead

[0269] For ease of writing, both the first annealing site (part of the bead-bound DNA barcode) and the first annealing site (part of the soluble "clamp oligo") are referred to as "the first annealing site," even though they do not have the same base sequence (instead, the base sequences are complementary to each other, with the result that the clamp oligo can hybridize to the first annealing site on the bead-bound growing DNA barcode, thereby serving as a template for a DNA polymerase to extend the bead-bound DNA barcode by copying what is on the clamp oligo.

[0270] Also, for ease of writing, both the second annealing site (part of the bead-bound DNA barcode) and the second annealing site (part of the soluble "clamp oligo") are referred to as "the second annealing site," even though they do not have the same sequence (but have complementary bases).

[0271] The bead-bound growing DNA barcode from 5' to 3' can contain the following sequence of nucleic acids:

[0272] bead- / first DNA barcode / first annealing site

[0273] Alternatively, the bead-bound growing DNA barcode from 5' to 3' can comprise nucleic acids that encode the step number, where the bead-bound growing DNA barcode has the following sequence of nucleic acids:

[0274] bead- / first DNA barcode / nucleic acid encoding step number / first annealing site

[0275] Alternatively, the bead-bound growing DNA barcode can contain nucleic acids that are themselves functional nucleic acids (sequencing primer annealing sites) as follows:

[0276] bead- / first DNA barcode / sequencing primer annealing site / first annealing site

[0277] Not shown in these in-text diagrams is an optional linker that mediates the coupling of the DNA barcode to the bead. The linker can take the form of a nucleic acid, or it can be made of some other chemical substance.

[0278] Step 2. Addition of a soluble clamp oligonucleotide, where this clamp oligonucleotide comprises a first annealing site and a second DNA barcode module, and a second annealing site.

[0279] Figure 7 Also shown is the step of using the hybridized clamp oligonucleotide as a template, where a DNA polymerase catalyzes the ligation of the second DNA barcode module and the second annealing site to the bead-bound growing DNA barcode. Figure 7 Shown is the enzymatic product, where a DNA polymerase catalyzes the use of the clamp oligonucleotide as a template, resulting in a longer growing bead-bound DNA barcode (growing by covalent attachment of the second DNA barcode and the second annealing site). Shown immediately below the text is the complex of the clamp oligonucleotide, which is hybridized to the bead-bound growing DNA barcode:

[0280] bead- / first DNA barcode / first annealing site

[0281] ...................... first annealing site / second DNA barcode / second annealing site

[0282] To repeat Figure 7 Some of the information shown, shown immediately below is the clamp oligonucleotide:

[0283] " first annealing site / second DNA barcode / second annealing site

[0284] Step 3. DNA polymerase and dNTPs are added to extend the bead-bound DNA barcode. Shown below is the bead-bound growing DNA barcode, with the splint oligo still hybridized to it, and wherein the bead-bound growing barcode is longer than before because now attached to the barcode is a nucleic acid that is itself a "second DNA barcode module" and a nucleic acid that is itself a "second annealing site". Figure 7 This step is also illustrated. The splint oligo is shown below the bead-bound growing barcode:

[0285] bead- / first DNA barcode / first annealing site / second DNA barcode / second annealing site

[0286] ......................... first annealing site / second DNA barcode / second annealing site

[0287] Step 4. The splint oligo is washed away. The splint oligo can be encouraged to dissociate from the bead-bound growing barcode by heating, i.e., by heating the entire picowell plate to, e.g., about 60 degrees Celsius, about 65 degrees Celsius, about 70 degrees Celsius, about 75 degrees Celsius, about 80 degrees Celsius (for about ten minutes), or, alternatively, by adding dilute NaOH to the picowell array, followed by neutralization.

[0288] Step 5. A second splint oligo is added, which, after the bead-bound growing splint oligo, can serve as a template for mediating DNA polymerase catalyzed ligation of a third DNA barcode and a third annealing site. Shown below (but not in Figure 7 ) is the second splint oligo, which is a soluble reagent:

[0289] second annealing site / third DNA barcode / third annealing site

[0290] Step 6. This oligo is allowed to anneal to the corresponding bead-bound "second annealing site" and DNA polymerase is allowed to extend the bead-bound oligo, so that it contains the following in addition: third DNA barcode / third annealing site /

[0291] Step 7. The second splint oligo is washed away.

[0292] Step 4. The following splint oligo is added (this particular addition is not shown in Figure 7 ).

[0293] third annealing site / fourth DNA barcode / fourth annealing site

[0294] This soluble oligonucleotide has a nucleic acid that can anneal to a "third annealing site" of the bead-bound oligonucleotide. Upon annealing, a DNA polymerase with four dNTPs will be used and will be used to extend the bead-bound oligonucleotide to encode yet another DNA barcode module (a fourth DNA barcode). The above step cycle is repeated throughout the splitting and merging process, which creates in parallel a library of compounds and associated DNA barcodes, where each DNA barcode is associated with a given compound (where each DNA barcode informs us of the chemical synthesis history of the associated compound). The above step cycle is stopped when the chemical synthesis of the library of compounds is complete. With the full library of bead-bound DNA barcodes chemistry, the beads can then be dispensed into picowells of a picowell array.

[0295] The DNA barcode of each bead also constitutes a DNA barcode that is associated with each picowell. The DNA barcode allows identification of the bead-bound compound. The sequencing method of the present disclosure occurs inside the picowell while the bead is still inside the picowell. In an exclusive embodiment, the present disclosure can exclude any sequencing method and can exclude any reagent for sequencing, where the bead-bound DNA template is not sequenced, or the bead-bound DNA template that is located inside the picowell is not sequenced.

[0296] Annealing site for sequencing primer. In one embodiment, each DNA barcode module in the full DNA barcode is operably linked to and in frame with its own sequencing primer annealing site, enabling the operator to perform a separate sequencing flow for each DNA barcode module (in this embodiment, it is preferred that each DNA barcode module can also be operably linked to its own nucleic acid that identifies (encodes) the synthesis step of the entire DNA barcode.

[0297] In another embodiment, each DNA barcode has only one sequencing primer annealing site, where this can be located at or near the 3' end of the bead-bound DNA barcode, and where the sequencing primer itself is soluble, which is added to the picowell and then hybridizes to the sequencing primer annealing site. Alternatively, if the sequencing primer is part of a DNA hairpin, this DNA hairpin is added by a "clamper oligonucleotide" in the last step of creating the bead-bound DNA barcode. Figure 7 Any annealing site of any sequencing primer is not shown.

[0298] Coupling of nucleic acid to bead through 3' end

[0299] While various embodiments disclosed in the present invention involve coupling DNA to a bead through the 5' end of the DNA, in other embodiments, DNA (such as DNA barcodes or DNA tags) can be coupled to a bead through their 3' end. The 3'-hydroxyl of DNA can be reactive under certain chemical synthesis conditions (e.g. Mitsunobu transformation), leading to 3' end damage and inability to participate in extension, ligation, or other steps. Thus, DNA tags can be ligated to a bead through their 3' end to prevent deleterious chemical reactions and to prevent damage to the DNA barcode.

[0300] Exclusive embodiments regarding the bead-bound DNA barcodes of the present disclosure. Excluded can be any polymeric composition of matter of a bead, microparticle, microsphere, resin, or substance, wherein a cascading DNA barcode is linked to the bead through a photocleavable linker or cleavable linker.

[0301] Excluded can be any bead, microparticle, microsphere, resin, or polymeric composition of matter that does not comprise: (1) a cascading DNA barcode coupled to a first location on the bead, (2) a compound coupled to a second location on the bead, and wherein the first location is different from the second location. In preferred embodiments, the "compound" is made from a plurality of chemical library monomers.

[0302] Excluded can be any bead, microparticle, microsphere, resin, or polymeric composition of matter that does not have an outer surface (or multiple outer surfaces) and also does not have an inner surface (or multiple inner surfaces or interior region), and wherein the bead does not comprise at least 10,000 substantially identical cascading DNA barcodes coupled to the bead, and wherein at least 90% of the at least 10,000 substantially identical cascading DNA barcodes are coupled to the outer surface. In other words, excluded can be any bead in which at least 90% of the coupled cascading DNA barcodes are not coupled to the outer surface.

[0303] Excluded can be any bead, microparticle, microsphere, resin, or polymeric composition of matter that is made substantially from polyacrylamide or that comprises any polyacrylamide.

[0304] Excluded can be any bead, microparticle, microsphere, hydrogel, resin, or polymeric composition of matter that contains a promoter (such as a T7 promoter), or that contains a polyA region, or that contains both a promoter and a polyA region.

[0305] Methods with only one annealing / polymerization cycle to generate bead-bound DNA barcodes with two DNA barcode modules. The disclosure encompasses systems, reagents, and methods in which bead-bound DNA barcodes comprise only one annealing / polymerization step. The following figure represents this embodiment, in which the first figure illustrates annealing of the clamp oligo, and the second figure illustrates filling using a DNA polymerase. The end result is a bead-bound DNA barcode that contains two DNA barcode modules. In this particular process, the bead-bound starting material can optionally comprise a linker (but preferably does not comprise any cleavable linker), optionally a nucleic acid that encodes information other than an identification compound, and optionally a functional nucleic acid such as a sequencing primer or DNA hairpin. Two figures are shown in the text (see below):

[0306] bead- / first DNA barcode / first annealing site

[0307] ........................ first annealing site / second DNA barcode / second annealing site

[0308] bead- / first DNA barcode / first annealing site / second DNA barcode / second annealing site

[0309] ......................... first annealing site / second DNA barcode / second annealing site

[0310] Methods with two annealing / polymerization cycles to generate bead-bound DNA barcodes with three DNA barcode modules. The disclosure encompasses bead-bound compositions, systems, and methods in which two different clamp oligos are used (first clamp oligo; second clamp oligo). In this case, the first clamp oligo comprises the following structure: first annealing site / second DNA barcode / second annealing site, and wherein the second oligo comprises the following structure: second annealing site / third DNA barcode / third annealing site.

[0311] Methods with three annealing / polymerization cycles to generate bead-bound DNA barcodes with four DNA barcode modules. The disclosure encompasses bead-bound compositions, systems, and methods in which three different clamp oligos are used (first clamp oligo; second clamp oligo; third clamp oligo). In this case, the first clamp oligo comprises the following structure: first annealing site / second DNA barcode / second annealing site, and wherein the second oligo comprises the following structure: second annealing site / third DNA barcode / third annealing site, and wherein the third oligo comprises the following structure: third annealing site / fourth DNA barcode / fourth annealing site.

[0312] Methods with four annealing / polymerization cycles to produce a bead-bound DNA barcode with five DNA barcode modules. The disclosure encompasses bead-bound compositions, systems, and methods in which four different splint oligos are used (a first splint oligo; a second splint oligo; a third splint oligo; a fourth splint oligo). In this case, the first splint oligo comprises the following structure: a first annealing site / a second DNA barcode / a second annealing site, and wherein the second oligo comprises the following structure: a second annealing site / a third DNA barcode / a third annealing site, and wherein the third oligo comprises the following structure: a third annealing site / a fourth DNA barcode / a fourth annealing site, and wherein the fourth oligo comprises the following structure: a fourth annealing site / a fifth DNA barcode / a fifth annealing site,

[0313] Embodiments with multiple annealing / polymerization steps to produce a bead-bound DNA barcode with multiple DNA barcode modules. The disclosure encompasses bead-bound compositions, systems, and methods related to cascade barcoding that use only one splint oligo (to make a 2-module DNA barcode), only two splint oligos (to make a 3-module DNA barcode), only three splint oligos (to generate a 4-module DNA barcode), only four splint oligos (to generate a 5-module DNA barcode), only five splint oligos (to generate a 6-module DNA barcode), only six splint oligos (to generate a 7-module DNA barcode), and so on.

[0314] Encompassed are bead-bound compositions, systems, and methods that use at least one splint oligo, at least two splint oligos, at least three splint oligos, at least four splint oligos, at least five, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 20 splint oligos, or fewer than 20, fewer than 15, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 3, fewer than 2 splint oligos. These numbers refer to the number of splint oligos themselves, as well as the number of steps of adding splint oligos, and also refer to the number of DNA modules added to the growing DNA barcode on the bead.

[0315] Reducing damage to DNA barcodes

[0316] Reduction of damage by using orthogonal DNA barcodes (rather than concatenated DNA barcodes). One approach to the subject matter of concatenated DNA barcodes and orthogonal DNA barcodes is to note the advantages that one has over the other. The advantages of orthogonal barcodes over concatenated barcodes are as follows. By ligating each monomer of a growing compound, ligated in parallel are chemical library monomers (for creating a chemical library) and DNA barcode modules (for creating a complete full-length DNA barcode).

[0317] For concatenated barcodes, if the ligation of any given module is not perfect (meaning not all ligation sites have successfully coupled with the desired module), then the order of the complete barcode will be incorrect. The statement “incorrect” means that the imperfect coupling results in a missing piece, and in this case, the user considers the complete product to be a complete and correct DNA barcode. In this context, the complete DNA barcode sequence will contain an error due to the failure of all DNA modules to ligate. In contrast, with orthogonal barcodes, each individual DNA module is covalently bound to its own unique ligation site on the bead. And where once a DNA module is ligated to a given site on the bead, there is no need to couple other DNA modules to the DNA module that is already bound to the bead.

[0318] Reduction of damage by using a crosslinking agent. The present disclosure provides reagents and methods for reducing damage to bead-bound DNA barcodes, and for reducing damage to partially synthesized bead-bound DNA barcodes. Each DNA barcode module, prior to ligation to a growing bead-bound DNA barcode, can be in the form of double-stranded DNA (dsDNA), where this dsDNA is treated with a DNA crosslinking agent such as mitomycin-C. After synthesis of the DNA barcode in the dsDNA form is complete, this dsDNA is converted to ssDNA. Conversion of dsDNA to ssDNA can be achieved in cases where one of the DNA strands has uracil (U) residues and where uracil-N-glycosylase catalyzes cleavage of DNA at the position of uracil residues (see U.S. Serial No. 62 / 562,905, filed September 25, 2017, which is incorporated by reference in its entirety). Figure 5 U.S. Serial No. 62 / 562,905). The above refers to damage caused to a growing DNA barcode by reagents used to make a bead-bound compound.

[0319] Reduction of damage by using double stranded DNA (dsDNA) to make DNA barcodes. Another way to reduce damage to bead-bound DNA barcodes and to partially synthesized DNA barcodes is by synthesizing DNA barcodes in the form of double stranded DNA, where each of the DNA barcode modules that are linked to each other are in the form of dsDNA, and where each of the two strands is stabilized by a DNA cap. To perform final sequencing of the intact DNA barcode, one of the strands is cleaved from the DNA cap and removed. The above refers to damage to growing DNA barcodes by reagents used to make bead-bound compounds (where the compound is a member of a chemical library).

[0320] Reduction of damage by inclusion of hairpins. Yet another way to reduce damage to bead-bound DNA barcodes is to synthesize DNA barcodes in a manner that self-assembles into hairpins, and where the first tine of the hairpin is annealed to the second tine of the hairpin.

[0321] If the DNA barcode being synthesized is in the form of double stranded DNA (dsDNA), solvents such as DCM, DMF, and DMA denature the DNA barcode. The above methods and reagents can prevent denaturation.

[0322] Reduction of damage by using sealed ends of dsDNA. Another way to reduce damage to bead-bound DNA barcodes and to partially synthesized DNA barcodes is to use double stranded DNA (dsDNA) and to seal the ends of this dsDNA by 7-aza-dATP and dGTP.

[0323] Reduction of damage by avoiding use of protein solvents, avoiding use of strong acids and strong bases, avoiding use of strong reducing agents and oxidizing agents. The types of chemistry that are compatible with the presence of deoxyribonucleic acid (DNA), whether bead-bound or not, can require the absence of protein solvents, avoidance of strong acid conditions, avoidance of strong bases (such as tert-butyllithium), avoidance of strong reducing agents (such as lithium aluminum hydride), avoidance of reagents that react with the bases of DNA (such as certain alkyl halides), and avoidance of certain oxidizing agents (see Luk and Sats (2014) “DNA-Compatible Chemistry” (Chapter 4) in A Handbook for DNA-Encoded Chemistry, 1st Edition, John Wiley and Sons, Inc.).

[0324] As described elsewhere, the term "DNA barcode" can refer to a polynucleotide that identifies a compound in its entirety, whereas a "DNA barcode module" can refer to only one of the monomers that make up the compound.

[0325] By using chemicals that are compatible with DNA, damage to nucleic acids is reduced. Satz et al. disclose various chemical methods that are 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). Although the Satz et al. description above relates to chemical reactions performed on DNA / chemical library member conjugates, the types of chemistry described that are compatible with DNA are also relevant, where organic chemistry is to be performed on beads that contain bead-bound compounds and bead-bound DNA.

[0326] Disclosed are reactions that are compatible with DNA for forming benzimidazole compounds, imidazolinone compounds, quinazolinone compounds, isoindolinone compounds, thiazole compounds, and imidazopyridine compounds (see Satz et al., Table 1, entries 1-6).

[0327] Further, protecting groups that are compatible with DNA are disclosed, including allyloxycarbonyl deprotection, BOC deprotection, t-butyl ester hydrolysis / methyl / ethyl ester hydrolysis, and nitro reduction of hydrazine and Raney nickel (see Satz et al., Table 1, entries 7-11).

[0328] Further, methods of coupling reagents to DNA are disclosed, where functional groups that have been attached to DNA undergo coupling. The methods include Suzuki coupling, an optimized protocol for Sonogashira coupling between alkynes and aryl halides, a new method for converting aldehydes to alkynes using dimethyl-1-diazo-2-oxopropyl phosphonate, a new method for adding triazole rings directly from purified alkynes, an improved method for the reaction of isocyanate building blocks with amine-functionalized DNA, where the improved reaction is with isocyanate reagents in pH 9.4 buffer (see Satz et al., Table 1, entries 12-15).

[0329] In addition, methods of coupling reagents to DNA are disclosed, where functional groups that have been attached to DNA undergo coupling. These include methods for conjugating primary amines to DNA, an optimized protocol for forming DNA-conjugated thioureas, methods for alkylating secondary amines and dialkylation of primary aliphatic amines, monoalkylation of primary amine DNA-conjugates (using heteroaryl halides as building blocks that can react with amine-functionalized DNA conjugates), and methods for Wittig reactions (see Satz et al., Table 1, entries 16-20).

[0330] DNA repair enzymes reduce damaged DNA. Various proteins comprising enzymes, DNA damage binding proteins, and helicases can be used to repair DNA damage. DNA repair proteins are commercially available that can repair oxidative damage, damage from radiation, damage from UV light, damage from formaldehyde adducts, and damage in the form of alkyl adducts. Glycosylases that remove damaged bases (but do not cleave ssDNA or dsDNA) can be used to repair 5-formyluracil, deoxyuridine, and 5-hydroxymethyluracil. T4 PDG can be used to repair pyrimidine dimers. hNEIL1 and Fpg can be used to repair oxidized pyrimidines, oxidized purines, purine sites, and apyrimidinic sites. EndoVIII can be used to repair oxidized pyrimidines and apyrimidinic sites. EndoV can be used to repair mismatches. HaaG is a glycosylase that can be used to repair alkylated purines. If a gap is left by the DNA repair enzyme, a gap in double stranded DNA where one or more consecutive deoxyribonucleotides are missing in one strand, then various DNA polymerases can be used to fill in the gap (see catalog (2018) New England Biolabs, Ipswich, MA).

[0331] 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 the use of NER and homologous recombination. Direct repair includes repair of cyclobutane pyrimidine dimers and 6-4 products by photolyase enzymes. Direct repair also includes removal of O 6 - methylguanine by DNA methyltransferase (methyltransferase) enzymes. See Sancar et al. (2004) Ann. Rev. Biochem. 73:39-85; Hu and Sancar (2017) J. Biol. Chem. 292:15588-15597. 6 - methyl. See Sancar et al. (2004) Ann. Rev. Biochem. 73:39-85; Hu and Sancar (2017) J. Biol. Chem. 292:15588-15597.

[0332] The present disclosure provides a system, reagents, and methods for repairing damage to bead-bound DNA barcodes by treatment with DNA repair enzymes or complexes of DNA repair proteins, and the like.

[0333] Damage is reduced by coupling the DNA to the bead through the 3' end. Certain chemical transformations can damage the 3'-hydroxyl of an exposed nucleic acid. For example, the Mitsunobu reaction allows the conversion of primary and secondary alcohols to esters, phenyl ethers, thioethers, and various other compounds, which can render the exposed 3' end unreactive to subsequent processing steps, or engage the now-modified 3' end in further chemical reactions. In some embodiments, DNA tags can be attached to beads through their 3' ends, so only the 5' end is exposed to solution.

[0334] The reagents, systems, and methods of the present disclosure encompass bead-bound nucleic acids, such as bead-bound DNA or bead-bound DNA tags, where the coupling to the bead involves the 3' end (or 3' end) of the DNA. If the ssDNA that includes a DNA barcode is coupled through the 3' end of the ssDNA, sequencing can be initiated by hybridizing only one sequencing primer, where the sequencing primer hybridizes upstream of the entire DNA barcode, and this hybridization is at or near the bead-bound end of the coupled ssDNA. As an alternative to using only one sequencing primer, multiple sequencing primers can be used, where each sequencing primer hybridizes upstream of a particular DNA barcode module. For example, if a given DNA barcode contains five DNA barcode modules, and the DNA is coupled to a bead through its 3' end, the DNA barcode can contain five different primer annealing sites, each of which is located upstream or immediately upstream of a given DNA barcode module.

[0335] Double-stranded DNA (dsDNA) coupling embodiments. In other embodiments, coupled to the bead is a dsDNA, where only the 3' end of one strand of the dsDNA is coupled to the bead. In 5' coupling embodiments involving dsDNA, what can be coupled is a dsDNA, where only the 5' end of one strand of the dsDNA is coupled to the bead.

[0336] (V) Coupling a compound to a bead

[0337] The present disclosure provides: (1) linkers to connect chemical library members to substrates such as beads; (2) linkers to connect nucleic acid barcodes to substrates such as beads; (3) cleavable linkers, for example cleavable by UV light, cleavable by enzymes such as proteases; (4) non-cleavable linkers; (5) bifunctional linkers; (6) multifunctional linkers; (7) multiple beads for ligation. For example, useful are 4-hydroxymethylbenzoic acid (HMBA) linkers, 4-hydroxymethylphenylacetic acid linkers (see Camperi, Marani, Cascone (2005) Tetrahedron Lett. 46:1561-1564).

[0338] A “non-cleavable linker” can be characterized as a linker that is not detectable using any reagent, condition, or environment used in the steps of a given organic chemistry flow. Alternatively, a “non-cleavable linker” can be characterized as a linker that cannot be cleaved unless by a reagent, condition, or environment that is unacceptably destructive to other reactants, products, or reagents of a given organic chemistry flow.

[0339] A bifunctional linker or other multifunctional linker can take the form of a fork (a fork used by humans to eat food), where the handle of the fork is attached to a bead, and each tine of the fork is linked to one of various chemicals. For example, one tine can be linked to a chemical library member. Another tine can be linked to a DNA barcode. Yet another tine of the fork can be linked to a metal ion.

[0340] With respect to the use of multiple beads, the present disclosure provides multi-bead embodiments such as: (1) a first bead containing a linked nucleic acid barcode linked to a second bead, where the second bead contains a linked chemical library member; (2) a first bead containing a linked nucleic acid barcode linked to a second bead, where the second bead contains a linked chemical library member, and where a third bead is linked (linked to one or both of the first bead and the second bead), and where the third bead contains a covalently linked reagent. The linked reagent can be an enzyme, where the enzyme is used to assay the activity of the linked chemical library member.

[0341] (VI) Coupling monomers together to make a compound

[0342] Exemplary chemical monomers. Figure 4 Amino acid derivatives are shown that are suitable for use as chemical monomers for the compositions and methods of the present disclosure. The figure indicates the source of the chemical, e.g., AnaSpec EGT Group, Fremont, CA; Sigma-Aldrich, St. Louis, MO; Acros Organics (part of Thermo Fisher Scientific) or Combi-Blocks, San Diego, CA.

[0343] Additional chemical monomers are shown in Figures 22-27 . Figures 22-27 Each of the figures in Figure 22 , the corresponding barcodes are ACGT, ACTC, AGAC, AGCG, AGTA, and ATAT. For compounds 7-10 Figure 23 , the corresponding barcodes are ATGA, CACG, CAGC, and CATA. For compounds 11-16Figure 24 ), the corresponding barcodes are CGAG, CGCT, CGTC, CTAC, CTGT, and GACT. For compounds 17-21 ( Figure 25 ), the corresponding barcodes are GAGA, GCAC, GCTG, GTAG, and GTCA. For compounds 22-26 ( Figure 26 ), the corresponding barcodes are GTGC, TAGT, TATC, TCAG, and TCGC. And for compounds 27-30 ( Figure 27 ), the corresponding barcodes are TCTA, TGAT, TGCA, and TGTG. These scheme barcodes are exemplary only. For any given compound library, a different set of DNA barcodes can be used to identify each chemical monomer used to build the compounds in the library.

[0344] Coupling reactions. The following describes the coupling of chemical monomers to the beads to each other, i.e., the first step is coupling the first chemical monomer directly to the bead via a cleavable linker, and wherein subsequent chemical monomers are linked to each other one by one. The conditions disclosed below are DNA compatible.

[0345] This describes the coupling of chemical monomers to the beads to each other, i.e., the first step is coupling the first chemical monomer directly to the bead via a cleavable linker, and wherein subsequent chemical monomers are linked to each other one by one. The conditions disclosed below are DNA compatible. Method for preparing three amino acid compounds on beads. Fmoc-protected resin (1 mg, Rapp Polymers, 10 um, TentaGel M-NH2, 0.23 mmol / g) modified with Fmoc-Photo-Linker, 4-{4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butyric acid) or another suitable linker with Fmoc protection was suspended in each well of a reactor plate (Merck Millipore Ltd, 0.45 um hydrophobic PTFE) in DMA (150 uL). Solvent was removed by applying vacuum to the bottom of the plate using a Resprep VM-96 vacuum manifold. Fmoc protecting group was removed by suspending the resin in 150 uL of a mixture of 5% piperazine and DMF, 2% DBU. The plate was sealed with Excel Scientific Alumna Seal and shaken at 40 °C for 15 minutes. Solvent was removed by applying vacuum and the deprotection procedure was repeated for 5 minutes. After filtration, each well was washed with 150 uL of 2X DMA, 3X DCM, 1X DMA, applying vacuum between each wash to remove solvent. Each well of resin was then acylated with the appropriate amino acid by adding 150 uL of a pre-activation mixture of 60 mM Fmoc-amino acid, 80 mM Oxyama, 200 mM DIC, and 80 mM 2,4,6-trimethylpyridine (stand at room temperature for 2 minutes). The plate was sealed again and shaken at 40 °C for 1 hour. After filtration, each well was washed with 150 uL of 2X DMA and 3X DCM. The beads in each well were resuspended in 150 ul of DCM and combined into a single vessel by pipetting. The combined beads were mixed thoroughly and then redistributed into the plate by pipetting equal amounts into the appropriate wells (1 mg / well). Solvent was removed by applying vacuum and each well was ready for the next appropriate step. For each additional amino acid coupling, the Fmoc deprotection step was repeated followed by the coupling step with the desired amino acid. If splitting and combining is required, the combining and redistribution method was repeated.

[0346] This describes a method for generating 3-mer amino acids on beads by a split-and-combine method. Fmoc-protected resin (1 mg, Rapp Polymers, 10 um, TentaGel M-NH2, 0.23 mmol / g) modified with Fmoc-Photo-Linker, 4-{4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butyric acid) or any other suitable linker was suspended in each well of a reactor plate (Merck Millipore Ltd, 0.45 um hydrophobic PTFE) in DMA (150 uL). Solvent was removed by applying vacuum to the bottom of the plate using a Resprep VM-96 vacuum manifold. Fmoc protecting group was removed by suspending the resin in 150 uL of a mixture of 5% piperazine and DMF, 2% DBU. The plate was sealed with Excel Scientific Alumna Seal and shaken at 40 °C for 15 minutes. Solvent was removed by applying vacuum and the deprotection procedure was repeated for 5 minutes. After filtration, each well was washed with 150 uL of 2X DMA, 3X DCM, 1X DMA, applying vacuum between each wash to remove solvent. Each well of resin was then acylated with the appropriate amino acid by adding 150 uL of a pre-activation mixture of 60 mM Fmoc-amino acid, 80 mM Oxyama, 200 mM DIC, and 80 mM 2,4,6-trimethylpyridine (stand at room temperature for 2 minutes). The plate was sealed again and shaken at 40 °C for 1 hour. After filtration, each well was washed with 150 uL of 2X DMA and 3X DCM. The beads in each well were resuspended in 150 ul of DCM and combined into a single vessel by pipetting. The combined beads were mixed thoroughly and then redistributed into the plate by pipetting equal amounts into the appropriate wells (1 mg / well). Solvent was removed by applying vacuum and each well was ready for the next appropriate step. For each additional amino acid coupling, the Fmoc deprotection step was repeated followed by the coupling step with the desired amino acid. If splitting and combining is required, the combining and redistribution method was repeated. The VM-96 vacuum manifold applies vacuum to the bottom of the plate to remove solvent. The Fmoc protecting group is removed by suspending the resin in 150 uL of a mixture of 5% piperazine and DMF, 2% DBU. The plate is sealed with an Excel Scientific Alumna Seal and shaken for 15 minutes at 40°C. The solvent is removed by applying vacuum and the deprotection procedure is repeated for 5 minutes. After filtration, each well is washed with 150 uL of 2X DMA, 3X DCM, IX DMA, applying vacuum between each wash to remove solvent. Each well of resin is then acylated with the appropriate AA by adding 150 uL of a pre-activation mixture of 60 mM Fmoc-amino acid, 80 mM Oxy ma, 200 mM DIC, and 80 mM 2,4,6-collidine (stand for 2 minutes at room temperature). The plate is sealed again and shaken for 1 hour at 40°C. After filtration, each well is washed with 150 uL of 2X DMA, 3X DCM, IX DMA. For each additional AA coupling, the Fmoc deprotection step is repeated first, followed by the coupling step with the desired AA. To analyze each successive coupling, a portion of 1 mg of the beads is suspended in 100 uL of DMSO and exposed to full power of a 365 nm LED for two hours. The resin is filtered out and the filtrate is injected into an Agilent 1100 series LCMS equipped with an Agilent Poroshell SB-C-18, 3.0 x 50 mm, 2.7 um column. A gradient of 5% CH3CN in 0.1% aqueous TFA to 100 CH3CN in 0.1% TFA is run over 4 minutes at a flow rate of 1.2 mL / min with monitoring at 220 nm.

[0347] Experiments to make non-amino acid side groups with lenalidomide After deprotection, this will be attached to the last amino acid. This is also done in rotation. (After Fmoc deprotection), the resin of each well is acylated with a mixture of 150 uL of 40 mM chloroacetic acid, 40 mM Oxy ma, 80 mM DIC, and 40 mM TMP in DMA (pre-mix for 5 minutes). The plate is sealed and shaken for 1 hour at 40°C. Each well is washed with 150 uL of 3X DMA, 3X DCM, and 2X DMA, respectively. The resin is then re-suspended in a DMA suspension of 100 mM K2CO3 and 100 mM Rev. The plate is sealed and shaken for 3 hours at room temperature. The resin is washed with 150 uL of 2X 50 / 50 DMA / water, 3X DMA, 3X DCM, and 2X DMA, respectively.

[0348] defines the synthetic fidelity of a compound attached to a given bead. This relates to the complete compound, where the compound is a member of a chemical library. Each compound can be made in part or in whole from chemical monomers. The following are the characteristics of a compound attached to a given bead. A given bead can be the product of a split-and-pool synthesis of a chemical library, where each bead has a unique compound.

[0349] Members of a chemical library can be synthesized on a solid support, such as a bead, by solid phase synthesis. Solid phase synthesis of chemical substances with peptide bonds is characterized by the use of one of two chemical groups. The first chemical group is N-alpha-9-fluorenyl-methoxy carbonyl (Fmoc, base-labile). The second chemical group is t-butyloxycarbonyl (tBoc, acid-labile) (see, Vagner, Barany, Lam (1996) Proc. Natl. Acad. Sci. 93:8194-8199). Fmoc and tBoc are protecting groups that can be used to protect a peptide substrate, where either the Fmoc group or the tBoc group is attached to the alpha-amino group (Sigler, Fuller, Verlander (1983) Biopolymers 22:2157-2162).

[0350] 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 bound to a given bead have the exact same chemical structure after synthesis is complete. Incomplete coupling can occur in one or more steps in the multi-step synthesis of a member of a chemical library. For this reason, the compositions of the present disclosure can be characterized and limited by one of the following limits or ranges.

[0351] The present disclosure also provides methods and reagents where 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 the chemical library bound to a given bead have the exact same chemical structure after synthesis is complete (these numbers do account for and reflect errors that can occur during solid phase synthesis, e.g., one growing compound cannot accept one chemical monomer. Additionally, these numbers do account for and reflect chemical damage to any monomer that can occur during solid phase synthesis).

[0352] In exclusive embodiments, the present disclosure can exclude any method or reagent that does not meet one of the above critical values for "exact same structure."

[0353] In alternative embodiments, two, 3, 4, 5, about 5-10, about 10-20, about 20-40, about 40-80 beads in a set of beads contain the same compound (not accounting for any errors in incorporation of chemical monomers during solid phase synthesis and not accounting for any chemical damage to chemical monomers that occurs during organic synthesis).

[0354] Click chemistry primer. According to Jewett et al., click reactions are defined as..those..selective, high yielding, and with good reaction kinetics. A subset of click reactions whose components are inert to the surrounding biological milieu are termed "bioorthogonal" (Jewett and Bertozzi (2010) Chem. Soc. Rev. 39: 1272-1279). "Click chemistry" can be used to join small units with heteroatoms (such as carbon-X-carbon) together. Click chemistry can be used alone, or in combination with other types of chemical reactions, to synthesize drugs or drug candidates. Click chemistry fits well with the flow used in combinatorial chemistry. Reactions in click chemistry are characterized by high yield, irreversibility, insensitivity to oxygen or water. Classes of chemical reactions used in "click chemistry" include: (1) cycloaddition reactions, especially from the 1,3-dipole family and hetero-Diels-Alder reactions; (2) nucleophilic ring opening reactions, such as reactions with strained heterocyclic molecules (such as epoxides, aziridines, and cyclic sulfates); (4) non-aldol type carbonyl chemistry; (5) addition to carbon-carbon multiple bonds, such as oxidation reactions and some Michael addition reactions. Click chemistry reactions are characterized by their high thermodynamic driving force, typically greater than 20 kcal / mol, whereas non-click chemistry reactions involve bond formation with only a modest thermodynamic driving force (Kolb and Sharpless (2003) Drug Discovery Today 8: 1128-1137, Kolb, Finn, Sharpless (2001) Angew. Chem. Int. Ed. 40: 2004-2021).

[0355] Tetrazine and trans-cyclooctene (TCO). Tetrazines, such as 1,2,4,5-tetrazine, can react with trans-cyclooctene (TCO) via a Diels-Alder cycloaddition reaction (Devaraj, Haun, Weissleder (2009) Angew. Chem. Int. 48: 7013-7016).

[0356] Hartig-Buchwald amination. Hartwig-Buchwald amination reactions can be used for solid phase synthesis of drugs. This amination reaction is used to synthesize carbon-nitrogen bonds, where the reaction involves the palladium-catalyzed coupling of aryl-halides with amines (R1-NH-R2) to form aryl products, where the amine replaces the halide, and where the nitrogen of the amino group is directly attached to the aromatic ring. The end result is a product that involves a carbon atom (of the aryl group) and a nitrogen atom (of the amino group). In other words, the reaction converts aryl halides to the corresponding anilines. Hartwig-Buchwald amination is compatible with a variety of amines and is well suited for combinatorial chemistry (Zimmermann and Brase (2007) J. Comb. Chem. 9:1114-1137).

[0357] Huisgen cycloaddition. Huisgen 1,3-dipolar cycloaddition reactions involve alkyne and organic azide. Alkyne has the structure R-C ＝ CH. Azide has the structure R-N + = N = N - . Copper catalysts speed up the Huisgen cycloaddition reaction. Huisgen reactions operate by “click chemistry” or “click reactions”. Under catalysis by copper, Huisgen reactions can produce 1,2,3-triazole nuclei that are suitable for making small molecule drugs. At least when in protected form, Huisgen reactions are compatible with the presence of amino acid side chains. Molecules made with 1,2,3-triazoles can have bonds that are similar to amide bonds of polypeptides, so these molecules can be surrogates for peptide bonds (Angell and Burgess (2007) Chem. Soc. Rev. 36:1674-1689).

[0358] Peptide nucleic acids (PNAs). The present disclosure provides methods of resolution and combination chemistry, combinatorial chemistry, or solid phase chemistry for the synthesis of peptide nucleic acids. Peptide nucleic acids are analogs of oligonucleotides. They resist hydrolysis by nucleases. They can bind tightly to target RNA sequences. Uptake of peptide nucleic acids into cells can be enhanced by “cell penetrating peptides” (Turner, Ivanova, Gait (2005) Nucleic Acids Res. 33:6837-6849; Koppelhus (2008) Bioconjugate Chem. 19:1526-1534). Peptide nucleic acids can be made 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).

[0359] The present disclosure encompasses bead-bound compounds, wherein the compound takes the form of only one monomer. For example, such bead-bound compounds can take the form of lenalidomide, or can take the form of lenalidomide with a linked carboxylic acid group, or the form of lenalidomide wherein the amino group has been modified with a small chemical moiety bearing a carboxylic group, or wherein the compound is a lenalidomide analog that is a stereoisomer or an enantiomer of lenalidomide.

[0360] (VII) Split and Pool and Parallel Synthesis

[0361] This relates to the use of a "split and pool" method to synthesize a library of compounds, and to a method wherein the "split and pool" method is used to simultaneously synthesize bead-bound compounds and bead-bound DNA barcodes. This also describes split and pool to form a mixed collection of compounds. At a later point, disclosed below is the coupling of non-amino acids and the preparation of PEG-modified beads.

[0362] The present disclosure provides split and pool synthesis for the production of chemical libraries. In one embodiment, the method involves the following steps: (a) dividing beads into different containers; (b) adding a different building block to each container. For example, in the case of using three containers, adding species A to the first container and allowing the reaction to proceed, adding species B to the second container; and adding species C to the third container, wherein the species covalently bind to the linking site on any bead in the container; (c) pooling all beads in one container; (d) dividing the beads into three containers, (e) adding a different building block to each container, wherein species A is added to the first container, species B is added to the second container, and species C is added to the third container, wherein the species covalently bind to the first species that has been linked previously (see, Stockwell (2000) Trends Biotechnol. 18:449-455).

[0363] The split and pool synthesis of the present disclosure comprises a DNA barcode coupling step prior to or after each chemical coupling step (to make a chemical library member), wherein the DNA barcode identifies the chemical species that is coupled in the step.

[0364] In exclusive embodiments, the present disclosure can exclude methods and reagents wherein the barcodes are linked prior to the linking of the chemical species for a given parallel synthesis step. Conversely, the present disclosure can exclude methods and reagents wherein the chemical species are linked prior to the linking of the barcodes for a given parallel synthesis step.

[0365] One feature of a library of chemically bound to beads prepared by splitting and pooling methods is that each bead will have only one compound attached to it. For example, in the case of incomplete coupling, if for a given splitting and pooling step, only 4,000 of 5,000 attachment sites are successfully coupled with the desired chemical species, then some heterogeneity will occur.

[0366] Parallel synthesis. In preferred embodiments of the disclosure, parallel synthesis can be used for the organic synthesis of compounds and associated DNA barcodes. In practical practice, the modification of a bead with more than one chemical monomer and the modification of the same bead with more than one DNA barcode module is not strictly parallel. In practical practice, a bead will receive one chemical unit (chemical monomer) and then receive a DNA barcode module that encodes for that particular chemical unit. The term "parallel" refers to the fact that as the polymer of chemical library monomers grows, the polymer of DNA barcode modules also grows. When all DNA barcode modules have been attached to a bead to form a concatenated structure or an orthogonal structure, the full-length DNA barcode is referred to as a "DNA barcode" (and not just a DNA barcode module).

[0367] Ratio of number of externally attached DNA barcodes to total number of attached chemical library members.

[0368] This refers to the external and internal surfaces of a bead. For a given bead with externally attached DNA barcodes (not considering the number of internally attached DNA barcodes) and attached chemical library members (attached to both the external surface and the internal surface), the ratio of number of externally attached DNA barcodes to total number of attached chemical library members can 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 exclusive embodiments, the disclosure can exclude any bead or any population of beads that meets one of the above values.

[0369] Homogeneity of DNA barcodes of a typical barcode; homogeneity of chemical library members of a typical bead

[0370] For any given bead (or for any population of beads), the disclosure provides for 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. of "chemical library homogeneity."

[0371] In less stringent embodiments, the present disclosure provides at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% “chemical library homogeneity” for any given bead or alternatively for any given population of beads.

[0372] Similarly, the present disclosure provides the aforementioned thresholds for assessing the homogeneity of a barcode (e.g., a DNA barcode).

[0373] Homogeneity of a DNA barcode and homogeneity of a chemical library member can be defined in terms of the total population percentage of exact sequences that conform to the planned and required sequence as outlined in the laboratory manual or notebook methods section.

[0374] In exclusive embodiments, the present invention can exclude any reagent, composition, or method that does not conform to one or more of the aforementioned thresholds.

[0375] In the context of assessing the homogeneity of a population of beads, for cases where homogeneity is desired to be maintained across the population of beads, the homogeneity of the sum of bead #1, bead #2, bead #3, bead #4, bead #5, bead #6, bead #7, etc. needs to be considered.

[0376] In exclusive embodiments, the present disclosure can exclude any bead or any population of beads where the homogeneity of a DNA barcode is no less than 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. Additionally, in exclusive embodiments, the present disclosure can exclude any bead or any population of beads where the homogeneity of a chemical library member is no less than 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.

[0377] Ratio of internally linked DNA barcodes to externally linked DNA barcodes

[0378] In some embodiments of the present disclosure, it can be desirable to manufacture and use beads with DNA barcodes that are primarily linked on the external surface. One reason not to make and use beads with internal DNA barcodes is that the permeability of DNA oligos to the interior space is low and the permeability of DNA ligases to the interior space is low (ligases used to link DNA modules to one another to generate the final DNA barcode). A reason not to make and use internal DNA barcodes for sequencing purposes is that the enzyme permeability required for DNA required for sequencing the amplified final barcode is low. Yet another reason not to make and use beads with internal DNA barcodes is to increase the interior space for linking chemical library members.

[0379] The present disclosure provides beads with DNA barcodes, wherein the ratio of internally linked DNA barcodes to externally linked DNA barcodes is about 0.1:100, about 0.2:100, about 0.4:100, about 0.8:100, about 1:100, about 2:100, about 4:100, about 8:100, about 10:100, about 20:100, about 40:100, about 50:100, about 60:100, about 70:100, about 80:100, about 90:100, about 1:1, etc.

[0380] Additionally, the present disclosure provides beads with DNA barcodes, wherein the ratio of internally linked DNA barcodes to externally linked 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, etc.

[0381] A population of beads in an aqueous suspension can be contacted with a substrate such as an array of picowells, resulting in the beads entering and occupying the picowells. The ratio of the number of beads in the suspension to the number of picowells in the substrate can be adjusted to achieve a desired occupancy. For example, if the suspension contains only one bead, then each picowell containing a bead will contain only one bead, with the remaining picowells containing no beads. If the suspension contains 20,000 beads, and if the substrate contains 200,000 picowells, then at least 180,000 picowells will be completely free 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.

[0382] In value embodiments, the ratio of the number of beads in the suspension to the number of picowells can be about 0.2:100, about 0.4:100, about 0.6:100, about 0.8:100, about 1:100, about 2:100, about 4:100, about 6:100, about 8:100, about 10:100, about 20:100, about 30:100, about 40:100, about 50:100, about 60:100; 80:100, about 100:100 (the same as 1:1), about 2:1, about 4:1, about 6:1, about 8:1, about 10:1, etc.

[0383] In exclusive embodiments, the present disclosure can exclude any method or system of one of the above values or ranges.

[0384] In a range embodiment, the ratio of the number of beads to the number of picowells in the suspension can be about 0.2: 100 to about 0.4: 100, about 0.4: 100 to about 0.6: 100, about 0.6: 100 to about 0.8: 100, about 0.6: 100 to about 1 : 100, about 1 : 100 to about 2: 100, about 2: 100 to about 4: 100, about 4: 100 to about 6: 100, about 0.6: 100 to about 8: 100, about 8: 100 to about 10: 100, about 10: 100 to about 20: 100, about 20: 100 to about 30: 100, about 30: 100 to about 40: 100, about 40: 100 to about 50: 100, about 50: 100 to about 60: 100, about 60: 100 to about 80: 100; about 80: 100 to about 100: 100 (same as 1 : 1), about 100: 100 (same as 1 : 1) to about 2: 1, about 2: 1 to about 4: 1, about 4: 1 to about 6: 1, about 6: 1 to about 8: 1, about 8: 1 to about 10: 1, etc.

[0385] In an exclusive embodiment, the present disclosure can exclude any method or system of one of the above values or ranges.

[0386] (VIII) Fabricating picowells

[0387] Combination of UV light, photomask, and photoresist for fabricating picowell array plates. Plates containing many micro- or picowells can be fabricated for use in the present 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. Picowells will be carved out of the photoresist by UV light. After the picowells are carved out of the flat sheet of photoresist, the photoresist resembles a typical metal pan that contains cups for baking pancakes, and wherein the cups in the pan for holding pancake batter have sloped sides. The UV light acts as a “non-crosslinker” because it breaks down the polymers of the photoresist. After UV treatment, a solvent is added to wash away the UV-treated photoresist, leaving clean picowells.

[0388] Rotation angle to create sloped walls. Picowells with sloped walls are created as follows. The photomask has many apertures, where each aperture corresponds to the desired bottom dimension of a picowell. The bottom dimension can contain a circumference, diameter, and shape, i.e., circular. The top dimension of the aperture is created by directing sloped UV light toward the aperture on the photomask while rotating the light source or rotating the platform of the sandwich of photomask / glass wafer / photoresist. While rotating, the light source is not at a 90-degree angle to the photomask / wafer / photoresist sandwich, but is slightly off the 90-degree point so that a sloped wall is cut into each picowell. The resulting picowell array plate containing many picowells can be used as is. Alternatively, the picowell array plate can be used as a mold to cheaply fabricate many picowell array plates.

[0389] Han et al. describe an apparatus and reagents for making micro well plates where the micro wells have sloped walls (see, Han et al. (2002) J. Semiconductor Technology and Science 2:268-272). A UV source, a contact stage, a tilt stage, and SU-8 photoresist are described. Fabrication begins with a single-side polished silicon wafer. SU-8 photoresist is coated on the wafer at a thickness of about 0.10 mm to 0.15 mm. The photoresist is then soft baked on a hot plate at 65 degrees for 10 minutes and then soft baked on a hot plate at 95 degrees for 30 minutes. The resulting photoresist / wafer sandwich is brought into contact with a UV mask using a contact stage. The term "tilted and rotated UV lithography" refers to a method for making a micro well array plate or a picowell array plate where each well has sloped walls. Here, the bottom diameter of the well is smaller and the top of the well (where the top edge of the well meets the plane of the plate) is larger. To expose with UV light, a turntable is used and where the UV light is tilted (Han et al., supra). The mask is brought into contact with the photoresist where each aperture in the mask is circular. Han et al., supra, describes how to make a truncated cone. A soft material such as PDMS (polydimethylsiloxane) can be poured over the array of cones and cured and then the PDMS layer peeled off to form a conical well. Figure 8 Pictures of the direction of the UV light, the UV mask, the photoresist structure, the wafer substrate, and the turntable are provided. Han et al. describes how to make a truncated cone. A soft material such as PDMS (polydimethylsiloxane) can be poured over the array of cones and cured and then the PDMS layer peeled off to form a conical well.

[0390] A mold is created for mass production of picowell array plates. If a picowell array plate has been fabricated, then epoxy can be poured over the plate, filling all the picowells, and connecting all the filled picowells to the epoxy platform. After the epoxy is cured, the solid platform with the array of picoprotrusions (the inverse of the desired picowells) is removed. The solid platform with the picoprotrusions is a reusable mold for the fabrication of many picowell array plates.

[0391] The process of making a replica from an epoxy mold (or from a mold of an array of cones made from any hard material) is called "hot embossing." Briefly, a substrate is heated to its glass transition temperature or softening temperature at which point the mold of the picoprotrusions is uniformly pressed onto the heated, soft material. After the picoprotrusions are transferred into the substrate as picorecesses, the mold can be separated from the substrate. The present invention preferably discloses the picicone and the picowell as patterns of the mold and the substrate, respectively.

[0392] Hot embossing, epoxy masters and photoresists like SU-8 photoresist are described (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; del Campo and Greiner (2007) J. Micromechanics and Microengineering 17: R81-R95).

[0393] Other micro-well plate embodiments. Plastic micro-well arrays can be fabricated by hot forming using silicon molds with micro-well arrays, e.g., 800,000 micro-well arrays. By using a non-pulsed dry etching process, high control can be achieved to form a tapered geometry and smooth sidewalls, and to achieve sub-micron tolerances. In contrast, methods using a pulsed dry etching process, such as the Bosch process, result in rough sidewalls and lack of control over lateral dimensions during etching.

[0394] Using a non-pulsed dry etching process, plastic arrays are made by hot forming plastic on a silicon master created by a non-pulsed, isotropic dry etching process using a chrome mask. The process uses three gases: Ar, SF6, and C4F8. The process is performed at an RF power between 1200 and 2000 Watts and a bias of 150 Watts. By varying the gas flow between the three gases, fine tuning of the taper of the silicon mold can be achieved with the production of smooth sidewalls. What is varied is the ratio of SF6 to C4F8, where varying the ratio results in, for example, a tapered wall of the mold (silicon post) existing at 18 degrees (very sloped wall), 9 degrees (slightly sloped wall), or 2 degrees (wall nearly perpendicular to the base) (see Perry, Henley, and Ramsey (October 26-30, 2014) "Development of Plastic Microwell Arrays for Improved Replication Fidelity", 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, San Antonio, Texas (pp. 1700-1703). th Int. Conference on Miniaturized Systems for Chemistry and Life Sciences), San Antonio, Texas (pp. 1700-1703).

[0395] In embodiments, the present disclosure provides a substrate, array, grid, microfluidic device, etc. comprising an array of microwells. In one embodiment, all of the microwells have substantially the same volume. The volume can be about 1 picoliter, about 2 picoliters, about 4 picoliters, about 6, about 8 picoliters, about 10 picoliters, about 20 picoliters, about 40 picoliters, about 60 picoliters, about 80 picoliters, about 100 picoliters, about 200 picoliters, about 400 picoliters, about 600 picoliters, about 800 picoliters, or about 1,000 picoliters.

[0396] Also, the volume can take the form of a range between any two adjacent values described above, e.g., a range of about 40 picoliters to about 60 picoliters. In addition, the volume can take the form of a range between any two values described above that are not immediately adjacent to each other in the above list.

[0397] Further, the volume can be about 1 nanoliter, about 2 nanoliters, about 4 nanoliters, about 6 nanoliters, about 8 nanoliters, about 10 nanoliters, about 20 nanoliters, about 40 nanoliters, about 60 nanoliters, about 80 nanoliters, about 100 nanoliters, about 200 nanoliters, about 400 nanoliters, about 600 nanoliters, about 800 nanoliters, or about 1,000 nanoliters, about 2,000 nanoliters, about 5,000 nanoliters, about 10,000 nanoliters, about 20,000 nanoliters, about 50,000 nanoliters, about 100,000 nanoliters, about 200,000 nanoliters, about 500,000 nanoliters, or about 1,000,000 nanoliters. In addition, the volume can take the form of a range between any two values described above that are not immediately adjacent to each other in the above list.

[0398] In exclusive embodiments, the present disclosure can exclude any substrate comprising microwells, or any array comprising microwells, wherein the volume of each microwell can be defined by one of the values described above, or can be defined by a range between any two values described above that are adjacent to each other, or can be defined by a range between any two values described above that are not adjacent to each other in the list.

[0399] Spherical plugs on picowells (also referred to as capping beads). The present disclosure provides a spherical plug for each well or each well of a substantially picowell array, or alternatively, a multi-well spherical plug. The purpose of the plug is to retain drugs, drug candidates, cellular contents, and metabolites in the wells. The plug also helps to isolate the contents of the picowells from each other. The spherical plug can not necessarily be perfectly spherical, as long as the purpose of covering the top (or opening or mouth) of the picowell is met. The wells can have a top diameter and a bottom diameter. The diameter of the spherical plug is about 10 microns, about 30 microns, about 35 microns, about 40 microns, about 45 microns, about 50 microns, about 55 microns, about 70 microns, about 90 microns, about 120 microns, or about 200 microns before the wells are capped. The plug can be added to cover the picowells by simply flowing the plug through the picowell array. The beads can be jammed at the top (or opening or mouth) of the picowell using centrifugation, pressure, agitation, or other methods to ensure a tight seal. In some embodiments, a solvent can be used to change the swelling and / or size of the capping beads. In some embodiments, the capping beads can be loaded into a solvent that shrinks the beads, and once replaced by assay buffer or a different solvent, the capping beads return to their original size or swell, thereby tightly sealing the picowells. In some embodiments, temperature can be used to swell or crush the capping beads for a better seal at the mouth of the picowell. Where needed, the capping beads can be fixed in place by one of the steps in a stepped picowell array and prevented from falling further into the picowell.

[0400] The capping bead can be the same type of bead that carries the compound of the disclosure, or can be a different type of bead. In some embodiments, the capping bead can actually be the compound with the bead itself. The capping bead can act as a passive cap, preventing or slowing the diffusion of molecules out of the picowell, or the bead can be an active bead, where the functional moiety attached to the capping bead can be used to capture reagents from the picowell. In some embodiments, the porous capping bead can passively capture metabolites released from a cell-based assay performed inside the picowell. In some embodiments, the capping bead can non-specifically capture cellular material, such as lipids, proteins, carbohydrates, and nucleic acids. In some embodiments, the capping bead can be functionalized with antibodies to specifically capture proteins released from healthy, diseased, cut, or fixed cells. In some embodiments, the capping bead can be functionalized with DNA or RNA oligonucleotides that specifically capture cellular nucleic acids. In some embodiments, the DNA or RNA functionalized capping bead can be used to capture microRNAs released from cells inside the capped picowell. In some embodiments, the picowell contains two beads, a compound containing a bead inside the picowell, and a capping bead that covers the mouth of the picowell. In some embodiments, the capping bead can also be a bead with a compound. In some embodiments, the capping bead captures material released from the composite bead. In some embodiments, the capping bead captures a sample of the compound released from the composite bead. In some embodiments, the capping bead captures a DNA barcode released from the composite bead. In some embodiments, the capping bead captures different types of analytes released from the picowell that they cap.

[0401] Relative hardness of the cap and the picowell. The preferred device is a microtiter plate, where each microtiter plate contains thousands of picowells in its bottom surface. The ability of the cap to properly seat or seal each picowell can be a function of the hardness of the plastic that makes up the well of the picowell and the inner walls of the picowell relative to the hardness of the cap.

[0402] The hardness of a plastic can be defined in terms of a "durometer" value. Hardness is defined and tested as the resistance of a material to indentation. The hardness of a spherical plug and the hardness of a picowell wall can be defined by its "durometer." The hardness can be, for example, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100. When attributing any of these hardness values to a plastic or other substance, it must also be stated which scale was used. For example, the scale can be the ASTM D2240 Type A scale, which is used for softer materials, or the ASTM D2240 Type D scale, which is used for harder materials (see, Silicon Design Manual, 6th Edition, Albright Technologies, Inc., Leominster, MA).

[0403] Picowell shape. In some embodiments, a picowell can be a cylindrical picowell, where the diameter of the cylinder is approximately similar at the top and bottom of the picowell. In some embodiments, a picowell can have a slight taper, where the top of the picowell is slightly larger than the bottom of the picowell. In some embodiments, a picowell can be a conical picowell, which deviates from the normal by anywhere between 1 degree and 30 degrees. In some embodiments, a picowell is a stepped picowell, where the picowell has discontinuous steps from the top diameter to the bottom diameter (as opposed to a tapered picowell, where the diameter changes smoothly from the top to the bottom). In some embodiments, a stepped picowell has a wide cylinder near the opening of the picowell and a narrower cylinder near the bottom of the picowell. In some embodiments, a stepped picowell can have multiple discontinuous steps from the top to the bottom. In some embodiments of a multi-step picowell, the diameter at each step can be larger than the diameter of the step below it. In some embodiments, a small bead can be deposited at the bottom of a stepped picowell, and a capping bead can be deposited at the topmost opening of a stepped picowell. In some embodiments, a picowell can contain more than 2 beads.

[0404] Method of making a stepped picowell. Figure 29A stepped picowell is disclosed. The illustrated embodiment has three compartments and two steps. The top compartment is the widest and is configured to receive a lid in which the majority of the top compartment is occupied by the lid in the case that the picowell is capped. The middle compartment is configured to be occupied primarily by reagents or by reagents only. The reagents can include buffers, enzyme substrates, one or more salts, and preservatives or stabilizers such as dithiothreitol, RNAse inhibitors, glycerol, or DMSO. The lowermost compartment is configured to be occupied by beads, i.e., beads coupled to DNA libraries and releasable compounds. In addition to carrying DNA barcodes and releasable compounds, the same bead can also carry a “response capture element.” The capping bead can be held in place and prevented from falling further into the picowell by one of the steps in the stepped picowell. In Figure 29 In the above, structure 1 is the lid, structure 2 is the bead, and structure 3 is the top region, which is above the first step. Structure 4 is the middle region, which can be used to place assay reagents. The middle region is above the second step. The assay reagents of the middle region can diffuse to the lowermost region. Structure 5 is the lowermost region, which can be used to place beads and to place one or more unit cells.

[0405] With respect to the space of the lowermost compartment occupied by the bead (assuming there is only one bead in the picowell), the diameter of the bead can be about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% of the diameter of the lowermost compartment (assuming the picowell is a circular well). If the picowell is not a circular well, the above values can refer to the widest dimension of the well. In exclusive embodiments, the present disclosure can exclude any system or bead that does not meet any of the above parameters.

[0406] Further, with respect to the space occupied by the beads (assuming that only one bead is present in a picowell), about 50% of the beads are in the lowermost compartment and about 50% of the same beads are in the middle compartment, where these parameters can also be: about 55% of the beads are in the lowermost compartment and about 65% of the same beads are in the middle compartment; about 60% of the beads are in the lowermost compartment and about 40% of the same beads are in the middle compartment; about 65% of the beads are in the lowermost compartment and about 45% of the same beads are in the middle compartment; about 70% of the beads are in the lowermost compartment and about 30% of the same beads are in the middle compartment; about 75% of the beads are in the lowermost compartment and about 25% of the same beads are in the middle compartment; about 80% of the beads are in the lowermost compartment and about 20% of the same beads are in the middle compartment; about 85% of the beads are in the lowermost compartment and about 15% of the same beads are in the middle compartment; about 90% of the beads are in the lowermost compartment and about 10% of the same beads are in the middle compartment; about 95% of the beads are in the lowermost compartment and about 5% of the same beads are in the middle compartment; and about 100% of the beads are in the lowermost compartment. To make these calculations, the space occupied by the beads (hypothetically) assumes that the beads are not porous. In exclusive embodiments, the present disclosure can exclude any system or beads that do not meet any of the above parameters.

[0407] As with the conical and cylindrical picowells, using a molding system is a preferred embodiment for creating stepped picowells. To this end, a mold containing an array of multilayered pillars is desired, which is subsequently stamped into a thermoplastic or other curable polymer substrate, thereby creating an impression of a stepped picowell. A layered columnar array with multiple steps, each with a different diameter (decreasing with increasing diameter) can be created through a multilayered photolithography process. In brief, a first layer of photoresist is exposed through a first mask to crosslink a first layer of a micro pillar array. A second layer of photoresist can be deposited directly on the first (previously exposed) layer, a second photomask can be used to crosslink a second pattern in the second photoresist later, etc. At the end of the multilayer patterning, the stack of resists can be developed to wash away the uncrosslinked areas, leaving behind an array of multilayered pillars. Detailed protocols for creating multilayered columnar arrays can be found in Francisco Perdigones et al., (January 8, 2011), "Microsystem Technologies for Biomedical Applications", Biomedical Engineering, Trends in Electronics, Anthony N. Laskovski, IntechOpen. Once an array of multilayered pillar arrays is created, a mold can be used to imprint an array of stepped picowells using standard processes.

[0408] Removing the capping beads. In many embodiments, it is advantageous to sample the capping beads to study reactions, analytes, or cellular responses to chemical perturbations within the picowells. In some embodiments, the capping beads can be removed from the mouths of the picowells by inverting the picowell array and using mechanical agitation. In some embodiments, solvents can be used to shrink the picowells, making them more easily removed from the mouths of the picowells. In some embodiments, a higher density liquid than the capping beads can be added to the top of the picowell array, causing the capping beads to rise and float on the high density medium due to buoyancy.

[0409] In some embodiments, the capping beads can be crosslinked to one another, converting the capping beads into a capping sheet that can be peeled off the top of the picowell array. In some embodiments, a crosslinking gel can be poured over the capped picowells, where the crosslinking gel crosslinks to the capping beads as well as to itself, embedding the capping beads into a peelable crosslinked sheet.

[0410] The relative positions of the picowells are preserved in the form of a peeled layer. It will be appreciated that in this embodiment, the relative positions of the capping beads to each other and to the picowells are preserved in the peeled layer, as when the capping beads sink into the peelable gel layer. This allows for direct linkage between the picowells, the assays in the picowells, the beads in the picowells, and any material captured in the capping beads.

[0411] In some embodiments, fiducial markers can be used to orient the relative features of the picowell array to the capping beads in the peeled layer.

[0412] Fiducial markers that enable registration and alignment of the picowells. Arranging the picowells in an irregular array can easily identify shifts and drifts during imaging of the picowell array. In some embodiments, the picowells are arranged in an irregular order to facilitate detection of optical and mechanical drifts during imaging. In some embodiments, the picowell array contains fiducial markers to help identify shifts and drifts during imaging. In some embodiments, the fiducial markers are easily identifiable shapes, patterns, or features interspersed among the picowells of the picowell array. In some embodiments, a small number of the picowells themselves can be arranged in an easily identifiable pattern to enable easy registration in the event of optical or mechanical drifts during imaging. In some embodiments, external markers, such as fluorescent beads, can be sprinkled on the picowell array to provide a fiducial pattern.

[0413] Capped-pad embodiments. At least in some forms or instances, the capped-pad embodiments can take the form of a "capped-pad film." Instead of sealing the openings at the top of the picowells, for example, to prevent evaporation of any cell culture media or enzyme assay media that can be present in the picowells, sealing can be achieved by a pad. Preferably, the pad is sized to cover all of the picowells in a given picowell array. Alternatively, the pad can be sized to cover a predetermined portion of the picowells in the array. The pad can be affixed to the top of the picowell plate, covering the picowells and also covering the generally flat top surface of the picowell plate that lies between the picowells. Secure contact can be achieved by one or more of the following: (i) a constant pressure is maintained, for example, by a hard-rubber press plate placed on top of the pad and acting as a weight on top of the pad; (ii) the pad is used in conjunction with a weight, such as a hard-rubber press plate; (iii) a reversible chemical adhesive that can be applied across the pad (in the case that the pad is not an absorbent pad). When the pad is an absorbent pad, the pad contains circular absorbent pads surrounded by a reversible chemical adhesive. Herein, the pad is in contact with and aligned to the picowell array such that the circular absorbent pads cover only the openings of each picowell without "spilling over" the openings to contact the flat surface of the picowell plate.

[0414] A pad for contact with the substantially flat surface of a pico well plate can be provided, as well as a membrane for capless sealing of the pico well. Flat sheet membranes, such as Dow Film Tex, GE Osmonics, Microdyn Nadir, Toray, TriSep, Synder, Novamem, Evonik, and Aquaporin flat sheet membranes, are available from Sterlitech Corporation of Kent, Washington. These include membranes made from polyamide-TFC, cellulose acetate, polyamide-urea-TFC, cellulose acetate blend, polyazetidinium-TFC, PES, composite polyamide-TFC, PES, PAN, PVDF, PSUH, RC, PESH, polyether ether ketone, polyimide, etc. Pore sizes 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 microns, 0.030 microns, 0.05 microns, 0.10 microns, 0.20 microns, etc. These cutoffs can allow selective collection of certain classes of compounds, while excluding others, with respect to the systems, compositions, reagents, and methods of the present disclosure. For example, certain of the above membranes can allow small molecule metabolites to pass through and be absorbed by the absorbent pad, while excluding proteins and other macromolecules. Flat sheet membranes that are impermeable to all molecules, including water, metal ions, salts, metabolites, proteins, and nucleic acids, can also be used in the systems, compositions, and methods of the present disclosure.

[0415] Reversible adhesion can be mediated by "molecular Velcro," for example, a metalloporphyrin-containing polymer with a pyridine-containing polymer (Sievers, Namyslo, Lederle, Huber (2018) eXPRESS Polymer Letters 12:556-568). Other molecular Velcro adhesives involve L-3,4-dihydroxyphenylalanine, complementary strands of ssDNA (one type of ssDNA covalently attached to the flat upper surface of a pico-well plate, and another type of ssDNA covalently attached to a pad), copolymers containing catechol side chains, etc. (see Sievers et al., supra). Additionally, reversible adhesion can be mediated by gallium adhesives, where the degree of adhesion can be controlled by slight changes in temperature (Metin Sitti (May 18, 2016) Switch and Stick. The chemical element gallium can be used as a new type of reversible adhesive, which can easily turn its adhesion on and off. (Max-Planck-Gesellschaft) Yet another reversible adhesive is available from DSM-Niaga Technologies, Zwolle, Netherlands.

[0416] Absorptive substances (non-specific absorbers; specific absorbers). Absorptive substances that can be incorporated into the pad to provide absorption properties include "molecular sieve" beads, such as and ion exchange beads made from DEAE cellulose, carboxymethyl cellulose, phosphocellulose, or any combination of the above, all incorporated into an absorptive pad. Absorptive ligands include those for high pressure liquid chromatography (HPLC) (see BioRad catalog, Hercules, CA). Specific absorbers include responsive capture elements, such as poly(dT), which can capture mRNA by hybridization to polyA tails. Additionally, responsive capture elements include exon-targeting RNA probes, antibodies, and aptamers. Each or any combination of these can be covalently attached to the pad to form an absorptive pad, where contacting the absorptive pad to the top surface of the pico-well enables capture of aqueous assay medium or aqueous cell culture medium that can be located inside the pico-well.

[0417] (IX) Depositing beads into the pico-well

[0418] A picowell-bearing plate can take the form of a 96-well plate, where each of the 96 wells contains thousands of picowells. Additionally, a picowell-bearing plate can take the form of a 24-well plate, where each of the 24 wells contains thousands of picowells. For a 96-well plate, a 0.1-0.2 mL suspension of beads in water or an aqueous solution can be used to fill each well. For a 24-well plate, about 0.5 mL of a suspension of beads in water or an aqueous solution can be used to fill each well. The suspension can be added using a common pipette with a disposable tip. The number of beads in the suspension can be such that about one-third of the picowells contain one bead, about one-third of the picowells contain two beads, and about one-third of the picowells contain no beads or more than two beads. Additionally, the number of beads in the suspension can be such that, of the wells that do contain one or more beads, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the wells contain only one bead.

[0419] After the beads have settled, excess liquid can be removed by contacting a pipette tip to the wall of each well of a 96-well plate, or by contacting a pipette tip to the wall of each well of a 24-well plate and drawing it out.

[0420] With respect to assay reagents, in cases where the picowells are used to perform a reaction, such as DNA sequencing, a biochemical assay, or an assay in which cells are cultured, assay reagents can be added to the picowells that already contain the settled beads. As described above, the assay reagents are added with a pipette, which is used for the initial addition of the bead suspension. After the assay reagents have equilibrated with the solution already present in each picowell, 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 drawn out with a pipette tip that is in contact with the wall of each of the 96 wells of a 96-well plate, or with the wall of each of the 24 wells of a 24-well plate.

[0421] Flow cell embodiment of a picowell array. A picowell array can be part of a flow cell, where a fluidic chamber with an inlet and an outlet is mounted on top of the picowell array. In this embodiment, the beads, cells, and other assay materials of the present disclosure can flow in from the inlet and out through the outlet. As the beads flow through the flow cell, the beads can be embedded in the picowells using gravity or centrifugal force.

[0422] (X) sequencing nucleic acids bound to beads in picowells

[0423] The bead-bound nucleic acids can be sequenced while still attached to the beads. Alternatively or additionally, the bead-bound nucleic acids can be sequenced after the DNA barcodes have been cleaved from the beads.

[0424] DNA barcodes are cleaved from the beads prior to sequencing. In some embodiments, the present disclosure can encompass methods wherein the bead-bound DNA barcodes are cleaved from the beads, thereby releasing the DNA barcodes in soluble form prior to amplification, prior to sequencing, or prior to any type of sequence identification technique, such as hybridization to nucleic acid probes.

[0425] Exclusive embodiments. In embodiments, the present disclosure can exclude any method, associated reagents, systems, compositions, or beads wherein the bead-bound DNA barcodes are understood prior to amplification, prior to sequencing, or prior to any type of sequence identification technique, such as hybridization to nucleic acid probes. Additionally, the present disclosure can exclude any method wherein the polynucleotides comprising the DNA barcodes, or the nucleic acids comprising only a portion of the DNA barcodes, are cleaved prior to amplification, prior to sequencing, or prior to any type of sequence identification technique, such as hybridization to nucleic acid probes.

[0426] Polymerase chain reaction (PCR); quantitative PCR (qPCR). PCR methods and qPCR methods follow a 3-step method that includes: (1) denaturing the DNA template at high temperature, annealing the primers at a reduced temperature, and finally extending the primers by DNA synthesis, as catalyzed by DNA polymerase (Gadkar and Filion (2014) Current Issues Mol. Biol. 16:1-6). qPCR is also known as “real-time PCR” (Kralik and Ricchi (2017) Frontiers Microbiology 8: pages 1-9.

[0427] Recent modifications or improvements to PCR methods and qPCR methods include the use of helicase-dependent amplification (HDA), the use of internal amplification controls, the use of locked nucleic acids (LNA), and the use of binding inhibitor-additive (Gadkar and Filion (2014) Current Issues Mol. Biol. 16:1-6). Locked nucleic acids have the advantage of recognizing and binding their targets with extremely high precision.

[0428] qPCR can simultaneously amplify and quantify targeted 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 brief description of apparent typical conditions for performing qPCR (Refsland, Stenglein, Harris (2010) Nucleic Acids Res. 38:4274-4284).

[0429] Guidance can be provided for designing and validating PCR primers, as well as variables such as annealing temperature (Ta), melting temperature (Tm), extension step temperature, buffer type, etc. (Bustin and Huggett (2017) Biomolecular Detection Quantification 14:19-28).

[0430] Rolling circle amplification (RCA). DNA attached to a bead can be amplified. Amplified forms of DNA are easier to sequence than unamplified DNA. In the rolling circle amplification method, DNA tags (DNA barcodes) are made single stranded. Once single stranded, a splint oligo is added to bridge the ends of the tag DNA, which is then extended and ligated. The use of a DNA polymerase (negative 5' 3' exonuclease activity) ensures that the DNA catalyzes ligation of the extended splint oligo. The circularized DNA can then be subjected to rolling circle amplification by the addition of a strand displacing DNA polymerase (such as phi29 DNA polymerase). The ability to perform rolling circle amplification (RCA) on DNA barcode tags allows for the use of synthetic chemistry that can cause damage to DNA, as any surviving DNA molecules can be thermally amplified to sufficient quantities to be easily sequenced. DNA can be made single stranded by exonuclease digestion, nicking, and melting at high temperatures, or treatment with sodium hydroxide.

[0431] More details of rolling circle amplification (RCA) are revealed by the following steps that can be used to perform RCA.

[0432] Step One: Start with a bead-bound ssDNA. If the bead-bound DNA is initially double-stranded from (dsDNA), the strand not used for RCA can be prepared to replace the thymine (T) residues with residues of uracil (U) on or near the bead-bound end. If the dsDNA is prepared in this way, the uracil-N glycosylase can be used to cleave the uracil residues, leaving behind an unstable sugar phosphate as part of the DNA backbone, where this unstable position can be cleaved by nuclease treatment (Ostrander). The bead-bound ssDNA is now ready for the next step.

[0433] Step Two: Add a "splint oligo" to the bead-bound ssDNA. The splint oligo is designed to hybridize to about 10-20 base pairs at the end (5' end) of the ssDNA (covalently attached to the bead) and also to about 10-20 base pairs at the free end (3' end) of the bead-bound ssDNA. The splint oligo does not need to be immediately adjacent to the bead-bound end of the ssDNA and the free end of the bead-bound ssDNA. All that is needed is to tether the distal ends of the bead-bound ssDNA sequence together to form one large loop.

[0434] Step Three: Add Sulfolobus DNA polymerase IV so that this polymerase uses the large loop of ssDNA as a template to create a complementary large loop, which is covalently attached to the splint oligo at one end.

[0435] Step Four: Covalently close the complementary large loop using a DNA ligase, where the result is a circular ssDNA. It is this closed ssDNA that "rolls" during RCA.

[0436] Step Five: Add a DNA polymerase with strand displacement activity and add dNTPs. The added DNA polymerase covalently attaches dNTPs to the bead-bound ssDNA and extends the end of the bead-bound ssDNA to create a complementary copy of the contents of the "rolling circle," which is then further extended to create a complementary copy of the contents of yet another complementary "rolling circle," which is extended even further to create a complementary copy of the contents of still another "rolling circle." In this potentially infinite amplification process, the strand displacement activity of the DNA polymerase makes possible the continued activity of the DNA polymerase.

[0437] Optionally, the method of this disclosure includes: real-time monitoring of rolling circle amplification (RCA) via fluorescent molecular beacons (Nilsson, Gullberg, Raap (2002) Nucleic Acid Research 30:e66(7)). Reagents for RCA are available from Sigma-Aldrich (St. Louis, Missouri) and Sygnis TruePrime Technologies (Heidelberg, Germany). RCA kits) and GE Healthcare (TempliPhi The amplification kit is available. Fluoresceins and quenchers are available from Thermo Fisher Scientific (Carlsbad, CA), Molecular Probes (Eugene, Oregon), Cayman Chemical (Ann Arbor, Michigan), and Sigma-Aldrich (St. Louis, Missouri).

[0438] Step 6. Using ssDNA amplified via RCA as a template for PCR amplification, primers are added, a thermostable DNA polymerase is added, and the PCR product is subsequently sequenced using next-generation sequencing.

[0439] In one aspect of this disclosure, the ssDNA amplified by RCA is cleaved from the beads prior to PCR amplification for preparing the PCR product. In another aspect of this disclosure, PCR amplification for preparing the PCR product can be performed without cleaving the ssDNA amplified by RCA from the beads.

[0440] As described by Baner et al., “through the RCA reaction, strands representing many tandem copies of the cyclic complement can be generated” (Baner, Nilsson, and Landegren (1998), Nucleic Acid Research 26:5073-5078). Bacillus subtilis phi29 DNA polymerase is a suitable enzyme due to its strand substitution activity and high productivity. Li et al. similarly characterized RCA as follows: in RCA, a circular template is isothermally amplified by the DNA polymerase phi29, which possesses strand substitution properties. The long single-stranded DNA product contains thousands of repetitive sequences: (Li and Zhong (2007), Analytical Chemistry 79:9030-9038).

[0441] Sequencing of the DNA barcodes of the present disclosure can be performed using U.S. Patent No. 8,632,975 to van der Horn, which is incorporated by reference herein in its entirety, without implying any limitation thereto. Additionally, the DNA barcodes of the present disclosure can be sequenced, for example, by using methods of sequencing by synthesis, such as the Sanger sequencing method, or by using methods of “next generation sequencing.”

[0442] Illumina method for DNA sequencing. The Illumina method for DNA sequencing is as follows. DNA can be fragmented to a size range of 100-400 base pairs (bp) by sonication (Hughes, Magrini, Demeter (2014) PLoS Genet. 10:el004462). In the Illumina method, a DNA library is made in which fragments of DNA from one cell or from multiple cells are modified by DNA adaptors (attached to the ends of the fragments). The reaction product takes the form of a sandwich in which the DNA to be sequenced is in the center of the sandwich. The reaction product takes the form of: (first adaptor) - (DNA to be sequenced) - (second adaptor). The adaptor-DNA-adaptor complex is then bound to yet another adaptor, where the other adaptor is covalently attached to a solid surface. The solid surface can be a flat plate. The solid surface has a lawn of adaptors sticking out of the flat surface. The adaptors have a DNA sequence that is complementary to one of the adaptors in the sandwich. In effect, the lawn contains two types of adaptors, where one of the adaptors binds (hybridizes) to one of the adaptors in the complex and non-covalently tethers the complex to the plate. These can be referred to as the “first lawn binding adaptor” and the “second lawn binding adaptor”. The primary task of the DNA polymerase is to use the tethered (but non-covalently bound) DNA as a template to create a daughter strand that, when DNA polymerization occurs, takes the form of a covalent attachment to the “first lawn binding adaptor”. This covalent bond is created through the catalytic action of the DNA polymerase. After the daughter strand is completely synthesized, the end (the end sticking out of the media) contains a DNA sequence that is complementary to the second adaptor in the sandwich described above. This complementary DNA sequence allows the end of the newly synthesized daughter DNA to bend and hybridize to the “second lawn binding adaptor”. How the two adaptors of the sandwich are used, and how the “first lawn binding adaptor” and the “second lawn binding adaptor” are used, has been described above.

[0443] The reaction cycle is then performed many times, with the result being a cluster of amplified versions of the original dsDNA. In effect, the cluster takes the form of covalently linked (tethered) ssDNA molecules, where all of these ssDNA molecules correspond to only one strand of the original dsDNA (the dsDNA isolated from a live cell or tissue). This cluster of tethered ssDNA molecules is referred to as a "polymer colony." The polymer colony is produced by a technique called "bridge amplification." Ultimately, after bridge amplification and formation of the polymer colony, the reverse strand covalently linked to the solid surface is cleaved from its tether, washed away, and discarded, leaving only the forward strand.

[0444] Information on the method 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; "Illumina Sequencing by Synthesis" (5-minute video on YouTube).

[0445] Sequencing by Oligonucleotide Ligation and Detection (SOLiD sequencing). SOLiD can measure the intensity of fluorescence of dye-labeled molecules to determine the sequence of a DNA fragment. A library of DNA fragments is prepared from the sample to be sequenced and used to prepare a population of clonal beads (only one species fragment on the surface of each magnetic bead). The fragments that are ligated to the beads are given a universal P1 adaptor sequence, so the starting sequence of each fragment is known and identical. PCR is performed and the resulting PCR products that are ligated to the beads are covalently bound to a glass slide.

[0446] Primers are then hybridized to the P1 adaptor sequence in the template library. A set of four fluorescently labeled di-base probes compete for ligation to the sequencing primer. The specificity of the di-base probes is achieved by interrogating each 1st and 2nd base in each ligation reaction. Multiple cycles of ligation, detection, and cleavage are performed, where the number of cycles determines the final read length. After a series of ligation cycles, the extension products are removed and the template is reset with primers complementary to the n-1 position to perform a second round of ligation cycles (see Wu et al. (2010) Nature Methods 7:336-337).

[0447] pH-based DNA sequencing. pH-based DNA sequencing is a system and method in which base incorporation is determined by measuring hydrogen ions, which are byproducts of the polymerase-catalyzed extension reaction. DNA templates, each with an operatively bound primer and polymerase, are loaded into a reaction chamber or micro-well, followed by repeated cycles of deoxynucleotide triphosphate (dNTP) addition and washing. The DNA templates are templates that are linked as a clonal population to a solid support. With each such incorporation, hydrogen ions are released, collectively by a large number of templates, resulting in a detectable change in the local pH of the reaction chamber (see Pourmand (2006) Proc Natl Acad Sci USA 103:6466-6470). The present disclosure can exclude pH-based DNA sequencing.

[0448] With respect to concatenated DNA barcodes, the entire concatenated DNA barcode can be sequenced in one run (where sequencing of the entire concatenated DNA barcode requires only one sequencing primer). Alternatively, some or all of the DNA barcode modules that make up the concatenated DNA barcode can be individually sequenced (each individually sequenced DNA barcode module has its own sequencing primer). With respect to orthogonal DNA barcodes, each DNA barcode module that makes up the orthogonal DNA barcode requires its own dedicated sequencing primer, as each DNA barcode module is attached to the bead’s own site.

[0449] Exclusive embodiments. In embodiments, the present disclosure can exclude any system, device, combination of devices, and method that involves microfluidics, aqueous droplets that reside in an oil medium, and the creation of aqueous droplets in which a first channel containing a water reagent is connected to a second channel containing oil to create an aqueous droplet that is passed through the oil medium by a third channel from the connection region. Microfluidic devices and reagents are described (see, e.g., Brouzes, Medkova, Savenelli (2009) Proc Natl Acad Sci USA 106:14195-14200; Guo, Rotem, Hayman (2012) Lab Chip 12:2146-2155; Debs, Utharala, Balyasnikova (2012) Proc Natl Acad Sci USA 109:11570-11575; Sciambi and Abate (2015) Lab Chip 15:47-51).

[0450] In other exclusive embodiments, any reagent, composition, nucleic acid, or bead comprising a "DNA headpiece" or any reagent, composition, nucleic acid, or bead covalently linked to a "DNA headpiece" can be excluded. MacConnell, Price, Paegel (2017) Anal. Chem. 19: 181-192 provides examples of DNA headpieces, in which beads are partially functionalized with azido-DNA headpiece.

[0451] Other exclusive embodiments related to sequencing methods and sequencing reagents. In embodiments, the disclosure can exclude reagents, systems, or methods that do not involve the use of "reversible terminators" in DNA sequencing. Additionally, any reagent, system, or method that does not comprise a methoxy protecting group can be excluded. Also, any reagent, system, or method related to DNA sequencing can be excluded, but in which the DNA being sequenced is not covalently bound to a bead when detecting and collecting polynucleotide sequence information. Further, any reagent, system, or method that amplifies a DNA template prior to performing a sequencing reaction, such as amplification by PCR techniques or rolling circle techniques, can be excluded. In embodiments, any method of concatenated barcode encoding (e.g. nucleic acid barcode encoding) (all information about the synthesis of a chemical library member residing on a single nucleic acid) can be excluded. In another aspect, any method of orthogonal barcode encoding (e.g. nucleic acid barcode encoding) (information about the synthesis of a given monomer of a chemical library is spread across multiple ligation sites on a bead) can be excluded. In exclusive embodiments related to DNA ligases, the disclosure can exclude any reagent, system, or method that uses a DNA ligase for ligation of modules of nucleic acid barcodes.

[0452] Fluorophores, quenchers, and FRET-based assays. The disclosure provides fluorophores and quenchers for use in screening members of a chemical library or for use in characterizing an isolated member of a chemical library. FRET is resonance energy transfer.

[0453] Assays can be performed on bead-bound chemical libraries. Additionally, assays can be performed on free chemical library members shortly after cleavage from the bead, i.e. in the same micro-well as the bead or in the vicinity of the same hydrogel substrate as the bead. Also, assays can be performed on soluble chemical library members that were never attached to any bead or have been cleaved from a bead and then purified.

[0454] Fluorophores suitable for use as reagents of the present 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 Joe dye (4'-5'-dichloro-2',7'-dimethoxy-6-carboxyrhodamine), SYBR green I (absorption 497 nm, emission 520 nm), 6-carboxyfluorescein (6-FAM) (absorption 492 nm, emission 518 nm), 5-carboxyfluorescein (5-FAM) (absorption 492 nm, emission 518 nm), FITC, and rhodamine. Quenchers include TAMRA quencher, black hole quencher 1 (BHQ1), black hole quencher 2 (BHQ2), and DABCYL quencher. Note that as disclosed elsewhere in this patent document, TAMRA can be a fluorophore as well as a quencher.

[0455] Guidelines for reagents for FRET-based assays, where the FRET reagents comprise a fluorophore and a quencher (see Johansson (2006) “Choosing reporter-quencher pairs for efficient quenching” Methods Mol. Biol. 335: 17-29). An example of a FRET-based assay comprises measuring the activity of signal peptidase (SpsB) with the substrate “SceD peptide”. The FRET pair attached to the peptide is 4-(4-dimethylaminophenylazo) 5-((2-aminoethyl)amino)-l-naphthalenesulfonic acid (see Rao et al. (2009) FEBS J. 276:3222-3234). Another example comes from an assay for HIV-1 protease using the peptide substrate KVSLNFPIL. The donor / acceptor FRET pair is EDANS (donor) and DABCYL (acceptor). The EDANS fluorescence can be quenched by the DABCYL through resonance energy transfer to the non-fluorescent DABCYL (see Meng et al. (2015) J. Biomol. Screen. 20:606-615). Yet another example comes from an assay for botulinum toxin. The activity of SNAP-25 can be measured by using the substrate BoNT-A. For the FRET-based assay, the substrate has a N-terminal attached fluorescein-isothiocyanate (FITC) and a C-terminal attached quencher 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).

[0456] The present disclosure provides reagents, compositions, and methods for screening libraries of compounds to discover and identify enzyme inhibitors, enzyme activators, and to discover compounds that can increase the rate of in vivo degradation of a given protein. These reagents, compositions, and methods can use FRET-based assays, and alternatively, assays other than FRET-based assays can be used.

[0457] Molecular beacons are described (see Baruch, Jefferey, Bogyo (2004) Trends Cell Biology 14:29-35). Molecular beacons are a reagent in which a fluorophore is bound to a quencher through a linker. The linker can be cleaved by a nuclease, thereby measuring nuclease activity. The present disclosure provides methods for screening a chemical library to identify nuclease inhibitors, and, alternatively, to identify nuclease activators. Feng et al. describe the use of molecular beacons and FRET-based assays to measure the activity of various nucleases (Feng, Duan, Liu (2009) Angew Chem. Int. Ed. Engl. 48:5316-5321). Feng et al. show the use of FRET-based assays to measure the activity of various restriction enzymes.

[0458] (XI) Releasing a bead-bound compound

[0459] Cleavable linkers. Non-cleavable linkers are provided. In addition, cleavable linkers are provided (see Holmes and Jones (1995) J. Org Chem. 60:2318-2319; Whitehouse et al. (1997) Tet. Lett. 38:7851-7852 and Yoo and Greenberg (1995) J. Org. Chem. 60:3358-3364, as cited by Gordon et al. (1999) J. Chem. Technology Biotechnology 74:835-851). Cleavable linkers also include acyl sulfonamide linkers that reside in basic hydrolysis, and activated N-alkyl derivatives that cleave under mild conditions, and traceless linkers based on aryl-silicon bonds, and traceless linkers based on silyl ether bonds (described on pages 839 and 842 of Gordon et al. (1999) J. Chem. Technology Biotechnology 74:835-851). In addition, tartaric acid-based linkers are provided that, upon cleavage, produce a C-terminal aldehyde, where cleavage is by periodate oxidation (see Paulick et al. (2006) J. Comb. Chem. 8:417-426).

[0460] Figure 3 Various cleavable linkers suitable for use in the compositions and methods of the present invention are disclosed. Figure 3Table 1 is taken from Yinliang Yang (2014) “Design of Cleavable Linkers and Applications in Chemical Proteomics” Thesis for the degree of Doctor of Philosophy in Biochemistry, Technical University of Munich. From Figure 3 It is known that the preferred cleavable linkers of the present disclosure are linkers a, c, d, p, q, r and t. Linker p was used for the experimental results disclosed herein. The cleavage conditions for these 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 are not expected to damage the beads, not damage the bead-bound compound or not damage any chemical library member (unit) of the bead-bound compound.

[0461] Chemically cleavable linkers compatible with click-chemistry. Qian et al. (2013) describe a number of cleavable linkers that are compatible with click chemistry (Qian, Martell, Pace (2013) ChemBioChem. 14: 1410-1414). These comprise a linker group with an azo bond, where the azo bond can be cleaved by hydrazine. This linker has the following structure: R1-phenyl1-N=N-phenyl2-R2. The first phenyl ring has a hydroxyl group in the para position to R1, and the second phenyl ring has a carbonyl group connected to R2, where the carbonyl group is in the para position to the azo moiety.

[0462] Photo-labile cleavable linkers. The present disclosure encompasses photo-cleavable linkers with an o-nitrobenzyl group. This group can be cleaved by irradiation at 330-370 nm (see, Saran and Burke (2007) Bioconjugate Chemistry 18: 275-279; Mikkelsen, Grier, Mortensen (2018) J. Am. Chem. Soc. DOI: 10.1021). A linker with a short photolysis time compared to the o-nitrobenzyl linker is the 2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC) linker. A variation of the o-nitrobenzyl linker is the o-nitrobenzyl amino linker. When attached to a peptide chain, and subsequently cleaved, this linker releases an amide. Linkers with an o-nitroveratryl group can be used, which have a shorter photolysis time and higher release yield compared to the unsubstituted o-nitrobenzyl linker. Also useful are the benzoyl linker, the benzoin linker and the pivaloyl linker (see Mikkelsen et al. (2018) J. Am. Chem. Soc. DOI: 10.1021).

[0463] A linker with a photo-cleavable ether linkage is available. This photo-cleavable linker can be used where the linker is attached to the bead and the cleavable group is an “R group” and after cleavage the released group takes the form of ROH (see Glatthar and Giese (2000) Organic Letters 2:2315-2317). A linker with a photo-cleavable ester linkage is also available (see Rich et al. (1975) 97:1575; Renil and Pillai (1994) Tetrahedron Letters 35:3809-3812; Holmes (1997) Journal of Organic Chemistry 62:2370-2380, as cited in Glatthar and Giese, supra). Ether linkages in linkers can be cleaved by acid, base, oxidation, reduction, and fluoride sensitive silyl-oxygen bonds and photolysis (Glatthar and Giese, supra).

[0464] Another photo-cleavable linker that has been used to attach a peptide (R1) and a nucleic acid (R2) is as follows. R1 is attached directly to the methylene portion of the benzyl group. In the para position to the methylene group is a ring-attached nitro group. In the meta position to the methylene portion is a ring-attached ethyl group. The 1-carbon of the ethyl group bears a phosphate. The R2 group is attached to the oxygen atom of this phosphate (Olejnik et al. (1999) Nucleic Acids Research 27:4626-4631).

[0465] Akerblom et al. disclose a photo-labile linker of the alpha linker-methyl 2- nitrobenzyl type, containing amino, hydroxyl, bromo, and methyl groups, as well as 4- nitrophenoxyl-activated hydroxyl and amino groups (see Akerblom and Nyren (1997) Molecular Diversity 3:137-148). Cathepsin B can cleave a linker with the target sequence “valine-citrulline” (Dal Corso, Cazzamalli, Neri (2017) Bioconjugate Chemistry 28:1826-1833).

[0466] Enzymatically cleavable linkers. Linkers that can be cleaved with enzymes such as proteases (see Leriche, Chisholm, Wagner (2012) Bioorganic Medicinal Chem. 20:571-582). A chymotrypsin cleavable linker (see Maltman, Bejugam, Flitsch (2005) Organic Biomolecular Chem. 3:2505-2507). A tobacco etch virus protease cleavable linker (see Weerapana, Speers, Cravat (2007) Nature Protocols 2:1414-1425; Dieterich, Link, Graumann (2006) Proc. Natl. Acad. Sci. USA 103:9482-9487). The linker sequences LVPRG and LVPRGS can be cleaved by thrombin (Jenny, Mann, Lundblad (2003) Protein Expression Purification 31 :1-11). A plasmin cleavable linker (Devy, Blacher, Noel (2004) FASEB J. 18:565-567).

[0467] Bead-bound release monitors. The present disclosure provides novel and unique release monitors that are capable of assessing the release of bead-bound compounds. The release monitors take the form of a bead-bound complex of a fluorophore and a quencher, wherein the fluorophore is attached to the bead via a cleavable linker. Preferably, the cleavable linker is a photocleavable linker. Preferably, the bead-bound release monitor is located in a dedicated picowell that does not contain any other type of bead. Upon cleavage of the photocleavable linker, the fluorophore is released from the bead, diffuses into the medium in the picowell, and is kept at a distance from the bead-bound quencher, with the result that there is an increase in fluorescence that is directly proportional to the amount released. The increase in fluorescence allows the concentration of free fluorophore in the picowell to be calculated, and more importantly, the amount of compound released from other beads located in other wells.

[0468] In summary, the bead-bound release monitor is located in its own dedicated well, wherein the other wells contain bead-bound compounds that are candidate drugs.

[0469] Figure 8A simplified version of a preferred and non-limiting example of a bead-bound release monitor is disclosed. The release monitor takes the form of a quencher that is immobilized in the vicinity of a fluorophore, resulting in 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%, etc. In a picowell, one bead is dedicated to serve as a release monitor, while another bead or beads are used to attach compounds and DNA libraries. Exposure of all beads in a picowell to UV light results in simultaneous cleavage of the fluorophore and the compound. QSY7 is a preferred quencher. The structure and CAS number of QSY7 are as follows (shown below):

[0470] CAS Name / Number: Xanthenium, 9-[2-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-1-piperidinyl]sulfonyl]phenyl]-3,6-bis(methylphenylamino)-, chloride 304014-12-8

[0471] The increase in fluorescence resulting from the separation of the fluorophore from the quencher can be used to infer the concentration of the simultaneously released compound in the picowell. Additionally, the increase in fluorescence resulting from the separation of the fluorophore from the quencher can be used to infer the number of molecules residing in free form in the picowell (molecules that were present as a compound, which was formerly a bead-bound compound). In a more preferred embodiment, the release monitor includes a quencher and a fluorophore, where cleavage results in the release of the fluorophore (without releasing the quencher). This embodiment provides lower background noise compared to the less preferred embodiment below. In the less preferred embodiment, cleavage results in the release of the quencher, where the readout takes the form of an increase in fluorescence from the bead-bound fluorophore.

[0472] After UV-induced release of the compound from the bead, the release monitor provides a measure of the concentration of the soluble compound to the user. In preferred embodiments, one type of bead is dedicated to serve as a release monitor. By "dedicated" it is meant that the bead does not contain a bead-bound compound, nor does it contain a bead-bound DNA library.

[0473] As a general recommendation, one cannot infer that a compound has become a soluble compound just because it has been released from a bead by cleavage of a photosensitive linker. First, note that just because a compound is considered "hydrophobic" or is considered "water-insoluble" does not mean that no molecules are free to move in a solvent. For example, even cholesterol has a measurable solubility in water (see Saad and Higuchi (1965) "Water Solubility of Cholesterol" J. Pharmaceutical Sciences 54: 1205-1206). Moreover, the biochemical efficacy of a water-insoluble compound bound to a bead can be enhanced by surfactants, detergents, additives (such as DMSO), or carriers (such as human serum albumin). Thus, in the case where the picowell contains one of the above agents, or alternatively, in the case where the water-insoluble compound is released near the plasma membrane of a living cell cultured inside the picowell, the release monitor can be used to assess the overall concentration of a compound with limited or no water solubility.

[0474] Figure 9 A simplified version of the preferred embodiment of a bead-bound release monitor is disclosed, while Figure 10 The full and detailed construction of this preferred embodiment of a bead-bound release monitor is disclosed.

[0475] Figure 30 Data demonstrating the use of a bead-bound release monitor is provided, where the bead is in a picowell. The bead-bound fluorophore (bound using a photocleavable linker) is TAMRA (excitation wavelength 530 nm; emission wavelength 570 nm). The plot shows the time course of release of the fluorophore from the bead. This shows the operation of the bead-bound release monitor, with fluorescence data taken at t=0 seconds, t=l second, t=l l seconds, and t=71 seconds. Figure 30 Also included are enlarged insets showing two of the four smaller plots. The incubation of Cathepsin-D (a cysteine protease) with the "Peptide Q-Fluor substrate" and the beads results in Figure 30 The reagents were placed in wells at 4°C. The fluorophore was cleaved from the bead using 365 nm ultraviolet light, thereby releasing the fluorophore and separating it from the quencher. The purpose of this assay was to assess the time course of release that occurs in separate wells, where the separate wells contain different types of beads. The different types of beads have the same photocleavable linker, but this photocleavable linker is attached to pepstatin-A. The release of pepstatin-A can bind to and inhibit a cysteine protease in the same assay medium. This setup of bead-bound pepstatin-A and cysteine protease can be used as a positive control.

[0476] UV exposure through 20x objective. Images acquired with gain = 5; exposure 400 msec. Excitation at 530 nm at TAMRA. TAMRA emission at 570 nm.

[0477] Figure 35 More details of the enzymatic assay are disclosed, wherein the bead-bound pepstatin-A is released, and wherein the released pepstatin-A causes enzyme inhibition. Photocleavable Fmoc-valine (negative control) 10 pm TentaGel beads are shown mixed with photocleaved Fmoc-valine (negative control) 10 pm TentaGel beads in PBST buffer. This bead population is introduced into picowells, then the buffer is exchanged into a protease inhibition assay, containing Cathepsin D protease and peptide Q-Fluor substrate (λ ex = 480 nm, λ em = 525 nm). The wells are air-clamped, the entire slide is exposed to UV (365 nm, 77 J / cm 2 ), cleaving the photocleavable linker, releasing the compound to approximately 13 mM. The flow cell is incubated (30 min, 37 °C). The wells containing the positive control beads should inhibit the peptide proteolysis by Cathepsin D, resulting in a low fluorescent signal. The wells containing the negative control beads should show no Cathepsin D inhibition, and the fluorescent intensity should be similar to the empty wells.

[0478] The terms quencher and fluorophore can change for a given chemical, depending on other chemicals in the immediate vicinity. Although TAMRA used in the lab data for the bead-bound release monitor is a fluorophore, in other contexts, TAMRA can be a quencher. TAMRA acts as a quencher in a probe containing FAM and TAMRA.

[0479] ​Experimental setup and additional explanation of lab data. The present disclosure provides data for controlled 5(6)-carboxytetramethylrhodamine (TAMRA) concentrations in phosphate buffer (10 mM phosphate, 154 mM sodium, pH 8.0) in filled picowells separated by air. Fluorescent images were captured (exposure times 10 msec, 2 msec, respectively) and the well area was quantified by averaging the pixel intensity (n > 100) to 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 mM, 10 mM, 30 mM, 60 mM, 100 mM TAMRA). This standard curve was prepared under two different conditions, namely taking photographic images at either 2 msec exposure or 10 msec exposure. The experiment used to prepare the standard curve was performed in picowells, but no beads were used in this experiment (only known amounts of TAMRA). The photographic images are not shown in this patent document because the data takes the form of a standard curve, which can also be referred to as a calibration curve.

[0480] The experimental setup contained the following. For protocol X), TentaGel-Lys (PCL1-Tamra)-QSY7 bead structure. QSY7 (gray) quenches the Tamra fluorophore (orange) while being covalently attached to the bead via a photocleavable linker (purple). Illumination with UV (365 nm) provides quantitative release of the compound in situ.

[0481] Figure 31A A photograph of catalysis of aspartyl protease on quencher fluorophore substrate is disclosed. Figure 31B Emission data produced after catalysis of aspartyl protease on quencher fluorophore substrate is disclosed. Greater fluorescence means the enzyme is more catalytically active. Less fluorescence means the enzyme is less catalytically active, that is, the enzyme is more inhibited by a free inhibitor, wherein the inhibitor is released from the bead and wherein freedom is obtained by cleaving the photocleavable linker. Images were captured after UV release and cathepsin D assay incubation (λ ex = 480 nm, λ em = 525 nm). Wells containing positive control beads can be spectrally identified by the Cy5 fluorophore (λ ex = 645 nm, λ em = 665 nm, orange false color). Slices were analyzed with a line graph across the open well volume, wells containing negative control beads did not cause cathepsin-D inhibition. The detection volume in wells containing positive control beads was dark, indicating strong inhibition. The assay volume in the empty well was comparable to wells containing negative control beads.

[0482] Figure 32The following flow is shown. Further to protocol X), picowell substrates (46 pL per well) are enclosed in a flow cell, the wells are wetted under vacuum, a suspension of TentaGel-Lys(PCL1-TAMRA)-QSY7 beads is introduced, and air is drawn through the flow cell partitioning each well (top). The flow cell is illuminated with a UV LED (λ mean 365 nm) with controlled light flux, allowed to equilibrate (20 minutes) Figure 32 ), in detail, Figure 32 A cross-sectional view of a picowell is shown, demonstrating the step of wetting the picowell in the flow cell, the step of introducing the beads in suspension into the picowell (so that there is one bead per picowell), the step of drawing air across the flow cell to reduce excess dispersion solution (resulting in the meniscus falling below the planar top surface of the picowell plate), the step of controlled UV exposure (365 nm) (resulting in some TAMRA release), and the step of detecting the fluorescent signal by fluorescence microscopy (excitation 531 / 40 nm) (emission 594 / 40 nm) exciting the TAMRA to luminesce. The notation "slash 40" refers to the bandwidth, that is, it means that the cutoff filter restricts the light to the following ranges: 531 nm plus 20 nm and minus 20 nm, and for 594 nm, plus 20 nm and minus 20 nm (this slash notation can be used for the excitation wavelength, and also for the emission wavelength).

[0483] The inventors obtained a photograph showing the following data (see Figure 33 ). Fluorescent emission (λ ex 531 / 40 nm, λ em 593 / 40) of the fluorophore (TAMRA) released from 10 μm TentaGel-Lys(PCL1-TAMRA)-QSY7 beads after exposure to a UV LED (365 nm) in a picowell flow cell. A) No significant emission above background without exposure to UV (0 J / cm 2 ) prior to exposure to UV. TAMRA release reached equilibrium (20 minutes) after exposure to (B) 25 J / cm 2 , (C) 257 J / cm 2 , (D) 489 J / cm 2 , (E) 721 J / cm 2 , (F) 953 J / cm 2 UV, then imaged using appropriate exposure times. Fluorescent emission was measured in the volume surrounding each bead to measure TAMRA concentration Figure 33). The notation“slash 40” refers to bandwidth, i.e., it means that the cutoff filter restricts light to the following range: 531 nm plus 20 nm and minus 20 nm (this slash notation can be used for excitation wavelengths, and also for emission wavelengths).

[0484] Below is the inventor’s interpretation of some fluorescence data from a test and use bead-bound release monitor (see Figure 34 ) after exposure to UV (365 nm). Image analysis used the average pixel intensity of the solution surrounding the bead-filled well (n > 14), normalized to the image exposure time, and then correlated to a standard curve of known TAMRA concentrations in the picowell. Error bars represent 1 sigma, calculated from RSD%. UV-released compound concentrations were 1.1 µM (RSD% 8.9), 54.3 µM (RSD% 5.2), 142 µM (RSD% 4.2), 174 µM (RSD% 7.7), 197.3 µM (RSD% 10.1) Figure 34 )

[0485] Biochemical assays (not cell-based assays) for compounds of (XII)

[0486] Various biochemical assays can be performed using beads within picowells. Non-limiting examples include binding assays, enzyme assays, catalytic assays, fluorescence-based assays, luminescence-based assays, scatter-based assays, and the like. Examples are detailed below.

[0487] Biochemical assays sensitive to protease and peptidase inhibitors. If the goal is to detect and then develop drugs that inhibit proteases, the screening assay can use a particular protease or peptidase, a suitable cleavable substrate, and a mixture of color-based assays or fluorescence-based assays sensitive to the degree of inhibition by the candidate drug compound. For example, one reagent can be a bead-bound compound, where the activity of the compound has not yet been tested. Another reagent can take the form of a bead-bound chymostatin, an established HIV-1 protease inhibitor (Hilton and Wolkowicz (2010) PLoS One 5:el0940 (7 pages)). Yet another reagent can be a cleavable substrate for HIV-1 protease, and where cleavage by HIV-1 protease results in a color change or a fluorescence change. Where the particular assay (in a given microwell) results in a color difference (or fluorescence difference), a positive screening candidate drug is identified. The cleavable substrate takes the form of a susceptible peptide covalently bound to and flanked by a quencher and a fluorophore. Prior to cleavage, the fluorophore does not fluoresce due to the nearby quencher, but after cleavage, fluorescence is enabled (see Lood et al. (2017) PLoS One 12:el073919 (11 pages); Ekici et al. (2009) Biochemistry 48:5753-5759; Carmona et al. (2006) Nat Protoc 1 :1971-1976). The reagents and methods of the present disclosure encompass the above-disclosed technology.

[0488] Enzyme-based screening assay for compounds that inhibit ubiquitin ligases, where the reagents include MDM2 (enzyme) and p53 (substrate). Applicants have worked tests based on the following technology. MDM2 regulates the amount of p53 in a cell. MDM2 is overexpressed in certain cancers. MDM2 is an enzyme, as shown in the following statement: “In vitro studies have shown that purified MDM2 is sufficient to ubiquitinate... p53” (Leslie et al. (2015) J. Biol. Chem. 290:12941-12950). Applicants’ goal is to find inhibitors of MDM2, where these inhibitors are expected to decrease ubiquitination of p53 and thus reduce subsequent degradation of p53. Given the expected increase in p53 in a cell, inhibitors with the above properties are expected to be useful in treating cancer.

[0489] Applicants used the following enzyme-based assay to evaluate the effect of lenalidomide on MDM2 / HDM2-mediated ubiquitination of p53. Applicants used the reagents in 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 with a covalently bound antibody. The bead was M NH2 (catalog number M30102, Lapointe Polymer, Germany), and the antibody is an anti-human p53 monoclonal antibody biosynthesized in mice. MDM2 is an E3 ligase that can use p53 as a substrate, where MDM2 catalyzes the ubiquitination of p53.

[0490] Activating p53 reduces the goal of cancer. The following description suggests a relationship between MDM2, a transcription factor known as “p53,” and anti-cancer treatments. The description is: “MDM2 is an E3 ubiquitin ligase that ubiquitinates p53, targeting it for proteasomal degradation” (Ortiz, Lozano (2018) Oncogene 37:332-340). p53 has tumor suppressor activity. MDM2 can inhibit p53 activity. According to Wu et al., MDM2 is a “p53-binding protein” (see Wu, Buckley, Chernov (2015) Cell Death Disease 6:e2035). If a compound prevents the ubiquitination of p53 by, for example, preventing the interaction between MDM2 and p53, then the compound is expected to act as an anticancer drug.

[0491] Purpose of the screening assay. The purpose of the screening assay is to find compounds that affect p53 ubiquitination, for example, compounds that stimulate p53 ubiquitination and compounds that inhibit p53 ubiquitination. In particular, the purpose is to find compounds that are inhibited or activated, where their effect is through MDM-2 and El ligase, E2 ligase, or E3 ligase. MDM2 means "mouse double minute". MDM2 is known as an "E3 ubiquitin ligase". When MDM2 occurs in a cell, there is evidence that its catalytic activity for p53 ubiquitination requires many other proteins, such as CUL4A, DDB1, and RoCl (see Banks, Gavrilova (2006) Cell Cycle 5: 1719-1729; Nag et al. (2004) Cancer Res. 64: 8152-8155). Banks et al. have described the physical interaction involving p53 and MDM2 as "Discovery of L2DTL, PCNA, and DDB1 / CUL4A complex physical interactions with p53 tumor suppressor and its regulator MDM2 / HDM2" (Banks, Gavrilova (2006) Cell Cycle 5: 1719-1729). Nag et al. have also described the physical interaction involving p53 and MDM2 as "Cul4A functions as an E3 ligase and is involved in proteolysis of multiple regulatory proteins through the ubiquitin proteasome pathway". In this document, we show that Cul4A associates with MDM2 and p53 (Nag et al. (2004) Cancer Res. 64: 8152-8155).

[0492] Desired readout from the bead-based assay from p53 ubiquitination modulators. Where the screening compound results in a positive screen hit, i.e., there is more AF488 fluorescence, this means that an activator has been found. And where the screening compound results in a positive screen hit, i.e., there is less fluorescence, this means that an inhibitor has been found. Compounds that inhibit p53 ubiquitination indicate that the compound can be used to treat cancer. In addition, compounds that specifically inhibit p53 ubiquitination, i.e., the compound does not inhibit ubiquitination of other proteins, or the compound does not inhibit ubiquitination of other proteins as severely as p53, also indicate that the compound can be used to treat cancer.

[0493] Materials. Materials include E3 ligase kit K-200B from Boston Biochem. Boston Biochem's catalog describes the kit as: Mdm2 / HDM2 Ubiquitin Ligase Kit - p53 Substrate. The following relates to Mdm2, which is part of the kit. The kit does not include cereblon. Lenalidomide and similar compounds can bind to cereblon or Mdm2, with the end result being activation of ubiquitin ligase. Materials also include Diamond White Glass Microscope Slides, 25 mm x 75 mm (Globe Scientific, Paramus, NJ). Corning Stirrer / Hot Plate (setting from zero to ten) 698 Watts, model PC-420. N-hydroxy-succinimide (NHS). Methyltetrazine (mTET). Alexa Fluor 488 (AF488) (Thermo Fisher Scientific). TentaGel Beads M NH2 (Catalog No. M30102) (Rapp Polymere). Parafilm (Sigma-Aldrich, St. Louis, MO). Figure 8 The structure of Alexa 488 is shown. Information on Alexa Fluor 488-nanogold-streptavidin (Nanoprobes, Yaphank, NY) shows the structure of Fluor 488 (AF488).

[0494] Cell-based assays for compounds of Formula (XIII)

[0495] Cell-based assays performed in picowells can use human cells, non-human cells, human cancer cells, non-human cancer cells, bacterial cells, parasitic cells (such as Plasmodium cells). In addition, cell-based assays can be performed with "killed but metabolically active" human or non-human cells, that is, their genomes have been cross-linked to allow for metabolism but prevent cell division (see U.S. Patent Publication No. 2007 / 0207170 to Dubensky, which is incorporated by reference herein in its entirety). Also, cell-based assays can be performed on apoptotic cells, necrotic cells, or dead cells. Cell-based assays of bacterial cells can be used to screen for antibiotics. Human cells infected with viruses can be used to screen for anti-viral agents. Combinations of cells are provided for use in cell-based assays. For example, combinations of dendritic cells and T cells are provided to screen for and identify compounds that stimulate antigen presentation or, alternatively, impair antigen presentation.

[0496] Cell-based assays can be based on primary cultures of cells, for example, cells obtained from a biopsy of normal tissue, a biopsy of a solid tumor or a hematological biopsy, or a biopsy of circulating solid tumor cells. In addition, cell-based assays can be based on cells that have been passaged one or more times.

[0497] Cell-based assays performed in picowells can use cultures containing only one cell or containing two cells, three cells, four cells, five cells, or about 2 cells, about 3 cells, about 4 cells, about 5 cells, or more cells, or less than 3 cells, less than 4 cells, less than 5 cells, etc.

[0498] Applicants performed a work test based on the following technology. This describes a cell-based assay for screening exemplary embodiment compounds, where lenalidomide (test compound) inhibits ubiquitin-mediated transcription factor proteolysis. The transcription factors include Ikaros and Aiolos.

[0499] The present disclosure provides a cell-based assay that screens compounds on bead-bound compounds and where the screening is performed using a plate with a plurality of picowells. Components of the cell-based assay include picowells that are used to house bead-bound chemical libraries, where each bead is essentially linked to only one uniform type of compound. The compounds are released by a cleavable linker. Mammalian cells are cultured in the picowells. The picowells also contain culture media. A non-limiting example of the current use of lenalidomide is a proof-of-principle example that can be used to screen chemical libraries to find other compounds that modulate ubiquitination of a given target protein.

[0500] Brief description of the cell-based assay. Recombinant cells are used as reagents to detect and screen for compounds that induce proteolysis of green fluorescent protein (GFP), where the readout for identifying positive screening compounds is the case where green cells become colorless cells or cells with reduced green color. With respect to the mechanism of this cell-based assay, the mechanism of action of lenalidomide to cause green cells to become colorless cells or cells with reduced green color is that lenalidomide binds to a protein called “cereblon”. In cells, cereblon is part of a complex of proteins called “E3 ubiquitin ligases”. Cereblon is a direct target of the anticancer drugs lenalidomide, thalidomide, and pomalidomide. The normal and constitutive activity of E3 ubiquitin ligases and its relationship to cereblon is described as “cereblon. Facilitates [target protein] proteasomal degradation by engaging E3 ubiquitin ligases”.. (See Akuffo et al. (2018) The Journal of Biological Chemistry 293:6187-6200). In contrast to the normal activity of E3 ubiquitin ligases, when drugs such as lenalidomide, thalidomide, or pomalidomide are added, the result is “lenalidomide, thalidomide, and pomalidomide. e18556 facilitate ubiquitination and degradation of [substrates] by E3 ubiquitin ligases. Each of these drugs induces degradation of the transcription factors IKZF1 and IKZF3” (Kronke et al. (2015) Nature 523:183-188).

[0501] With respect to terminology, cereblon has been described as part of a protein complex, which is referred to as an “E3 ligase,” and also as an “E3 ubiquitin ligase.” In general, cereblon itself is not referred to as an “E3 ligase.” The following excerpts reveal how the term “cereblon” is used. According to Akuffo et al. (2018) Biochem J 293:6187-6200, “Upon binding to thalidomide, the E3 ligase substrate receptor cereblon is recruited by the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase to facilitate proteome destruction [of a substrate].” Consistently, Yang et al. (2018) Biochem J 293:10141 10157 disclose that “Cereblon acts as a substrate receptor for the cullin 4 RING E3 ligase to mediate ubiquitination of proteins [substrates].” Zhu et al. (2014) Blood 124:536 545 state that “Thalidomide binds to CRBN [cereblon] to alter the function of the E3 ubiquitin ligase complex, which is composed of CRBN, DDB1, and CUL4.” Lopez-Girona et al. (2012) Leukemia 26:2326-2335 state that “Studies identified the E3 ligase protein cereblon (CRBN) as a direct molecular target of thalidomide. CRBN and DDB1 form a functional E3 ligase complex with Cul4A and Roc1.”

[0502] To view an overview of the cell-based assay designed and used by the Applicant, the first step is the addition of lenalidomide to the cells. The last step is the degradation of IKZF1 and IKZF3. When IKZF1 appears as a fusion protein with GFP, the last step is the entire fusion protein is degraded by the proteasome. Similarly, when IKZF3 appears as a fusion protein with GFP, the last step is this entire fusion protein is degraded by the proteasome. As a result of the GFP degradation, a cell that was once a green fluorescent cell becomes a non-fluorescent cell.

[0503] Detailed description of cell-based assays. This involves the protein name (protein complex) of the E3 ubiquitin ligase, the protein name that binds to this complex, and the protein name that is the target of this complex. For these names, published literature is inconsistent. Sometimes a protein is referred to by the name of the protein, and sometimes a protein is referred to by the name of the gene that encodes the protein. Thus, the following description uses protein names together with gene names, such as “cereblom” (protein name) and “CRBN” (gene name). In addition, “Ikaros” is a protein name, while the gene name is IKZF1. In addition, “Aiolos” is a protein name, while the gene name is IKZF3. “Cullin-Ring Ligase-4” is a protein name, and the gene name is CRL4. “Regulator of cullin-1” is a protein name, 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 a protein name, and the gene name is CUL4A. See Schafer, Ye, Chopra (2018) Ann. Rheum. Dis. DOI: 10.1136; Chen, Peng, Hu (2015) Sci. Rep. 5: 10667; Matyskiela et al. (2016) Nature 535: 252-257; Akuffo et al. (2018) Biochem. J. 293: 6187-6200).

[0504] E3 ubiquitin ligases catalyze the transfer of ubiquitin residues to target proteins, with the result that the target proteins are sent to the proteasome for degradation. E3 ligases catalyze the attachment of ubiquitin to one or more lysine residues of a target protein. Humans express about 617 different E3 ubiquitin ligases (see Shearer et al. (2015) Molecular Cancer Res. 13: 1523-1532). E3 ubiquitin ligases are complexes of the proteins DNA damage binding protein-1 (DDB1); Cullin-4 (CUL4A or CUL4B); Regulator of Cullins-1 (RoC1); and RING Box-domain protein (RBX1). As noted above, RoC1 and RBX1 are the same protein (see Jia and Sun (2009) Cell Division 4: 16. DOI: 10.1186). When cereblon (CRBN) is added to the E3 ubiquitin ligase complex, the resulting larger complex is called: CRL4 CRBN(Matyskiela et al. (2016) Nature 535:252-257). The term“CRL4” means“Cullin-4 RING Ligase” (Gandhi et al. (2013) J. Haematol. 164:233-244; Chamberlain et al. (2014) Nature Struct. Mol. Biol. 21 :803-809). When reading the literature for cereblon, one needs to take into account the differences in the above terms.

[0505] The following is a longer version of the short summary above. What is shown below is another form of the terms, namely the terms:“CRL4 CRBN E3 ubiquitin ligase”. The longer description integrates the various names and activities of the entities more fully. The relationship between cereblon (CRBN) and the E3 ubiquitin ligase complex has been described as:“cereblon (CRBN) promotes proteasomal degradation of [target proteins] by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase” (Akuffo et al. (2018) Biochem. J. 293:6187 6200). With respect to anticancer drugs, lenalidomide, thalidomide, and pomalidomide. Ubiquitination and degradation of substrates are facilitated by E3 ubiquitin ligases. Each of these compounds binds CRBN CRBN, CRL4 CRBN E3 ubiquitin ligase substrate adaptor, and each of these drugs induces degradation of the transcription factors IKZF1 and IKZF3” (Kronke et al. (2015) Nature 523:183-188).

[0506] This involves a cell-based assay in which any given microwell, nanowell, or picowell contains a bead, where the bead has a covalently attached compound, where the compound is attached by a cleavable linker, and where the well contains one or more cultured mammalian cells. The response to the compounds of the disclosure and to the candidate drug can be assessed by one or more biomarkers.

[0507] Biomarkers include diagnostic biomarkers, biomarkers that predict whether a particular patient will respond (get better) to a particular drug, and biomarkers that predict whether a particular patient will develop unacceptable toxicity to a particular drug (Brody, T. (2016) Clinical Trials: Study Design, Endpoints and Biomarkers, Drug Safety, and FDA and ICH Guidelines, 2nd Edition, Elsevier, San Diego, CA). The present disclosure takes advantage of yet another biomarker, a biomarker that monitors a patient’s response to a given drug after the drug treatment has already begun. For example, the following relates to the biomarker peroxiredoxin 6 (PRDX6) and lung cancer. According to Hughes et al., “PRDX6 levels in cell culture media of cell lines increased after treatment with gefitinib compared to vehicle.” The accumulation of PRDX6 over time was positively correlated with gefitinib sensitivity. Serum PRDX6 levels increased significantly within the first 24 hours of treatment. Changes in serum PRDX6 during gefitinib treatment provided an advantage over imaging-based strategies to monitor anti-EGFR drug response.” Note the comment that biomarkers have an advantage over more direct measures of response efficacy, i.e., using “imaging” to detect a reduction in tumor size and number (Hughes et al. (2018) Cancer Biomarkers 22:333-344). Other biomarkers that monitor response to anti-cancer drugs include CA125 that monitors response of ovarian cancer to platinum therapy and serum HSPB1 that monitors response of ovarian cancer to chemotherapy (see Rohr et al. (2016) Anticancer Res. 36:1015-1022; Stope et al. (2016) Anticancer Res. 36:3321-3327).

[0508] Cytokine expression. Responses can be assessed by measuring expressed cytokines, such as IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha. These particular cytokines can be measured simultaneously using gold nanostructures with antibodies that specifically recognize one of these cytokines, where detection involves plasmonic 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 by fluorescent antibodies in a device containing microwells (Zhu, Stybayeva (2009) Anal. Chem. 81 :8150-8156). The above methods can be used as reagents and methods of the present disclosure.

[0509] In some embodiments, antibodies to cytokines can be attached to the walls of the picowells, where any cytokine released or differentially released from the cells as a function of drug exposure can be captured by the antibodies attached to the walls of the picowells. The captured cytokines can be identified by a second set of labeled antibodies. In some embodiments, antibodies to cytokines can be attached to capping beads. The capping beads can then be embedded into a cross-linked hydrogel sheet, which can be peeled off and subjected to further analysis, for example, by ELISA, mass spectrometer, or other analytical techniques.

[0510] Apoptosis. Surface enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) can be used to measure real-time data of single cell apoptosis and early events of apoptosis (see Stojanovic, Schasfoort (2016) Sensing Bio-Sensing Res. 7:48-54; Loo, Lau, Kong (2017) Micromachines 8:338. DOI: 10.3390). Stajanovic, supra, detects the release of cytochrome C, EpCam, and CD49e from cells. Loo et al., supra, measures the release of cytochrome C from cells, where the detection involves a DNA aptamer (the DNA aptamer functions like an antibody). Zhou et al. use SERS to detect early apoptosis in single cells, where the measured is phosphatidylserine on the cell membrane (see Zhou, Wang, Yuan (2016) Analyst 141 :4293-4298). In addition to collecting data about apoptosis, SERS can be used to evaluate drug activity by collecting data about stages of mitosis, metabolite release, 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 occurs in cell apoptosis (see Kang, Austin, El-Sayed (2014) ACS Nano 8:4883-4892). Plasmon resonance (SERS) can distinguish cancer cells from normal cells by measuring the percentage of alpha helix form versus beta sheet form of mitotic proteins (Panikkanvalappil, Hira, El-Sayed (2014) J. Am. Chem. Soc. 136:159-15968). The above methods are suitable as reagents and methods of the present disclosure.

[0511] Apoptosis of cultured cells can also be measured by methods that do not use plasmon resonance, but rather use immunocytochemistry with an antibody against cleaved caspase-3 (Shih et al. (2017) Mol. Cancer Ther. 16:1212-1223).

[0512] General information about cell-based assays. The cell-based assays of the present disclosure can be used to test responses from human cancer cells, solid tumor cells, blood cancer cells, human stem cells, human liver cells, pathogenic bacteria, infectious bacteria, human cells infected with bacteria, human cells infected with viruses, etc. The assays can detect morphological responses of the cells, such as migration, as well as genetic and biochemical responses.

[0513] The assays of the present disclosure can be designed to detect responses of cells located inside pico-wells, or to detect responses of cells located outside pico-wells, such as in a nutrient media located above a pico-well array. Additionally, the assays of the present disclosure can be designed to detect responses of cells, where the cells and beads are located in a media, where the cells are located in a media and the beads are located above or below the media, where the cells are located on top of a media and the beads are located above or inside or below the media.

[0514] The present disclosure provides a population of cells in a pico-well 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 population of cells are located inside the pico-wells (as opposed to being located in any area above the pico-wells). In embodiments, the proportion of cells residing inside the wells can be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or any range defined by two of these numbers, such as “about 60% to about 90%, with the remainder located in a layer of nutrient media above the array of wells.”

[0515] Cell matrix. For bioactivity assays of cells where the cells are exposed to compounds released from beads or the cells are exposed to bead-bound compounds, suitable matrices include those comprising one or more of the following: poly-D-lysine (PDL), poly-L-lysine (PLL), poly-L-ornithine (PLO), vitronectin, osteonectin, collagen, peptides containing RGD sequences, polypeptides containing RGD sequences, laminin, laminin / fibronectin complex, laminin / entactin complex, etc. Suitable matrices also include products available from Corning, Inc., such as Peptide Cell and tissue adhesives, et al. See Corning Life Sciences (2015) Corning Cell Culture Surfaces, Tewksbury, MA (20 pages), De Castro, Orive, Pedraz (2005) J. Microencapsul 22:303-315. In exclusive embodiments, the present disclosure can exclude any composition or method comprising one of the above matrices or one of the above polymers.

[0516] In embodiments, the present disclosure provides an array in which a single picowell contains a bead, one or more cells, and either a solution (without any matrix) or a matrix or a combination of a solution and a matrix. The matrix can be a hydrogel, polylysine, vitronectin, et al.

[0517] The activity of the bead-bound compound or bead-released compound can be assayed. The assay to assess activity can comprise activating or inhibiting an enzyme, activating or inhibiting a cell signaling cascade or individual cell signaling protein, binding to an antibody (or to a complementarity determining region (CDR) of an antibody, to a variable region of an antibody), inhibiting binding of a ligand or substrate to an enzyme (or to an antibody or to a variable region of an antibody).

[0518] For the above assays, the readout can be determined by a fluorescence assay, e.g., involving a fluorophore linked to a quencher (F-Q). The linker can be designed to be cleaved by an endoprotease, a DNase, an RNase, or a phospholipase (see Stefflova, Zheng (2007) Frontiers Bioscience 12:4709-4721). The term "molecular beacon" refers to such F-Q molecules, but "molecular probe" is also used to refer to constructs that induce separation of F and Q by hybridization, such as assay (Tyagi and Kramer (1996) Nature Biotechnol. 14:303-308; Tsourkas, Behlke, Bao (2003) Nucleic Acids Res. 15:1319-1330).

[0519] Transcriptional profiling analysis in response to drug exposure. DNA barcodes of the present disclosure can be modified to contain a response capture element, where the response capture element captures the response of the cell to the perturbation encoded by the encoding portion of the barcode. In some embodiments, the DNA barcode can terminate in a poly-T segment (a plurality of repeats of thymidine nucleosides), and the poly-T sequence can be used to capture poly-A tailed mRNA released from lysed cells. In some embodiments, the response capture sequence can be complementary to a gene of interest, capturing the expression profile of the desired gene by hybridization to the bead of that embodiment. In some embodiments, the picowell can contain a single cell picowell whose transcriptional profile is captured on a bead. In some other embodiments, multiple cells can be contained in a picowell whose transcriptional profile is captured.

[0520] In one exemplary workflow, the following procedure can be followed to capture the transcriptional response of cells to a drug. (a) Picowells designed to capture a single cell per well are provided. (b) DNA barcode beads loaded with a compound are introduced to the picowells, such that there is one bead per picowell. (c) The compound is released on the bead in each picowell by an appropriate method (UV treatment of a compound linked by a UV cleavable linker, diffusion in the case where the bead is immersed in the compound, acid cleavable, base cleavable, temperature cleavable, etc. appropriate to the bead of that embodiment). (d) The picowells can be isolated from each other by a capping bead that retains the contents in the picowell, or by other means (e.g. an air barrier or oil barrier on top of the picowell). (e) The cells in the picowell are incubated for a period of time in the presence of the compound released from the bead. (f) After an appropriate period of time, e.g. 1 hour, 2 hours, 5 hours, 9 hours, 12 hours, 15 hours, 18 hours, one day, 3 days, one week, two weeks, one month, or another appropriate assay-based time, the cells are lysed by a lysis method. The lysis method can involve the addition of a detergent, repeated cycles of freezing and thawing, heating, addition of a membrane disrupting peptide, mechanical agitation, or other appropriate means. (g) Once lysed, the contents of the cells are exposed to the bead within the picowell, at which point the response capture element on the bead of the picowell is able to capture its designated response. In some embodiments, the response capture is a poly-T sequence, which captures the complete mRNA profile of the cell (or cells) within each picowell. In some embodiments, the response capture element is designed to capture a specific DNA or RNA sequence from the cell. In some embodiments, the transcriptional response of the cell can be captured as a function of the dose (or concentration) of the compound.

[0521] (XIV) Perturbation response analysis of cells

[0522] The methods described herein can include a perturbation library and a cell library. In some embodiments, the perturbations and cells are incubated in a closed environment. During or after incubation, a barcode identifying the perturbation ("perturbation barcode") can be transferred to the cells with which it was incubated. The methods can further include releasing (i.e., isolating or removing) the cells from the perturbations, and subjecting the cells to a second confinement in which cellular contents and the perturbation barcode are captured. In some embodiments, the second confinement can contain a cell-specific barcode that can be used to subsequently study the cellular contents and the perturbation barcode, thereby correlating the perturbation with the cellular response.

[0523] In some embodiments, the methods described herein can include two reactions, a perturbation reaction encoded by a perturbation barcode and a measurement reaction. In the perturbation reaction, a cell can be subjected to a perturbation. In the measurement reaction, a cellular response due to the perturbation can be measured. In some embodiments, the measurement reaction can include a measurement barcode. In other embodiments, the methods can include bringing the perturbation barcode into the measurement reaction, and capturing the measurement barcode and the perturbation barcode included in the measurement reaction, thereby correlating the perturbation with the cellular response.

[0524] The methods described herein can include a perturbation reaction encoded by a perturbation barcode. The perturbation reaction includes subjecting a cell to a perturbation, and then measuring a cellular response of the cell to the perturbation subjected. The perturbation barcode can be decoded before or after measuring the cellular response, thereby correlating the identity of the perturbation with the measured cellular response.

[0525] In some embodiments, the method comprises providing a DNA-encoded, bead-bound compound library, wherein the compounds can be released from the beads, contacting the DNA-encoded, bead-bound compound library with a library of cells, wherein the contacting can be performed by confining the beads to one or more cells in a first finite volume, releasing the compounds from the beads, and incubating the compounds with the cells in the first finite volume. In some embodiments, the DNA barcodes identifying the compounds can be released from the beads and ligated to the cells, either simultaneously or after the incubation. The cells with the ligated DNA barcodes can then be released from the first finite volume and confined again in a second finite volume, wherein the second finite volume has reagents for lysing the cells and a mechanism for capturing the cellular contents and the bead-specific barcodes carried by the cells. In some embodiments, the mechanism for capturing the cellular contents and the barcodes can involve the use of capture barcodes, which can be used to uniquely identify individual cells or small clusters of cells. In some embodiments, the capture barcodes a...

Claims

1. A method for identifying a transcriptomic change in a cell induced by a compound, wherein the compound is included in an assay of a combinatorial chemical library, the method comprising: generating an assay array, wherein the array comprises: a plurality of finite volumes, wherein each finite volume is separated from other finite volumes, and wherein each finite volume comprises a cell of interest; a plurality of beads, wherein each bead comprises: a plurality of substantially identical compounds, such that each bead comprises a unique compound from the combinatorial chemical library, and each compound in the combinatorial chemical library is selected as a potential drug candidate; a plurality of functionalized oligonucleotides, wherein each functionalized oligonucleotide comprises an oligonucleotide portion that encodes a structure of the unique compound or a synthetic step used to make the unique compound; and an RNA capture element, wherein a single bead is disposed in each finite volume; contacting the cell in each finite volume with the compound released from the bead into the finite volume and maintaining the contact for a period of time sufficient to generate a transcriptomic change in RNA expressed by the cell in response to the contact; capturing RNA from the cell in each finite volume by lysing the cell and contacting the RNA with the RNA capture element on the bead; identifying the captured RNA from at least a portion of the plurality of beads and assessing any transcriptomic change in the captured RNA; and identifying the structure of any compound that generates the transcriptomic change.

2. The method of claim 1, further comprising f) assessing whether the compound demonstrates potential as a drug candidate based on the level of the captured RNA compared to a control.

3. The method of claim 1, wherein the RNA capture element comprises a microRNA (miRNA).

4. The method of claim 1, wherein the plurality of substantially identical compounds are linked to the bead by a cleavable linker.

5. The method of claim 1, wherein the cell is lysed by adding a detergent, repeatedly adding freeze / thaw cycles, heating, adding a membrane disrupting peptide, mechanical agitation, or any combination thereof.

6. The method of claim 1, wherein the method comprises sequencing the functionalized oligonucleotides.

7. The method of claim 1, wherein the RNA is messenger RNA (mRNA).

8. The method of claim 7, wherein the transcriptomic change comprises one or more changes in mRNA in the cell.

9. The method of claim 1, wherein the cell of interest is selected from: (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 Plasmodium cell; (vii) a cell that has metabolic activity but has a cross-linked genome and is unable to undergo cell division; (ix) a mammalian cell infected with a virus; and (x) a human cell.

10. The method of claim 1, wherein the RNA capture element on the bead comprises a poly-T segment configured to capture a poly-A tailed mRNA molecule released from a lysed cell; and the plurality of functionalized oligonucleotides terminate at the poly-T segment.

11. The method of claim 1, wherein the plurality of functionalized oligonucleotides comprises an orthogonal DNA barcode, wherein each DNA barcode module making up the orthogonal DNA barcode is linked to a different location on the bead.

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