Chemical library encoded in oligonucleotide

Abstract

To provide a system for high-throughput screening using a library of beads-bonded compounds.SOLUTION: Provided are beads with a covalently attached compound and a covalently attached DNA bar code, and a method for using the beads. The beads have many substantially the same copies of the compound, and many substantially the same copies of the DNA bar code. The compound consists of one or more chemical monomers, the DNA bar code takes a mode of a bar code module, and each module is capable of specifying a corresponding and corresponding chemical monomer. A nucleic acid bar code can have a chain-like structure or an orthogonal structure. A method for sequencing a beads-bonded nucleic acid bar code, a method for cutting off a compound from beads, and a method for evaluating the biological activity of a discharged compound are provided.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to high-throughput screening using libraries of compounds, where the compounds are bound to or contained within beads, each bead containing multiple copies of one type of compound, and the beads also contain DNA tags that code the identity or synthetic history of the compounds contained within or on the beads. The present disclosure also relates to high-throughput assays carried out in picowells, where the picowells contain compound-loaded beads and assay materials. The present disclosure further relates to releasing the bead-bound compounds and screening them for biological activity. In general, the present disclosure contemplates assays in which beads are used as delivery vehicles for compounds, and methods for making such compound-loaded beads.

[0002] The present disclosure relates to bead-bound compounds, each compound being made from one or more monomers belonging to a chemical library.The present disclosure also relates to bead-bound DNA barcodes, i.e., nucleic acids (not related to genetic code) whose sequence is a code for each nucleic acid refer to one specific chemical library monomer.The present disclosure further relates to releasing the bead-bound compounds and then screening the released compounds for biological activity.

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

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 562,905, filed September 25, 2017, and U.S. Provisional Patent Application No. 62 / 562,912, further filed September 25, 2017, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0005] Combinatorial chemistry, for example involving split pool chemistry, can be used to synthesize large quantities of compounds. Compounds made in this way find use in the field of medicinal chemistry, where compounds can be screened for various biochemical activities. These activities include binding to one or more proteins, where the proteins are known at the time the screening test is performed. Alternatively, the proteins bound by the compound being tested are identified only after a binding event is detected. Compounds can also be screened for their activity to inhibit or activate known proteins (this is not just screening for "binding" activity). Alternatively, compounds can be screened for their activity to inhibit or activate cellular functions, where the molecular target is unknown to the researcher at the time of screening.

[0006] Screening of compounds, such as those belonging to the vast library of chemicals created by split-pooling, can be facilitated by performing the screening using an array of thousands of microwells, nanowells, or picowells. Furthermore, screening can be facilitated by providing a different compound to each picowell via beads, each bead containing hundreds of copies of the same compound, and the same bead further containing hundreds of copies of a "DNA barcode" that can be used to identify compounds bound to the same bead. Furthermore, screening of compounds can be further facilitated by using cleavable linkers, which allow for the controlled release of compounds from the beads, which can then be used in biochemical or cell-based assays within the same picowell.

[0007] Assaying compounds in very small, restricted volumes, such as droplets, picowells, or microfluidic environments, is widely beneficial, for example, because the amount of assay reagents required is small, and therefore does not need to be limited to compounds generated in combinatorial chemistry. Any method that can load compounds onto beads and elute them from the beads at a later time point can be used to deliver bead-bound compounds to assays in small, restricted volumes. The addition of nucleic acid barcodes to beads can carry the identity of the compounds present within the beads along the assay volume. The method can perform very fast throughput assays without the need for robotics or spatial indexing of compounds within microtiter plates. Millions to billions of compounds can be held in one small vial, and the identity of the compound is tagged to the same bead that contains each individual compound.

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

[0009] Traditional phenotypic screening in cells involves creating models of diseased cells, contacting the cells with various drug libraries, and monitoring for correction of the disease phenotype by measurable assays. Such screening methods are termed phenotypic screening because the underlying biological mechanisms are not necessarily understood initially, but measurable phenotypic changes indicative of a healing response are considered the relevant metric. Currently, a vast number of cell lines and disease models are available that reflect various baseline and diseased cell states. Also, numerous compound libraries and biological drug candidates are available. An obvious screening campaign combining different cell models with different drug candidates to look for phenotypic responses has technical limitations as the assays are limited to microtiter plate formats and imaging methods, both of which severely limit throughput.

[0010] One way to overcome throughput limitations is to employ high-throughput single-cell screening approaches to drug discovery (see, e.g., Heath et al., Nat Rev Drug Discov. 15:204-216, 2016). In these approaches, single cells are separated and isolated into compartments, and individual assays can be performed on each of the cells. Genomic analysis by mRNA sequencing of single cells, for example using droplet encapsulation, is a common method to reveal intricate 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 herein by reference in their entirety). State-of-the-art single-cell analysis platforms are capable of quantification of mRNA transcripts at single-cell resolution, characterizing and fingerprinting cells based on their transcriptional state. This approach allows comparisons between tissue samples extracted from controls or prepared in experiments, and the examination of the transcriptional and therefore protein expression status of single cells. Measurement of single-cell mRNA by transcriptome sequencing and profiling is an important approach to investigate the molecular mechanisms of drug efficacy, resistance, and therapeutic target discovery, as well as the lineage phenotype of cells during disease progression (see, for example, Chu et al., Cell Biol and Toxicol 33:83-97, 2017; Wang, Cell Biol Toxicol 32:359-361, 2016; and Wang et al., Cell Biol Toxicol 33:423-427, 2017). The application of single-cell RNA sequencing is used to define cell-to-cell heterogeneity evidenced by cell-to-cell variability in the transcriptome, which is highly relevant to drug efficacy and specificity, transcriptional probability, transcriptome plasticity, and genome evolution. Encapsulation within picowells has also been demonstrated (see, e.g., Gierahn et al., Nat Methods 14:395-398, 2017).Using similar isolation methods, single-cell protein measurements are also possible (Butnik et al., BioRxiv, Jan. 2017, Su et al., Proteomics 17:3-4, 2017).

[0011] Despite the rapid increase in high-throughput single-cell RNA sequencing (RNA-seq) methods, including commercialized versions of automated platforms such as Fluidigm C1, 10XGenomics, or 1CellBiO systems, the application of single-cell RNA profiling for target-agnostic high-throughput drug screening and target discovery is constrained by the lack of methods that can efficiently partition different drugs into different cells. Although cells or tissues can be incubated under different perturbations in well plates, followed by single-cell analysis and comparison between transcriptional profiles, the number of drugs that can be tested is limited by the capacity of the plate. Moreover, the need to isolate and prepare barcoded mRNA from each sample and perform comprehensive RNA profiling on all samples also creates a major bottleneck. Summary of the Invention

[0012] Briefly, the disclosure provides a system for screening compounds comprising: (a) a picowell array plate including a plurality of picowells, each picowell having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, and a wall being between the upper hole and the floor; (b) beads disposed within the picowells, the beads including a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds; and (c) the beads including bead-bound DNA barcodes in the form of either concatenated DNA barcodes or orthogonal DNA barcodes, where the DNA barcodes are in the form of concatenated DNA barcodes, the concatenated DNA barcodes are generated by a method of (i) using click chemistry, or (ii) using repeated cycles of steps, the repeated cycles of steps generating a plurality of bead-bound DNA barcodes in the form of orthogonal DNA barcodes, the concatenated DNA barcodes being generated by a method of (i) using click chemistry, or (ii) using ... (d) using a splint oligonucleotide (sprint oligo) hybridizable to a DNA barcode module, where hybridization is mediated by an annealing site on the splint oligo and a corresponding complementary annealing site on the partially fabricated DNA barcode, where the annealed splint oligo is used as a template to extend the partially fabricated DNA barcode using a DNA polymerase, where the splint oligo comprises bases complementary to the DNA barcode module that is polymerized into the partially fabricated DNA barcode; (d) each one of a plurality of substantially identical bead-bound compounds comprises one or more chemical library monomers, where each bead-bound DNA barcode module identifies a corresponding chemical library monomer, where the term "compound" is used to refer to a completed product comprising one or more chemical library members, where the completed DNA barcode identifies the compound.

[0013] The floor of a microwell, nanowell, or picowell need not be flat. The floor may be curved, like the bottom of a glass test tube or metal centrifuge tube. The floor may also be cone-shaped, like a conical centrifuge tube. The floor may be flat, but may include notches, for example, notches that facilitate movement of assay or cell culture solutions near the bottom of any beads located in the picowell. In flat floor embodiments, the systems and methods may require a flat floor.

[0014] Concatenated DNA barcodes can be made entirely by organic chemistry methods, e.g., click chemistry, and orthogonal DNA barcodes can be made entirely by organic chemistry methods, e.g., including click chemistry.

[0015] Also provided is the above system, further including a plurality of caps, each cap capable of fitting into the opening of a different picowell, and each cap capable of minimizing or preventing evaporation of fluid within the picowell, and minimizing or preventing leakage of fluid within the picowell.

[0016] Further encompassed are systems as above in which the concatenated DNA barcodes are generated by a method that uses (i) both click chemistry and repeated cycles of steps using splint oligos, (ii) both click chemistry and non-click chemistry chemical methods, (iii) click chemistry alone, or (iv) only repeated cycles of steps using splint oligos. In this particular embodiment, the "concatenated DNA barcodes" in question do not include any chemical couplers used to directly attach nucleic acids to beads.

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

[0018] In response element embodiments, provided is a system as described above, wherein at least one bead disposed in at least one picowell comprises at least one response capture element associated with said at least one bead. Also contemplated is a system as described above, wherein at least one bead disposed in at least one picowell comprises at least one response capture element associated with said at least one bead, wherein at least one response capture element comprises (a) poly(dT) or (b) an exon-targeting RNA probe.

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

[0020] The following discloses "consisting only of" and "comprising" embodiments, which apply to the number of bead-bound DNA barcode modules that make up the DNA barcode. Provided are embodiments of DNA barcodes that consist of only one DNA barcode module, or only two DNA barcode modules, or only three DNA barcode modules, or only four DNA barcode modules, etc., or the DNA barcode comprises at least one DNA barcode module, or at least two DNA barcode modules, or at least three DNA barcode modules, or at least four DNA barcode modules, etc.

[0021] Also included is a bead-bound concatenated DNA barcode comprising: (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 a DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, a third annealing site, a fourth DNA barcode module, a fourth annealing site, and a fifth DNA barcode module; or (v) a first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, a third annealing site, a fourth DNA barcode module, a fourth annealing site, a fifth DNA barcode module, a fifth annealing site, and a sixth DNA barcode module.

[0022] Further contemplated is the above system, further comprising a primer binding site capable of binding to 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, wherein the primer binding site is located 3 prime to the first DNA barcode module, 3 prime to the second DNA barcode module, 3 prime to the third DNA barcode module, 3 prime to the fourth DNA barcode module, 3 prime to the fifth DNA barcode module, or 3 prime to the sixth DNA barcode module, or wherein the primer binding site is located between the first and second DNA barcode modules, or between the second and third DNA barcode modules, or between the third and fourth DNA barcode modules, or between the fourth and fifth DNA barcode modules, or between the fifth and sixth DNA barcode modules.

[0023] Further provided are systems as above, wherein the primer binding site is located between the first and second DNA barcode modules, or between the second and third DNA barcode modules, or between the third and fourth DNA barcode modules, or between the fourth and fifth DNA barcode modules, or between the fifth and sixth DNA barcode modules. In embodiments relating to the location of the primer binding site relative to the upstream DNA barcode module and relative to the downstream DNA barcode module, provided are systems as above, wherein a primer binding site is located between each and every pair of consecutive DNA barcode modules.

[0024] Further provided is the above system, wherein the bead comprises a DNA barcode that is an orthogonal DNA barcode, the bead comprises an outer surface, the orthogonal DNA barcode comprises: (a) a first nucleic acid comprising a first DNA barcode module and an annealing site for a sequencing primer, the first nucleic acid being attached 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, the second nucleic acid being attached 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, the second nucleic acid being attached to the bead at a third location, wherein the first, second, and third locations on the bead are each located at different locations on the outer surface of the bead.

[0025] In encoding embodiments, provided are systems as described above, wherein the DNA barcode does not identify any chemical library monomer, but instead includes one or more nucleic acids that specify (a) the class of compound that is cleavably attached to the bead, (b) the step number in a multi-step pathway of organic synthesis, (c) the date the bead-bound compound was synthesized, (d) the disease that the bead-bound compound is intended to treat, (e) the cellular event that the bead-bound compound is intended to stimulate or inhibit, or (f) the reaction conditions used to bind a given chemical library monomer to the bead.

[0026] In linker embodiments, provided are the above systems, wherein each of the plurality of substantially identical bead-bound compounds is coupled to the bead via a cleavable linker. Also provided are the above systems, wherein each of the plurality of substantially identical bead-bound compounds is coupled to the bead via a photocleavable linker. Also provided are the above systems, wherein each of the plurality of substantially identical bead-bound compounds is coupled to the bead via a non-cleavable linker.

[0027] In an embodiment of TentaGel®, provided is the above system, wherein at least one bead comprises a grafted copolymer consisting of a low cross-linked polystyrene matrix to which polyethylene glycol (PEG) is grafted.

[0028] In an emission monitor embodiment, the present disclosure provides a system as described above, wherein at least one picowell contains an emission monitor bead and does not contain any other type of bead.

[0029] the emission monitor bead comprises a bead-bound quencher and a bead-bound fluorophore, the bead-bound quencher is quenchingly positioned in close proximity to the bead-bound fluorophore and is capable of quenching at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or at least 99.5%, or at least 99.9%) of the fluorescence of the bead-bound fluorophore, the bead-bound fluorophore is attached via a first photocleavable linker, the picowell containing the emission monitor bead is a first picowell, the first picowell contains a first solution, Exposing the first picowell to cleavage conditions allows the photocleavable linker to be cleaved, releasing the fluorophore into a first solution in the first picowell, the exposing results in the fluorophore diffusing throughout the first solution in the first picowell, a fluorescent signal obtained by shining light on the first picowell containing the first solution containing the diffused fluorophore is used by a user to calculate a percentage release of the bead-bound fluorophore from the emission monitor beads, resulting in a calculated percentage release, a second picowell contains a bead-bound compound coupled with a photocleavable linker of the same type as the first photocleavable linker, the second picowell contains a second solution,

[0030] The release percentage value calculated from the release monitor beads in the first picowell allows for calculation of the concentration of released compound in the second solution in the second picowell.

[0031] In embodiments relating to the identity of all compounds bound to a given bead, or the identity of all DNA barcodes bound to a given bead, provided is the system as described above, wherein at least one bead comprises a plurality of substantially identical bead-bound DNA barcodes, the plurality being between 10 million and 100 million copies of the substantially identical bead-bound DNA barcodes. Also provided is the system as described above, wherein at least one bead comprises a plurality of substantially identical bead-bound compounds, the plurality being between 10 million and 100 million copies of the substantially identical bead-bound compounds.

[0032] In embodiments relating to cells (e.g., mammalian cells, cancer cells, bacterial cells), provided is a system as described above, wherein at least one picowell contains at least one cell, and a plurality of substantially identical bead-bound compounds are coupled to at least one bead via a cleavable linker, and cleaving the cleavable linker releases the bead-bound compounds from the beads to generate released compounds, and the released compounds are capable of contacting the at least one cell. In other cellular embodiments, provided is a system as described above, wherein at least one picowell contains at least one cell, a plurality of substantially identical bead-bound compounds are coupled to at least one bead via a cleavable linker, and cleaving the cleavable linker releases the bead-bound compounds from the beads to generate released compounds, and the released compounds can contact the at least one cell, 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 malaria parasite cell, (vii) a cell that is metabolically active but has a cross-linked genome and is incapable of undergoing cell division, or (ix) a mammalian cell infected with a virus.

[0033] In an embodiment of the device, provided is the above system, wherein each picowell has an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, a wall being between the upper hole and the floor, the hole being circular, the floor being circular, the wall being in the form of a truncated cone, the hole having a first diameter, and the floor having a second diameter, the first diameter being larger than the second diameter.

[0034] In other device related embodiments, provided is a picowell, each picowell having a top hole defining an opening at the top of the picowell, a bottom defined by a floor, the top hole being separated from the floor, a wall being between the top hole and the floor, the hole being circular, the floor being circular, the wall being in the form of a truncated cone, the hole having a first diameter and the floor having a second diameter, the first diameter being greater than the second diameter, and further including a cap that fits snugly over the hole, the hole being comprised of a polymer having a greater durometer (harder) and the cap being made from a polymer having a smaller durometer (softer), and the relative durometers of the cap and hole are such that the cap can be reversibly and snugly fitted over the hole. and the cap is (i) a cap whose sole purpose is to plug the picowell and prevent leakage, (ii) a passive cap, capable of absorbing metabolic products released by cells in a cell culture medium as they are cultured in the picowell, (iii) an active cap, in the form of beads containing a plurality of essentially identical compounds, each of the plurality of essentially identical compounds being attached to the beads with a cleavable linker, or (iv) an active cap, in the form of beads containing a plurality of identical reagents, each of the plurality of essentially identical reagents being attached to the beads with a cleavable linker. Provided is the above system, wherein the cap is spherical or the cap is non-spherical.

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

[0036] In a biochemical assay embodiment, included is a system as described above comprising at least one picowell, wherein the at least one picowell comprises beads comprising a plurality of substantially identical compounds and a plurality of substantially identical barcodes, and wherein the at least one picowell comprises an assay medium comprising a substrate for cereblon E3 ubiquitin ligase, such as cereblon E3 ubiquitin ligase, Ikaros or Aiolos, and wherein the system is capable of reducing intracellular concentrations of Ikaros or Aiolos to allow screening of compounds that activate the E3 ubiquitin ligase activity of cereblon.

[0037] In another biochemical assay embodiment, contemplated is a system as described above comprising at least one picowell, wherein the at least one picowell comprises beads comprising a plurality of substantially identical compounds and a plurality of substantially identical barcodes, and wherein the at least one picowell comprises an assay medium comprising a substrate for MDM2 E3 ubiquitin ligase, such as MDM2 E3 ubiquitin ligase, p53, and wherein the system allows for screening of compounds that activate the E3 ubiquitin ligase activity of MDM2, thereby increasing the intracellular concentration of p53.

[0038] In more barcoding embodiments, provided are systems as described above, wherein the DNA barcode does not encode any chemical library monomer, but instead includes one or more nucleic acids that specify one or more of: (a) a class of compound that is cleavably attached to the bead; (b) a step in a multi-step pathway of organic synthesis where the bead-bound nucleic acid corresponds to a given chemical monomer used to make the bead-bound compound, and the bead-bound nucleic acid corresponding to the given chemical monomer identifies that chemical monomer; (c) the date the bead-bound compound was synthesized; (d) a disease that the bead-bound compound is intended to treat; or (e) a cellular event that the bead-bound compound is intended to stimulate or inhibit.

[0039] In an embodiment lacking any headpiece, provided is the above system, wherein 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 there is no optional headpiece present linking any of the bead-bound compounds with any of the bead-bound DNA barcodes.

[0040] Further contemplated are systems as above, in which 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. Further contemplated are systems as above, in which 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.

[0041] Further provided is a system as described above, wherein the concatenated DNA barcode comprises at least one nucleic acid that is a DNA barcode module, or a system as described above, wherein the concatenated DNA barcode comprises only one nucleic acid that is a DNA barcode module.

[0042] In an embodiment of a sequencing primer annealing site, provided is a system as described above, wherein the concatenated DNA barcode comprises at least one nucleic acid that is a DNA barcode module, and at least one functional nucleic acid that is (a) capable of being used as an annealing site for a sequencing primer, (b) capable of forming a hairpin structure, the hairpin structure comprising a sequencing primer, an annealing site for the sequencing primer, and a bead in the hairpin structure, the bead being 5 prime to the sequencing primer and 3 prime to the annealing site for the sequencing primer, or (c) is a spacer nucleic acid.

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

[0044] In an embodiment reciting functional language for splint oligos, provided are beads comprising concatenated DNA barcodes, the concatenated DNA barcodes comprising (a) a first annealing site of a first splint oligonucleotide (sprint oligo) and a first annealing site of a first splint oligonucleotide, the splint oligo comprising three nucleic acids, the three nucleic acids being capture nucleic acids that hybridize to the first annealing site, a capture nucleic acid that hybridizes to the second DNA barcode module, and a nucleic acid that is a second annealing site, and (b) a second annealing site of a second splint oligo and a second annealing site of a second splint oligo, the second splint oligo comprising three nucleic acids, the three nucleic acids being capture nucleic acids that hybridize to the second annealing site, a nucleic acid that is a third DNA barcode module, and a nucleic acid that is a third annealing site.

[0045] In another embodiment including functional language related to the splint oligo, provided is the above bead, further comprising a third annealing site of the third splint oligo, wherein the third splint oligo includes three nucleic acids, the three nucleic acids being a capture nucleic acid that hybridizes to the third annealing site, a fourth DNA barcode module nucleic acid, and a fourth annealing site nucleic acid.

[0046] Additionally, in yet another embodiment including functional language related to the splint oligo, provided is the above bead, further comprising one or more of: (i) a fourth DNA barcode module and a fourth annealing site of the fourth splint oligo, wherein the fourth splint oligo comprises three nucleic acids, the three nucleic acids being a capture nucleic acid that hybridizes to the fourth annealing site, a fifth DNA barcode module, and a fifth annealing site; (ii) a response capture element; and (iii) a release monitor.

[0047] In linker embodiments, included are beads as described above in which concatenated DNA barcodes are attached to the beads but are not (i) attached to the beads via any photocleavable linker, (ii) attached to the beads by any enzymatically cleavable linker, or (iii) attached to the beads by any type of cleavable linker.

[0048] In embodiments relating to separate binding locations, provided are beads as described above, where the concatenated DNA barcodes are bound to a first location on the bead, and the bead also comprises a compound bound to a second location on the bead, and the first location is not the same as the second location.

[0049] In surface embodiments (inner and outer surfaces), provided are the above beads, wherein the bead comprises an outer surface and an inner surface, the bead comprises at least 10,000 substantially identical concatenated DNA barcodes attached to the bead, and at least 90% of the at least 10,000 substantially identical concatenated DNA barcodes are attached to the outer surface.

[0050] In an exclusive embodiment that distinguishes this disclosure from other embodiments, provided is the above bead that does not contain any polyacrylamide, and the concatenated DNA barcode (i) does not contain any nucleic acid that is a promoter, (ii) does not contain any nucleic acid that is polyA, or (iii) does not contain any nucleic acid that is a promoter and does not contain any nucleic acid that is polyA.

[0051] In an embodiment of an emission monitor bead, the disclosure provides an emission monitor bead capable of functioning in an aqueous medium, the emission monitor bead comprising a bead-bound quencher and a bead-bound fluorophore, the bead-bound quencher being quenchingly positioned in close proximity to the bead-bound fluorophore and capable of quenching at least 50% of the fluorescence of the bead-bound fluorophore, the bead-bound fluorophore being attached via a first photocleavable linker, the picowell comprising the emission monitor bead is a first picowell, the first picowell comprises a first solution, exposing the first picowell to cleavage conditions allows the photocleavable linker to be cleaved, and the fluorophore is quenched to the first picowell. Releasing and exposing to a first solution in the well results in the fluorophore diffusing throughout the first solution in the first picowell, a fluorescent signal obtained by shining light on the first picowell containing the first solution containing the diffused fluorophore is used by a user to calculate a release percentage of the bead-bound fluorophore from the release monitor beads resulting in a calculated release percentage, a second picowell contains a bead-bound compound attached to a photocleavable linker of the same type as the first photocleavable linker, the second picowell contains a second solution, and the calculated release percentage value from the release monitor beads in the first picowell can calculate the concentration of the released compound in the second solution in the second picowell. In other emission monitor embodiments, provided are emission monitor beads in which the fluorophore is TAMRA and the quencher is QSY7, and emission monitor beads having the structure shown in FIG. 9, and emission monitor beads having the structure shown in FIG. 10, and emission monitor beads capable of quenching at least 90%, at least 98%, at least 99%, or at least 99.9%.

[0052] In an embodiment of the manufacturing method, included is a method for synthesizing an emission monitor bead, the emission monitor bead comprising a bead, a quencher, a fluorophore, and a photocleavable linker connecting the fluorophore to the bead, the method comprising, in this order: (i) providing a resin; (ii) coupling a lysine linker to the resin, where the reagent comprising the lysine linker is L-Fmoc-Lys(4-methyltrityl)-OH; (iii) removing the Fmoc protecting group; (iv) coupling the quencher using a reagent where the quencher is quencher-N-hydroxysuccinimide (quencher-NHS) as a source of quencher; (v) removing the 4-methyltrityl protecting group using a reagent where the trifluoroacetic acid comprises; (vi) coupling a photocleavable linker to the epsilon amino group of the lysine, where the photocleavable linker is provided by a reagent of Fmoc-photocleavable linker-OH; and (vii) coupling a fluorophore. The above embodiments are also provided, but the order of the steps is not taken into consideration. In other method embodiments, the above method is provided, wherein the fluorophore is TAMRA and the quencher is QSY7.

[0053] In a method related to the utility of the release monitor beads, provided is a method of controlling the concentration of a compound in a solution present in a picowell, the method being applied to a bead-bound compound in the picowell, the picowell containing the solution, the bead-bound compound being bound to the bead via a cleavable linker, the method including: (a) exposing the bead-bound compound to conditions that result in cleavage of the cleavable linker, releasing the bead-bound compound from the beads and generating a released compound, where diffusion or dispersion of the released compound in the solution following release results in a substantially uniform concentration of the compound in the solution; (b) conditions including light that is capable of cleaving the cleavable linker; (c) conditions adjusted to generate a determined concentration of substantially uniform concentration; and (d) the determined concentration is performed taking into account the concentration of the released fluorophore released from the bead-bound release monitor. Further 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 by the duration of the exposure, as well as the above methods, wherein the concentration of emitted fluorophore emitted from the bead-bound emission monitor is determined contemporaneously with effecting release of the bead-bound compound from the bead to produce the emitted compound, and the above methods, wherein the concentration of emitted fluorophore emitted from the bead-bound emission monitor is determined at a time substantially prior to effecting release of the bead-bound compound from the bead to produce the emitted compound.

[0054] The term "determined" can refer to a concentration that is pre-determined and determined to be the desired concentration prior to exposing the beads to light, or it can refer to a concentration that is determined in "real time", i.e., simultaneously with exposing the beads to light.

[0055] In embodiments of the cap, included is a cap in combination with a picowell plate including a plurality of picowells, the cap being capable of being used with a picowell plate including a plurality of picowells, each of the plurality of picowells being definable by a hole, a floor, and a wall, the wall being defined by a hole at an upper portion and a floor at a lower portion, the hole being circular, the floor being circular, the wall being in the form of a surface of a frustum of a cone, the hole having a first diameter, the floor having a second diameter, the first diameter being greater than the second diameter,

[0056] The cap is a spherical cap capable of fitting snugly into the hole, the hole being comprised of a polymer having a larger durometer (harder) and the cap being made from a polymer having a smaller durometer (softer), the relative durometers of the cap and hole allowing the spherical cap to reversibly fit snugly into the hole, the cap being (i) capable of plugging the picowell to prevent leakage, (ii) a passive cap capable of absorbing metabolic products released by cells in a cell culture medium when the cells are cultured in the picowell, (iii) an active cap in the form of beads comprising a plurality of essentially identical compounds, each of the plurality of essentially identical compounds being coupled to the beads with a cleavable linker, and cleavage of the cleavable linker releasing at least a portion of the plurality of compounds from the beads, and (iv) an active cap in the form of beads comprising a plurality of identical reagents, each of the plurality of essentially identical reagents being coupled to the beads with a cleavable linker, and cleavage of the cleavable linker releasing at least a portion of the plurality of reagents from the beads.

[0057] In an embodiment of a porous cap, provided is a picowell plate and a plurality of porous caps in combination with a solid polymer coating, each of the plurality of porous caps including an upper surface and a lower surface, the picowell plate including a plurality of picowells, at least one porous cap contacts the picowells and reversibly and snugly fits therewith, each of the upper surfaces of the picowell plate and the plurality of porous caps is covered with a solid polymer coating, the solid polymer coating contacts and is adhesively attached to at least a portion of the upper surface of each cap, and (i) each of the plurality of picowells is capable of holding an aqueous solution, and a product of a reaction is generated in the solution, and the product is a solid polymer coating. At least a portion of the absorbed reaction product is absorbed by the underside of each of the plurality of porous caps; (ii) a solution of a polymerizable reagent capable of polymerization is poured over the plurality of porous caps in combination with the picowell plate, where the polymerizable reagent polymerizes to form a substantially flat surface coating substantially all of an upper surface of the picowell plate, thereby fixing the polymerized reagent to each of the plurality of porous caps; and (iii) all of the plurality of porous caps are removable from the plurality of picowells by a peeling action, whereby adhesion is maintained between the plurality of porous caps and the polymerized reagent, resulting in an array of caps partially adhered to an upper surface of each cap embedded in the polymerized reagent, and the underside of each cap is available for analysis of the absorbed reaction product.

[0058] This provides an embodiment of a manufacturing method for directing enzymatic synthesis of DNA barcodes using splint oligos. Provided is a method of making bead-bound concatenated DNA barcodes, the bead-bound concatenated DNA barcodes comprising a plurality of DNA barcode modules, optionally one or more functional nucleic acids, optionally one or more identity-encoding nucleic acids encoding an identity other than the identity of the chemical library monomers, the method comprising: (a) providing a first annealing site on a first splint oligonucleotide (sprint oligo), the first splint oligo capable of serving as a template for a DNA polymerase to catalyze polymerization into a bound polynucleotide, the first splint oligo capable of hybridizing to nucleotides complementary to those of the hybridized first splint oligo, the polymerized nucleotides complementary to those of the hybridized first splint oligo after polymerization being bound to a bead-bound second splint oligo. The method includes the steps of: (a) providing a bead with a bound polynucleotide comprising a first DNA barcode module and a first annealing site, the first DNA barcode module and the first annealing site, (b) providing the bead with a polynucleotide bound to the first splint oligo and hybridizing the first splint oligo to the bound polynucleotide, (c) adding a DNA polymerase and deoxynucleotide triphosphates (dNTPs) where the bound polynucleotide has a free 3' end and polymerization is to the free 3' end, and allowing the DNA polymerase to catalyze the polymerization of the dNTPs to the bound polynucleotide, and (d) washing away the first splint oligo. Also contemplated is the above method, where the first splint oligo is comprised of a first annealing site, a second DNA barcode module, and a second annealing site.

[0059] In a further method of manufacturing example, provided is the above method, wherein the first splint oligo is comprised of a nucleic acid encoding a first annealing site, a second DNA barcode module, a second annealing site, and a first sequencing primer annealing site, wherein the first sequencing primer annealing site is capable of hybridizing to a sequencing primer to provide 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.

[0060] Further contemplated are methods as above, wherein the first splint oligo, DNA polymerase, and dNTPs are all added at the same time, or wherein the first splint oligo, DNA polymerase, and dNTPs are each added at separate times.

[0061] With regard to internal versus external locations on the beads, provided is the above method, wherein the beads are comprised of external and internal locations, the bead-bound concatenated DNA barcodes are bound to the beads substantially at locations on the outside of the beads and slightly at locations on the inside of the beads, the beads also comprise a plurality of bound compounds, all of the plurality of bound compounds having substantially identical structures when compared to one another, and the beads are comprised of substantially hydrophobic polymers.

[0062] In a further method embodiment, provided is (a) a first DNA barcode module, a first annealing site, a second DNA barcode, and a second annealing site, the second splint oligo capable of functioning as a template for a DNA polymerase that catalyzes polymerization of the attached first longer polynucleotide, the second splint oligo capable of hybridizing to nucleotides complementary to those of the hybridized second splint oligo, and the polymerized nucleotides complementary to those of the hybridized second splint oligo after polymerization include a bead-bound third DNA barcode module and a third annealing site. The above method further comprises the steps of (a) providing a bead with a bound first longer polynucleotide; (b) providing a polynucleotide bound to the second splint oligo to the bead and hybridizing the second splint oligo to the bound first longer polynucleotide; (c) adding a DNA polymerase and deoxynucleotide triphosphates (dNTPs), wherein the bound longer polynucleotide has a free 3' end and polymerization is to the free 3' end, and allowing the DNA polymerase to catalyze the polymerization of the dNTPs to the bound longer polynucleotide; and (d) washing away the second splint oligo.

[0063] This relates to sequential numbering of the first DNA barcode module, the second DNA barcode module, the third DNA barcode module, etc., to produce an overall DNA barcode. This also relates to repeating a cycle of the method steps multiple times in the production of an overall DNA barcode. Provided is the above method, wherein each of the plurality of DNA barcode modules is identified or named by a number, and the method further comprises repeating the enumerated steps, where for the first iteration, the name of the DNA barcode module is incremented by adding one number to its existing name, the name of the annealing site is incremented by adding one number to its existing name, the name of the splint oligo is incremented by adding one number to its existing name, and the name of the "first longer polynucleotide" is changed by adding one number to its existing name, comprising repeating the enumerated steps one iteration, or two iterations, or three iterations, or four iterations, or five iterations, or more than five iterations, or more than ten iterations.

[0064] Also contemplated is the above method comprising a plurality of splint oligos, each splint oligo comprising a sequence primer annealing site, the sequence primer annealing site capable of hybridizing to a sequencing primer, resulting in a hybridized sequence primer, the hybridized sequencing primer capable of directing sequencing of at least one bead-bound DNA barcode module and at least one bead-bound DNA barcode module.

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

[0066] Briefly, the disclosure provides a system for screening compounds comprising: (a) a picowell array plate including a plurality of picowells, each picowell having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, and a wall being between the upper hole and the floor; (b) at least one bead disposed within at least one picowell, the at least one bead comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds; and (c) at least one bead comprising a DNA barcode in the form of either a concatenated DNA barcode or an orthogonal DNA barcode. The system includes a barcode, and when the DNA barcode takes the form of a concatenated DNA barcode, the concatenated DNA barcode is generated using a method of (i) using click chemistry, or (ii) using repeated cycles of steps, wherein the repeated cycle steps include using a splint oligo for annealing to the partially generated DNA barcode, and the annealed splint oligo is used as a template to extend the partially generated DNA barcode using a DNA polymerase, and the splint oligo includes bases complementary to the DNA barcode module that is polymerized into the partially generated DNA barcode.

[0067] In another embodiment, provided is a system as described above, wherein the DNA barcode comprises: (a) one or more DNA barcode modules, each of the one or more DNA barcode modules encoding information regarding the identity of a chemical library monomer; (b) optionally, one or more functional nucleic acids; and (c) optionally, one or more nucleic acids encoding information that is a type of information other than information regarding the identity of the chemical library monomer.

[0068] Further provided is a system as described above, further including a plurality of caps, each capable of fitting into the opening of a different picowell, each capable of minimizing or preventing evaporation of fluid within the picowell, and each capable of minimizing or preventing leakage of fluid within the picowell.

[0069] Also included is the above system, further including a plurality of spherical caps, each capable of fitting into a hole in the picowell, the hole being circular, each capable of minimizing or preventing evaporation of the fluid within the picowell, and each capable of minimizing or preventing leakage of the fluid within the picowell.

[0070] Also contemplated is the above system, where at least one bead comprises a DNA barcode in the form of a concatenated DNA barcode, the concatenated DNA barcode comprising: (i) a sequencing primer binding site; (ii) a first DNA barcode module; (iii) a first annealing site to which the first oligonucleotide splint can hybridize, which can be used to direct enzymatic synthesis of a second DNA barcode module; (iv) a second DNA barcode module; (v) a second annealing site to which the second oligonucleotide splint can hybridize, which can be used to direct synthesis of a third DNA barcode; (vi) a third DNA barcode module; (vii) a third annealing site to which the third oligonucleotide splint can hybridize, which can be used to synthesize a fourth DNA barcode.

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

[0072] More specifically, included 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 competitive inhibition assay, or wherein the assay comprises an interaction of an unlinked compound with other assay reagents, and wherein the unlinked compound is a compound released from the bead surface, or wherein the compound is released by cleaving a cleavable linker connecting the compound to the bead, or wherein the assay occurs in multiple confined volumes, typically one bead being distributed in each confined volume.

[0073] In another embodiment, it is further contemplated that the restricted volume comprises an aqueous droplet; or The above method, wherein the aqueous droplets are suspended in an oil or hydrophobic liquid medium, or the restricted volumes comprise picowells, or the picowells are organized in a regular array, or a plurality of restricted volumes are organized in a regular array.

[0074] Moreover, further included are the above methods, in which the limited volume comprises a layer of aqueous medium attached around the beads, and the beads are suspended in a hydrophobic medium, and the above methods, in which the assay reagent is washed away before sequencing the oligonucleotide.The above methods, in which the sequencing step (d) is performed before the assay step (b).Also provided are the above methods, in which the oligonucleotides on the beads are removed after the sequencing step but before the assay step.Furthermore, contemplated are the above methods, in which the removal of the oligonucleotides comprises enzymatic digestion, chemical cleavage, pyrolysis, or physical shearing, and the above methods, in which the binding assay comprises the binding of RNA molecules to the beads, and the above methods, in which the signal from the beads comprises the sequencing of the bound RNA molecules.

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

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

[0077] In a system embodiment, a system for screening a compound library for a compound having a desired activity is provided, comprising: (a) a sample compartment for receiving beads having a plurality of compounds attached thereto and encoded with oligonucleotides; (b) a plurality of encapsulation compartments in the sample compartment, each encapsulation compartment nominally comprising a single bead dispersed in an assay medium, the assay medium further comprising a reagent whose interaction with the compound on the bead is assayed and results in a measurable signal; (c) a detector for measuring the signal; (d) a sequencing platform; and (e) a user interface for receiving one or more commands from a user. Also provided is the above system, wherein the encapsulation compartment comprises a droplet. In another aspect, provided is the above system, wherein the encapsulation compartment comprises a picowell, or further comprises an assay reagent, or the detector comprises an optical detector, or the sequencer comprises an optical detector.

[0078] In one aspect, the disclosure features a method of 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 confined volume, where the onset and dose of the perturbation are controlled; (c) incubating the cell with the nucleic acid-encoded perturbation for a specified period of time; and (d) introducing into the cell a nucleic acid encoding the nucleic acid-encoded perturbation.

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

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

[0081] In some embodiments of this aspect of the disclosure, confining the cells and perturbations comprises droplet encapsulation, emulsion encapsulation, picowell encapsulation, macrowell encapsulation, physical attachment, bubble encapsulation, or microfluidic confinement.

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

[0083] In certain embodiments, the incubation comprises exposing the cells to the perturbation after cleaving the perturbation from the substrate or after cleaving the nucleic acid from the substrate, in some embodiments, the incubation comprises exposing the cells to the perturbation without cleaving the perturbation from the substrate or without cleaving the nucleic acid from the perturbation.

[0084] In some embodiments, introducing a nucleic acid encoding a nucleic acid-encoded perturbation into a cell comprises attaching the nucleic acid to the cell surface of the cell. In certain embodiments, attaching the nucleic acid to the cell surface of the cell comprises inserting the nucleic acid into the cell membrane. In certain embodiments, attaching the nucleic acid to the cell surface of the cell comprises attaching the nucleic acid to a biomolecule on the cell surface. In certain embodiments, the biomolecule is a protein or a carbohydrate. In other embodiments, attaching the nucleic acid to the cell surface of the cell comprises attaching via any tag on the nucleic acid.

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

[0086] In yet another aspect, the disclosure features a method of perturbing a cell, encoding the cell with the identity of the perturbation, and measuring the cell's response to the perturbation. The method includes: (a) contacting the cell with a bead-bound DNA-encoded library in a first confined volume, the bead-bound DNA-encoded library including one or more copies of combinatorially synthesized compounds and one or more copies of an encoding nucleic acid tag, the compounds and the encoding nucleic acid being attached to the beads, and the encoding nucleic acid encoding the identity of the compound; (b) releasing the compounds in the library from the beads and incubating the compounds in the library with the cells in the first confined volume; (c) optionally releasing the encoding nucleic acid tag from the beads in the first confined volume; (d) capturing the encoding nucleic acid tag on the cell surface of the cell, whereby the cell is exposed to the compounds in the library and the identity of the exposed compounds is captured on the cell surface; and (e) releasing the cell from the first confined volume, whereby the encoding nucleic acid tag is attached to the cell and the encoding nucleic acid tag encodes the identity of the compound that the cell was exposed to. (f) capturing the previously perturbed, nucleic acid-tagged cells with response detection beads in a second restricted volume, where the cells are exposed to lysis conditions that expose the cellular contents of the cells to the response capture beads, where the response capture beads comprise a capture probe that captures the cellular contents and a nucleic acid tag that encodes the perturbation in the previously perturbed, nucleic acid-tagged cells; (g) incubating the response capture beads with the lysed cells in the second restricted volume, thereby capturing both the cellular contents and the nucleic acid tag that encodes the perturbation to the response capture beads; (h) optionally converting the cellular response to the perturbation into a nucleic acid signal, where the cellular response to the perturbation is not a nucleic acid signal; and (i) sequencing the nucleic acid tag attached to the response capture beads, whereby the identity of the perturbation is correlated to the cellular response to the perturbation.

[0087] In yet another aspect, the disclosure provides a method for detecting a cell's cellular response to a perturbation contained in the functionalized perturbation bead, comprising: (a) providing an array of picowells and a library of functionalized perturbation beads, wherein a picowell is capable of accommodating a single cell and a single functionalized perturbation bead, each functionalized perturbation bead comprising a plurality of different substantially identical releasable compounds and a plurality of nucleotide barcodes encoding the compounds, the nucleotide barcodes being functionalized barcodes capable of capturing cellular contents of the cell, the cellular contents of the cell comprising a cellular response to a perturbation contained in the functionalized perturbation bead; (b) capturing a single cell in each picowell of the picowell array; (c) capturing a single functionalized perturbation bead in the picowell containing the single cell; and (d) releasing compounds from the functionalized perturbation bead and incubating the cell with the released compounds. (e) incubating the cells with a mixture of the functionalized oligonucleotides on the functionalized perturbation beads, where the capturing includes hybridization and enzymatic extension to combine a nucleotide barcode with a nucleic acid element of the cellular content, thereby forming a hybrid of the nucleotide barcode and the nucleic acid element of the cellular content; and (g) releasing the hybrids, collecting the hybrids from a library of functionalized perturbation beads, and sequencing the hybrids, thereby relating the perturbation to the cellular response to the perturbation. [Brief description of the drawings]

[0088] [Figure 1] Concatenated beads. In concatenated beads, the DNA barcode takes the form of all DNA barcode modules connected to each other in a single strand, along with any other nucleic acid that carries functions such as primer annealing sites, spacers, or information about the date of manufacture. The numbers in this diagram are not structure numbers. The numbers indicate the sequence of the "DNA barcode modules" in the DNA barcode. [Diagram 2] Orthogonal beads. In orthogonal beads, the DNA barcode takes the form of all the DNA barcode modules, which do not occur together in a single strand, but instead occur individually linked at different locations on the bead. The numbers in this diagram are not structure numbers. The numbers indicate the sequence of the "DNA barcode modules" within the DNA barcode. [Figure 3-1] Conditions for cleavable linkers, cleavage (UV light or chemical), and cleavage products. Information from Yinliang Yang (2014) Design of Cleavable Linkers and Applications in Chemical Proteomics. Technische Universitat Munchen Lehrstuhl fur Chemie der Biopolymere. The alphabet to the left of each linker is from this reference. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] Exemplary amino acid derivatives for the compositions and methods of the present disclosure. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Diagram 5] Photographs showing increasing degradation of the fusion protein with increasing concentrations of added lenalidomide in HeLa cells. Top: Expression of IKZF1 / GFP fusion protein. Bottom: Expression of mScarlett® control. Lenalidomide was added at 0, 0.1, 1.0, or 10 micromolar. [Figure 6] Photographs showing increasing degradation of the fusion protein with increasing concentrations of added lenalidomide in HeLa cells. Top: Expression of IKZF3 / GFP fusion protein. Bottom: Expression of mScarlett control. Lenalidomide was added at 0, 0.1, 1.0, or 10 micromolar. [Figure 7]Methods and reagents for making bead-bound DNA barcodes. The most accurate description of "DNA barcode" is the sum of all information contained in the sum of all DNA barcode modules. However, for convenience, the term "DNA barcode" is used herein to refer to the sum of all information of all DNA barcode modules and any additional nucleic acid that provides information such as step number, or chemical monomers of the general formula that make up the bead-bound compound, and further any additional nucleic acid that serves a function such as linker, sequencing primer binding site, hairpin with sequencing primer binding site, or spacer. When the DNA barcode is made at least in part by click chemistry, the DNA barcode may include residual chemical groups from the click chemistry reaction. [Figure 8] Structure of Alexa Fluor® 488. The purpose of this diagram is to identify the compound without using the trade name. [Figure 9] A simplified diagram of a bead-bound release monitor. The release monitor provides the user with a measurement of the concentration of soluble compounds and subsequently the UV-induced release of the compounds from the beads. In a preferred embodiment, one type of bead is dedicated to being a release monitor, i.e., the bead does not contain bead-bound compounds, nor does it contain a bead-bound DNA library. "PCL" is a photocleavable linker. [Figure 10] Detailed view of the bead release monitor. [Figure 11-1] Chemical synthesis of bead release monitor. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 12] Amine-functionalized beads with bifunctional linkers, where the linkers contain lysine residues. [Figure 13] Steps in the chemical synthesis of type 1 carboxyl-modified lenalidomide. [Figure 14] Steps in the chemical synthesis of type 2 carboxyl-modified lenalidomide. [Figure 15] Steps in the chemical synthesis of type 3 carboxyl-modified lenalidomide. [Figure 16] Lenalidomide analogues. [Figure 17-1] Chemical synthesis steps of deoxycytidine analogues suitable for click chemistry synthesis of DNA barcodes. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 17-4] Same as above. [Figure 18] A cap for placing over the top of a picowell and sealing the picowell. FIG. 18A shows an active cap, where a compound is releasable via a cleavable linker. FIG. 18B shows another type of active cap, where a reagent such as an antibody is attached. The attached reagent can be permanently linked, linked with a cleavable linker, or attached via hydrogen bonds, and can be released simply by exposure to the solution in the picowell and then diffusing away from the active cap into the solution. FIG. 18C shows a passive cap, where it can be used to absorb, adsorb, collect, or capture metabolites from the solution in the picowell. The absorbed metabolites can then be analyzed. [Figure 19-1] FIG. 19A is a picowell plate without caps covering the picowells. FIG. 19B is a picowell plate with caps covering each picowell. FIG. 19C is a polyacrylamide solution poured over the picowell plate, with one cap safely and securely fastened over each picowell. The polyacrylamide then penetrates the porous caps and solidifies, forming a stable bond to each cap. FIG. 19D then peels the solidified polyacrylamide "roof" off the picowell plate, resulting in each cap. Metabolites absorbed into each cap can then be analyzed, displaced from the picowell solution. Preferably, the solution poured over the picowell plate and over the beads will be a hydrogel, and preferably the beads are made from a hydrogel. [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0089] In exclusive embodiments, the present disclosure may exclude systems, microtiter plates, microtiter plates with microwells, nanowells, or picowells, and related methods, in which at least one well is capped and a liquid polymer solution is poured over the plate and over the capped wells. Also excluded are those above in which the liquid polymer polymerizes to form a solid polymer that adheres to each cap. Also excluded are methods and resulting compositions in which the solid polymer is torn and the attached caps are removed. [Figure 20] A map of the circular plasmid used to integrate the IKZF1 gene into the genome of cells. The plasmid is IKZF1 mNEON-p2a-mScarlet-w3-2FB (9081 base pairs). IKZF1 encodes the Ikarus protein. [Figure 21] Map of the circular plasmid used to integrate the IKZF3 gene into the genome of cells. The plasmid is IKZF3 mNeon-p2a-mScarlet-w3-2FB (9051 bp). IKZF3 encodes the Aiolos protein. [Figure 22-1] Chemical monomers (compounds 1–6) and their DNA barcodes. [Figure 22-2] Same as above. [Figure 23] Chemical monomers (compounds 7–10) and their DNA barcodes. [Figure 24-1] Chemical monomers (compounds 11–16) and their DNA barcodes. [Figure 24-2] Same as above. [Figure 25-1] Chemical monomers (compounds 17–21) and their DNA barcodes. [Figure 25-2] Same as above. [Figure 26-1] Chemical monomers (compounds 22–16) and their DNA barcodes. [Figure 26-2] Same as above. [Figure 27-1] Chemical monomers (compounds 27–30) and their DNA barcodes. [Figure 27-2] Same as above. [Figure 28] Sequencing of bead-bound DNA barcodes. This figure shows the intensity of the fluorescent signal for each of the five consecutive bases that are part of the bead-bound DNA barcode. [Figure 29] Picowell with steps. [Diagram 30] Time course of fluorophore release from beads, showing activation of the bead-bound release monitor and acquisition of emission data at t=0 s, t=1 s, t=11 s, and t=71 s. [Diagram 31] Release data following catalysis of aspartyl proteases on quencher-fluorophore substrates. [Diagram 32] 1 shows a cross-section of a picowell, showing the various steps. [Diagram 33] Titration data showing how increasing UV dose increases cleavage of the fluorophore from the bead. In layman's terms, this shows how a more powerful swing of the axe affects cleaving the fluorophore from the bead (the power of the UV dose is measured in Joules / cm2). The "Exposure" designation refers only to the parameter when the picture is taken. This is simply the exposure time when the picture is taken (this does not refer to the exposure time of the light that does the cleavage, or the light that does the excitation). [Diagram 34] TAMRA concentration vs. luminous flux. Shown is the concentration of free TAMRA followed by its release after exposure to 365 nm UV light. [Figure 35-1] 1 provides a hand-drawn diagram of a quencher-fluorophore substrate and the cleavage of this substrate by the enzyme, resulting in inhibition of the enzyme. Also shown are the molecular structures of bead-bound pepstatin-A and bead-bound Fmoc-valine (negative control). [Figure 35-2] Same as above. [Diagram 36]and conditioning the beads for final capture of mRNA from lysed cells, followed by production of a cDNA library. This diagram also originates in one of our provisional applications (Compositions and methods for screening compound libraries on single cells), to which priority is claimed. [Figure 37] Cells are tagged with DNA barcodes via lipids embedded in the cell membrane. This diagram also occurs in one of the provisional applications (Compositions and methods for screening complex libraries on single cells), from which priority is claimed for this application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include the corresponding plural references unless the context clearly dictates otherwise. All references cited herein are incorporated by reference to the same extent as if each individual patent, published patent application, figure, drawing, sequence listing, compact disc, etc. was specifically and individually indicated to be incorporated by reference.

[0091] Abbreviation Table 1 provides abbreviations and non-limiting definitions. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0092] Reagents, kits, enzymes, buffers, live cells, equipment, etc. are available (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).

[0093] Barcoded gel beads, non-barcoded gel beads, and microfluidic chips are available from 1CellBio, Cambridge, Mass. Guidance and equipment for flow cytometry is available (e.g., FACSCalibur®, BD Biosciences, San Jose, Calif., see BD FACSAria II® User Guide, Part No. 643245, Rev. A, December 2007, page 344).

[0094] A "labeled" composition is detectable, directly or indirectly, by spectroscopic, photochemical, fluorometric, biochemical, immunochemical, isotopic, or chemical methods, as well as methods involving plasmonic nanoparticles. For example, useful labels include: 32 P, 33 P, 35 S, 14 C. 3 H, 125I, stable isotopes, epitope tags, fluorescent dyes, Raman tags, electron-dense reagents, substrates, or enzymes, for example, as used in enzyme-linked immunosorbent assays, or fluorescein (Rozinov and Nolan (1998) Chem. Biol. 5:713-728).

[0095] Table of contents for detailed description (I) Beads (II) One bead, one compound (OBOC) (III) Binding of nucleic acids to beads (IV) DNA barcode (V) Binding compounds to beads (VI) Linking chemical monomers together to make compounds (VII) Split-pool synthesis and parallel synthesis (VIII) Build a Picowell (IX) Depositing beads into picowells (X) Sequencing of bead-bound nucleic acids in picowells (XI) Releasing bead-bound compounds from beads Biochemical Assay of Compound XII Cell-based assays for compounds (XIII) (XIV) Perturbation-response analysis in cells (I) Beads

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

[0097] TentaGel® is characterized by "PEG chains of up to 20 kilodaltons molecular weight immobilized on functionalized and cross-linked polystyrene. Graft copolymers with PEG chains of about 2000-3000 daltons have proven optimal in terms of kinetic speed, mobility, swelling, and resin capacity" (Rapp Polymere, Germany). The present disclosure therefore provides beads or resins in the form of graft copolymers with PEG chains of about 2000-3000 daltons. With regard to swelling, Comellas et al. provide guidance for measuring the swelling capacity of beads when immersed in, for example, DCM, DMF, methyl alcohol, water, or buffers used in enzyme assays (Comellas et al (2009) PLoS ONE.4:e6222 (page 12)). The units of swelling are milliliters per gram of beads.

[0098] In an alternative bead embodiment, the present disclosure uses a resin in which a PEG spacer is attached to a polystyrene backbone via an alkyl linkage, the resin being microsphere and monosized (TentaGel® M resin).

[0099] In a further alternative bead embodiment, the present disclosure uses resins with PEG spacers attached to a polystyrene backbone via alkyl linkages, and the resin types exist in two bifunctional species, the first surface modified resin in which reactive sites on the outer surface of the bead are protected orthogonally to reactive sites in the inner volume of the bead, and the second hybrid resin in which cleavable and non-cleavable ligands are present on this support and has been developed for sequential cleavage (TentaGel® B resin).

[0100] Furthermore, in another embodiment, the present disclosure uses a resin in which a PEG spacer is attached to a polystyrene backbone via an alkyl linkage, and the macrobead resin exhibits very large particle size and high capacity (TentaGel® MB resin). The present disclosure also uses a resin in which a PEG spacer is attached to a polystyrene backbone via a benzyl ether linkage. This resin can be used for immunological procedures or synthesis for PEG-modified derivatives (PEG-linked PEG-modified compounds) (TentaGel® PAP resin).

[0101] Additionally, the beads can be HypoGel® 200 resin, which is a composite of oligoethylene glycol (MW 200) grafted to a low cross-linked polystyrene matrix (Fluka Chemie GmbH, CH-9471 Buchs, Switzerland).

[0102] In some embodiments, amino-functionalized polystyrene beads without PEG linkers may further be used, for example mono-sized polystyrene M NH2 microbeads (5, 10, 20 micrometers, etc. diameter, from Rapp Polymere, 72072 Tubingen, Germany).

[0103] In some embodiments, compounds may be encapsulated in pores or chambers or tunnels within the beads without covalent attachment to the beads. Compounds may diffuse or be forced into such pores of the beads by various means. In some embodiments, compounds may be loaded into the beads by diffusion. In some embodiments, high temperature may be used to swell the beads and load the compounds into the beads. In some embodiments, high pressure may be used to force the compounds into the beads. In some embodiments, a solvent that swells the beads may be used to load the compounds into the beads. In some embodiments, a vacuum or low pressure may be used to partition the compounds into the beads. In some embodiments, gentle or vigorous physical agitation may be used to load the compounds into the beads.

[0104] In such embodiments where the compound is loaded onto the bead without covalent attachment, the compound may be unloaded from the bead by diffusion. In some embodiments, the compound may be removed from such beads using, without limitation, temperature, pressure, solvent, pH, salt, buffer, or detergent, or a combination of such conditions. In some embodiments, the physical integrity of the beads, for example with non-crosslinked polymerized beads, may be used to release the compound contained within such beads.

[0105] In exclusive embodiments, the present disclosure may exclude any bead, bead-compound complex, or any method, including one of the beads described above.

[0106] The beads of the present disclosure further include the following: Merrifield resin (chloromethyl polystyrene); PAM resin (4-hydroxymethylphenylacetamidomethyl polystyrene); MBHA resin (4-methylbenzhydrylamine); Brominated Wang resin (alpha-bromo pryopiophenone); 4-nitrobenzophenone oxime (Kaiser) resin; Wang resin (4-hydroxymethylphenoxymethyl polystyrene; PHB resin (p-hydroxybenzyl alcohol; HMPA resin (4-hydroxymethylphenoxyacetic acid); HMPB resin (4-hydroxymethyl-3-methoxyphenoxy butanoic acid); 2-chlorotrityl resin; 4-carboxytrityl resin; Rink acid resin (4-[(2,4-dimethoxyphenyl) hydroxyphenyl]phenyl) hydroxyphenyl] ... 4-Sulfamoylbenzoyl resin "Kenner's Safety" resin (N-(4-Sulfamoylbenzoyl)aminomethyl-polystyrene); FMP-resin (4-(4-Formyl-3-Methoxyphenoxy)-ethyl) (see ChemFiles Resins for Solid-Phase Peptide Synthesis Vol. 3 (page 32) (Fluka Chemie GmbH, CH-9471 Buchs, Switzerland)).

[0107] Beads of the present disclosure further include the beads described above used as passive encapsulants for compounds (passively holding compounds without covalent linkage to the compound) and further include non-functionalized polystyrene beads, silica beads, alumina beads, porous glass beads, polyacrylamide beads, titanium oxide beads, alginate beads, ceramic beads, PMMA (polymethylmethacrylate) beads, melamine beads, zeolite beds, polylactide beads, deblock copolymer micelles, dextran beads, etc. Many of the beads listed in this paragraph can be purchased from vendors such as Microspheres-Nanospheres, Cold Spring, NY 10516, USA.

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

[0109] In all embodiments where passive encapsulation is used to deliver compounds, DNA tags may further be passively loaded, or alternatively, DNA tags may be covalently attached to beads, vesicles, or droplets.

[0110] In exclusive embodiments, the present disclosure may exclude beads or resins made with any of the above chemicals, or made with derivatives of any one of the above chemicals.

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

[0112] Also provided are non-spheroid beads that can be defined in terms of the above values ​​and ranges. For example, one of the axes, or one of the primary dimensions (e.g., sides), or one of the secondary dimensions (e.g., diagonals) can include the above range values. In an exclusive embodiment, the present disclosure can exclude any reagent, composition, system, or method that includes a spheroid bead (or a non-spheroid bead) that falls into one or more of the above values ​​or ranges.

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

[0114] Bead Permeability. In embodiments, the present disclosure provides beads with various ranges or degrees of permeability. Permeability can be measured as the percentage of the volume of the bead that is accessible by the solvent, the unit of measurement being the percentage of the surface of the bead that takes the form or pores, or the unit of measurement being the percentage of the interior of the bead that takes the form of channels, networks, or chambers that are in fluid communication with the surface of the bead (and the external medium). The present disclosure can include porous beads, or alternatively, can exclude porous beads.

[0115] US Patent No. 9,062,304 to Rothberg discloses beads with exterior and interior regions. "Internal surface (pore surface)", "suitable pores that will exclude larger molecules", and optionally "developed differential functionalization of the interior and exterior surfaces" with various pore sizes, polymers such as poly(styrene sulfonate) and polystyrene are shown. Figure 1 to Rothberg provides a photograph of the surface of the beads and the pores of the beads. US Patent No. 9,745,438 to Bedre provides a transmission electron microscope image of a porous bead. US Patent No. 5,888,930 to Smith provides a scanning electron microscope photograph of a cross section of a porous bead. Spherical beads with small pores on the surface and larger pores in the interior are shown, the beads being made from, for example, polystyrene, polyacrylonitrile, polycarbonate, cellulose, or polyurethane. Cooke, U.S. Patent No. 5,047,437, discloses the pore morphology of spherical poly(acrylonitrile) copolymers with skinless surfaces (FIG. 1) and beads with an external skin on the surface (FIG. 5). Tsao, U.S. Patent No. 4,090,022, discloses the porous openings and internal spaces of cellulose beads.

[0116] Each of the patents referenced above, including all figures, is incorporated herein in its entirety as if each were individually incorporated by reference in its entirety.

[0117] Without implying any limitation, the exterior surface of a bead or particle can be determined by tightly wrapping the entire bead or particle in an elastic film. The bead or particle can be wrapped via a thought experiment, or the wrapped bead can be depicted in a diagram or photograph, or the bead can actually be wrapped. Without implying any limitation, the exterior surface of the bead is that portion of the bead that physically contacts the wrapper.

[0118] For example, the disclosure provides beads having pores that occupy 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. The disclosure also provides beads in which the volume of the internal channels or networks occupies 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 beads, and the internal channels or networks are in fluid communication with the exterior surface (and external medium) of the beads.

[0119] Additionally, the disclosure provides beads having pores that occupy less than 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40% or less of the surface area. Also, the disclosure provides beads having an internal channel or network volume that occupies less than 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or less of the total volume of the bead, and the internal channel or network is in fluid communication with the exterior surface (and external medium) of the bead.

[0120] Iron core beads. The present disclosure encompasses iron core beads or magnetic beads. These beads are manipulated using magnets to move them from one reaction vessel to another or from one vessel to another. These beads can be used to enhance robotic manipulation. Methods for 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).

[0121] In exclusive embodiments, the present disclosure can exclude any bead, or any population of beads, that meets one of the above values ​​or ranges.

[0122] Loading of compounds onto beads In many experiments, it is advantageous to load the beads with pre-synthesized compounds, and the beads can be used as a vehicle to deliver the compounds to the assay. Many of the standard techniques used for drug delivery to biological specimens can be adapted to deliver 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).

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

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

[0125] One Bead, One Compound (OBOC) The method used to produce combinatorial libraries involves three steps: (1) preparing the library, (2) screening the compounds in the library, and (3) determining the structures of the compounds, e.g., all the compounds or only those compounds that provided interesting results in the screen (see Lam et al (1997) The One-Bead-One-Compound Combinatorial Library Method. Chem. Rev. 97:411-448). The advantage of synthesizing compounds via bead-bound synthesis is that compounds can be made rapidly by a "split pool" method.

[0126] OBOC Combined with Encoding Strategy. Another feature of OBOC is that each bead further includes an encoding strategy in addition to the compound. When bead-bound nucleic acids are used to encode compounds that are bound to the same bead, the term "encoding" does not refer to a genetic code. Instead, the term "encoding" means that the user possesses a legend, key, or code that associates each of the thousands of short nucleic acid sequences with a single bead-bound compound.

[0127] A dramatic variation of the use of beads with bead-bound compounds and bead-bound nucleic acids, where the nucleic acid codes for the relevant compound, is as follows. A dramatic variation is to produce a library of complexes, where each member of the library takes the form of a complex of a small molecule and a DNA moiety, where the DNA moiety codes for the small molecule). The complexes are soluble and not bead-bound. After screening with cells or purified proteins, the complexes remain bound to the cells or purified proteins, allowing the complexes to be isolated and the complexed nucleic acids to be sequenced to ultimately identify the compound (see Satz et al (2015) Bioconjugate Chemistry. 26:1623-1632).

[0128] Here, as in most of this patent document, the term "encode" does not refer to the genetic code, but instead to the fact that researchers use specific nucleic acid sequences to indicate specific known structures of compounds attached to them.

[0129] Instead of using a coding strategy such as the use of DNA barcodes, the beads that are screened positive (thereby indicating the compounds that are screened positive) can be subjected to Edman degradation or mass spectrometry to identify the bead-bound compounds (see Shih et al (2017) Mol. Cancer Ther. 16:1212-1223). If the bead-bound compounds are peptides, MALDI mass spectrometry can be used to directly determine the sequence of the positively screened peptide compounds. Direct sequencing is possible because cleavage and ionization occur simultaneously under laser irradiation (Song, Lam (2003) J. Am. Chem. Soc. 125:6180-6188).

[0130] One subtle point in performing split-pool synthesis of combinatorial libraries is that the compounds can be produced such that all share a common motif. This strategy has been described as "generating libraries of motifs rather than libraries of compounds" (Sepetov et al (1995) Proc. Natl. Acad. Sci. 92:5426-5430; see Lam et al, supra at 418).

[0131] To provide a representative example of a large bead, the bead is 0.1 mm in diameter and contains approximately 10 13 (Lam et al., supra). After preparation of a library of bead-bound compounds, each bead can be used in an individual assay, which measures biochemical activity, or alternatively, binding activity. The assays can be "on-bead" assays, or alternatively, the compounds can be detached from the beads and used in solution phase assays (Lam et al., supra).

[0132] Parameters for any type of bead include its tendency to swell in a given assay medium, whether the polymer of the bead is hydrophobic or hydrophilic, the identification of attachment sites on the bead for attachment of each compound, and the issue of whether a spacer such as polyethylene glycol is used to provide some separation of each compound from the surface of the bead and provide internal volume for the bead.

[0133] Considering the need to attach compounds to beads, but far from the hydrophobic surface of the beads, Lam et al., supra, disclose that polyoxyethylene grafted styrene (TentaGel®) has the advantage that the functionalizable groups are at the ends of the polyoxyethylene chains and are therefore far from the hydrophobic polystyrene. Beads with water-soluble linkers include TentaGel, and polydimethylacrylamide beads (PepSyn® gel, Cambridge Research Biochemicals, Northwich, UK).

[0134] The parameter of internal volume can provide an advantage and is necessary to prevent interactions between the bead-bound DNA barcode and the target of the bead-bound compound. To exploit this advantage, beads can be manufactured such that the DNA barcode is located inside the bead, whereas the compounds to be screened are attached to the surface of the bead (Lam et al., supra, at 438-439). This advantage of internal volume may be irrelevant if the bead-bound compounds are attached by a cleavable linker and if assays for the compounds are performed only on the compounds that have been cleaved and released.

[0135] Appell et al. provide a non-limiting example of a split-pool method for synthesizing chemical libraries followed by screening to detect active compounds (Appell et al (1996) J. Biomolecular Screening. 1:27-31). Library beads are placed one in each well in an array of wells on a first microwell, nanowell, or picowell plate. The beads are exposed to light to cleave approximately 50% of the bead-bound compounds, releasing them into solution within the wells. The released compounds are then transferred to a second microwell plate and assayed to detect wells containing active compounds, thereby identifying 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, thus providing the synthetic history and structure of the active compound" (Appell et al, supra).

[0136] In a cell-based screening assay to screen for bead-bound compounds, Shih et al. provide a new type of bead (Shih et al (2017) Mol. Cancer Ther. 16:1212-1223). This new type of bead contains a bead-bound compound that is a member of a library of "synthetic death ligands directed to ovarian cancer". The bead is further decorated with biotin, and two chemicals are added that create a sandwich, which maintains the adhesion of the cell to the bead. The sandwich contains streptavidin and a biotin-LXY30 complex, which connects the bead to the receptor for LXY30, a well-known protein on the cell surface, namely, integrin. The method of Shih et al. mentioned above led to the discovery of a new molecule ("LLS2") that can kill cancer cells. In the above method, a bead-bound compound is used, and the compound binds to the cell (even if the compound is still via bead binding). Cho et al. made a similar one bead, one compound library, and the compounds screened were sufficient to bind to cells (without any need for the sandwich described above) (Cho et al (2013) ACS Combinatorial Science. 15:393-400). The goal of the Cho et al. report was to discover RGD-containing peptides that bind to integrins expressed by cancer cells. The reagents and methods disclosed above are useful in the present disclosure.

[0137] Attaching nucleic acids to beads (orthogonal; concatenated) One way to understand the topic of concatenated and orthogonal barcoding is to note the significance one has over the other. The advantages of orthogonal barcoding over concatenated barcoding are as follows: With the attachment of each monomer of the growing compound, it is the DNA barcode module that is attached in parallel. In concatenated barcoding, if the attachment of a given module is incomplete (i.e., if all attachment sites were not successfully coupled with the required module), the sequence of the completed barcode will not be accurate. By "inaccurate" we mean incomplete coupling, meaning chunks that may be missing from a small chunk that was supposed to be the completed correct DNA barcode. Now, because attachment of all modules failed, the completed barcode sequence will contain errors. In contrast, orthogonal barcoding covalently binds each individual module to its own unique attachment site on the bead. Also, once a module is attached to a given site on the bead, no additional modules will be connected to the modules already attached.

[0138] 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 can be in the form of double-stranded DNA (dsDNA), which is treated with a DNA crosslinker, such as mitomycin C, before being attached to the growing bead-bound DNA barcode. After completion of the synthesis of the DNA barcode in dsDNA form, the dsDNA is converted to ssDNA. The conversion of dsDNA to ssDNA is effective when one of the DNA strands has a uracil (U) residue, and the cleavage of the DNA at the uracil residue is catalyzed by uracil-N-glycosidase (see FIG. 5 of Serial No. 62 / 562,905, filed September 25, 2017, which is incorporated herein by reference in its entirety). The above refers to the damage inflicted on the growing DNA barcode by the reagents used to make the bead-bound compounds.

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

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

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

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

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

[0144] In an alternative embodiment, the method may use an intermediate between "concatenated DNA barcoding" and "orthogonal DNA barcoding", which involves blocks of DNA barcodes, i.e., 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 the DNA modules that specify the full-length compound).

[0145] FIG. 1 discloses an exemplary and non-limiting diagram of a chain-like structured bead. The bead contains multiple DNA barcodes (each made of a DNA barcode module) and multiple compounds (each made of a chemical library monomer). For simplicity, the term "DNA barcode" is used to refer to all nucleic acids that are "DNA barcode modules" and polymers that contain all nucleic acids that provide some function. The function can be an annealing site for a sequencing primer or can be used to identify a step in the chemical synthesis of the bead-bound compound. FIG. 1 further illustrates bead-bound compounds, each compound made of several chemical library members, each chemical library member represented by a square, circle, or triangle. FIG. 1 illustrates that each DNA barcode module is numbered consecutively from 1 to 8, which correspond to each of the eight shapes (square, circle, triangle). For clarity, nucleic acids that perform a function (and do not represent or "code" for a specific chemical unit) are not shown in the figure.

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

[0147] In Figure 2, for clarity, the functional nucleic acid attached to each DNA barcode module is not shown. Of course, each DNA barcode module needs a nucleic acid that specifies the position of the chemical library monomer in the completed full-length compound. In the example shown in Figure 2, the position needs to be the first, second, third, fourth, fifth, sixth, seventh, or eighth.

[0148] In one embodiment, the chemical monomer is attached first, followed by the corresponding DNA barcode module. In an alternative embodiment, the DNA barcode module is attached first, followed by the corresponding chemical monomer. Alternatively, one may follow the procedure of organic synthesis, which may use either "one embodiment" or "alternative embodiment." In yet another alternative embodiment, the method provides for block-wise addition of several blocks of chemical monomers attached to the beads in parallel with the attachment of several blocks of DNA barcode modules.

[0149] In exclusive embodiments, those that can be excluded are reagents, compositions, and methods that use block-wise addition of chemical monomers, DNA barcode modules, or both chemical monomers and DNA barcode modules to beads.

[0150] This pertains to nucleic acids that may be present in the bead-bound polynucleotide, including nucleic acids that serve to "code" or identify monomers of the bead-bound compound. In an exclusive embodiment, the present disclosure may exclude nucleic acids that code for "step-specific DNA sequencing primer sites." In this situation, for each chemical monomer present in the compound, there is a corresponding DNA barcode module. Each DNA barcode module is flanked by at least one corresponding primer binding site, i.e., a "step-specific DNA sequencing primer site." Also excluded may 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.

[0151] Furthermore, the present disclosure may include a nucleic acid that functions as a spacer. For example, the spacer may create a distance along a polynucleotide chain between a first site that is the annealing site of a sequencing primer and a second site that identifies a chemical monomer. The present disclosure may also use a nucleic acid that repeats or confirms the information provided by another nucleic acid. The present disclosure may also use a nucleic acid that codes for a PCR primer binding site. A polynucleotide that has a PCR primer binding site has two PCR primer binding sites, and both of these sites are designed to have the same melting temperature (the melting temperature when the PCR primer anneals to the PCR primer binding site), so that the PCR primer binding site can be distinguished from the sequencing primer.

[0152] In an exclusive embodiment, the present disclosure can exclude nucleic acids that function as spacers or as sole spacers. The present disclosure can also exclude nucleic acids that repeat or confirm 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.

[0153] Additionally, the disclosure may specify the date that a chemical library was created, or specify steps in the chemical synthesis of a particular compound, or exclude nucleic acids that serve as primer annealing sequences.

[0154] Providing a sequencing primer for a specific DNA barcode module. The present disclosure provides a DNA barcode comprising a DNA barcode module and one or more sequencing primer annealing sites. Each DNA barcode module can have its own dedicated sequencing primer binding site. Alternatively, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive DNA barcode modules can be sequenced using one specific sequencing primer binding site, as can be present on a bead-bound DNA barcode.

[0155] Below we describe the situation where each DNA barcode module has its own dedicated sequencing primer binding site. The present disclosure provides bead-bound concatenated barcodes comprising a primer binding site capable of binding a DNA sequencing primer, the primer binding site 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, the primer binding site being located 3 prime to a first DNA barcode module with no other DNA barcode modules between the first DNA barcode module and the primer binding site, 3 prime to a second DNA barcode module with no other DNA barcode modules between, 3 prime to a third DNA barcode module with no other DNA barcode modules between, 3 prime to a fourth DNA barcode module with no other DNA barcode modules between, 3 prime to a fifth DNA barcode module with no other DNA barcode modules between, or 3 prime to a sixth DNA barcode module with no other DNA barcode modules between.

[0156] Coding sequences and sequences complementary to coding sequences. The present disclosure can include any one, any combination, or all of the coding sequences disclosed above or elsewhere in this document. In an exclusive embodiment, it can exclude any one, any combination, or all of the coding sequences disclosed above or elsewhere in this document. It can also exclude double-stranded nucleic acids that code for any one, any combination, or all of the coding sequences described above or elsewhere in this document.

[0157] Orthogonal DNA barcodes (each DNA barcode module is attached to a separate location on the bead) Orthogonal bead synthesis. In orthogonal synthesis, each DNA module is covalently attached to a separate site on the bead, so that the entire DNA barcode is provided by multiple DNA modules. When a DNA barcode has an orthogonal structure, the DNA barcode modules are not attached to each other, instead, each and every DNA barcode molecule has its own bead attachment site dedicated to that particular DNA barcode module.

[0158] A nucleic acid that specifies the number of synthesis steps of each DNA barcode module. In an embodiment, the orthogonal DNA barcode comprises a short nucleic acid that specifies the first step of the compound synthesis. In this embodiment, due to the parallel attachment of the first chemical monomer and the first DNA barcode module, the first DNA barcode module actually takes the form of a complex of two nucleic acids: a short nucleic acid that denotes "step 1" connected to the first DNA barcode module. All nucleotides of this complex are in frame with each other and can be read in a sequencing assay, although the first short nucleic acid can be optionally attached to the first DNA barcode module via a spacer nucleic acid.

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

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

[0161] Orthogonal structures offer the following advantages over concatenated structures: In the case of concatenated synthesis (where all DNA barcode modules are attached to each other in one continuous polymer), failure to complete any of the synthesis intermediate coupling steps may compromise the meaning of the final completed concatenated DNA barcode. In contrast, in orthogonal synthesis (where each and every DNA barcode module is attached to a dedicated site on the bead), failure to attach any of the DNA barcode modules will only result in an empty attachment site on the bead, and will not compromise the meaning of any of the other attached DNA barcode modules. In a preferred embodiment, each attached DNA barcode module includes an attached second nucleic acid, which identifies a step (a step during the parallel synthesis of DNA barcodes and compounds).

[0162] For orthogonal synthesis, it is permissible to use up all attachment sites on the beads (sites for attaching growing chemical library members). However, for orthogonal synthesis, the chemistry must be designed such that the first attachment of many DNA barcode modules results in only partial use of the entire population of attachment sites on the beads. The following provides an optional limit to the depletion of sites during chemical synthesis of orthogonal barcodes. For unmodified beads, the total number of sites available for attaching DNA barcode modules is 100%.

[0163] The extent to which the synthesis of an orthogonal constituent bead (with respect to the first DNA barcode) depletes attachment sites on a given bead. The following relates to attaching a first DNA barcode module. In embodiments, attachment of a first DNA barcode module depletes about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the DNA barcode attachment sites on the bead. In other embodiments, less than about 2%, less than about 5%, less than about 10%, less than about 20%, less than about 30%, less than about 40%, or less than about 50% of the DNA barcode attachment sites on the bead are depleted. In still other embodiments, attachment of the first DNA barcode module consumes 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 DNA barcode attachment site.

[0164] With respect to limitations, attachment of the last DNA barcode module that constitutes a particular DNA barcode consumes less than 20% of the sites, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 95%, or less than 98% of the sites.

[0165] Exclusionary embodiments can exclude beads or methods that meet any of the above values ​​or ranges, and exclusionary embodiments can exclude beads or methods that do not meet any of the above values ​​or ranges.

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

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

[0168] In other, but not all, embodiments, which may be excluded, are bead-bound DNA barcodes, where the completed DNA barcode does not contain any double-stranded DNA (dsDNA) and only contains single-stranded DNA (ssDNA).

[0169] The extent to which the synthesis of an orthogonal component bead (with respect to the second DNA barcode) depletes attachment sites on a given bead. The following relates to attaching a second DNA barcode module. In embodiments, the attachment of a second DNA barcode module (in the case of the creation of an orthogonal component bead) depletes about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% of the remaining free DNA barcode attachment sites on the bead. 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 attachment sites on the bead are depleted. In still other embodiments, attachment of the first DNA barcode module consumes 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 attachment sites.

[0170] Exclusionary embodiments can exclude beads or methods that meet any of the above values ​​or ranges, and exclusionary embodiments can exclude beads or methods that do not meet any of the above values ​​or ranges.

[0171] The above embodiments, as well as the above exclusive embodiments, are also applicable to methods involving attaching a third DNA module barcode, or involving attaching a fourth DNA module barcode, or involving attaching a fifth DNA barcode module, etc.

[0172] Concatenated DNA barcodes (all DNA barcode modules are present in one strand or polymer, and the entire strand or polymer is attached to one location on the bead).

[0173] Synthesis of bead-bound concatenated DNA barcodes. The present disclosure provides bead-bound concatenated DNA barcodes, where a bead comprises a plurality of concatenated DNA barcodes, where most or nearly all of the plurality of concatenated DNA barcodes have essentially the same structure. The concatenated DNA barcodes can comprise one or more DNA barcode modules, where the order of these DNA barcode modules (from the bead-attached end to the distal end) along the entire DNA barcode is the same order in time that the bead-bound concatenated DNA barcode is synthesized. Also, the order of these DNA barcode modules along the entire DNA barcode is the same order in time that the corresponding chemical library monomers are attached to the growing bead-bound compounds.

[0174] The concatenated DNA barcode can include, in that order, a linker that is used to attach the entire concatenated DNA barcode to a bead, and it can include, in that order, a first DNA barcode module, a first annealing site, a second DNA barcode module, a second annealing site, a third DNA barcode module, and a third annealing site.

[0175] One order of the sequencing primer hybridizing sites in the bead-bound DNA barcode. In an embodiment of the sequencing primer hybridizing sites, the 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, a third sequencing primer binding site, etc.

[0176] Alternative ordering of sequencing primer hybridizing sites occurring within bead-bound DNA barcodes. In an embodiment of alternative sequencing primer hybridizing sites, the 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, a third annealing site, etc.

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

[0178] Synthesis in blocks. In alternative embodiments, the growing compound and the growing DNA barcode module sequences can be synthesized in blocks. For example, a block comprised of 2 chemical library units can be attached to a bead in parallel with the attachment of a block comprised of a corresponding 2-DNA barcode module. Similarly, a block comprised of 3 chemical library units can be attached to a bead in parallel with the attachment of a block comprised of a corresponding 3 DNA barcode. Block syntheses with 4 blocks, 5 blocks, 6 blocks, 7 blocks, 8 blocks, 9 blocks, 10 blocks, etc. are also provided. Each of these block transfer embodiments can also be excluded by the present disclosure. The block-by-block transfer of DNA barcode monomers can be performed orthogonally, with a unique attachment point for receiving each successive block of DNA barcode monomers. Alternatively, block-by-block transfer of DNA barcode monomers can be performed to generate a concatemeric structure (all DNA barcode modules occur only as one continuous linear polymer).

[0179] Also, in the case of split-pool synthesis of bead-bound DNA barcodes and bead-bound compounds in parallel, synthesis can occur in blocks, where a block can take the form of two or more chemical library monomers, and a block can take the form of two or more DNA barcode modules.

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

[0181] Distance from primer annealing site to DNA barcode module. For the purpose of sequencing bead-bound DNA barcode, i.e., for the purpose of sequencing all DNA barcode modules forming a DNA barcode, a polynucleotide comprising a first nucleic acid which is an annealing site of a sequencing primer, and a second nucleic acid which is a DNA barcode module, the first nucleic acid may be immediately upstream of the second nucleic acid. Alternatively, the first nucleic acid may be upstream of the second nucleic acid, and the first and second nucleic acids are separated from each other by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides, or by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides. The separation can use a nucleic acid that simply functions as a spacer, or alternatively, the separation can use a third nucleic acid that encodes information such as the number of a step in a multi-step pathway of organic synthesis, or the number of a class of compound, or a disease that may be treated with the bead-bound compound, or a date, or a lot number.

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

[0183] Also capable of binding is a polynucleotide in the form of a first nucleic acid that is a first DNA barcode module attached to a second nucleic acid that is a first sequencing primer binding site that allows an operator to determine the sequence of the first DNA barcode module.

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

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

[0186] A preferred method for synthesizing DNA barcodes is presented below and uses the same reaction cycle to incrementally attach each DNA barcode module.

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

[0188] Step 2. [Tetrazine]-[first DNA barcode module]-[azide] is added to the bead, and the TCO group is condensed with the tetrazine group. The result is the following construct: BEAD-TCO-tetrazine-first DNA barcode module-azide. In fact, this construct does not contain any TCO or tetrazine, but the condensation product that is generated when TCO is condensed with tetrazine.

[0189] Step 3. Optional washing.

[0190] Step 4. Add DBCO-TCO to cap the azide and create a TCO terminus. The result is the following structure:

[0191] BEAD-TCO-Tetrazine-First DNA barcode module-Azide-DBCO-TCO

[0192] Step 5. Optional washing.

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

[0194] [tetrazine]-[second DNA barcode module]-[azide] to the bead, condensing the TCO group with the tetrazine group. The result is the following construct: BEAD-TCO-tetrazine-1st DNA barcode module-azide-DBCO-TCO-[tetrazine]-[2nd DNA barcode module]-[azide] The above scheme involves a series of steps for incrementally adding more DNA barcode modules, which are added in parallel with the addition of more chemical monomers. As noted elsewhere, this "parallel" synthesis can involve attaching a chemical monomer followed by connecting a DNA barcode module that specifies that monomer, or alternatively, attaching a DNA barcode module followed by attaching a chemical monomer specified by that particular chemical monomer.

[0195] Compounds for click chemistry synthesis of DNA barcodes FIG. 17 discloses the chemical synthesis of a compound suitable for connecting deoxycytidine residues (dC) during the synthesis of DNA barcode modules, and ultimately the entire DNA barcode. The starting material is N4-acetyl-2'-deoxy-5'-O-DMT cytidine. The abbreviation "DMT" stands for 4,4-dimethoxytrityl. The end product of this multi-step route of organic synthesis has a cytosine moiety, a triphosphate group, and a propargyl group attached to the 3' position of the ribose group. The propargyl group is used in click chemistry to condense with an azide group to generate a covalent bond. The result after condensation is that the remaining chemicals (which never occur naturally in nucleic acids) arise as "traces" from the click chemistry being performed. What is available is a DNA polymerase that can be used for sequencing-by-synthesis of DNA barcodes created by click chemistry, where the DNA polymerase can move across the traces and the traces do not cause sequencing errors. TBAI is tetrabutylammonium iodide.

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

[0197] An option to create a DNA barcode that contains a terminal nucleic acid that encodes a DNA hairpin. This involves a DNA barcode that contains a 3-prime end, a nucleic acid with an annealing site for a sequencing primer, a bend in the form of about 4 bases that are not base-paired, and an annealing site for the sequencing primer and a sequencing primer that can be bent to form base pairs. Again, the sequencing primer anneals to the annealing site of the sequencing primer, and the actual sequencing reaction starts from the 3' end of the annealed sequencing primer.

[0198] When performing the final step of synthesizing the DNA barcode and attaching the final DNA barcode module to the growing bead-bound DNA barcode, the "sprint oligo" can include a sequence encompassing a DNA hairpin (the DNA hairpin includes, in this order, an annealing site for the sequencing primer, a number of nucleotides that are not base-paired to each other or to any nearby base sequences, and the sequencing primer). After annealing the "sprint oligo," DNA polymerase and dNTPs are then added and polymerization occurs at the 3' end of the growing DNA barcode, with what polymerizes using the splint oligo as a template being, in this order, (1) an annealing site for the sequencing primer, (2) a bend in the hairpin in the form of four or five deoxyribonucleotides that do not base-pair with one another, and (3) the sequencing primer.

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

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

[0201] For convenience, the first annealing site that is part of the bead-bound DNA barcode and the first annealing site that is part of the soluble "sprint oligo" will both be referred to as the "first annealing site" even though they do not have the same sequence of bases (instead, the sequences of bases are complementary to each other, so that the splint oligo can hybridize to the first annealing site of the bead-bound DNA barcode, thus acting as a template for DNA polymerase to extend the bead-bound DNA barcode by copying what is on the splint oligo.

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

[0203] The bead-bound DNA barcode from the 5' to the 3' end may comprise nucleic acids in the following order: Beads / First DNA barcode / First annealing site / Alternatively, the bead-bound grown DNA barcode from the 5' end to the 3' end can include nucleic acids encoding step numbers, the bead-bound grown DNA barcode having nucleic acids in the following order: Beads / first DNA barcode / nucleic acid encoding step number / first annealing site / Alternatively, the bead-bound grown DNA barcode can comprise a nucleic acid that is a functional nucleic acid (sequencing primer annealing site), as shown below: Beads / First DNA barcode / Sequencing primer annealing site / First annealing site / Not shown in the figures in these texts 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 can be made of some other organic chemical.

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

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

[0206] Beads / First DNA barcode / First annealing site / First annealing site / second DNA barcode / second annealing site To repeat some of the information shown in Figure 7, shown immediately below is the splint oligo:

[0207] "First annealing site / second DNA barcode / second annealing site" Step 3. Add DNA polymerase and dNTPs to extend the bead-bound DNA barcode. Below is the bead-bound DNA barcode, the splint oligo is still hybridized and has a "second DNA barcode module" nucleic acid and a "second annealing site" nucleic acid attached, so the bead-bound growing barcode is longer than before. Figure 7 further illustrates this step. The splint oligo is displayed below the bead-bound barcode: Bead / 1st DNA barcode / 1st annealing site / 2nd DNA barcode / 2nd annealing site

[0208] First annealing site / second DNA barcode / second annealing site Step 4. Wash the splint oligos. The splint oligos can be encouraged to dissociate from the bead-bound growing barcodes by heating, i.e., by heating the entire picowell plate to, for example, about 60° C., about 65° C., about 70° C., about 75° C., about 80° C. for about 10 minutes, or, alternatively, neutralize by adding dilute NaOH to the picowell array.

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

[0210] 2nd annealing site / 3rd DNA barcode / 3rd annealing site / Step 6. Allow this oligonucleotide to anneal to the corresponding bead-bound "second annealing site" and allow DNA polymerase to extend the bead-bound oligonucleotide, thereby containing the complement: "third DNA barcode / third annealing site /

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

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

[0213] 3rd annealing site / 4th DNA barcode / 4th annealing site / This soluble oligonucleotide has a nucleic acid that can anneal to the "third annealing site" of the bead-bound oligonucleotide. Once annealed, a DNA polymerase with four dNTPs is utilized to extend the bead-bound oligonucleotide to encode yet another DNA barcode module (the fourth DNA barcode). The cycle of the above steps is repeated throughout the split-pool procedure to create a library of compounds and associated DNA barcodes in parallel, each DNA barcode associated with a given compound (each DNA barcode informs the chemical synthesis history of the associated compound). When the chemical synthesis of the library of compounds is completed, the cycle of the above steps is stopped. With the completed bead-bound DNA barcode chemical library controlled, the beads can then be dispensed into the picowells of the picowell array.

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

[0215] Sequencing primer annealing site. In one embodiment, each DNA barcode module in the completed DNA barcode is operably linked to a unique sequencing primer annealing site, thus providing the operator with the ability to perform a separate sequencing procedure on each DNA barcode module (in this embodiment, each DNA barcode module is also preferably operably linked to a unique nucleic acid that specifies (encodes) a step in the synthesis of the entire DNA barcode).

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

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

[0218] Exclusionary embodiments of the bead-bound DNA barcodes of the present disclosure, which may be excluded, are any bead, microparticle, granule, resin, or polymer composition material in which the concatenated DNA barcodes are linked to the beads via a photocleavable or cleavable linker.

[0219] Exclusions may include materials of beads, microparticles, granules, resins, or polymer compositions that do not contain both: (1) concatenated DNA barcodes attached to a first location on a bead; and (2) a compound attached to a second location on a bead, where the first location is not the same as the second location. In a preferred embodiment, the "compound" is made of a plurality of chemical library monomers.

[0220] Excluded may be any bead, microparticle, granule, resin, or polymer composition material that does not have an exterior surface (or exterior surface) and an interior surface (or interior surface, or interior region), and the bead does not include at least 10,000 substantially identical concatenated DNA barcodes attached thereto, and at least 90% of the at least 10,000 substantially identical concatenated DNA barcodes are attached to the exterior surface. In other words, excluded may be any bead in which at least 90% of the attached concatenated DNA barcodes are not attached to the exterior surface.

[0221] Excluded are materials that are beads, microparticles, granules, resins, or polymer compositions made substantially of or including any polyacrylamide.

[0222] Excluded are materials that are beads, microparticles, granules, hydrogels, resins, or polymer compositions, and that contain a promoter, such as the T7 promoter, or that contain a polyA region, or that contain a promoter and a polyA region.

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

[0224] Beads / First DNA barcode / First annealing site / First annealing site / second DNA barcode / second annealing site Bead / 1st DNA barcode / 1st annealing site / 2nd DNA barcode / 2nd annealing site

[0225] First annealing site / second DNA barcode / second annealing site A method using two cycles of annealing / polymerization to generate a bead-bound DNA barcode with three DNA barcode modules. The present disclosure encompasses bead-bound compositions, systems, and methods in which two different split oligos are used (first splint oligo; second splint oligo). In this situation, the first splint oligo consists of a structure of first annealing site / second DNA barcode / second annealing site, and the second splint oligo consists of a structure of second annealing site / third DNA barcode / third annealing site.

[0226] A method using three cycles of annealing / polymerization to generate a bead-bound DNA barcode with four DNA barcode modules. The present disclosure encompasses bead-bound compositions, systems, and methods, where three different split oligos are used (first splint oligo; second splint oligo; third splint oligo). In this situation, the first splint oligo consists of a structure of first annealing site / second DNA barcode / second annealing site, the second splint oligo consists of a structure of second annealing site / third DNA barcode / third annealing site, and the third splint oligo consists of a structure of third annealing site / fourth DNA barcode / fourth annealing site.

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

[0228] Embodiments using multiple steps of annealing / polymerization to generate bead-bound DNA barcodes with multiple DNA barcode modules. The present disclosure encompasses bead-bound compositions, systems, and methods related to concatenated barcodes that use only one splint oligo (creating a 2-module DNA barcode), only two splint oligos (creating a 3-module DNA barcode), only three splint oligos (creating a 4-module DNA barcode), only four splint oligos (creating a 5-module DNA barcode), only five splint oligos (creating a 6-module DNA barcode), only six splint oligos (creating a 7-module DNA barcode), etc.

[0229] Included are bead-bound compositions, systems, and methods that use at least 1 splint oligo, at least 2 splint oligo, at least 3 splint oligo, at least 4 splint oligo, at least 5 splint oligo, 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 oligo, or less than 20, less than 15, less than 10, less than 8, less than 6, less than 4, less than 3, less than 2 splint oligo. These numbers refer to the splint oligo itself, as well as the number of steps of adding splint oligo, and the number of DNA modules added to the growing bead-bound DNA barcode.

[0230] Reduce damage to DNA barcodes Using orthogonal DNA barcodes to reduce damage (instead of concatenated DNA barcodes). One way to understand the topic of concatenated DNA barcodes and orthogonal DNA barcodes is to note the significance that one has over the other. The advantages of orthogonal barcoding over concatenated barcoding are as follows: With the attachment of each monomer of a growing compound, attached in parallel are compound library monomers to create a chemical library, and DNA barcode modules to create a completed full-length DNA barcode.

[0231] In concatenated barcoding, if the attachment of any given module is incomplete (i.e., not all attachment sites are successfully coupled with the required module), the sequence of the completed barcode will not be accurate. By "inaccurate" we mean that incomplete coupling results in chunks being missing, and the completed product was assumed by the user to be a complete and correct DNA barcode. Here, the completed DNA barcode sequence will contain errors, since attachment of all DNA modules was unsuccessful. In contrast, orthogonal barcoding covalently binds each individual DNA module to its own unique attachment site on the bead. Also, once a DNA module is attached to a predefined site on the bead, there is no need to further bind DNA modules to the DNA modules already bound to the bead.

[0232] Damage is reduced by using crosslinking agents. 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 can be in the form of double-stranded DNA (dsDNA), which is treated with a DNA crosslinking agent such as mitomycin C, before being attached to the growing bead-bound DNA barcode. After completion of the synthesis of the DNA barcode in dsDNA form, the dsDNA is converted to ssDNA. The conversion of dsDNA to ssDNA is effective when one of the DNA strands has a uracil (U) residue, and the cleavage of the DNA at the uracil residue is catalyzed by uracil-N-glycosidase (see FIG. 5 of Serial No. 62 / 562,905, filed September 25, 2017, which is incorporated herein by reference in its entirety). The above refers to the damage inflicted on the growing DNA barcode by the reagents used to make the bead-bound compounds.

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

[0234] Inclusion of a hairpin reduces damage Yet another method for reducing damage to bead-bound DNA barcodes is to synthesize the DNA barcode via self-assembly to form a hairpin, where the DNA barcode self-assembles by annealing a first prong of the hairpin to a second prong of the hairpin.

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

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

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

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

[0239] Damage to nucleic acids is reduced by using DNA-compatible chemistries. Satz et al. disclose various chemistries that are compatible with bead-bound nucleic acids (Satz et al (2015) Bioconjugate Chemistry. 26:1623-1632; amended by Satz et al (2016) Bioconjugate Chem. 27:2580-2580). While the above description by Satz et al. is directed to chemical reactions carried out on DNA / chemical library member complexes, the types of DNA-compatible chemistries described are also relevant, and organic chemistry is carried out on beads, including bead-bound compounds and bead-bound DNA.

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

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

[0242] Additionally, methods have been disclosed for coupling reagents to DNA, where conjugation occurs at functional groups already attached to the DNA. Methods include the Suzuki coupling, an optimized procedure for the Sonogashira coupling between alkynes and aryl halides, conversion of aldehydes to alkynes using dimethyl-1-diazo-2-oxopropylphosphonate, a new method for the addition of triazoles directly from purified alkynes, improved reactions occurring with isocyanate reagents in pH 9.4 buffer, and an improved method for the reaction of isocyanate building blocks with amine-functionalized DNA (see Satz et al., Table 1, entries 12-15).

[0243] Additional methods for coupling reagents to DNA have been disclosed where the coupling occurs at functional groups already attached to the DNA. These include methods for attaching primary amines to DNA, optimized procedures for forming DNA-conjugated thioureas, methods for alkylating secondary amines and bis-alkylating aliphatic primary amines, mono-alkylating primary amine-DNA conjugates using hetaryl halides as building blocks that can react with amine-functionalized DNA conjugates, and methods for the Wittig reaction (see Satz et al., Table 1, entries 16-20).

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

[0245] A variety of 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 recombinational repair. Interstrand DNA crosslinks can be repaired using a combination of NER and homologous recombination. Direct repair includes the repair of cyclobutane pyrimidine dimers and 6-4 products via photolyase enzymes. Direct repair includes the repair of O-terminal cleavage products by DNA methyltransferases. 6- O from methylguanine 6-This includes the removal of methyl. See Sancar et al (2004) Ann. Rev. Biochem. 73:39-85; Hu, Sancar (2017) J. Biol. Chem. 292:15588-15597.

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

[0247] Coupling DNA to beads via their 3' ends reduces damage. Certain chemical transformations can damage exposed 3'-hydroxyl groups of nucleic acids. For example, the Mitsunobu reaction allows the transformation of primary and secondary alcohols into esters, phenyl ethers, thioethers, and various other compounds, which renders exposed 3' ends insensitive to subsequent processing steps, or the now altered 3' ends may participate in further chemical reactions. In some embodiments, DNA tags can be attached to beads via their 3' ends, so that only the 5' ends are exposed to the solution.

[0248] The disclosed reagents, systems, and methods encompass bead-bound nucleic acids, such as bead-bound DNA or bead-bound DNA tags, with coupling to the bead involving the 3' terminus (or 3' end) of the DNA. If the ssDNA that constitutes the DNA barcode is attached via the 3' end of the ssDNA, sequencing can be initiated by hybridizing only one sequencing primer, which hybridizes upstream of the entire DNA barcode, at or near the bead-bound end of the attached ssDNA. Instead of using only one sequencing primer, multiple sequencing primers can be used, with each sequencing primer hybridizing upstream to a specific DNA barcode module. For example, if a given DNA barcode contains five DNA barcode modules, the DNA is attached to the bead via the 3' end, and the DNA barcode can include five different primer annealing sites, with each primer annealing site located just upstream or immediately upstream of a given DNA barcode module.

[0249] In another embodiment, it is dsDNA that is bound to the bead, and the 3' end of only one of the strands in the dsDNA is bound to the bead. In a 5' coupling embodiment involving dsDNA, it is dsDNA that can be bound, and the 5' end of only one of the strands of the dsDNA is bound to the bead.

[0250] (V) Coupling of compounds to beads The present disclosure provides (1) linkers that attach chemical library members to substrates such as beads, (2) linkers that attach nucleic acid barcodes to substrates such as beads, (3) cleavable linkers, e.g., cleavable by UV light, cleavable by enzymes such as proteases, (4) non-cleavable linkers, (5) bifunctional linkers, (6) multifunctional linkers, and (7) beads that can be used for linking. For example, available linkers are 4-hydroxymethylbenzoic acid (HMBA) linkers, 4-hydroxymethylphenylacetic acid linkers (see Camperi, Marani, Cascone (2005) Tetrahedron Letters. 46:1561-1564).

[0251] A "non-cleavable linker" may be characterized as a linker that is not detectably cleaved by any reagent, condition, or environment used during a given organic chemistry procedure step. Alternatively, a "non-cleavable linker" may be characterized as a linker that cannot be cleaved except by reagents, conditions, or environments that are unacceptably destructive to other reactants, products, or reagents of a given organic chemistry procedure.

[0252] A bifunctional linker, or other multifunctional linker, can take the form of a fork (the fork humans use to consume food), with the handle of the fork attached to a bead, and each tine of the fork linked to one of a variety of 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 can be linked to a metal ion.

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

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

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

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

[0257] A method for making three amino acid compounds on Tentagel® beads is described. Fmoc-protected resin (1 mg, Rapp Polymere GmbH, 10 um, TentaGel M-NH2, 0.23 mmol / g) modified with Fmoc-Photo-Linker, 4-{4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butanoic acid), or another suitable linker with Fmoc protection, was suspended in each well of a reaction plate (Merck Millipore Ltd, 0.45 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. The Fmoc protecting group was removed by suspending the resin in 150 uL of a mixture of 5% piperazine, 2% DBU in DMF. The plate was sealed with Excel Scientific Alumna Seal and shaken for 15 minutes at 40°C. Vacuum was applied to remove the solvent and the deprotection procedure was repeated for 5 min. After filtration, each well was washed with 150uL each of 2XDMA, 3XDCM, and 1XDMA, with vacuum applied between each wash to remove the solvent. Each well of resin was then acylated with the appropriate amino acid by adding 150uL of a pre-activation mixture of 60mM Fmoc-amino acid, 80mM Oxyma, 200mM DIC, and 80mM 2,4,6-trimethylpyridine that had been left at room temperature for 2 min. The plate was resealed and shaken at 40 degrees for 1 hour. After filtration, each well was washed with 150uL each of 2XDMA and 3XDCM. The beads in each well were resuspended in 150ul of DCM and the contents of each well were combined into one vessel via pipetting. The combined beads were mixed thoroughly and redistributed across the plate by pipetting equal amounts into the appropriate wells (1mg / well). The solvent is removed by applying a vacuum and each well is ready for the next appropriate step. For each additional amino acid coupling, the Fmoc deprotection step is first repeated, followed by the coupling step with the desired amino acid. If a split pool is required, the coupling and redistribution method is repeated.

[0258] A method for making trimer amino acids on beads by split pool method is described. Fmoc-protected resin (1 mg, Rapp Polymere GmbH, 10 um, TentaGel M-NH2, 0.23 mmol / g) modified with Fmoc-Photo-Linker, 4-{4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butanoic acid), or any other suitable linker, was suspended in each well of a reaction 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. The Fmoc protecting group was removed by suspending the resin in 150 uL of a mixture of 5% piperazine, 2% DBU in DMF. The plate was sealed with Excel Scientific Alumna Seal and shaken for 15 minutes at 40°C. Vacuum was applied to remove the solvent, and the deprotection procedure was repeated for 5 min. After filtration, each well was washed with 150 uL each of 2XDMA, 3XDCM, and 1XDMA, with vacuum applied between each wash to remove the solvent. Each well of resin was then acylated with the appropriate AA by adding 150 μL of a pre-activation mixture of 60 mM Fmoc-amino acids, 80 mM Oxyma, 200 mM DIC, and 80 mM 2,4,6-trimethylpyridine that had been left at room temperature for 2 min. The plate was resealed and shaken at 40 °C for 1 h. After filtration, each well was washed with 150 uL each of 2XDMA, 3XDCM, and 1XDMA. 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 1 mg portion of beads was suspended in 100 uL DMSO and exposed to the full output of the 365 nm LED for 2 h. The resin is filtered and the filtrate is injected into an Agilent 1100 Series LCMS equipped with an Agilent Poroshell SB-C-18, 3.0X50mm, 2.7um column. A gradient from 5% CH3CN in 0.1% TFA in water to 100 CH3CN in 0.1% TFA over 4 minutes was run at a flow rate of 1.2mL / min and monitoring at 220nm.

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

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

[0261] Members of a chemical library can be synthesized on a solid support, such as on beads, via solid-phase synthesis. Solid-phase synthesis of chemicals with peptide bonds is characterized by the use of one of two chemical groups: the first is N-alpha-9-fluorenyl-methyloxycarbonyl (Fmoc, base labile); the second is tert-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 peptide substrates, with the Fmoc or tBoc group attached to the alpha-amino group (Sigler, Fuller, Verlander (1983) Biopolymers. 22:2157-2162).

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

[0263] Further provided by the present disclosure are methods and reagents whereby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% of the members of a chemical library bound to a given bead have the exact same chemical structure after complete synthesis (these numbers take into account and reflect errors that may occur during solid phase synthesis, e.g., the failure of one growing compound to receive one of the chemical monomers. These numbers also take into account and reflect chemical damage to any monomers that may occur during solid phase synthesis).

[0264] In exclusive embodiments, the present disclosure may exclude any method or reagent that does not meet one of the above cutoff values ​​for "exactly the same structure."

[0265] In alternative embodiments, 2 beads, 3 beads, 4 beads, 5 beads, about 5-10 beads, about 10-20 beads, about 20-40 beads, about 40-80 beads contain similar equivalent compounds in the population of beads (not considering any errors in the incorporation of chemical monomers during solid phase synthesis and not considering chemical damage that occurs to the chemical monomers during organic synthesis).

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

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

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

[0269] Huisgen Cycloaddition. The Huisgen 1,3-dipolar cycloaddition reaction involves an alkyne and an organic azide. The alkyne has the structure RC=CH. The azide is reacted with RN + =N=N - The structure of the 1,2,3-triazole-based 1,2-triazole-based 1,2-triazole derivative is shown in Figure 1. The copper catalyst accelerates the rate of the Huisgen cycloaddition reaction. The Huisgen reaction works via "click chemistry" or "click reaction". When catalyzed by copper, the Huisgen reaction can generate a 1,2,3-triazole core suitable for making small molecule agents. The Huisgen reaction is compatible with the presence of amino acid side chains, at least in protected form. Molecules made with 1,2,3-triazoles can have bonds similar to the amide bonds of polypeptides, and therefore these molecules can be substitutes for peptide bonds (Angell and Burgess (2007) Chem. Soc. Rev. 36:1674-1689).

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

[0271] The present disclosure encompasses bead-bound compounds, which are in the form of only one monomer.For example, the bead-bound compounds can be in the form of lenalidomide, or in the form of lenalidomide with a carboxylic acid group attached, or in the form of lenalidomide with an amino group modified with a small chemical moiety that has a carboxylic acid group, or the compounds are stereoisomers or enantiomers of lenalidomide, that is, lenalidomide analogs.

[0272] (VII) Split-pool synthesis and parallel synthesis It describes the use of the "split pool" method to synthesize libraries of compounds, and how the "split pool" method is used to simultaneously synthesize bead-bound compounds and bead-bound DNA barcodes. It also describes splitting and pooling to create mixed sets of compounds. At some later point, disclosed below is the coupling of non-amino acids, and the preparation of polyethylene glycol (PEG) modified beads.

[0273] The present disclosure provides a split-pool synthesis for generating chemical libraries. In one embodiment, the method involves (a) dividing beads into different containers, (b) adding different components to each container. For example, if three containers are used, add and react species A to the first container, species B to the second container, and species C to the third container, where the species covalently bind to the attachment sites of the beads in the containers, (c) pool all beads into one container, (d) divide the beads into three containers, and (e) add different components to each container, where 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, where the species covalently bind to the first species that was previously attached (see Stockwell (2000) Trends Biotechnol. 18:449-455).

[0274] The split pool synthesis of the present disclosure includes a DNA barcode coupling step either before or after each chemical coupling step (to create a chemical library member), where the DNA barcode identifies the chemical being coupled in that step.

[0275] In exclusive embodiments, the present disclosure may exclude methods and reagents where a barcode is attached prior to attachment of a chemical for a given step of a parallel synthesis. Conversely, the present disclosure may exclude methods and reagents where a chemical is attached prior to attachment of a barcode for a given step of a parallel synthesis.

[0276] One feature of bead-bound chemical libraries prepared by split-pooling is that only one compound is attached to each bead. If coupling is incomplete, e.g., only 4,000 of 5,000 attachment sites are successfully coupled to the desired species in a given split-pooling step, some heterogeneity will occur.

[0277] Parallel synthesis. In a preferred embodiment of the present disclosure, parallel synthesis can be used for the organic synthesis of compounds and associated DNA barcodes. In practice, the modification of a bead with one or more chemical monomers and the modification of the same bead with one or more DNA barcode modules are not strictly parallel. In practice, a bead receives one or more chemical units (chemical monomers) and subsequently receives a DNA barcode module that codes for that particular chemical unit. The term "parallel" refers to the fact that as the polymer of the chemical library monomers grows, the polymer of the DNA barcode module also grows. When all the DNA barcode modules are attached to the bead and form a concatenated or orthogonal structure, the full-length DNA barcode is referred to as a "DNA barcode" (not just the DNA barcode module).

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

[0279] This relates to the exterior and interior surfaces of the beads. For a given bead having externally attached DNA barcodes (without considering the number of internally attached DNA barcodes) and attached chemical library members (attached to both the exterior and interior surfaces), the ratio of the number of externally attached DNA barcodes to the 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 present disclosure may exclude any bead, or any population of beads, that meets one of the above values.

[0280] Uniformity of DNA barcodes on typical beads;Uniformity of chemical library members on typical beads The present disclosure provides for a "chemical library uniformity" for any given bead (or any population of beads) of 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.

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

[0282] Similarly, the present disclosure provides the above cutoff values ​​for assessing the uniformity of barcodes, such as DNA barcodes.

[0283] Uniformity of DNA barcodes and uniformity of chemical library members can be defined as the percentage of the total population that matches the exact sequence as planned in the methods section of a laboratory manual or notebook, as desired.

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

[0285] When assessing the uniformity of a population of beads, one should consider the total uniformity of bead #1, bead #2, bead #3, bead #4, bead #5, bead #6, bead #7, etc. in situations where uniformity is desired across the entire population of beads.

[0286] In exclusive embodiments, the present disclosure can exclude any bead, or any population of beads, where the uniformity of the DNA barcodes is not at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99.5%, etc. Also, in exclusive embodiments, the present disclosure can exclude any bead, or any population of beads, where the uniformity of the chemical library members is not at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99.5%, etc.

[0287] Ratio of internally and externally attached DNA barcodes In some embodiments of the present disclosure, it may be desirable to manufacture and use beads with DNA barcodes attached mainly to the outer surface.One reason for not making and using beads with internal DNA barcodes is that the penetration of DNA oligomers into the inner space is low, and the penetration of DNA ligase (the ligase for connecting DNA modules to each other to make a completed DNA barcode) into the inner space is low.For sequencing purposes, the reason for not making and using internal DNA barcodes is that the penetration of the enzyme required to amplify the DNA required for the final sequencing of the barcode is low.Another reason for not making and using beads with internal DNA barcodes is the cost of the inner space for attaching members of a chemical library.

[0288] The present disclosure provides beads associated with DNA barcodes, where the ratio of internally attached DNA barcodes to externally attached 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.

[0289] The present disclosure also provides beads associated with DNA barcodes, where the ratio of internally attached DNA barcodes to externally attached DNA barcodes is less than 0.1:100, less than 0.2:100, less than 0.4:100, less than 0.8:100, less than 1:100, less than 2:100, less than 4:100, less than 8:100, less than 10:100, less than 20:100, less than 40:100, less than 50:100, less than 60:100, less than 70:100, less than 80:100, less than 90:100, less than 1:1, etc.

[0290] A population of beads in an aqueous suspension can be contacted with a substrate, such as a picowell array, such that the beads enter and occupy the picowells. The ratio of the number of beads in the suspension and the number of picowells in the substrate can be adjusted to achieve a desired occupancy rate. For example, if the suspension contains only one bead, all picowells containing beads will contain only one bead, and the remaining picowells will not contain any beads. If the suspension contains 20,000 beads and the substrate contains 200,000 picowells, at least 180,000 picowells will be completely emptied of beads, and most picowells containing beads will contain only one bead. A small fraction of the occupied picowells will contain two beads.

[0291] In valuable embodiments, the ratio of beads to picowells in the suspension 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 (which is the same as 1:1), about 2:1, about 4:1, about 6:1, about 8:1, about 10:1, and the like.

[0292] In exclusive embodiments, the present disclosure may exclude any method or system that falls within one of the above values ​​or ranges.

[0293] In a range of embodiments, the ratio of the number of beads to the number of picowells in the suspension is 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 The ratio may be from about 20:100 to about 20:100, from about 20:100 to about 30:100, from about 30:100 to about 40:100, from about 40:100 to about 50:100, from about 50:100 to about 60:100, from about 60:100 to 80:100, from about 80:100 to about 100:100 (same as 1:1), from about 100:100 (same as 1:1) to about 2:1, from about 2:1 to about 4:1, from about 4:1 to about 6:1, from about 6:1 to about 8:1, from about 8:1 to about 10:1, and the like.

[0294] In exclusive embodiments, the present disclosure may exclude any method or system that falls within one of the above values ​​or ranges.

[0295] (VIII) Build a Picowell Combination of UV light, photomask, and photoresist to make picowell array plates. Plates containing many microwells or picowells can be made for use in this disclosure as follows. Briefly, a three-layer sandwich is assembled. The top layer is photoresist. The middle layer is a glass wafer. The bottom layer is a photomask. The picowells will be 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 dish containing a cup for baking muffins, with the cup of the dish used to hold the muffin batter having slanted sides. The UV light acts as a "non-crosslinker" as it breaks down the polymers in the photoresist. After UV treatment, a solvent is added to wash away the UV-treated photoresist, leaving behind clean-looking picowells.

[0296] Tilt and rotate to create the tilted walls. Picowells with tilted walls are created as follows: A photomask has many holes, each hole corresponding to the desired bottom dimension of the picowell. The bottom dimensions can include circumference, diameter, and shape, i.e., circular shape. The top dimension of the well is created by directing a tilted UV light into the photomask holes while rotating the light source or rotating the stage holding the sandwich (photomask / glass wafer / photoresist sandwich). In rotating, the light source is not at a 90 degree angle to the photomask / wafer / photoresist sandwich, but instead is tilted slightly from the 90 degree position to carve out the tilted walls of each picowell. The resulting picowell array plate containing many picowells can be used as is. Alternatively, this picowell array plate can be used as a mold to inexpensively create many picowell array plates.

[0297] Han et al. describe equipment and reagents for fabricating microwell plates in which the microwells have slanted walls (see Han et al (2002) J. Semiconductor Technology and Science. 2:268-272). Described are a UV light source, a contact stage, a tilt stage, and SU-8 photoresist. Fabrication begins with a single-sided polished silicon wafer. SU-8 photoresist is coated onto the wafer to a thickness of approximately 0.10-0.15 mm. The photoresist is then gently baked on a 65 degree hot plate for 10 minutes, then gently baked on a 95 degree hot plate for 30 minutes. The resulting photoresist / wafer sandwich is contacted with a UV mask using a contact stage. The term "tilt and rotate UV lithography" refers to a method of fabricating microwell array plates or picowell array plates, in which each well has slanted walls. Here, the floor of the well has a small diameter and the top of the well (where the top edge of the well contacts the flat surface of the plate) has a wider diameter. To expose with UV light, a rotating stage is used and the UV light is tilted (Han et al., supra). The mask is contacted with the photoresist where each of the holes in the mask is circular. Figure 8 in Han et al., supra, provides a photograph of the direction of the UV light, the UV mask, the photoresist structure, the wafer substrate, and the rotating stage. Han et al. describe a method for fabricating the truncated cones. A soft material such as PDMS (polydimethylsiloxane) is poured over the conical array and, upon curing, the PDMS layer peels off to form the conical wells.

[0298] A mold is created that will be used for mass production of picowell array plates. When picowell array plates are being manufactured, epoxy can be poured across the plate, filling all the picowells and contacting all filled picowells with the epoxy platform. Once the epoxy has solidified, the solid platform with the array of picoprotrusions attached (picoprotrusions are the inverse of the picowells of interest) is removed. The solid platform with picoprotrusions is a reusable mold that can be used to manufacture many picowell array plates.

[0299] The procedure of making replicas from a cone array mold made of an epoxy mold (or any hard material) is called "hot embossing". Briefly, the substrate material is heated to its glass transition or softening temperature, at which point the mold with the picoprotrusions is pressed uniformly into the heat-softened material. The picoprotrusions are transferred to the substrate material as picoindentations, after which the mold can be separated from the substrate. This disclosure preferably discloses picocones and picowells as patterns on the mold and substrate, respectively.

[0300] Hot embossing, epoxy masters, and photoresists such as 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).

[0301] Other microwell plate embodiments. Plastic microwell arrays can be fabricated via thermoforming using a silicon mold containing an array of microwells, for example an array of 800,000 microwells. Tapered shapes, smooth sidewalls, and a high degree of control resulting in sub-micron tolerances can be produced using a non-pulsed dry etching process. In contrast, methods using pulsed dry etching processes such as the Bosch process can result in rough sidewalls and a lack of control over lateral dimensions during etching.

[0302] A non-pulsed dry etching process is used to fabricate plastic arrays by thermoforming plastic onto a silicon master that has been 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 done with 1200-2000 watts of RF power, and 150 watts of bias. By varying the gas flow between the three gases, the taper of the silicon mold can be fine-tuned to create smooth sidewalls. What is changed is the ratio of SF6 to C4F8, and the result of changing the ratio is tapered walls of the mold (silicon pillars) that exist at a slope of, for example, 18 degrees (highly sloped walls), 9 degrees (slightly sloped walls), or 2 degrees (walls nearly perpendicular to the substrate) (Perry, Henley, and Ramsey (Oct.26-30, 2014) Development of Plastic Microwell Arrays for Improved Replication Fidelity.18 th Int. Conference on Miniaturized Systems for Chemistry and Life Sciences. San Antonio, TX (see pages 1700-1703).

[0303] In an embodiment, the present disclosure provides a substrate, array, grid, microfluidic device, etc., that includes an array of microwells. In one embodiment, all of the microwells have essentially the same volume. This volume can be about 1 femtoliter, about 2, about 4, about 6, about 8, about 10, about 20, about 40, about 60, about 80, about 100, about 200, about 400, about 600, about 800, or about 1,000 femtoliters.

[0304] Additionally, the volume can take the form of a range between any two of the above adjacent values, such as a range of about 40 femtoliters to about 60 femtoliters, or a range between any two of the above values ​​that are not directly adjacent to one another in the above list.

[0305] Additionally, the volume can be about 1 picoliter, about 2, about 4, about 6, about 8, about 10, about 20, about 40, about 60, about 80, about 100, about 200, about 400, about 600, about 800, or about 1,000, about 2,000, about 5,000, about 10,000, about 20,000, about 50,000, about 100,000, about 200,000, about 500,000, or about 1,000,000 picoliters. The volume can also be in the form of a range between any two of the above values ​​that are not directly adjacent to one another in the above list.

[0306] In exclusive embodiments, the disclosure may exclude any substrate comprising microwells, or any array comprising microwells, wherein the volume of each microwell is definable by one of the values ​​above, or by any range of two of the values ​​above that are adjacent to each other, or by any range of two of the values ​​above that are not adjacent to each other in the list.

[0307] Spherical plugs (also known as capped beads) on picowells. The present disclosure provides spherical plugs, or alternatively porous spherical plugs for each or substantially all wells of a picowell array. The purpose of the plug is to keep the drug, drug candidates, cellular contents, metabolites in the well. The plug also serves to isolate the contents of the picowell from each other. The spherical plug may not need to be perfectly spherical, as long as the purpose of covering the top (or opening, or mouth) of the picowell can be fulfilled. The wells can have a top diameter and a bottom diameter. The diameter of the spherical plug before capping the well is about 10 micrometers, about 30, about 35, about 40, about 45, about 50, about 55, about 70, about 90, about 120, or about 200 micrometers. The plug can be added to cover the picowell by simply flowing it across the picowell array. Centrifugation, pressure, agitation, or other methods can be used to pack the beads into the top (or mouth or hole) of the picowell and tightly seal it. In some embodiments, a solvent can be used to swell and / or change the size of the capped beads. In some embodiments, the capped beads are loaded into a solvent that shrinks the beads, and when replaced with an assay buffer or a different solvent, the capped beads return to their original size or swell, thereby tightly sealing the picowell. In some embodiments, temperature can be used to swell or shred the capped beads to obtain a better seal at the mouth of the picowell. If necessary, the capped beads can be held in place and prevented from falling further into the picowell by one of the steps in the stepped picowell array.

[0308] The capped beads may be the same type of bead carrying the compounds of the present disclosure, or may be different types of beads. In some embodiments, the capped beads may actually be compounds with the beads themselves. The capped beads may function as passive caps that prevent or slow diffusion of molecules from the picowells, or the beads may be active beads and capture reagents from the picowells using functional moieties attached to the capped beads. In some embodiments, the porous capped beads may passively capture metabolites released from cell-based assays performed in the picowells. In some embodiments, the capped beads may non-specifically capture cellular material such as lipids, proteins, carbohydrates, and nucleic acids. In some embodiments, the capped beads may be functionalized with antibodies to specifically capture proteins released from healthy, diseased, lysed, or fixed cells. In some embodiments, the capped beads may be functionalized with DNA or RNA oligonucleotides that specifically capture cellular nucleic acids. In some embodiments, DNA or RNA functionalized capped beads may be used to capture microRNA released from cells in the capped picowells. In some embodiments, the picowell contains two beads, a compound-containing bead inside the picowell and a capped bead covering the mouth of the picowell. In some embodiments, the capped bead is also a bead with a compound. In some embodiments, the capped bead captures material released from the complex beads. In some embodiments, the capped bead captures a sample of a compound released from the complex beads. In some embodiments, the capped bead captures DNA barcodes released from the complex beads. In some embodiments, the capped beads capture different types of analytes released from within the picowells they cap.

[0309] Relative Hardness of Caps and Picowells. A preferred device is a microtiter plate, with each microtiter plate containing thousands of picowells on its bottom surface. The ability of a cap to properly seat or seal each picowell may be a function of the hardness of the plastic that makes up the holes in the picowells and the interior walls of the picowells relative to the hardness of the cap.

[0310] 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 being pressed into place. The hardness of a spherical plug, and the hardness of the walls of a picowell, can be defined in terms of 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 ascribing any of these durometer values ​​to a plastic or other material, the scale used should also be specified. 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 (Silicon Design Manual, 6 th ed., Albright Technologies, Inc., Leominster, MA).

[0311] Picowell Shape. In some embodiments, the picowell may be a cylindrical picowell, where the diameter of the cylinder is approximately the same at the top and bottom of the picowell. In some embodiments, the picowell may have a slight taper, where the top of the picowell is slightly larger than the bottom of the picowell. In some embodiments, the picowell may be a conical picowell, where the angle is anywhere between 1 degree and 30 degrees off normal. In some embodiments, the picowell is a stepped picowell, where the picowell has a discontinuous step from the diameter at the top to the diameter at the bottom (as opposed to a conical picowell, where the diameter changes smoothly from top to bottom). In some embodiments, the stepped picowell has a wide cylinder near the opening of the picowell, and a narrow cylinder near the bottom of the picowell. In some embodiments, the stepped picowell may have multiple discontinuous steps from top to bottom. In some embodiments of a multi-stepped picowell, the diameter of every step may 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 capped bead can be deposited at the top opening of the stepped picowell, hi some embodiments, a picowell can contain more than two beads.

[0312] A method for making a stepped picowell. FIG. 29 disclosed a stepped picowell. The embodiment shown has three compartments and two steps. The top compartment is the widest and is configured to receive the cap in a situation where the picowell is capped, with most of the top compartment occupied by the cap. The middle compartment is configured to be occupied primarily by reagents or by reagents alone. The reagents can include buffers, enzyme substrates, one or more salts, and preservatives or stabilizers such as dithiothreitol, RNAse inhibitors, glycerol, or DMSO. The bottom compartment is configured to be occupied by beads, i.e., beads with both DNA libraries and releasable compounds attached. In addition to having DNA barcodes and releasable compounds, the same beads can also have a "response capture element." It holds the capped beads in place and prevents them from falling further into the picowell by one of the steps in the stepped picowell. In FIG. 29, structure 1 is the cap, structure 2 is the beads, and structure 3 is the top region located just above the first step. Structure 4 is the central region and can be used to place assay reagents. The central region is just above the second step. The assay reagents in the central region can diffuse to the lowest region. Structure 5 is the lowest region and can be used to place beads and place one or more cells.

[0313] For the space of the lowest 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 lowest 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 an exclusive embodiment, the present disclosure can exclude systems or beads that do not meet any of the above parameters.

[0314] Furthermore, for the space occupied by the beads (assuming that there is only one bead in the picowell), about 50% of the beads are in the lowest section, and about 50% of the same beads are in the middle section, and these parameters can also be about 55% lowest and about 65% middle, about 60% lowest and about 40% middle, about 65% lowest and about 45% middle, about 70% lowest and about 30% middle, about 75% lowest and about 25% middle, about 80% lowest and about 20% middle, about 85% lowest and about 15% middle, about 90% lowest and about 10% middle, about 95% lowest and about 5% middle, and about 100% lowest section. To perform these calculations, the space occupied by the beads is (hypothetically) assumed to be non-porous. In an exclusive embodiment, the present disclosure can exclude systems or beads that do not meet any of the above parameters.

[0315] As with conical and cylindrical picowells, the use of a molding system is one preferred embodiment for making stepped picowells. For this purpose, a mold containing an array of multilayer pillars is desired, which when stamped into a thermoplastic or other curable polymer substrate can form the imprint of the stepped picowell. A layered pillar array with multiple steps, each step of a different diameter (smaller the higher), can be formed in a multilayer lithography process. Briefly, to crosslink the first layer of the micropillar array, a first layer of a first photoresist is exposed through a first mask. A second layer of photoresist can be deposited directly on the first layer (previously exposed) and a second pattern of the second photoresist can be crosslinked later using a second photomask. At the end of the multilayer patterning, a stack of resists can be developed to wash away the uncrosslinked areas, leaving behind an array of multilayer pillars. A detailed protocol for making multilayer pillar arrays can be found in Francisco Perdigones et al., (January 8 th, 2011). Microsystem Technologies for Biomedical Applications, Biomedical Engineering, Trends in Electronics Anthony N. Laskovski, IntechOpen. Once the array of multi-layer pillar arrays is fabricated, standard processes can be used to imprint the stepped picowell array using a mold.

[0316] Remove the capped beads. In many embodiments, it is advantageous to harvest the capped beads to study reactions, analytes, or cellular responses to chemical perturbations within the picowells. In some embodiments, the capped beads can be removed from the mouths of the picowells by inverting the picowell array and using mechanical agitation. In some embodiments, a solvent can be used to shrink the picowells so they can be easily removed from the mouths of the picowells. In some embodiments, a liquid with a higher density than the capped beads can be added to the top of the picowell array, causing the capped beads to rise by buoyancy and float on top of the dense medium.

[0317] In some embodiments, the capped beads can be crosslinked to one another, converting the capped beads into a capped sheet that can be peeled off from the top of the picowell array, hi some embodiments, a bridging gel can be poured over the capped picowells, and the bridging gel crosslinks to the capped beads and themselves, causing the capped beads to become embedded in the crosslinked sheet, which can be peeled off.

[0318] The relative positions of the picowells are preserved in the form of a peeled layer. It should be understood that in embodiments where the capped beads are interdigitated with a gel layer that can be peeled away, the relative positions of the capped beads relative to each other and to the picowells are preserved in the peeled layer. This allows direct connection between the picowells, the assays in the picowells, the beads within the picowells, and any material captured by the capped beads.

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

[0320] Fiducial markers to allow for alignment and alignment of the picowells. Arranging the picowells in an irregular array allows easy identification of shifts and drifts during imaging of the picowell array. In some embodiments, the picowells are arranged in an irregular order to facilitate detection of optical and mechanical drifts during imaging. In some embodiments, the picowell array includes 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 picowells may themselves be arranged in an easily identifiable pattern, allowing for easy alignment in the event of optical or mechanical drifts during imaging. In some embodiments, external markers such as fluorescent beads may be misted onto the picowell array to provide a fiducial pattern.

[0321] Cap-Free Mat Embodiments. Cap-free mat embodiments can take the form of a "capless film" in at least some forms or examples. Instead of sealing the openings at the top of the picowells, they can be sealed through the mat, for example to prevent evaporation of any cell culture medium or enzyme assay medium that may be in the picowells. Preferably, the mat is sized to cover all the picowells in a given picowell array. Alternatively, the mat can be sized to cover a given section of the picowells in the array. The mat can be secured to the top of the picowell plate to cover the picowells and also cover the generally flat top surface of the picowell plate between the picowells. A secure contact can be achieved by one or more of (i) maintaining a constant pressure, for example, by a hard rubber platen placed on top of the mat and acting as a weight on top of the mat, (ii) using a mat connected to a weight such as a hard rubber platen, or (iii) a reversible chemical adhesive that can be applied to the entire mat (if the mat is not an absorbent mat). When the mat is to be an absorbent mat, it includes a circular absorbent pad surrounded by a reversible chemical adhesive, where the mat is aligned in contact with the picowell array such that the circular absorbent pad covers only the opening of each picowell and does not "spill over" the opening and contact the flat surface of the picowell plate.

[0322] Membranes are available for use as mats for contacting the substantially flat surface of picowell plates and for use in capless sealing of picowells. 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 Corp, Kent, Washington. These include membranes made of polyamide-TFC, cellulose acetate, polyamide-urea-TFC, cellulose acetate blends, polypiperazine-amide-TFC, PES, composite polyamide-TFC, PES, PAN, PVDF, PSUH, RC, PESH, polyetheretherketone, and polyimide. Pore ​​sizes in terms of molecular weight cut off include 150 Da, 200 Da, 300 Da, 500 Da, 900 Da, 600 Da, 1,000 Da, 2,000 Da, 3,000 Da, 5,000 Da, 10,000 Da, 50,000 Da, 20,000 Da, 30,000 Da, 70,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 400,000 Da, 500,000 Da, 800,000 Da, 3500 Da, 0.005 micrometers, 0.030 micrometers, 0.05 micrometers, 0.10 micrometers, 0.20 micrometers, etc. In the context of the disclosed systems, compositions, reagents, and methods, these cutoff values ​​can allow for the selective collection of certain classes of compounds to the exclusion of other classes of compounds. For example, some of the membranes described above can allow small molecule metabolites to pass through and be absorbed by the absorbent mat, 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, are also available for use in the disclosed systems, compositions, and methods.

[0323] Reversible adhesion is mediated by "molecular Velcro", e.g., metalloporphyrin-containing and pyridine-containing polymers (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 top surface of the picowell plate and the other type of ssDNA covalently attached to the mat), copolymers containing catechol side chains, etc. (See Sievers et al., supra). Also, 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 could be used as a new reversible adhesive that allows its adhesive effect to be switched on and off with ease. Max-planck-Gesellschaft). Yet another reversible adhesive is available from DSM-Niaga Technology in Zwolle, The Netherlands.

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

[0325] (IX) Depositing beads into picowells The plate with picowells can take the form of a 96-well plate, each of these 96 wells containing thousands of picowells. The plate with picowells can also take the form of a 24-well plate, each of these 24 wells containing thousands of picowells. In the case of a 96-well plate, each well can be filled using 0.1-0.2 mL of a suspension of beads in water or an aqueous solution. In the case of a 24-well plate, each well can be filled using 0.5 mL of a suspension of beads in water or an aqueous solution. The suspension can be added using a regular pipette with a disposable tip. The number of beads in the suspension can result in about one-third of the picowells containing only one bead, about one-third of the picowells containing two beads, and about one-third of the picowells containing either no beads or two or more beads. The number of beads in the suspension can also depend on the circumstances, such that at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the wells containing one or more beads contain only one bead of those wells.

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

[0327] With regard to assay reagents, if the picowells are to be used to perform reactions, e.g., DNA sequencing, biochemical assays, or assays of cultured cells, assay reagents can be added to the picowells that already contain the precipitated beads. As described above for the initial addition of the bead suspension, the addition of the assay reagents is done using a pipette. After the assay reagents have equilibrated with the solution already in each picowell, any excess solution in each of the 96 wells of a 96-well plate, or in each of the 24 wells of a 24-well plate, can be drawn off with a pipette tip touching the wall of each of the 96 wells of a 96-well plate, or the wall of each of the 24 wells of a 24-well plate.

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

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

[0330] Cleaving the DNA barcodes from the beads prior to sequencing. In some embodiments, the present disclosure can encompass methods in which the bead-bound DNA barcodes are cleaved from the beads, thereby releasing the DNA barcodes in a soluble form prior to amplification, or prior to sequencing, or prior to any type of sequence identification technique, such as hybridization with a nucleic acid probe.

[0331] EXCLUSIVE EMBODIMENTS. In embodiments, the present disclosure may exclude any method, associated reagent, system, composition, or bead, in which the bead-bound DNA barcode is cleaved prior to amplification, or prior to sequencing, or prior to any type of sequence identification technique, such as hybridization with a nucleic acid probe. The present disclosure may also exclude any method in which a polynucleotide comprising a DNA barcode is cleaved, or a nucleic acid comprising only a portion of a DNA barcode is cleaved prior to amplification, or prior to sequencing, or prior to any type of sequence identification technique, such as hybridization with a nucleic acid probe.

[0332] Polymerase Chain Reaction (PCR); Quantitative PCR (qPCR). PCR and qPCR methods rely on a three-step method that (1) denatures the DNA template at high temperature, anneals the primers at reduced temperature, and finally extends the primers via DNA synthesis as catalyzed by DNA polymerase (Gadkar and Filion (2014) Curr. Issues Mol. Biol. 16:1-6). qPCR is also referred to as "real-time PCR" (Kralik and Ricchi (2017) Frontiers Microbiology. 8 (page 9)).

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

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

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

[0336] Rolling Circle Amplification (RCA). DNA can be amplified while still attached to the beads. The amplified form of DNA is easier to sequence than the unamplified DNA. In rolling circle amplification, DNA tags (DNA barcodes) are made single stranded. Once single stranded, splint oligos are added to bridge the ends of the tag DNA, followed by extension and ligation of the splint oligos. Using a DNA polymerase (minus 5'→3' exonuclease activity) ensures that the DNA has a ligatable junction after catalyzing the extension of the splint oligo. The circularized DNA can then be subjected to rolling circle amplification by adding a strand displacing DNA polymerase, such as phi29 DNA polymerase. The ability to perform rolling circle amplification (RCA) on DNA barcode tags allows the use of synthetic chemicals that may damage DNA, as any surviving DNA molecules can be thermally amplified to sufficient quantities to easily sequence. DNA can be made single stranded by exonuclease digestion, nicking and melting at high temperature, or treatment with sodium hydroxide.

[0337] The details of rolling circle amplification (RCA) are elucidated in the following procedure that can be used to perform RCA.

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

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

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

[0341] Step 4: DNA ligase is used to covalently close the complementary giant loop, generating a circular ssDNA. It is this closed ssDNA ring that does the "rolling" during RCA.

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

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

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

[0345] In one embodiment of the disclosure, the RCA amplified ssDNA is cleaved from the beads prior to PCR amplification to generate PCR products. In another embodiment of the disclosure, the PCR amplification to generate PCR products can be generated without cleaving the RCA amplified ssDNA from the beads.

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

[0347] The DNA barcodes of the present disclosure can be sequenced using the methods of Vander Horn, U.S. Patent No. 8,632,975, which is incorporated herein by reference in its entirety, without implying any limitation.The DNA barcodes of the present disclosure can also be sequenced by methods using sequencing by synthesis, such as, for example, Sanger sequencing, or by methods using "next-generation sequencing."

[0348] 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: e1004462). In the Illumina method, a DNA library is created and fragments of DNA from cells or from cells are modified with DNA adapters (attached to the ends of the fragments). The reaction product takes the form of a sandwich, with the DNA to be sequenced in the center of the sandwich. The reaction product takes the form of (first adapter)-(DNA to be sequenced)-(second adapter). The adapter-DNA-adapter complex is associated with yet another adapter, and this other adapter is covalently attached to a solid surface. The solid surface can be a flat plate. The solid surface has a lawn of many adapters protruding from the plane. The adapter has a DNA sequence complementary to one of the adapters that is in the sandwich. In fact, the lawn contains two types of adaptors, one of which binds (hybridizes) to one of the adaptors in the complex, non-covalently tethering the complex to the plate. These can be referred to as the "first lawn-bound adaptor" and the "second lawn-bound adaptor." The first task of the DNA polymerase is to use the tethered (but non-covalently bound) DNA as a template to create a daughter strand, and as DNA polymerization occurs, the daughter strand becomes covalently attached to the "first lawn-bound adaptor." This covalent link is generated by the catalytic action of the DNA polymerase. After the daughter strand is fully synthesized, the distal end (the end that protrudes into the medium) contains a DNA sequence complementary to the second adaptor in the sandwich referred to above. This complementary DNA sequence allows the distal end of the newly synthesized daughter DNA to bend and hybridize to the "second lawn-bound adaptor." Described above is how both adapters in the sandwich are used, as well as how both a "first lawn joining adapter" and a "second lawn joining adapter" are used.

[0349] Then, cycles of the reaction are carried out many times, resulting in a cluster of amplified versions of the original dsDNA. In fact, the cluster takes the form of covalently attached (tethered) ssDNA molecules, all of which correspond to only one of the strands of the original dsDNA (the dsDNA isolated from the living cell or tissue). This cluster of tethered ssDNA molecules is called a "polony". The generation of the polony is by a technique called "bridge amplification". Finally, after bridge amplification and the creation of the polony, the reverse strand, which is covalently attached to the solid surface, is cleaved from its tether, washed away and discarded, leaving only the forward strand.

[0350] Information regarding the Illumina® methodology 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).

[0351] Sequencing by Oligonucleotide Ligation and Detection (SOLiD Sequencing). SOLiD measures the fluorescence intensity from dye-labeled molecules to determine the sequence of DNA fragments. A library of DNA fragments is prepared from the sample to be sequenced and used to prepare a clonal bead population (only one type of fragment on the surface of each magnetic bead). The fragments attached to the beads are given a universal P1 adapter sequence attached so that the starting sequences of all fragments are both known and identical. PCR is performed and the bead-attached PCR products are covalently bound to the slide.

[0352] The primer then hybridizes to the P1 adaptor sequence in the library template. A set of four fluorescently labeled dibase probes compete for ligation to the sequencing primer. The specificity of the dibase probes is achieved by interrogating every first and second base of each ligation reaction. Multiple cycles of ligation, detection, and cleavage are performed, with the number of cycles determining the length of the final read. Following a series of ligation cycles, the extension product is removed and the template is reset with a primer complementary to the n-1 position of the second ligation cycle (see Wu et al (2010) Nature Methods. 7:336-337).

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

[0354] For concatenated DNA barcodes, the entire concatenated DNA barcode can be sequenced in one run (only one sequencing primer is required to sequence the entire concatenated DNA barcode). Alternatively, some or all of the DNA barcode modules that make up the concatenated DNA barcode can be subjected to sequencing separately (each separately sequenced DNA barcode module gets its own sequencing primer). For orthogonal DNA barcodes, each DNA barcode module that makes up the orthogonal DNA barcode requires its own dedicated sequencing primer, since each DNA barcode module is attached to a unique site on the bead.

[0355] EXCLUSIVE EMBODIMENTS: In embodiments, the present disclosure may exclude any systems, devices, combinations of devices, and methods involving microfluidics, aqueous droplets present in an oil medium, the aqueous droplets being created by connecting a first channel containing an aqueous reagent with a second channel containing oil to generate aqueous droplets that travel through the oil medium via a third channel originating from the connecting region. Microfluidic devices and reagents are described (see, e.g., Brouzes, Medkova, Savenelli (2009) Proc. Natl. Acad. Sci. 106:14195-14200; Guo, Rotem, Hayman (2012) Lab Chip. 12:2146-2155; Debs, Utharala, Balyasnikova (2012) Proc. Natl. Acad. Sci. 109:11570-11575; Sciambi and Abate (2015) Lab Chip. 15:47-51).

[0356] In other exclusive embodiments, any reagent, composition, nucleic acid, or bead that constitutes the "DNA headpiece" or is covalently attached to the "DNA headpiece" can be excluded. MacConnell, Price, Paegel (2017) ACS Combinatorial Science. 19:181-192 provides an example of a DNA headpiece, where beads are functionalized with an azide DNA headpiece.

[0357] Additional exclusionary embodiments regarding sequencing methods and sequencing reagents. In an embodiment, the present disclosure can exclude any reagent, system, or method that does not involve the use of "reversible terminators" in DNA sequencing. Also excluded can be any reagent, system, or method that does not include a methoxy protecting group. Additionally, excluded can be any reagent, system, or method that involves DNA sequencing, but where the DNA being sequenced is not covalently attached to a bead when information about the order of the polynucleotide is detected and collected. Additionally, excluded can be any reagent, system, or method that amplifies the DNA template before performing the sequencing reaction, for example, by PCR or rolling circle technology. In an embodiment, excluded can be any method of barcoding, such as, for example, concatenated (all information about the synthesis of the members of a chemical library present on one single nucleic acid) nucleic acid barcoding. In another aspect, excluded can be any method of barcoding, such as, for example, orthogonal (information about the synthesis of a given monomer of a compound library distributed across multiple attachment locations on a bead) nucleic acid barcoding. In exclusive embodiments relating to DNA ligase, the present disclosure can exclude any reagent, system, or method that uses DNA ligase to connect modules of nucleic acid barcodes.

[0358] Fluorophores, quenchers, and FRET-based assays. The present disclosure provides fluorophores and quenchers for screening members of a chemical library or for characterizing isolated members of a chemical library. FRET is Förster resonance energy transfer.

[0359] Assays can be performed on bead-bound chemical libraries, assays can be performed on free chemical library members immediately after cleavage from the beads, i.e., in the same microwells as the beads or in the same hydrogel matrix vicinity as the beads, and assays can be performed on soluble chemical library members that are not attached to any beads or that have been cleaved from the beads and then purified.

[0360] Fluorophores suitable for use as reagents in 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, Ill.), 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-carboxyfluorescein), SYBR Green I (absorbs at 497 nm, 520 nm, and 540 nm, respectively). Fluorescent quenchers include fluorophore and quencher. Quenchers include TAMRA quencher, black hole quencher-1 (BHQ1), black hole quencher-2 (BHQ2), and DABCYL quencher. As disclosed elsewhere in this patent document, it is noted that TAMRA can be both a fluorophore and a quencher.

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

[0362] The present disclosure provides reagents, compositions, and methods for discovering and identifying enzyme inhibitors, enzyme activators, and screening libraries of compounds to discover compounds that can enhance the rate of in vivo degradation of a given protein. These reagents, compositions, and methods can use FRET-based assays, or alternatively, can use assays other than FRET-based assays.

[0363] Molecular beacons have been described (see Baruch, Jeffery, Bogyo (2004) Trends Cell Biology. 14:29-35). Molecular beacons are reagents in which a fluorophore is attached to a quencher via a linker. The linker may be cleavable by a nuclease, thus measuring nuclease activity. The present disclosure provides methods for screening chemical libraries to identify nuclease inhibitors, or alternatively, to identify nuclease activators. Feng et al. described 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. demonstrated the use of FRET-based assays to measure the activity of various restriction enzymes.

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

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

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

[0367] Photocleavable Linkers The present disclosure encompasses photocleavable linkers that have an o-nitrobenzyl group. This group can be cleaved by irradiation at 330-370 nm (see Saran and Burke (2007) Bioconjugate Chem. 18:275-279; Mikkelsen, Grier, Mortensen (2018) ACS Combinatorial Science. DOI:10.1021). A linker that has a shorter photolysis time than 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 are available, which have shorter photolysis times and greater release yields than unsubstituted o-nitrobenzyl linkers. Phenacyl, benzoin, and pivaloyl linkers are also available (see Mikkelsen et al (2018) ACS Combinatorial Science. DOI:10.1021).

[0368] Linkers with photocleavable ether bonds are available. These photocleavable linkers can be used when the linker is attached to a 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). Linkers with photocleavable ester bonds are also available (see Rich et al (1975) 97: 1575; Renil and Pillai (1994) Tetrahedron Lett. 35: 3809-3812; Holmes (1997) J. Org. Chem. 62: 2370-2380, cited by Glatthar and Giese, supra). The ether bond of the linker can be cleaved by acid, base, oxidation, reduction, and fluoride sensitive silyl-oxygen bonds, and by photolysis (Glatthar and Giese, supra).

[0369] Another photocleavable linker that has been used to link peptides (R1) and nucleic acids (R2) is as follows: R1 is connected directly to the methylene moiety of a benzyl group. Para to the methylene group is a nitro group with a ring attached. Meta to the methylene moiety is an ethyl group with a ring attached. One carbon of the ethyl group carries a phosphate. To the oxygen atom of this phosphate is attached the R2 group (Olejnik et al (1999) Nucleic Acids Res. 27:4626-4631).

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

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

[0372] Bead-bound emission monitor. The present disclosure provides a novel and unique emission monitor capable of assessing the emission of bead-bound compounds. The emission monitor takes the form of a bead-bound complex of a fluorophore and a quencher, where the fluorophore is connected to the bead via a cleavable linker. Preferably, the cleavable linker is a photocleavable linker. Preferably, the bead-bound emission monitor is placed in a dedicated picowell, where the picowell does not contain other types of beads. When the photocleavable linker is cleaved, the fluorophore is released from the bead, diffuses into the medium in the picowell, and achieves some distance from the bead-bound quencher, resulting in an increase in fluorescence proportional to the amount of emission. The increase in fluorescence allows the calculation of the concentration of free fluorophore in the picowell, and more importantly, the amount of compound released from other beads in other wells.

[0373] Briefly, a bead-bound release monitor is placed in its own dedicated well, while other wells contain bead-bound compounds that are drug candidates.

[0374] FIG. 8 discloses a simplified version of a preferred, non-limiting example of a bead-bound emission monitor. The emission monitor takes the form of a quencher held in close proximity to the 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 the picowell, one bead is dedicated to the emission monitor, and the other bead or beads are used to attach the compound and to attach the DNA library. Exposing all beads in the picowell to UV light simultaneously cleaves the fluorophore and the compound. QSY7 is a preferred quencher. The structure and CAS number of QSY7 are as follows (see below):

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

[0376] The increase in fluorescence resulting from the separation of the fluorophore from the quencher can be used to infer the concentration of the simultaneously released compound in the picowell. The increase in fluorescence resulting from the separation of the fluorophore from the quencher can also be used to infer the number of molecules present in free form in the picowell (molecules in the form of the compound that was previously a bead-bound compound). In a more preferred embodiment, the emission monitor comprises a quencher and a fluorophore, and cleavage results in the release of the fluorophore (and no release of the quencher). This embodiment provides lower background noise than the less preferred embodiment below. In a less preferred embodiment, cleavage releases the quencher, and the readout takes the form of an increase in fluorescence from the bead-bound fluorophore.

[0377] The release monitor provides the user with a measurement of the concentration of soluble compounds and subsequently the UV-induced release of the compounds from the beads. In a preferred embodiment, one type of bead is dedicated to being a release monitor. By "dedicated" we mean that the beads do not contain bead-bound compounds or bead-bound DNA libraries.

[0378] As a general proposition, it should not be inferred that a compound is now a soluble compound just because it has been released from the bead by cleavage of the photolabile linker. First, it should be noted that just because a compound is considered "hydrophobic" or "water-insoluble" does not mean that the molecule does not move freely in the 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). Furthermore, the biochemical availability of bead-bound water-insoluble compounds can be increased by additives such as surfactants, detergents, DMSO, or carriers such as human serum albumin. Thus, the release monitor can be used to assess the overall concentration of a compound that has limited or no water solubility under conditions in which the picowell contains one of the above agents, or alternatively, the water-insoluble compound is released near the plasma membrane of live cells cultured inside the picowell.

[0379] FIG. 9 discloses a simplified version of a preferred embodiment of a bead-bound release monitor, and FIG. 10 discloses the complete and detailed construction of this preferred embodiment of a bead-bound release monitor.

[0380] FIG. 30 provides data showing the use of a bead release monitor, where the beads are in picowells. The bead-bound fluorophore, attached using a photocleavable linker, is TAMRA (excitation wavelength 530 nm, emission wavelength 570 nm). The figure shows the time course of release of the fluorophore from the beads. It shows the actuation of the bead-bound release monitor, acquiring fluorescence data at t=0 s, t=1 s, t=11 s, and t=71 s. FIG. 30 also includes insets showing enlargements of the smaller figures for two of the four smaller figures. FIG. 30 was obtained from incubation of the aspartyl protease cathepsin-D with "Peptide Q-Fluoro Substrate" and beads. The reagents were placed in the wells at 4 degrees. Using 365 nm ultraviolet light, the fluorophore was cleaved from the beads, thereby releasing the fluorophore and separating it from the quencher. The purpose of this assay was to evaluate the time course of release performed in separate wells, where the separate wells contained different types of beads. The different types of beads had the same photocleavable linker, which was attached to pepstatin-A. The release of pepstatin-A can be inhibited by binding with aspartyl protease present in the same assay medium. This setup with bead-bound pepstatin-A and aspartyl protease serves as a positive control.

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

[0382] FIG. 35 discloses further details regarding the enzyme assay, where bead-bound pepstatin-A is released and the released pepstatin-A results in enzyme inhibition. 10 μm TentaGel beads displaying photocleavable pepstatin-A (positive control) and a covalently bound Cy5 label were mixed with 10 μm TentaGel beads displaying photocleavable Fmoc-valine (negative control) in PBST buffer. This bead population was introduced into a picowell, whereupon the buffer was added to Cathepsin D protease and Peptide Q-fluorescent substrate (λ ex = 480 nm, λ em The wells were encapsulated with air and the entire slide was then exposed to UV (365 nm, 77 J / cm). 2 ), cleaving the photolabile linker and releasing the compound to reach approximately 13 μM. The flow cells were incubated (30 min, 37° C.). Wells containing positive control beads should inhibit peptide proteolysis by cathepsin-D and have a low fluorescent signal. Wells containing negative control beads should not show any cathepsin-D inhibition and should have a similar fluorescent intensity to the empty wells.

[0383] The terms quencher and fluorophore can vary for a given chemical, depending on other chemicals occurring in close proximity. TAMRA, used in the bead-bound emission monitor laboratory data, is the fluorophore, but in other situations, TAMRA may be the quencher. TAMRA functions as a quencher in TaqMan® probes, including FAM and TAMRA.

[0384] Further description of the experimental setup and laboratory data. This disclosure provides data on controlled 5(6)-carboxytetramethylrhodamine (TAMRA) concentrations in phosphate buffer (10 mM phosphate, 154 mM sodium, pH 8.0) in filled picowells partitioned with air. The captured fluorescence images (10 ms, 2 ms exposure) and well area quantified by average pixel intensity (n≧100) generate a standard curve of concentration vs. fluorescence intensity. The above data takes the form of a standard curve, showing the fluorescence at various predefined concentrations of free TAMRA (2, 10, 30, 60, 100 mM TAMRA). This standard curve was prepared under two different conditions, i.e., photographic images were taken with 2 ms exposure or 10 ms exposure. The experiment used to prepare the standard curve was performed in picowells, but without any beads (known amount of TAMRA) in this experiment. Since the data simply takes the form of a standard curve, which may also be referred to as a calibration curve, photographic images are not shown in this patent document.

[0385] The experimental set-up includes the following: For Scheme X), TentaGel-Lys(PCL1-Tamra)-QSY7 bead structure. QSY7 (grey), which quenches the Tamra fluorophore (orange), is covalently attached to the bead via a photocleavable linker (purple). Irradiation from UV (365 nm) provides quantitative release of the compound in situ.

[0386] FIG. 31 discloses luminescence data after catalysis of an aspartyl protease on a quencher-fluorophore substrate. Higher fluorescence means that the enzyme is more catalytically active. Lower fluorescence means that the enzyme is less catalytically active, i.e., the enzyme is more inhibited by the free inhibitor, which is released from the beads, the release being obtained by cleavage of the photocleavable linker. Images were captured after UV emission and incubation of the cathepsin-D assay (λ ex = 480 nm, λ em = 525 nm). Wells containing positive control beads can be spectrally identified by the Cy5 fluorophore (λex = 645 nm, λ em = 665 nm, orange false color). Sections were analyzed with a line plot across the open well volume; wells containing negative control beads induce no cathepsin-D inhibition. The assay volume in wells containing positive control beads is dark, indicating strong inhibition. The assay volume in empty wells is comparable to wells containing negative control beads.

[0387] FIG. 32 illustrates the procedure that follows. Further to Scheme X), picowell substrate (46 pL per well) is sealed into a flow cell, the wells are wetted under vacuum, a suspension of TentaGel-Lys(PCL1-TAMRA)-QSY7 beads is introduced, and air is drawn across the flow cell to compartmentalize each well (top). The flow cell is illuminated with a UV LED (λ 2 ) with a controlled light flux before a fluorescence microscope image is taken to quantify the released compound (TAMRA) concentration (bottom). 平均値 365 nm) and equilibrated (20 min) (Figure 32). In detail, Figure 32 shows a cross-section of a picowell and illustrates the steps of wetting the picowell in the flow cell, beads in suspension are introduced over the picowell resulting in one bead per picowell, air is drawn across the flow cell to reduce excess dispersion and allow the meniscus to fall below the surface of the flat top surface of the picowell plate, controlled UV exposure (365 nm), resulting in the release of some TAMRA, and detection of the fluorescent signal with a fluorescent microscope to induce emission from TAMRA (excitation at 531 / 40 nm) (emission at 594 / 40 nm). The "slash 40" notation refers to bandwidth, i.e., this means that the cutoff filter limited the light to the range of 531 nm plus 20 nm and minus 20 nm, and 594 nm, plus 20 nm and minus 20 nm (this slash notation can also be used for excitation and emission wavelengths).

[0388] We have acquired photographs showing the following data (see FIG. 33): Fluorescence emission (λ) of the fluorophore (TAMRA). ex 531 / 40nm, λem 593 / 40) was released from 10 μm TentaGel-Lys(PCL1-TAMRA)-QSY7 beads after exposure to a UV LED (365 nm) in a Picowell flow cell. A) Due to the FRET quenching effect of QSY7, the FRET was released from 10 μm TentaGel-Lys(PCL1-TAMRA)-QSY7 beads before UV exposure (0 J / cm 2 ) there is no significant emission above background. TAMRA emission is (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 Following UV exposure, equilibrium (20 min) was allowed to be reached and then imaged using the appropriate exposure time. To measure TAMRA concentration, fluorescence emission was measured within the volume surrounding each bead (Figure 33). The "slash 40" notation refers to bandwidth, i.e., this means that the cutoff filter restricted the light to a range of 531 nm plus and minus 20 nm (this slash notation can also be used for excitation and emission wavelengths).

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

[0390] (XII) Biochemical Assays of Compounds (Non-Cell-Based Assays) A variety of biochemical assays are possible using the beads in the picowells. Non-limiting examples include binding assays, enzyme assays, catalytic assays, fluorescence-based assays, luminescence-based assays, scattering-based assays, etc. Examples are provided below.

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

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

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

[0394] The purpose of activating p53 to reduce cancer. The relationship between MDM2, a transcription factor called "p53", and anti-cancer therapy is proposed by the following statement: "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. p53 activity is inhibited by MDM2. According to Wu et al., MDM2 is a "p53 binding protein" (see Wu, Buckley, Chernov (2015) Cell Death Disease. 6: e 2035). For example, by blocking the interaction between MDM2 and p53, compounds will prevent ubiquitination of p53 and are predicted to function as anti-cancer agents.

[0395] The purpose of the screening assay. The purpose of the screening assay is to find compounds that affect p53 ubiquitination, such as compounds that stimulate p53 ubiquitination and compounds that inhibit p53 ubiquitination. In particular, the purpose is to find compounds that inhibit or activate, and their effects are mediated by MDM-2 and either E1 ligase, E2 ligase, or E3 ligase. MDM2 stands for "mouse double minute chromosome." MDM2 is referred to as an "E3 ubiquitin ligase." When MDM2 occurs in cells, evidence suggests that many other proteins, such as CUL4A, DDB1, and RoC1, are required for its activity to catalyze p53 ubiquitination (see Banks, Gavrilova (2006) Cell Cycle. 5:1719-1729, Nag et al (2004) Cancer Res. 64:8152-8155). Banks et al. described a physical interaction involving p53 and MDM2, "We found that L2DTL, PCNA, and the DDB1 / CUL4A complex physically interact with the p53 tumor suppressor and its regulators MDM2 / HDM2" (Banks, Gavrilova (2006) Cell Cycle. 5:1719-1729). Nag et al. further described a physical interaction involving p53 and MDM2, "Cul4A functions as an E3 ligase and is involved in the proteolysis of several regulatory proteins via the ubiquitin-proteasome pathway. Here we show that Cul4A associates with MDM2 and p53" (Nag et al (2004) Cancer Res. 64:8152-8155).

[0396] Desired readout from bead-based assay of modifiers of p53 ubiquitination. Compounds are screened to produce positive screening hits, i.e., AF488 fluorescence is increased, which means that an activator has been found. Compounds are screened to produce positive screening hits, i.e., fluorescence is reduced, which means that an inhibitor has been found. Compounds that inhibit p53 ubiquitination suggest that the compound can be used to treat cancer. Compounds also specifically inhibit p53 ubiquitination, i.e., the compound does not inhibit the ubiquitination of other proteins, or the compound inhibits the ubiquitination of other proteins, but less severely than p53, which also suggests that the compound can be used to treat cancer.

[0397] Materials. Materials include E3 Ligase Kit K-200B from Boston Biochem. The Boston Biochem catalog describes kits such as Mdm2 / HDM2 Ubiquitin Ligase Kit - p53 Substrate. The following is regarding Mdm2, which is part of this kit. This kit does not include cereblon. Lenalidomide and similar compounds can bind to either cereblon or Mdm2, with the end result being activation of ubiquitin ligase. Materials further include Diamond White Glass Microscope Slides, 25mmx75mm (Globe Scientific, Paramus, NJ). Corning Stirrer / Hotplate (Settings 0-10) 698 Watts, Model PC-420. N-Hydroxy-Succinimide (NHS). Methyltetrazine (mTET). AlexaFluor488 (AF488) (ThermoFisher Scientific). TentaGel beads M NH2 (catalog number M30102) (Rapp Polymere GmbH). Parafilm (Sigma-Aldrich, St. Louis, MO). Figure 8 shows the structure of Alexa Fluor® 488. The structure of Alexa Fluor 488 (AF488) is shown in the product information for AlexaFluor488-Nanogold-Streptavidin (Nanoprobes, Inc., Yaphank, NY).

[0398] Cell-based assays for compounds (XIII) The cell-based assays performed in the picowells can use human cells, non-human cells, human cancer cells, non-human cancer cells, bacterial cells, cells of parasites such as malaria parasite cells. Also, the cell-based assays can be performed with "killed but metabolically active" human or non-human cells, i.e., their genomes are crosslinked to allow metabolism but prevent cell division (U.S. Patent Publication No. 2007 / 0207170 to Dubensky, incorporated herein by reference in its entirety). Additionally, the cell-based assays can be performed with apoptotic, necrotic, or dead cells. Cell-based assays using bacterial cells can be used to screen for antibiotics. Virus-infected human cells can be used to screen for antiviral agents. Combinations of cells are provided for the cell-based assays. For example, combinations of dendritic cells and T cells are provided to screen and identify compounds that stimulate antigen presentation or, alternatively, impair antigen presentation.

[0399] Cell-based assays can be based on primary cultures of cells obtained, for example, from a biopsy of normal tissue, a biopsy from a solid tumor, or a hematological cancer, or from circulating solid tumor cells, and cell-based assays can be based on cells that have been passaged one or more times.

[0400] 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 two cells, about three cells, about four cells, about five cells, or multiple cells, or less than three cells, less than four cells, less than five cells, etc.

[0401] Applicants have conducted practical testing based on the following technique, which describes a cell-based assay for screening compounds of exemplary embodiments in which lenalidomide (a test compound) inhibits ubiquitin-mediated proteolysis of transcription factors. The transcription factors include Ikaros and Aiolos.

[0402] The present disclosure provides a cell-based assay for screening compounds on bead-bound compounds, the screening being performed in a plate with many picowells. The components of the cell-based assay include picowells for holding bead-bound chemical libraries, each bead having substantially only one uniform type of compound attached thereto. The compounds are released via cleavable linkers. Mammalian cells are cultured in the picowells. The picowells further comprise culture medium. The presently disclosed non-limiting example of lenalidomide is a proof-of-principle example that can be used to screen chemical libraries to discover other compounds that control the ubiquitination of a given target protein.

[0403] A brief description of the cell-based assay. Recombinant cells are used as reagents to detect and screen compounds that induce proteolysis of green fluorescent protein (GFP), and the readout identifying a positive screening compound is the situation where green cells become colorless or less green. Regarding the mechanism of this cell-based assay, the mechanism of action of lenalidomide where green cells become colorless or less green is generated is that lenalidomide binds to a protein called "cereblon." Intracellularly, cereblon is part of a complex of proteins called "E3 ubiquitin ligase." Cereblon is the direct target of the anticancer drugs lenalidomide, thalidomide, and pomalidomide. The normal constitutive activity of E3 ubiquitin ligases, and its relationship to cereblon, has been described as "cereblon promotes proteosomal degradation [of target proteins] by binding E3 ubiquitin ligases" (see Akuffo et al (2018) J. Biol. Chem. 293:6187-6200). In contrast to the normal activity of E3 ubiquitin ligases, the addition of drugs such as lenalidomide, thalidomide, or pomalidomide results in "lenalidomide, thalidomide, and pomalidomide promoting(s) ubiquitination and degradation of substrates by E3 ubiquitin ligases, and each of these drugs induces degradation of the transcription factors IKZF1 and IKZF3" (Kronke et al (2015) Nature. 523:183-188).

[0404] Regarding terminology, cereblon has been described as being part of a complex of proteins referred to as an "E3 ligase" and also as an "E3 ubiquitin ligase." Generally, cereblon itself is not referred to as an "E3 ligase. The following excerpt illustrates how the word "cereblon" is used. According to Akuffo et al (2018) J. Biol. Chem. 293:6187-6200, "binding of thalidomide to the E3 ligase substrate receptor cereblon promotes proteosomal destruction [of the substrate] by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase." Consistently, Yang et al (2018) J. Biol. Chem. 293:10141-10157 disclose that "cereblon functions as a substrate receptor for the cullin-4 RING E3 ligase to mediate protein [substrate] ubiquitination." Zhu et al (2014) Blood. 124:536-545 state that "Thalidomide binds to CRBN [cereblon] and alters the function of an E3 ubiquitin ligase complex composed of CRBN, DDB1, and CUL4." Lopez-Girona et al (2012) Leukemia. 26:2326-2335 state that "Studies have identified the E3 ligase protein cereblon (CRBN) as a direct molecular target of thalidomide, and CRBN and DDB1 form a functional E3 ligase complex with Cul4A and Roc1."

[0405] To put the cell-based assay devised and used by applicants in perspective, the first step is the addition of lenalidomide to the cells. The final step is that IKZF1 and IKZF3 become impaired. If IKZF1 occurs as a fusion protein with GFP, the final step is that the entire fusion protein is degraded by the proteasome. Similarly, if IKZF3 occurs as a fusion protein with GFP, the final step is that the entire fusion protein is degraded by the proteasome. GFP degradation results in the conversion of previously green fluorescent cells to non-fluorescent cells.

[0406] A lengthy description of cell-based assays. It relates the protein names of E3 ubiquitin ligases (a complex of proteins), the names of proteins that bind to this complex, and the names of proteins that are targets of this complex. The published literature is inconsistent regarding these names. Sometimes proteins are referred to by their protein names, and sometimes the name of the gene that codes for the protein is used to refer to the protein. For this reason, the following reports use protein and gene names together, such as "cereblon" (protein name) and "CRBN" (gene name). Also, "Ikaros" is the protein name and the gene name is IKZF1. Also, "Aeolus" is the protein name and IKZF3 is the gene name. "Cullin-ring finger ligase-4" is the protein name and the gene name is CRL4. "Regulator of cullin-1" is the 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 the name of the protein and the name of the gene is CUL4A. See Schafer, Ye, Chopra (2018) Ann. Rheum. Dis. DOI:10.1136; Chen, Peng, Hu (2015) Scientific Reports. 5:10667; Matyskiela et al (2016) Nature. 535:252-257; Akuffo et al (2018) J. Biol. Chem. 293:6187-6200).

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

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

[0409] This is relevant for cell-based assays in which any given microwell, nanowell, or picowell contains beads, the beads having covalently linked compounds attached via cleavable linkers, and the wells contain one or more cultured mammalian cells. Responses to the compounds and drug candidates of the present disclosure can be assessed by one or more biomarkers.

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

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

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

[0413] Apoptosis. Real-time data on apoptosis, and early events in apoptosis of single cells, can be measured using surface-enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) (see Stojanovic, Schasfoort (2016) Sensing Bio-Sensing Res. 7:48-54; Loo, Lau, Kong (2017) Micromachines. 8:338. DOI:10.3390). Stajanovic, supra, detects the release of cytochrome C, EpCam, and CD49e from cells. Loo et al., supra, measure the release of cytochrome C from cells, and detection involves a DNA aptamer, which functions like an antibody. Zhou et al. used SERS to detect early apoptosis in single cells, and it is the phosphatidylserine on the cell membrane that is measured (see Zhou, Wang, Yuan (2016) Analyst. 141:4293-4298). In addition to collecting data on apoptosis, SERS can be used to assess drug activity by collecting data on the stage of mitosis, release of metabolites, and expression of biomolecules associated with 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 apoptosis (see Kang, Austin, El-Sayed (2014) ACS Nano. 8:4883-4892). Plasmon resonance (SERS) can distinguish between cancer cells and normal cells by measuring the proportion of mitotic proteins in alpha-helical and beta-sheet forms (Panikkanvalappil, Hira, El-Sayed (2014) J. Am. Chem. Soc. 136:159-15968). The above methods are suitable as reagents and methods for the present disclosure.

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

[0415] General Information Regarding Cell-Based Assays: The cell-based assays of the present disclosure may be used to test responses from human cancer cells, cells from solid tumors, cells from hematological cancers, human stem cells, human liver cells, pathogens, infectious bacteria, human cells infected with bacteria, human cells infected with viruses, etc. The assays can detect morphological responses of cells, such as migration, as well as genetic and biochemical responses.

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

[0417] The present disclosure provides a population of cells in a picowell array. In embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the cell population is present inside the picowell (not in any area located at the top of the picowell). In embodiments, the percentage of cells present inside the well, with the remainder located in a layer of nutrient medium located at the top of the array of 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 a range defined by two of these values, such as the range "from about 60% to about 90%."

[0418] Matrices for cells. In assays of biological activity of cells, cells are exposed to compounds released from the beads or cells are exposed to compounds bound to the beads, suitable matrices include those containing one or more of poly-D-lysine (PDL), poly-L-lysine (PLL), poly-L-ornithine (PLO), vitronectin, osteopontin, collagen, peptides containing RGD configurations, polypeptides containing RGD configurations, laminin, laminin / fibronectin complexes, laminin / entactin complexes, etc. Suitable matrices also include products available from Corning, Inc., such as PuraMatrix® Peptide Hydrogel®, Cell-Tak® cell and tissue adhesive, Matrigel®, etc. See Corning Life Sciences (2015) Corning Cell Culture Surfaces, Tewksbury, MA (page 20); 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.

[0419] In embodiments, the present disclosure provides an array, where each picowell contains beads, 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, MatriGel®, etc.

[0420] Activity of the bead-bound or bead-released compounds can be performed. Assays to assess activity include activation or inhibition of an enzyme, activation or inhibition of a cell signaling cascade or individual cell signaling proteins, binding to an antibody (or complementarity determining region (CDR) of an antibody, variable region of an antibody), inhibition of binding of a ligand or substrate to an enzyme (or antibody, or variable region of an antibody).

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

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

[0423] In one exemplary workflow, the following procedure may track the transcriptional response of cells to drugs: (a) provide picowells designed to capture a single cell per well; (b) introduce compound-loaded DNA barcoded beads into the picowells such that one bead is present per picowell; (c) release the compound from the beads in each picowell by an appropriate method (UV treatment of the compound attached via a UV-cleavable linker, diffusion when the beads are immersed in an acid-cleavable, base-cleavable, temperature-cleavable compound, etc., as appropriate for the embodiment beads); (d) the picowells may be isolated from each other via capped beads that retain the contents within the picowells, or by other means such as an air or oil barrier on top of the picowells; (e) incubate the cells in the picowells in the presence of the compound released from the beads for a period of time. (f) After a suitable time, for example 1 hour, 2 hours, 5 hours, 9 hours, 12 hours, 15 hours, 18 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, or another suitable time based on the assay, the cells are lysed by a lysis method. The lysis method may involve the addition of detergent, repeated cycles of freezing and thawing, heating, the addition of membrane disrupting peptides, mechanical agitation, or other suitable means. (g) Upon lysis, the contents of the cells are exposed to the beads in the picowells, at which point the response capture elements on the beads in the picowells allow for the capture of their intended response. In some embodiments, the response capture is a poly-T sequence that captures the complete mRNA profile of the cell (or cells) in each picowell. In some embodiments, the response capture element is designed to capture a specific DNA or RNA sequence from the cells. In some embodiments, the transcriptional response of the cells may be captured as a function of the dose (or concentration) of the compound.

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

[0425] In some embodiments, the methods described herein may include two responses, a perturbation response encoded by a perturbation barcode and a measurement response. In a perturbation response, a cell may be subjected to a perturbation. In a measurement response, a cellular response as a result of the perturbation may be measured. In some embodiments, the measurement response may include a measurement barcode. In other embodiments, the method may include carrying over the perturbation barcode to the measurement response and relating the perturbation to the cellular response by capturing both the measurement barcode and the perturbation barcode included in the measurement response.

[0426] The methods described herein can include a perturbation response encoded by a perturbation barcode. The perturbation response includes subjecting a cell to a perturbation and subsequently measuring the cellular response of the cell to the applied perturbation. The perturbation barcode can be decoded either before or after measuring the cellular response, thus relating the identity of the perturbation to the measured cellular response.

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

[0428] In some embodiments, the method includes capturing individual cells in droplets. The cells may contain nucleic acid barcodes on their cell membranes to uniquely identify the perturbations the cells experience. The cells may be lysed in the droplets, and the cell's mRNA and the nucleic acid barcodes bound to the cell membrane may be captured with a set of barcoded capture oligonucleotides ("capture barcodes"). Each droplet may contain a different unique barcoded capture oligonucleotide, and the barcodes within one droplet have a substantially identical span of sequence. The mRNA and the barcodes bound to the cell membrane may be copied into the droplet barcoded oligonucleotides using reverse transcriptase. The nucleic acid material may be pooled together from the droplets by disrupting the droplets. All the nucleic acid material may be sequenced to study the transcriptional profile of the individual cells and relate it to the perturbations associated with the transcriptional profile.

[0429] The methods described herein may include subjecting a library of cells to two barcoded restrictions, a perturbation restriction and a lysis restriction. Cells may be restricted individually or as small clusters. The barcodes may include a barcode introduced into the cells while perturbing the cells and a barcode introduced while lysing the cells. In some embodiments, the perturbation barcode may be carried by the cells to the lysis step. In some embodiments, the lysis barcode may be applied to the cell contents and / or the perturbation barcode, resulting in the establishment of a barcoded cell contents that relates the cell contents to the perturbation the cell experiences. In some embodiments, the perturbation beads may further include a response capture probe. In this case, instead of two compartmentalization steps, a single picowell compartmentalization step may be sufficient. In such embodiments, the composite barcode may be functionalized to be able to capture the cell response. In some embodiments, the perturbation barcode terminates in a poly(T) segment to which the poly(A) tail of an mRNA molecule may hybridize.

[0430] In some embodiments, the workflow for single cell perturbation response analysis is as follows: (1) provide functionalized perturbation beads, where the perturbation barcode terminates with a capture sequence, which may include a set of poly(T) nucleotides for capture of mRNA, or other suitable capture probes for capturing other cellular responses; (2) capture a library of cells in a picowell array; (3) capture the library of functionalized perturbations in the same picowell, where in some embodiments a single cell and a single functionalized bead are captured per well, and in other embodiments a cluster of cells may be captured in a picowell; (4) optionally cover the picowell with an oil medium to prevent cross-contamination of reagents between wells; and (5) extract the compounds from the perturbation beads. (5) releasing the beads from the picowells and incubating the cells in each well with the compound released from the perturbation beads, (6) lysing the cells in the picowells by flowing a lysis buffer over the picowells, (7) capturing the mRNA or other cellular response directly onto the tip of the perturbation barcode, (8) copying the cellular response into the perturbation barcode using a polymerase or reverse transcriptase, (9) releasing the beads from the picowells by sonication, then cleaving the elongated perturbation barcode from the released beads or simply cleaving the perturbation barcode from the beads while they are still in the picowells, (10) subjecting the cleaved nucleotides (elongated perturbation barcodes) to an appropriate library preparation method and sequencing the prepared nucleotides. In some embodiments, the sequenced nucleotides include two zones: the perturbation barcode identifying the perturbation / compound to which the cells were subjected, and a response zone corresponding to the mRNA expression of cells subjected to the perturbation / compound identified by the perturbation barcode. This workflow is shown in Figure 36, optionally with an imaging step for process QC. Reverse transcriptase methods can serve to extend the captured RNA onto bead-attached DNA, thereby transferring cell content information to the functionalized beads. The beads can then be pooled, extracted, and analyzed on a sequencer. In some embodiments, DNA from a single cell can be further captured to specific primers on the functionalized beads.In such embodiments, the polymerase may replace the reverse transcriptase.

[0431] In some embodiments, perturbation and capture of cellular response can occur in two different limits, as described in FIG. 37. The perturbation barcode can be transferred to the cell surface before being subjected to the cellular response capture limit. The cellular response capture also involves capturing the perturbation barcode carried on the cell surface, thereby directly relating the cellular response to the perturbation to which the cell is exposed. In some embodiments, the capture of cellular response can be achieved by the Drop-seq method. In some embodiments, the capture of cellular response can occur on any commercially available single cell analysis instrument, such as the 10X Genomics single cell instrument, the Raindance single cell analysis protocol, the BioRad single cell isolation instrument, the Mission Bio single cell analysis protocol, the GigaGen instrument and protocol, and / or any other commercially available single cell analysis instrument or service.

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

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

[0434] In some embodiments, the constraint used in the methods described herein comprises a droplet constraint. In some embodiments, the droplet comprises an aqueous droplet in an oil matrix. In some embodiments, the dropl...

Claims

1. A system for screening compounds, comprising: (a) a picowell array plate including a plurality of picowells, each picowell having a top hole defining an opening at a top of the picowell, a bottom defined by a floor, the top hole being separated from the floor by a wall, the wall being between the top hole and the floor; (b) beads disposed within the picowells, the beads comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds; (c) the bead comprises a bead-bound DNA barcode in the form of either a concatenated DNA barcode or an orthogonal DNA barcode, and when the DNA barcode is in the form of a concatenated DNA barcode, the concatenated DNA barcode comprises: (i) using click chemistry, or (ii) using repeated cycles of steps, the repeated cycles of steps including using a splint oligonucleotide (sprint oligo) capable of hybridizing to a partially engineered bead-bound DNA barcode, the hybridization being mediated by an annealing site on the splint oligo and a corresponding complementary annealing site on the partially engineered bead-bound DNA barcode; the annealed splint oligo is used as a template to extend the partially fabricated DNA barcode using a DNA polymerase, the splint oligo includes bases complementary to the DNA barcode module that is polymerized onto the partially fabricated bead-bound DNA barcode, and the splint oligo also includes bases complementary to the annealing site that is polymerized onto the partially fabricated bead-bound DNA barcode; (d) each one of the plurality of substantially identical bead-bound compounds comprises one or more chemical library monomers, each bead-bound DNA barcode module identifies a corresponding chemical library monomer, the term "compound" being used to refer to a completed product comprising one or more chemical library members, and the completed DNA barcode identifying the compound.

2. 2. The system of claim 1, further comprising an oligonucleotide sequencing primer capable of inducing sequencing of one or more DNA barcode modules comprised in the bead-bound DNA barcode, and optionally, the system comprises a DNA sequencing instrument, wherein the DNA sequencing instrument is not a luminescence-based sequencer and is not a pH-based DNA sequencing instrument.

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

4. 2. The system of claim 1, wherein the at least one bead disposed in the at least one picowell comprises at least one response capture element associated with the at least one bead.

5. The at least one of the beads disposed within the picowell is at least one response capture element coupled to said at least one bead, said at least one response capture element comprising: (a) poly(dT); (b) exon-targeting RNA probes; (c) an antibody, or The system of claim 1 , further comprising (d) an aptamer.

6. the DNA barcode is either a concatenated DNA barcode or an orthogonal DNA barcode, the DNA barcode comprises one or more DNA barcode modules, each of the one or more DNA barcode modules encoding information identifying a chemical library monomer, the concatenated DNA barcode or the orthogonal DNA barcode comprising: (a) one or more functional nucleic acids, and 2. The system of claim 1, further comprising: (b) one or both of one or more nucleic acids encoding information other than the identity of the chemical library monomers.

7. The bead-bound concatenated DNA barcode is (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 2. The system of claim 1, comprising: (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.

8. The bead comprises a DNA barcode that is an orthogonal DNA barcode, the bead comprises an exterior surface, the orthogonal DNA barcode comprising: (a) a first nucleic acid comprising a first DNA barcode module and an annealing site for a sequencing primer, the first nucleic acid being bound 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, the second nucleic acid being bound 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 bound to the bead at a third location; The system of claim 1 , wherein the first, second, and third locations on the bead are each located at different locations on an exterior surface of the bead.

9. The concatenated DNA barcodes are (i) both click chemistry and repeated cycles of the steps using the splint oligo; (ii) both click chemistry and non-click chemistry methods; (iii) click chemistry alone, or (iv) The system of claim 1, produced by a method that uses only repeated cycles of the step of using the splint oligo.

10. 2. The system of claim 1, wherein each of the plurality of substantially identical bead-bound compounds is coupled to the bead via a cleavable linker, or via a cleavable linker that is a photocleavable linker, or via a non-cleavable linker.

11. 10. The system of claim 1, wherein the at least one bead comprises a grafted copolymer consisting of a low cross-linked polystyrene matrix to which polyethylene glycol (PEG) is grafted.

12. At least one picowell contains at least one cell; the plurality of substantially identical bead-bound compounds are coupled to the at least one bead via a cleavable linker, and cleaving the cleavable linker releases the bead-bound compounds from the bead to generate released compounds; The released compound is capable of contacting the at least one cell, the at least one cell being (i) a mammalian cell that is not a cancer cell; (ii) mammalian cancer cells, (iii) dead mammalian cells; (iv) apoptotic mammalian cells; (v) necrotic mammalian cells; (vi) bacterial cells; (vii) malaria parasite cells, (vii) cells that are metabolically active but have a bridged genome and are incapable of undergoing cell division; or (ix) The system according to claim 1, which is a mammalian cell infected with a virus.

13. each picowell having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, and a wall being between the upper hole and the floor; 2. The system of claim 1, wherein the hole is circular, the floor is circular, the wall is in the form of a truncated cone, the hole has a first diameter, the floor has a second diameter, and the first diameter is greater than the second diameter.

14. each picowell having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor, and a wall being between the upper hole and the floor; the hole is circular, the floor is circular, the wall is in the form of a truncated cone, the hole has a first diameter, the floor has a second diameter, the first diameter is greater than the second diameter; further comprising a cap that fits snugly into the hole, the hole being constructed from a polymer having a greater durometer (harder) and the cap being made from a polymer having a lesser durometer (softer), the relative durometers of the cap and hole allowing the cap to reversibly and snugly fit into the hole; The cap, (i) a cap whose sole purpose is to plug the picowell and prevent leakage; (ii) a passive cap, capable of absorbing metabolic products released by cells in a cell culture medium when the cells are cultured in said picowell; (iii) an active cap, which takes the form of a bead containing a plurality of essentially identical compounds, a cap, wherein each of said plurality of essentially identical compounds is attached to said bead with a cleavable linker; (iv) The system of claim 1, wherein the cap is an active cap, the cap taking the form of a bead containing a plurality of identical reagents, each of the plurality of essentially identical reagents being attached to the bead with a cleavable linker.

15. The system of claim 14 further comprising at least one spherical cap.

16. The system of claim 14 further comprising at least one aspherical cap.

17. The DNA barcode does not encode any chemical monomer, but instead (a) a class of compounds that are cleavably attached to the bead; (b) a step in a multi-step pathway of organic synthesis, in which a bead-bound nucleic acid corresponds to a given chemical monomer used to make a bead-bound compound, and said bead-bound nucleic acid corresponding to a given chemical monomer identifies that chemical monomer; (c) the day the bead-bound compound was synthesized; (d) a disease for which the bead-bound compound is intended to be treated; (e) the bead-bound compound stimulates or inhibits a cellular event; or 10. The system of claim 1, further comprising: (f) one or more nucleic acids that specify one or more of the reaction conditions used to bind a given chemical library monomer to said bead.

18. The system of claim 1, wherein there is no headpiece that links any of the bead-bound compounds with any of the bead-bound DNA barcodes.

19. the concatenated DNA barcode comprising at least one nucleic acid that is a DNA barcode module; and (a) can be used as an annealing site for a sequencing primer; (b) capable of forming a hairpin structure, the hairpin structure comprising a sequencing primer in the hairpin structure, an annealing site for the sequencing primer, and a bead, the bead being 5 prime to the sequencing primer and 3 prime to the annealing site for the sequencing primer; or (c) at least one functional nucleic acid that is a spacer nucleic acid.

20. the orthogonal DNA barcode comprises a plurality of DNA barcode modules, each of the DNA barcode modules being linked to a different site on the bead, either directly or via a linker, and each of the plurality of DNA barcode modules is (a) capable of serving as an annealing site for a sequencing primer; (b) capable of forming a hairpin structure, the hairpin structure comprising a sequencing primer in the hairpin structure, an annealing site for the sequencing primer, and a bead, the bead being 5 prime to the sequencing primer and 3 prime to the annealing site for the sequencing primer; or (c) at least one functional nucleic acid that is a spacer nucleic acid.

21. 1. A method of controlling a concentration of a compound in a solution present in a picowell, the method being applied to a bead-bound compound in a picowell, the picowell containing a solution, the bead-bound compound being attached to the beads via a cleavable linker, the method comprising: (a) exposing the bead-bound compound to conditions that result in cleavage of the cleavable linker, the conditions comprising light capable of cleaving the cleavable linker; (b) allowing release of the bead-bound compound from the beads to produce a released compound, wherein following release, diffusion or dispersion of the released compound throughout the solution results in a substantially uniform concentration of the compound in the solution. (c) adjusting the conditions to produce a determined concentration of the substantially uniform concentration, wherein the determined concentration is performed taking into account the concentration of emitted fluorophore emitted from a bead-bound emission monitor.

22. The conditions are adjusted by adjusting one or more of the wavelength of light, the intensity of light, and by the duration of exposure, and optionally (i) the concentration of released fluorophore released from a bead-bound release monitor is determined simultaneously with the release of said bead-bound compound from said beads to produce a released compound; or 22. The method of claim 21, wherein (ii) the concentration of released fluorophore released from a bead-bound release monitor is determined at a time substantially prior to resulting in release of the bead-bound compound from the bead and generating a released compound.

23. 1. A cap in combination with a picowell plate containing a plurality of picowells, the cap is capable of being used with the picowell plate; each of the plurality of picowells may be defined by a hole, a floor, and a wall, the wall being defined by the hole at a top and the floor at a bottom, the hole being circular, the floor being circular, and the wall taking the form of a surface of a frustum of a cone; the hole has a first diameter and the floor has a second diameter, the first diameter being greater than the second diameter; the cap is a spherical cap capable of fitting snugly into the hole, the hole being constructed from a polymer having a larger durometer (harder) and the cap being made from a polymer having a smaller durometer (softer); The relative durometers of the cap and hole allow the spherical cap to reversibly and snugly fit into the hole, the cap comprising: (i) the picowell can be plugged to prevent leakage; (ii) a passive cap, capable of absorbing metabolic products released by cells in a cell culture medium when the cells are cultured in the picowell; (iii) an active cap, in the form of a bead containing a plurality of essentially identical compounds; A cap, wherein each of the plurality of essentially identical compounds is attached to a bead with a cleavable linker, at least one of the plurality of picowells contains an aqueous medium, and cleavage of the cleavable linker releases at least some of the plurality of essentially identical compounds from the bead into the aqueous medium.

24. a picowell array plate including a generally planar top portion, a plurality of picowells, each picowell having an upper hole defining an opening at the top of the picowell, a bottom defined by a floor, the upper hole being separated from the floor by a wall, the wall being between the upper hole and the floor; and optionally a bead disposed in at least one of said plurality of picowells, said bead comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds, The picowell array plate further comprises a mat capable of positively covering the openings over at least one or all of the plurality of picowells or substantially positively covering the openings over at least one or all of the plurality of picowells, the positive covering being reversible, and the mat optionally (a) an absorbent surface capable of absorbing any metabolites, biochemicals, or proteins that may be contained in one or more of the plurality of picowells when placed in contact with the top general planar surface of the picowell array plate; (b) a system including one or all of the adhesive surfaces capable of maintaining reversible adhesion with the top general planar surface of the picowell array plate.

25. 1. A method of determining a signal from an assay and a sequencing readout on a bead, thereby identifying one or more compounds of interest from said assay, comprising: (a) providing a plurality of beads, each bead comprising a plurality of compounds and a plurality of oligonucleotides attached to the bead that are substantially associated with one another, the plurality of oligonucleotides attached to each bead identifying the plurality of compounds attached to the same bead; (b) performing the assay with the plurality of compounds attached to the beads; (c) determining at least one signal reflective of the performance of said compound in said assay of step b; (d) sequencing the plurality of oligonucleotides attached to the beads without removing the oligonucleotides from the beads, thereby determining a sequencing readout for each bead; (e) identifying the compound attached to the bead by the sequencing readout of step d and correlating it with the assay performance contained in the determined signal of step c, wherein the beads having a signal from the assay and the sequencing readout identify and correlate the compound of interest.

26. 1. A method for screening a compound library for compounds having desired properties, comprising: (a) providing a plurality of beads, each bead comprising a plurality of oligonucleotides attached to a surface of the bead and a plurality of substantially related compounds attached to a surface of the bead, wherein the sequences of the oligonucleotides attached to the bead encode the identities of the plurality of substantially related compounds attached to the surface of the bead; (b) incorporating said plurality of beads into an assay for a desired property of compounds in said compound library; (c) capturing a signal from at least one bead, said signal reflecting the performance of said compound on said bead in said assay; and (d) sequencing the plurality of oligonucleotides attached to the at least one bead from which an assay signal has been further captured without removing the oligonucleotides from the bead; and (e) identifying at least one compound from the sequencing readout of step (d) and correlating it with a corresponding assay performance captured in the signal of step (c).

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

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

29. 26. The method of claim 25, wherein the multiple compounds are attached to the bead surface by linking multiple compound components in tandem, all of which together constitute the compound.

30. 30. The method of claim 29, wherein each DNA module and each compound component is assembled sequentially and alternatively.

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

32. 32. The method of claim 31 , wherein the cleavable linker is a photocleavable linker, a protease cleavable linker, or an acid cleavable linker.

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

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

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

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

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

38. 38. The method of any one of claims 25-37, wherein the method further comprises identifying a candidate target among a plurality of potential targets, and wherein the compound having the desired property binds to the candidate target.

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

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

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

42. 1. A method for screening a compound library for compounds having desired properties, comprising: (a) providing a plurality of beads, each bead comprising a plurality of oligonucleotides attached to a surface of the bead and a plurality of substantially related compounds attached to a surface of the bead, wherein the sequences of the oligonucleotides attached to the bead encode the synthetic history of the plurality of substantially related compounds attached to the surface of the bead; (b) incorporating said plurality of beads into an assay for a desired property of compounds in said compound library; (c) capturing a signal from at least one bead, said signal reflecting the performance of said compound on said bead in said assay; and (d) sequencing the plurality of oligonucleotides attached to the at least one bead from which an assay signal has been further captured without removing the oligonucleotides from the bead; and (e) identifying at least one compound from the sequencing readout of step (d) and correlating it with a corresponding assay performance captured in the signal of step (c).

43. 43. The method of claim 42, wherein the assay comprises a binding assay.

44. 43. The method of claim 42, wherein the assay comprises an activity assay.

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

46. 43. The method of claim 42, wherein the assay involves the interaction of unbound compounds with other assay reagents, the unbound compounds being compounds released from the bead surface.

47. 46. ​​The method of claim 45, wherein the compound is released by cleaving a cleavable linker that connects the compound to the bead.

48. The method of claims 42-47, wherein the assay occurs in a plurality of confined volumes, and nominally one bead is distributed in each confined volume.

49. 49. The method of claim 48, wherein the restricted volume comprises an aqueous droplet.

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

51. 49. The method of claim 48, wherein the restricted volume comprises a picowell.

52. 51. The method of claim 50, wherein the picowells are organized in a regular array.

53. 52. The method of claim 51 , wherein the plurality of confined volumes are organized into a regular array.

54. 49. The method of claim 48, wherein the restricted volume comprises a layer of aqueous medium adhered around the beads, the beads being suspended in a hydrophobic medium.

55. 43. The method of claim 42, wherein the assay reagents are washed away prior to sequencing the oligonucleotides.

56. 43. The method of claim 42, wherein said sequencing step (d) is performed prior to said assaying step (b).

57. 57. The method of claim 56, wherein the oligonucleotides on the beads are removed after the sequencing step but before the assay step.

58. 58. The method of claim 57, wherein said removal of said oligonucleotides comprises enzymatic digestion, chemical cleavage, thermal decomposition, or physical shearing.

59. 44. The method of claim 43, wherein the binding assay comprises binding of an RNA molecule to the beads.

60. 44. The method of claim 43, wherein the signal from the bead comprises sequencing of the bound RNA molecule.

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

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

63. The desired property is (i) inhibiting or stimulating the catalytic activity of an enzyme; (ii) stimulating a Th1-type immune response measurable by cell-based or in vivo assays; (iii) stimulating a Th2-type immune response measurable by cell-based or in vivo assays; (iv) inhibiting a Th1-type immune response measurable by cell-based or in vivo assays; (v) inhibiting a Th2-type immune response measurable by cell-based or in vivo assays; (vi) stimulating or inhibiting ubiquitin-mediated degradation of proteins, measurable by purified protein, cell-based assay, or in vivo assay.

64. 1. A system for screening a compound library for a compound having a desired activity, comprising: (a) a sample compartment for receiving oligonucleotide-encoded beads having a plurality of compounds attached thereto; (b) a plurality of encapsulation compartments within said sample compartment, each encapsulation compartment nominally comprising a single bead dispersed in an assay medium, said assay medium further comprising a reagent that is assayed for interaction with said compound on said bead to provide 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.

65. 65. The system of claim 64, wherein the encapsulated compartment comprises a droplet.

66. 65. The system of claim 64, wherein the encapsulation compartment comprises a picowell.

67. 65. The system of claim 64, wherein the encapsulated compartment further comprises an assay reagent.

68. 65. The system of claim 64, wherein the detector comprises an optical detector.

69. 65. The system of claim 64, wherein the sequencer comprises an optical detector.

70. 1. A method of perturbing a cell, comprising: (a) providing a nucleic acid-encoded perturbation and restricting a cell with said nucleic acid-encoded perturbation; (b) contacting the cell with a perturbation encoded by the nucleic acid in a confined volume, wherein the onset and dose of the perturbation are controlled; (c) incubating the cells with the nucleic acid-encoded perturbation for a specified period of time; (d) introducing into the cell the nucleic acid encoding the nucleic acid-encoded perturbation.

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

72. 72. The method of Claim 70 or 71, wherein the nucleic acid-encoded perturbations are a DNA-encoded library.

73. 73. The method of any one of claims 70-72, wherein the perturbation and the nucleic acid encoding the perturbation are unattached and free in solution.

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

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

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

77. 8. The method of claim 7, wherein the cleavable attachment is selected from the group consisting of a photocleavable attachment, a temperature cleavable attachment, a pH sensitive attachment, an acid cleavable attachment, a base cleavable attachment, a sound cleavable attachment, a salt cleavable attachment, a redox sensitive attachment, or a physically cleavable attachment.

78. 78. The method of any one of claims 70-77, wherein confining the cells and the perturbation comprises droplet encapsulation, emulsion encapsulation, picowell encapsulation, macrowell encapsulation, physical attachment, bubble encapsulation, or microfluidic confinement.

79. 79. The method of any one of claims 70-78, wherein control over 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 redox potential, or controlling mechanical agitation exposure.

80. 80. The method of any one of claims 75-79, wherein said incubation comprises exposing said cell to said perturbation after cleaving said perturbation from said substrate or after cleaving said nucleic acid from said substrate.

81. 80. The method of any one of claims 75-79, wherein said incubation comprises exposing said cell to said perturbation without cleaving said perturbation from said substrate or without cleaving said nucleic acid from said perturbation.

82. 82. The method of any one of claims 70-81, wherein introducing the nucleic acid encoding the nucleic acid-encoded perturbation into the cell comprises attaching the nucleic acid to a cell surface of the cell.

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

84. 83. The method of claim 82, wherein attaching the nucleic acid to a cell surface of the cell comprises attaching the nucleic acid to a biomolecule on the cell surface.

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

86. 86. The method of any one of claims 82 to 85, wherein attaching the nucleic acid to the cell surface of the cell comprises attaching via any tag on the nucleic acid.

87. 1. A method of perturbing a cell with a perturbation and encoding the cell with the identity of the perturbation, comprising: (a) providing a bead-bound DNA-encoded library; (b) restricting cells with the bead-bound DNA-encoded library, the bead-bound DNA-encoded library comprising one or more copies of combinatorially synthesized compounds and one or more copies of encoding nucleic acid tags, the compounds and the encoding nucleic acids being attached to beads, the encoding nucleic acids encoding the identities of the compounds, and the bead-bound DNA-encoded library and the cells being restricted to a restricted volume; (c) releasing the compound from the beads and incubating the compound with the cells in the restricted volume; (d) optionally releasing the encoding nucleic acid tag from the bead; (e) attaching the encoding nucleic acid tag to the cell, thereby maintaining the identity of the compound via the encoding nucleic acid tag attached to the cell.

88. 1. A method of perturbing a cell, encoding the cell with the identity of the perturbation, and measuring the response of the cell to the perturbation, comprising: (a) contacting a cell with a bead-bound DNA-encoded library in a first confined volume, the bead-bound DNA-encoded library comprising one or more copies of combinatorially synthesized compounds and one or more copies of encoding nucleic acid tags, the compounds and the encoding nucleic acids being attached to beads, and the encoding nucleic acids encoding the identities of the compounds; (b) releasing the compounds in the library from the beads and incubating the compounds in the library with the cells in the first confined volume; (c) optionally releasing the encoding nucleic acid tags from the beads within the first confined volume; and (d) capturing the encoding nucleic acid tag on the cell surface of the cell, whereby the cell is exposed to the compounds in the library and the identity of the exposed compounds is captured on the cell surface; (e) releasing the cells from the first confined volume, wherein the encoding nucleic acid tag is attached to the cells, the encoding nucleic acid tag encoding the identity of the compound to which the cells are exposed; and (f) capturing previously perturbed, nucleic acid-tagged cells with responsive detection beads in a second restricted volume, wherein the cells are exposed to lysis conditions that expose the cellular contents of the cells to responsive capture beads, the responsive capture beads comprising capture probes that capture the cellular contents and nucleic acid tags that encode the perturbation in the previously perturbed, nucleic acid-tagged cells; (g) incubating the response capture beads with the lysed cells in the second restricted volume, thereby capturing both the cellular contents and the nucleic acid tag encoding the perturbation to the response capture beads; (h) optionally converting the 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 tags attached to the response capture beads, thereby correlating the identity of the perturbation to the response of the cell to the perturbation.

89. 1. A method of perturbing a cell and capturing the response of the cell to the perturbation, comprising: (a) providing an array of picowells and a library of functionalized perturbation beads, wherein the picowell is capable of accommodating a single cell and a single functionalized perturbation bead, each functionalized perturbation bead comprising a plurality of different substantially identical releasable compounds and a plurality of nucleotide barcodes encoding said compounds, wherein the nucleotide barcodes are functionalized barcodes capable of capturing cellular contents of the cells, the cellular contents of the cells comprising a cellular response to the perturbation contained in the functionalized perturbation bead; (b) capturing a single cell in each picowell of the picowell array; (c) capturing a single functionalized perturbed bead in said picowell containing a single cell; (d) releasing the compound from the functionalized perturbed beads and incubating the cells with the released compound, wherein the compound between the picowells has minimal diffusion; (e) lysing the cells to release the intracellular contents; (f) capturing one or more components of the cellular contents onto functionalized oligonucleotides on the functionalized perturbed beads, the capturing comprising hybridization and enzymatic extension to combine nucleotide barcodes with nucleic acid elements of the cellular contents, thereby forming hybrids of the nucleotide barcodes and the nucleic acid elements of the cellular contents; (g) releasing the hybrids, collecting the hybrids from the library of functionalized perturbed beads, and sequencing the hybrids, thereby relating the perturbation to the cellular response to the perturbation.

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