Oligonucleotide-encoded chemical libraries, related systems, devices, and methods for detecting, analyzing, quantifying, and testing biological / genetic material

Abstract

The present application provides beads bearing covalently bound compounds and covalently bound DNA barcodes, as well as methods for using such beads. The beads bear multiple copies of substantially identical compounds and multiple copies of substantially identical DNA barcodes. The compounds are composed of one or more chemical monomers, and the DNA barcodes take the form of barcode modules, each corresponding to a corresponding chemical monomer and enabling its identification. The nucleic acid barcodes can have linked or independent structures. Methods are provided for sequencing the bead-bound nucleic acid barcodes to cleave the compounds from the beads and evaluate the biological activity of the released compounds.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of and claims the benefit of and priority to co-pending U.S. patent application Ser. No. 16 / 774,871, filed Jan. 28, 2020, the entire contents of which are incorporated herein by reference.

[0002] This application is also a continuation-in-part of, and claims the benefit of, and priority to, co-pending U.S. patent application Ser. No. 16 / 870,809, filed May 8, 2020, the entire contents of which are incorporated herein by reference.

[0003] Field of Disclosure 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 a single compound, and the beads also contain DNA tags that encode the identity or synthetic history of the compounds contained within or on the beads. The present disclosure also relates to high-throughput assays performed in picowells containing compound-loaded beads and assay materials. The present disclosure further relates to releasing bead-bound compounds and screening their biological activity. In general, the present disclosure contemplates assays that use beads as a delivery vehicle for compounds and methods for creating such compound-loaded beads.

[0004] The present disclosure relates to bead-bound compounds, each compound consisting of one or more monomers from a chemical library. The present disclosure also relates to bead-bound DNA barcodes, i.e., nucleic acids whose sequence each represents a code (regardless of genetic code) for one particular chemical library monomer. The present disclosure further relates to releasing the bead-bound compounds and screening the released compounds for biological activity.

[0005] The present disclosure also generally relates to methods for perturbing a cell or a group 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 purposes of high-throughput screening, target discovery, or diagnostics, and other similar applications.

[0006] The present disclosure further relates to a computer-implemented method, a computer-implemented system, and a computer-readable medium for respectively perturbing a cell and capturing the cell's response to the perturbation. [Background technology]

[0007] Combinatorial chemistry, including split-and-pool chemistry, can be used to synthesize large quantities of compounds. Compounds generated in this manner find applications in medicinal chemistry, where they can be screened for various biochemical activities. These activities include binding to one or more proteins, which are known at the time the screening test is performed. Alternatively, the protein to which the tested compound binds is identified only after a binding event is detected. Compounds can also be screened for the ability to inhibit or activate known proteins (this is not simply a screen for "binding" activity). Alternatively, compounds can be screened for the ability to inhibit or activate cellular functions when the molecular target is unknown to the researcher at the time of screening.

[0008] Screening of compounds, such as those belonging to large libraries of chemicals generated by split-and-pool methods, can be facilitated by performing screening in arrays of thousands of microwells, nanowells, or picowells. Screening can be further facilitated by providing a different compound in each picowell with beads, where each bead contains hundreds of copies of the same compound and the same bead contains hundreds of copies of a "DNA barcode" that can be used to identify compounds bound to the same bead. Compound screening is further facilitated by using cleavable linkers that allow for controlled release of compounds from the beads, and when the released compounds are used for biochemical or cell-based assays within the same picowell.

[0009] Assaying compounds in extremely small, confined volumes, such as droplets, picowells, or microfluidic environments, is generally beneficial, for example, because it requires fewer assay reagents and therefore does not require the limitation of combinatorially generated compounds. Any method that allows compounds to be loaded onto beads and subsequently eluted from the beads can be used to deliver bead-bound compounds to assays in small, confined volumes. Adding nucleic acid barcodes to beads allows the identity of compounds present within the beads to be carried into the assay volume. In this way, extremely high-throughput assays can be performed without the need for robotics or spatial indexing of compounds within microtiter plates. Millions to billions of compounds can be held in a single small vial, and the identity of the compound is tagged (with DNA) to the same bead containing each individual compound.

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

[0011] Traditional phenotypic screening of cells has involved creating models of cells in a disease state, contacting the cells with various drug libraries, and monitoring whether the disease phenotype is corrected by a measurable assay. Such screening methods are called phenotypic screening because measurable phenotypic changes that indicate a therapeutic response are considered relevant criteria, even though the underlying biological mechanisms are not necessarily understood at the outset. A vast number of cell lines and disease models that reflect various baseline and diseased cellular states are now available. Larger numbers of compound libraries and biological drug candidates are also available. Straightforward screening efforts that combine various cell models with various drug candidates to look for phenotypic responses have technical limitations. This is because assays are limited to microtiter plate formats and imaging tools, both of which are severely limited in throughput.

[0012] One way to overcome throughput limitations is to adopt high-throughput single-cell screening approaches for 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 where individual assays can be performed on each cell. Genetic analysis, for example, via single-cell mRNA screening using droplet encapsulation, is a well-known method that reveals intricate details hidden in combinatorial measurements (see, e.g., Macosko et al., Cell 161:1202-1214, 2015 and Ziegenhain et al., Mol Cell 65:631-643, 2017, the entire disclosures of which are incorporated herein by reference). Current state-of-the-art single-cell analysis platforms make it possible to characterize cells based on their transcriptional state and quantify mRNA transcripts at single-cell resolution to obtain fingerprints. This approach allows comparison between tissue samples extracted from subjects or prepared in experiments, and allows the examination of the transcription and therefore protein expression status of single cells.The measurement of single-cell mRNA by transcriptome sequencing and profiling is an important approach to examine not only the phenotype of cells during disease progression, but also the molecular mechanisms of drug efficacy, resistance, and therapeutic target discovery (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 has been used to reveal cell-to-cell heterogeneity, as evidenced by cell-to-cell variations in transcriptome.This cell-to-cell heterogeneity is highly related to drug efficacy and specificity, transcriptional randomness, transcriptome plasticity, and genome evolution.Encapsulation in picowells has also been demonstrated (see, e.g., Gierahn et al., Nat Methods 14:395-398, 2017). Single-cell protein measurements are also possible using similar isolation methods (Butnik et al., BioRxiv, Jan. 2017; Su et al., Proteomics 17:3-4, 2017).

[0013] Despite rapid advances in high-throughput single-cell RNA sequencing (RNA-seq) methods, including commercial versions of automated platforms such as Fluidigm C1, 10XGenomics, or the 1CellBiO system, the application of single-cell RNA profiling for target-agnostic high-throughput drug screening and target discovery remains limited by the lack of methods capable of efficiently separating different drugs into different cells. While it is possible to incubate cells or tissues under various perturbations in well plates, followed by single-cell analysis and comparison of transcript profiles, the number of drugs that can be tested is limited by the capacity of the plate. Furthermore, the need to prepare barcoded mRNA from each sample during isolation and then perform comprehensive RNA profiling for each sample creates a major obstacle.

[0014] One current challenge with large-scale assays such as those described herein is that a single bead is often required in a single well. While this is typically not an issue when employing 96-well plates, it becomes a significant problem when employing assay devices with over 100,000 wells. For example, Vann et al., U.S. Patent Application Publication No. 2003 / 0021734 (incorporated herein by reference in its entirety), discloses a robotic bead dispensing system designed to provide a single bead in a single well; however, this reference notes that in practice, more than one bead may enter a well, requiring visual inspection to ensure that the well contains only one bead (see paragraph

[0131] of Vann et al.).

[0015] Described herein are improved systems, devices, and methods for perturbing cells and capturing the cellular response to the perturbation that provide a bead dispensing device that deposits a single bead into a single well, when this is a necessary component of large-scale assays. Summary of the Invention

[0016] Briefly, the present disclosure provides a system for screening compounds, comprising: (a) a picowell array plate comprising a plurality of picowells, each picowell having a top aperture defining an opening at the top of the picowell and a bottom defined by a floor, the top aperture being separated from the floor with a wall between the top aperture and the floor; and (b) beads disposed in the picowells, the beads comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds, and (c) the beads comprise bead-bound DNA barcodes in the form of linked DNA barcodes or orthogonal DNA barcodes, and when the DNA barcodes are in the form of linked DNA barcodes, the linked DNA barcodes are made by a method using (i) click chemistry or (ii) a step-repeating cycle, the step-repeating cycle hybridizing the partially made bead-bound DNA barcodes. (d) using a splint oligonucleotide (sprint oligo) that can be hybridized to a bead-bound DNA barcode, 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) a plurality of substantially identical bead-bound compounds, each one comprising 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 mean a completed product comprising one or more chemical library members, where the completed DNA barcode identifies the compound.

[0017] The floor of a microwell, nanowell, or picowell need not be flat. The floor may be curved, such as the bottom of a glass test tube or a metal centrifuge tube. The floor may also be conical, such as a conical centrifuge tube. The floor may be flat, but may have notches, for example, notches that facilitate movement of assay or cell culture solution near the bottom of any beads in the picowell. In flat-bed embodiments, the systems and methods may require a flat floor.

[0018] The linked DNA barcodes can be made entirely by organic chemistry methods, such as click chemistry, and the orthogonal DNA barcodes can be made entirely by organic chemistry methods, including click chemistry.

[0019] Also provided is the above system, further comprising a plurality of caps, each cap capable of fitting into the opening of a different picowell, 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.

[0020] Additionally encompassed are the above systems in which the linked DNA barcodes are produced by methods that use (i) both click chemistry and repeat cycles using splint oligos, (ii) both click chemistry and non-click chemistry chemical methods, (iii) click chemistry alone, or (iv) only repeat cycles using splint oligos. For this particular embodiment, the "linked DNA barcodes" in question do not include any chemical couplers used to directly link nucleic acids to beads.

[0021] In spherical cap embodiments, the above system is provided, further comprising a plurality of spherical caps, each cap capable of fitting into the aperture of a picowell, the aperture of the picowell 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.

[0022] In response element embodiments, the above-described system is provided, wherein at least one bead disposed in at least one picowell comprises at least one response capture element linked to said at least one bead. Also contemplated is the above-described system, wherein at least one bead disposed in at least one picowell comprises at least one response capture element linked to said at least one bead, wherein at least one response capture element comprises (a) poly(dT) or (b) an exon-targeted RNA probe.

[0023] Also contemplated are systems as described above, wherein the DNA barcode is 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.

[0024] The following discloses "consisting only of" and "comprising" embodiments. This applies to the number of bead-bound DNA barcode modules that make up the DNA barcode. Embodiments are provided in which the DNA barcode consists of only one DNA barcode module, or only two DNA barcode modules, or only three DNA barcode modules, or only four DNA barcode modules, or in which the DNA barcode includes 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.

[0025] The bead-bound concatenated DNA barcodes may comprise: (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 Also encompassed are systems comprising: (i) 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 (ii) 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.

[0026] Further, the above system is contemplated, further comprising a primer binding site capable of binding to a DNA sequencing primer, wherein said primer binding site can direct 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 at the 3 prime of the first DNA barcode module, the 3 prime of the second DNA barcode module, the 3 prime of the third DNA barcode module, the 3 prime of the fourth DNA barcode module, the 3 prime of the fifth DNA barcode module, or the 3 prime of 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.

[0027] Further provided are systems as described above, wherein the primer binding site is located between the first and second DNA barcode modules, between the second and third DNA barcode modules, between the third and fourth DNA barcode modules, between the fourth and fifth DNA barcode modules, or between the fifth and sixth DNA barcode modules. In embodiments regarding the location of the primer binding site, the systems as described above are provided, wherein the primer binding site is located between every pair of consecutive DNA barcode modules with respect to the upstream DNA barcode module and with respect to the downstream DNA barcode module.

[0028] Further provided is the above system, wherein the bead comprises a DNA barcode that is an orthogonal DNA barcode, the bead comprising 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 and linked to the bead at a first position, (b) a second nucleic acid comprising a second DNA barcode module and an annealing site for a sequencing primer and linked to the bead at a second position, and (c) a third nucleic acid comprising a third DNA barcode module and an annealing site for a sequencing primer and linked to the bead at a third position, wherein the first, second, and third positions on the bead are each located at different positions on the exterior surface of the bead.

[0029] In coding embodiments, the above system is provided wherein the DNA barcode does not identify the chemical library monomer, but instead comprises one or more nucleic acids that identify (a) the class of compound cleavably attached to the bead, (b) the number of steps in a multi-step organic synthesis pathway, (c) the date the bead-attached compound was synthesized, (d) the disease the bead-attached compound is intended to treat, (e) the cellular event the bead-attached compound is intended to stimulate or inhibit, or (f) the reaction conditions used to link a given chemical library monomer to the bead.

[0030] In linker embodiments, the above system is provided where each of the plurality of substantially identical bead-bound compounds is linked to the bead by a cleavable linker. Also provided is the above system where each of the plurality of substantially identical bead-bound compounds is linked to the bead by a photocleavable linker. Also provided is the above system where each of the plurality of substantially identical bead-bound compounds is linked to the bead by a non-cleavable linker.

[0031] In an embodiment of TentaGel®, the above system is provided, wherein at least one bead comprises a graft copolymer consisting of a low-crosslinked polystyrene matrix to which polyethylene glycol (PEG) is grafted.

[0032] In an embodiment of a release monitor, the present disclosure provides a method for detecting a release monitor bead in which at least one picowell contains a release monitor bead and does not contain any other type of bead;

[0033] the emission monitor beads comprise a bead-bound quencher and a bead-bound fluorescent dye, the bead-bound quencher is quenchably located in close proximity to the bead-bound fluorescent dye 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 fluorescent dye, the bead-bound fluorescent dye is linked by a first photocleavable linker, the picowell containing the emission monitor beads is a first picowell, the first picowell contains a first solution, and the first picowell is subjected to cleavage conditions. the photocleavable linker can be cleaved by exposing the first picowell to a light source to cleave the photocleavable linker and release the fluorescent dye into a first solution in the first picowell, the exposure can cause the fluorescent dye to diffuse throughout the first solution in the first picowell, a fluorescent signal obtained by irradiating the first picowell containing the first solution containing the diffused fluorescent dye with light allows a user to use the fluorescent signal to calculate a percentage of release of the bead-bound fluorescent dye from the release-monitoring beads to provide a calculated percent release value; a second picowell contains a bead-bound compound linked with a photocleavable linker of the same type as the first photocleavable linker, the second picowell contains a second solution;

[0034] Provided is a system as above, wherein the calculated percent release value 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.

[0035] In embodiments relating to all identities of compounds bound to a given bead, or all identities of DNA barcodes bound to a given bead, the above system is provided wherein at least one bead comprises a plurality of substantially identical bead-bound DNA barcodes, the plurality being 10 million to 100 million copies of the substantially identical bead-bound DNA barcodes.Also provided is the above system wherein at least one bead comprises a plurality of substantially identical bead-bound compounds, the plurality being 10 million to 100 million copies of the substantially identical bead-bound compounds.

[0036] In embodiments relating to cells (e.g., mammalian cells, cancer cells, bacterial cells), the above system is provided, wherein at least one picowell contains at least one cell, and a plurality of substantially identical bead-bound compounds are attached to at least one bead by a cleavable linker, and the bead-bound compounds are released from the bead by cleaving the cleavable linker to produce released compounds, which can contact the at least one cell. In other cell embodiments, the above system is provided, wherein at least one picowell contains at least one cell, a plurality of substantially identical bead-bound compounds are attached to at least one bead by a cleavable linker, and cleavage of the cleavable linker releases the bead-bound compounds from the bead to produce released compounds, which can contact at least one cell, and the at least one cell is (i) a mammalian cell that is not a cancer cell, (ii) a mammalian cancer cell, (iii) a dead mammalian cell, (iv) an apoptotic mammalian cell, (v) a necrotic mammalian cell, (vi) a bacterial cell, (vii) a plasmodium cell, (vii) a cell that is metabolically active but has a crosslinked genome and is incapable of undergoing cell division, or (ix) a mammalian cell infected with a virus.

[0037] In a device embodiment, the above system is provided, wherein each picowell has a top aperture defining an opening at the top of the picowell and a bottom defined by a floor, the top aperture is separated from the floor, a wall is present between the top aperture and the floor, the aperture is circular, the floor is circular, the wall is in the form of a truncated cone, the aperture has a first diameter, and the floor has a second diameter, the first diameter being greater than the second diameter.

[0038] In other device-related embodiments, each picowell has a top aperture defining an opening at the top of the picowell and a bottom defined by a floor, the top aperture being separated from the floor, a wall being present between the top aperture and the floor, the aperture being circular, the floor being circular, and the wall being in the form of a truncated cone, the aperture 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 fitting snugly over the aperture, the aperture being made of a polymer having a greater durometer (hard) and the cap being made of a polymer having a smaller durometer (soft), and the relative durometers of the cap and aperture determine The above system is also provided, in which the cap fits reversibly and snugly into the aperture, and the cap is (i) a cap intended only to block the picowell and prevent leakage, (ii) a passive cap that can absorb metabolites 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 containing a plurality of essentially identical compounds, each of the plurality of essentially identical compounds being linked to the beads by a cleavable linker, or (iv) an active cap in the form of beads containing a plurality of identical drugs, each of the plurality of essentially identical drugs being linked to the beads by a cleavable linker.The above system is also provided, in which the cap is spherical or the cap is non-spherical.

[0039] In one embodiment, the system includes a picowell array plate including an upper, generally flat surface and a plurality of picowells, each picowell having an upper aperture defining an opening at the top of the picowell and a bottom defined by a floor, the upper aperture being separated from the floor by a wall, the wall being between the upper aperture and the floor, and optionally a bead disposed in at least one of the plurality of picowells, the bead comprising a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds, and the picowell array plate includes at least one of the plurality of picowells. The picowell array plate further includes a mat capable of tightly covering at least one or all of the top openings of the plurality of picowells, or capable of effectively tightly covering at least one or all of the top openings of the plurality of picowells, wherein the tight covering is reversible, and the mat may include 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 top, generally flat surface of the picowell array plate; and (b) an adhesive surface capable of maintaining reversible attachment to the top, generally flat surface of the picowell array plate.

[0040] Biochemical assay embodiments include a system comprising at least one picowell containing beads comprising a plurality of substantially identical compounds and a plurality of substantially identical barcodes, wherein at least one picowell contains an assay medium comprising cereblon E3 ubiquitin ligase and a substrate of cereblon E3 ubiquitin ligase, e.g., Ikaros or Aiolos, wherein the system is capable of screening for compounds that activate the activity of cereblon E3 ubiquitin ligase, thereby reducing the intracellular concentration of Ikaros or Aiolos.

[0041] Another biochemical assay embodiment contemplates a system as described above, comprising at least one picowell containing beads comprising a plurality of substantially identical compounds and a plurality of substantially identical barcodes, wherein at least one picowell contains an assay medium comprising an MDM2 E3 ubiquitin ligase and a substrate of the MDM2 E3 ubiquitin ligase, e.g., p53, wherein the system is capable of screening for compounds that activate the E3 ubiquitin ligase activity of MDM2, thereby increasing the intracellular concentration of p53.

[0042] In further barcode embodiments, the above systems are provided wherein the DNA barcode does not encode any chemical monomer, but instead comprises one or more nucleic acids that identify one or more of: (a) the class of compound cleavably attached to the bead; (b) the step in a multi-step pathway of organic synthesis where the bead-attached nucleic acid corresponds to a given chemical monomer used to make the bead-attached compound and the bead-attached nucleic acid corresponding to a given chemical monomer identifies that chemical monomer; (c) the date the bead-attached compound was synthesized; (d) the disease the bead-attached compound is intended to treat; or (e) the cellular event the bead-attached compound is intended to stimulate or inhibit.

[0043] In embodiments lacking any headpiece, the above system is provided, wherein at least one bead comprises a plurality of substantially identical bead-bound compounds and also a plurality of substantially identical bead-bound DNA barcodes, and wherein there is no headpiece present linking any of the bead-bound compounds to any of the bead-bound DNA barcodes.

[0044] Further contemplated are systems as described above, wherein at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the substantially identical bead-bound DNA barcodes have the same structure. Further contemplated are systems as described above, wherein at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the substantially identical bead-bound compounds have the same structure.

[0045] Further provided are systems as described above, wherein the linked DNA barcode comprises at least one nucleic acid that is a DNA barcode module, or systems as described above, wherein the linked DNA barcode comprises only one nucleic acid that is a DNA barcode module.

[0046] In a sequencing primer annealing site embodiment, the above system is provided, wherein the linked DNA barcode comprises at least one nucleic acid that is a DNA barcode module, and at least one functional nucleic acid that (a) can be used as an annealing site for a sequencing primer, (b) can form a sequencing primer, an annealing site for a sequencing primer, and a hairpin structure that includes a bend therein that is 5 prime to the sequencing primer and 3 prime to the annealing site for the sequencing primer, or (c) is a spacer nucleic acid.

[0047] In other sequencing primer embodiments, the above system is provided, wherein the orthogonal DNA barcode comprises a plurality of DNA barcode modules, each of which is linked to a different site on the bead, either directly or via a linker, and each of the plurality of DNA barcode modules comprises 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 sequencing primer, an annealing site for the sequencing primer, and a hairpin structure comprising a bend therein that is 5-prime to the sequencing primer and 3-prime to the annealing site for the sequencing primer, or (c) is a spacer nucleic acid.

[0048] In an embodiment reciting functional terms for splint oligos, beads are provided that contain linked DNA barcodes that include (a) a first annealing site for a first splint oligonucleotide (sprint oligo) that includes a first DNA barcode module and three nucleic acids: a nucleic acid that is a complement that hybridizes to the first annealing site, a nucleic acid that is a complement that hybridizes to the second DNA barcode module, and a nucleic acid that is a second annealing site; and (b) a second annealing site for a second splint oligo that includes a second DNA barcode module and three nucleic acids: a nucleic acid that is a complement that hybridizes 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.

[0049] In another embodiment including a functional expression for a splint oligo, the above bead is provided, further comprising a third DNA barcode module and a third annealing site for a third splint oligo comprising three nucleic acids: a nucleic acid that is a complement that hybridizes to the third annealing site, a nucleic acid that is a fourth DNA barcode module, and a nucleic acid that is a fourth annealing site.

[0050] Additionally, in yet another embodiment including a functional expression for a splint oligo, the above-described bead is provided, further comprising one or more of: (i) a fourth annealing site for a fourth splint oligo comprising a fourth DNA barcode module and a nucleic acid that is a complement that hybridizes to the fourth annealing site, a nucleic acid that is a fifth DNA barcode module, and a nucleic acid that is a fifth annealing site; (ii) a response capture element; and (iii) an emission monitor.

[0051] Linker embodiments include beads as described above in which the linked DNA barcode is linked to the bead, but is not linked to the bead by any photocleavable linker, (ii) by any enzyme-cleavable linker, or (iii) by any type of cleavable linker.

[0052] In embodiments with distinct linkage locations, beads as described above are provided, wherein the linked DNA barcode is linked to a first location on the bead, and the bead also includes a compound linked to a second location on the bead, wherein the first location is not the same as the second location.

[0053] In a surface (inner and outer surface) embodiment, the beads described above are provided, wherein the beads comprise an outer surface and an inner surface, the beads comprise at least 10,000 substantially identical linked DNA barcodes linked to the beads, and at least 90% of the at least 10,000 substantially identical linked DNA barcodes are linked to the outer surface.

[0054] In an exclusive embodiment that distinguishes the present disclosure from other embodiments, there is provided a bead that does not contain any polyacrylamide, wherein the linked DNA barcodes are (i) not containing any nucleic acids that are promoters, (ii) not containing any nucleic acids that are polyA, or (iii) not containing any nucleic acids that are promoters and not containing any nucleic acids that are polyA.

[0055] In an embodiment of a release monitor bead, the present disclosure provides a release monitor bead capable of functioning in an aqueous medium, the release monitor bead comprising a bead-bound quencher and a bead-bound fluorescent dye, the bead-bound quencher being quenchably located in close proximity to the bead-bound fluorescent dye and capable of quenching at least 50% of the fluorescence of the bead-bound fluorescent dye, the bead-bound fluorescent dye being attached by a first photocleavable linker, the picowell containing the release monitor bead is a first picowell, the first picowell comprises a first solution, and the first picowell can be exposed to cleavage conditions to cleave the photocleavable linker and release the fluorescent dye into the first solution in the first picowell, The fluorescent dye diffuses throughout the first solution in the first picowell, and a fluorescent signal obtained by irradiating the first picowell containing the first solution containing the diffused fluorescent dye allows a user to use the fluorescent signal to calculate the percent release of the bead-bound fluorescent dye from the emission monitor beads to obtain a calculated percent release value; a second picowell contains a bead-bound compound linked to a photocleavable linker of the same type as the first photocleavable linker, and the second picowell contains a second solution; the calculated percent release value from the emission monitor beads in the first picowell allows calculation of the concentration of the released compound in the second solution in the second picowell. Other emission monitor embodiments include emission monitor beads in which the fluorescent dye is TAMRA and the quencher is QSY7, emission monitor beads having the structure shown in Figure 9, emission monitor beads having the structure shown in Figure 10, and emission monitor beads with a quenching ability of at least 90%, at least 98%, at least 99%, or at least 99.9%.

[0056]

[0013] In a method of manufacture, a method for synthesizing a release-monitoring bead, the release-monitoring bead comprising a bead, a quencher, a fluorescent dye, and a photocleavable linker linking the fluorescent dye to the bead, the method comprising, in order: (i) providing a resin; (ii) coupling a lysine linker to the resin, the lysine linker-containing agent being L-Fmoc-Lys(4-methyltrityl)-OH; (iii) removing the Fmoc protecting group; (iv) coupling a quencher using an agent that is quencher-N-hydroxysuccinimide (quencher-NHS) as a quencher source; (v) removing the 4-methyltrityl protecting group using an agent that includes trifluoroacetic acid; (vi) coupling a photocleavable linker provided by the agent that is Fmoc-photocleavable linker-OH to the epsilon-amino group of the lysine; and (vii) coupling a fluorescent dye. The above embodiments, regardless of the order of the steps, are also provided. In another method embodiment, the above method is provided wherein the fluorescent dye is TAMRA and the quencher is QSY7.

[0057] In a method relating to the utility of release monitor beads, a method for controlling the concentration of a compound in a solution present in a picowell is provided, the method being applied to a bead-bound compound in the picowell, the picowell containing the solution, the bead-bound compound being linked to the bead by a cleavable linker, the method comprising (a) exposing the bead-bound compound to conditions that result in cleavage of the cleavable linker, causing the bead-bound compound to be released from the bead to produce a released compound, and following release, the released compound diffuses or disperses in the solution to provide a substantially uniform concentration of the compound in the solution, (b) the conditions comprising light capable of cleaving the cleavable linker, (c) the conditions being adjusted to provide a predetermined concentration of the substantially uniform concentration, and (d) the predetermined concentration is determined relative to the concentration of released fluorescent dye emitted from the bead-bound release monitor. Also provided are the above methods wherein the conditions are adjusted by adjusting one or more of the wavelength of light, the intensity of light, and the duration of light exposure; the above methods wherein the concentration of emitted fluorescent dye emitted from the bead-bound emission monitor is determined contemporaneously with releasing the bead-bound compound from the bead to form the released compound; and the above methods wherein the concentration of emitted fluorescent dye emitted from the bead-bound emission monitor is determined substantially prior to releasing the bead-bound compound from the bead to form the released compound.

[0058] The term "determined" can refer to a concentration that is predetermined and determined as the desired concentration before exposing the beads to light, or it can refer to a concentration that is determined in "real time," i.e., a concentration that is determined simultaneously with exposing the beads to light.

[0059] In an embodiment of the cap, the cap is in combination with a picowell plate including a plurality of picowells, the cap being usable with the picowell plate including the plurality of picowells, each of the plurality of picowells may be defined by an aperture, a floor, and a wall, the wall being defined by an aperture at a top and a floor at a bottom, the aperture being circular, the floor being circular, the wall being in the shape of a surface of a truncated cone, the aperture having a first diameter and the floor having a second diameter, the first diameter being greater than the second diameter;

[0060] The cap is a spherical cap that can fit snugly over the aperture, where the aperture is made of a polymer with a larger durometer (hard) and the cap is made of a polymer with a smaller durometer (soft), the relative durometers of the cap and aperture allowing the spherical cap to fit snugly over the aperture in a reversible manner, and the cap is (i) a passive cap that can block the picowell to prevent leakage and (ii) can absorb metabolites released by cells in cell culture medium when the cells are cultured in the picowell; (iii) an active cap that takes the form of beads containing multiple essentially identical compounds, each of the multiple essentially identical compounds linked to the beads by a cleavable linker, where cleavage of the cleavable linker releases at least some of the multiple compounds from the beads; and (iv) an active cap that takes the form of beads containing multiple identical drugs, each of the multiple essentially identical drugs linked to the beads by a cleavable linker, where cleavage of the cleavable linker releases at least some of the multiple drugs from the beads.

[0061] In a porous cap embodiment, there is provided a picowell plate and a plurality of porous caps in combination with a solid polymer coating, each of the plurality of porous caps comprising an upper surface and a lower surface, the picowell plate comprising a plurality of picowells, at least one porous cap contacting the picowells and reversibly and closely fitting thereto, each of the upper surfaces of the picowell plate and the plurality of porous caps being covered by a solid polymer coating, the solid polymer coating contacting and adhering to at least some of the upper surface of each cap, and (i) each of the plurality of picowells being capable of holding an aqueous solution in which reaction products are generated, at least some of the products being adsorbed onto the plurality of porous caps. A plurality of porous caps are provided, wherein (ii) a solution of a polymerizable agent that can be polymerized is poured onto the plurality of porous caps combined with the picowell plate, and the polymerizable agent polymerizes to form a substantially flat surface covering substantially all of the upper surface of the picowell plate, thereby immobilizing the polymerized agent on 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, and adhesion is maintained between the plurality of porous caps and the polymerized agent, resulting in an array of adhesive caps having a portion of their upper surface embedded in the polymerized agent and a portion of their lower surface accessible for analysis of absorbed reaction products.

[0062] This provides a method of manufacture embodiment for directing enzymatic synthesis of DNA barcodes using splint oligos. A method of making bead-bound concatenated DNA barcodes, where the bead-bound concatenated DNA barcodes comprise a plurality of DNA barcode modules, and may comprise one or more functional nucleic acids, and may comprise one or more identity-encoding nucleic acids that encode an identity of something other than the identity of a chemical library monomer, the method comprising: (a) applying to a bead a concatenated polynucleotide comprising a first DNA barcode module and a first annealing site, wherein the first annealing site is capable of hybridizing with a first splint oligonucleotide (sprint oligo), and the first splint oligo catalyzes the polymerization of a nucleotide complementary to the nucleotide of the hybridized first splint oligo into the concatenated polynucleotide; (b) attaching a linked polynucleotide to the bead along with the first splint oligo, wherein the polymerized nucleotide is complementary to the nucleotide of the hybridized first splint oligo after polymerization and comprises a second DNA barcode module and a second annealing site; (c) attaching a linked polynucleotide to the bead along with the first splint oligo and hybridizing the first splint oligo to the linked polynucleotide; (c) adding a DNA polymerase and deoxynucleotide triphosphates (dNTPs) to allow the DNA polymerase to catalyze the polymerization of the dNTPs to the linked polynucleotide, wherein the linked polynucleotide has a 3' free end and the polymerization is toward the 3' free end; and (d) washing away the first splint oligo. Also contemplated are methods as described above, wherein the first splint oligo comprises a first annealing site, a second DNA barcode module, and a second annealing site.

[0063] In a further method of manufacturing embodiment, the above method is provided, wherein the first splint oligo comprises 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 the sequencing primer to produce 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.

[0064] Additionally, methods described above in which the first splint oligo, DNA polymerase, and dNTPs are all added at the same time, or in which the first splint oligo, DNA polymerase, and dNTPs are each added at different times, are contemplated.

[0065] With respect to the internal and external locations on the beads, the above method is provided, wherein the beads comprise external and internal locations, the linked DNA barcodes attached to the beads are linked to the beads at locations that are substantially external to the beads and are linked to a lesser extent at internal locations on the beads, the beads also comprise a plurality of linked compounds all of which have substantially identical structures compared to one another, and the beads are substantially comprised of a hydrophobic polymer.

[0066] In a further method embodiment, the method includes (a) applying to a bead a linked first long polynucleotide comprising a first DNA barcode module, a first annealing site, a second DNA barcode, and a second annealing site, wherein the second annealing site is capable of hybridizing with a second splint oligo, and the second splint oligo is capable of serving as a template for a DNA polymerase that catalyzes polymerization of nucleotides complementary to the nucleotides of the hybridized second splint oligo into the linked first long polynucleotide, and wherein after polymerization, the polymerized nucleotides complementary to the nucleotides of the hybridized second splint oligo form a bead-bound first long polynucleotide. The above method further comprises the steps of (a) attaching the linked polynucleotide to the bead together with the second splint oligo and hybridizing the second splint oligo to the linked first long polynucleotide; (b) attaching the linked polynucleotide to the bead together with the second splint oligo and hybridizing the second splint oligo to the linked first long polynucleotide; (c) adding a DNA polymerase and deoxynucleotide triphosphates (dNTPs) to allow the DNA polymerase to catalyze the polymerization of the dNTPs to the linked long polynucleotide, wherein the linked long polynucleotide has a 3' free end and the polymerization is to the 3' free end; and (d) washing away the second splint oligo.

[0067] This relates to consecutive numbering of a first DNA barcode module, a second DNA barcode module, a third DNA barcode module, etc., for the production of an overall DNA barcode. This also relates to repeating a cycle of method steps multiple times in the production of an overall DNA barcode. Provided are methods as described above, wherein each of the plurality of DNA barcode modules is identified or named by a number, the method further comprising repeating the enumerated steps, wherein for the first iteration, the name of the DNA barcode module is incremented by adding one number to the existing name, the name of the annealing site is incremented by adding one number to the existing name, the name of the splint oligo is incremented by adding one number to the name of the existing distal end DNA barcode module, and the name of the "first long polynucleotide" is changed by adding one number to the existing name, and wherein the repetition of the enumerated steps includes 1 iteration, or 2 iterations, or 3 iterations, or 4 iterations, or 5 iterations, or more than 5 iterations, or more than 10 iterations.

[0068] Also contemplated is the above method, which includes a plurality of splint oligos, each of which includes a sequencing primer annealing site, and the sequencing primer annealing site is capable of hybridizing with a sequencing primer to provide a hybridized sequencing primer, and the hybridized sequencing primer is capable of directing sequencing of at least one bead-bound DNA barcode module and at least one bead-bound DNA barcode module.

[0069] This relates to embodiments involving splint oligos that direct DNA polymerases to synthesize functional nucleic acids and various types of informational nucleic acids. The above methods are provided in which at least one splint oligo comprises a functional nucleic acid or in which at least one splint oligo encodes information other than information about chemical library monomers. The above methods are also provided in which the method further comprises the step of ligating at least one DNA barcode module by click chemistry, without using any splint oligos.

[0070] Briefly, the present disclosure provides a system for screening compounds comprising: (a) a picowell array plate comprising a plurality of picowells, each picowell having a bottom defined by a floor and an upper aperture defining an opening at the top of the picowell, the upper aperture being separated from the floor with a wall between the upper aperture and the floor; and (b) at least one bead disposed in at least one picowell, the 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 linked DNA barcodes or orthogonal DNA barcodes. and where the DNA barcode is in the form of a concatenated DNA barcode, the concatenated DNA barcode is made by a method using either (i) click chemistry or (ii) a repeating cycle of steps, wherein the step in the repeating cycle includes using a splint oligo to anneal to the partially fabricated DNA barcode, wherein the annealed splint oligo is used as a template for extending the partially fabricated DNA barcode using a DNA polymerase, and wherein the splint oligo comprises bases complementary to the DNA barcode module that is polymerized into the partially fabricated DNA barcode.

[0071] In another aspect, the above system is provided, wherein the DNA barcode (a) comprises 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) may comprise one or more functional nucleic acids; and (c) may comprise one or more nucleic acids encoding a type of information other than information regarding the identity of a chemical library monomer.

[0072] Additionally, there is provided a system as described above, further comprising a plurality of caps, each of which can fit over the opening of a different picowell, each of which can minimize or prevent evaporation of fluid within the picowell, and each of which can minimize or prevent leakage of fluid within the picowell.

[0073] Also encompassed is the above system further comprising a plurality of spherical caps, each capable of fitting into the circular aperture of the 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.

[0074] Also contemplated are the above systems, where at least one bead comprises a DNA barcode in the form of a linked DNA barcode, wherein the linked DNA barcode comprises: (i) a sequencing primer binding site; (ii) a first DNA barcode module; (iii) a first annealing site capable of hybridizing with a first oligonucleotide splint that can be used to direct enzymatic synthesis of a second DNA barcode module; (iv) a second DNA barcode module; (v) a second annealing site capable of hybridizing with a second oligonucleotide splint that can be used to direct synthesis of a third DNA barcode; (vi) a third DNA barcode module; (vii) a third annealing site capable of hybridizing with a third oligonucleotide splint that can be used to synthesize a fourth DNA barcode.

[0075] In a method embodiment, a method is provided for screening a compound library for compounds having a desired property, the method comprising: (a) providing a plurality of beads, each bead comprising a plurality of oligonucleotides attached to its surface and a plurality of substantially related compounds attached to its surface, wherein the sequence of the oligonucleotides attached to the beads encodes the synthetic history of the plurality of substantially related compounds attached to its surface; (b) incorporating the plurality of beads in an assay for the desired property of compounds in the compound library; (c) capturing a signal from at least one bead that reflects the performance of the compound on the bead in the assay; (d) sequencing the plurality of oligonucleotides attached to at least one bead from which the assay signal was captured, without removing the oligonucleotides from the bead; and (e) identifying at least one compound from the sequencing read of step (d) and correlating this with the corresponding assay performance captured in the signal of step (c).

[0076] In further detail, the above methods are encompassed where the assay comprises a binding assay, or the assay comprises an activity assay, or the assay comprises a competitive binding assay or a competitive inhibition assay, or the assay comprises the interaction of the unbound compound with other assay reagents, and the unbound compound is the compound that is released from the surface of the bead, or the compound is released by cleaving a cleavable linker connecting the compound to the bead, or the assay is performed in defined volumes, with nominally one bead dispersed per defined volume.

[0077] In another embodiment, the limited volume comprises a droplet of water, or

[0078] Further contemplated are the above methods wherein the aqueous droplets are suspended in an oil medium or a hydrophobic liquid medium, or the limited volumes comprise picowells, or the picowells are organized in a regular array, or a plurality of limited volumes are organized in a regular array.

[0079] Further included are the above methods in which the limited volume comprises a layer of adhesive aqueous medium around the beads, the beads being suspended in a hydrophobic medium, the above methods in which assay reagents are washed away before sequencing the oligonucleotides, and the above methods in which sequencing step (d) is performed before 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, the above methods in which the removal of the oligonucleotides comprises enzymatic digestion, chemical cleavage, thermal degradation, or physical shearing, the above methods in which the binding assay comprises binding of RNA molecules to the beads, and the above methods in which the signal from the beads comprises sequencing of the bound RNA molecules are also contemplated.

[0080] In yet another aspect, there is provided a method as described above, wherein the binding assay comprises a fluorescently labeled binding assay, wherein the molecule that binds to the compound on the bead comprises a fluorescent dye, or a method as described above, wherein the binding assay comprises a nucleic acid labeled binding assay, wherein the molecule that binds to the compound on the bead comprises a nucleic acid tag, and further wherein the signal from the assay comprises sequencing of the nucleic acid tag attached to the molecule that binds to the compound on the bead.

[0081] Further, in embodiments of the methods related to properties, the above methods are provided, wherein the desired property includes one or more of: (i) inhibiting or stimulating the catalytic activity of an enzyme; (ii) stimulating a Th1-type immune response, which can be measured by a cell-based or in vivo assay; (iii) stimulating a Th2-type immune response, which can be measured by a cell-based or in vivo assay; (iv) inhibiting a Th1-type immune response, which can be measured by a cell-based or in vivo assay; (v) inhibiting a Th2-type immune response, which can be measured by a cell-based or in vivo assay; or (vi) stimulating or inhibiting ubiquitin-mediated degradation of a protein, which can be measured by purified protein, cell-based assay, or in vivo assay.

[0082] In one embodiment, a system for screening a compound library for compounds having a desired activity includes: (a) a sample compartment that receives a plurality of oligonucleotide-encoded beads to which compounds are bound; (b) a plurality of encapsulation compartments within the sample compartment, each encapsulation compartment nominally comprising a single bead dispersed in an assay medium, the assay medium further comprising reagents that are assayed for interaction with the compounds on the beads to produce a measurable signal; (c) a detector for measuring the signal; (d) a sequencing platform; and (e) a user interface that accepts one or more commands from a user. Also provided are systems as described above, in which the encapsulation compartments comprise droplets. In another aspect, the systems as described above are provided, in which the encapsulation compartments comprise picowells, further comprise assay reagents, the detector comprises an optical detector, or the sequencer comprises an optical detector.

[0083] In one aspect, the disclosure features a method for perturbing a cell by (a) providing a nucleic acid-encoded perturbation and confining a cell within 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) transferring a nucleic acid encoding the nucleic acid-encoded perturbation to the cell.

[0084] 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.

[0085] In some embodiments, the perturbation and the nucleic acid encoding the perturbation are unbound and free in solution. In some embodiments, the perturbation and the nucleic acid encoding the perturbation are bound to each other. In some embodiments, the perturbation and the nucleic acid encoding the perturbation are bound to the same substrate but are not bound to each other. In some embodiments, the bond of the perturbation to the substrate and the bond of the nucleic acid to the substrate are cleavable bonds. In certain embodiments, the cleavable bond is selected from the group consisting of a photocleavable bond, a temperature cleavable bond, a pH-sensitive bond, an acid cleavable bond, a base cleavable bond, an acoustic cleavable bond, a salt cleavable bond, a redox-sensitive bond, or a physically cleavable bond.

[0086] In some embodiments of this aspect of the disclosure, confining the cells and perturbations includes droplet encapsulation, emulsion encapsulation, picowell encapsulation, macrowell encapsulation, physical association, bubble encapsulation, or microfluidic confinement.

[0087] In some embodiments, controlling the perturbation includes controlling light exposure, controlling temperature exposure, controlling pH exposure, controlling exposure time, controlling sound exposure, controlling salt exposure, controlling chemical or physical redox potential, or controlling exposure to mechanical agitation.

[0088] In certain embodiments, incubating includes exposing the cell to the perturbation after cleaving the perturbation from the substrate or after cleaving the nucleic acid from the substrate, hi some embodiments, incubating includes exposing the cell to the perturbation without cleaving the perturbation from the substrate or without cleaving the nucleic acid from the perturbation.

[0089] In some embodiments, transferring a nucleic acid encoding a nucleic acid-encoded perturbation to a cell includes binding the nucleic acid to a cell surface of the cell. In certain embodiments, binding the nucleic acid to the cell surface of the cell includes intercalating the nucleic acid into the cell membrane. In certain embodiments, binding the nucleic acid to the cell surface of the cell includes binding the nucleic acid to a biomolecule on the cell surface. In certain embodiments, the biomolecule is a protein or a carbohydrate. In other embodiments, binding the nucleic acid to the cell surface of the cell includes binding through an optical tag on the nucleic acid.

[0090] In another aspect, the disclosure features a method for perturbing cells with a perturbation and encoding the cells with the identity of the perturbation. The method includes: (a) providing a bead-bound DNA-encoded library; (b) entrapping the cells 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 encoding nucleic acids are bound to the beads, and the encoding nucleic acids encode the identity of the compounds, and where the bead-bound DNA-encoded library and the cells are entrapping in a confining volume; (c) releasing the compounds from the beads and incubating the compounds with the cells in the confining volume; (d) optionally releasing the encoding nucleic acid tags from the beads; and (e) binding the encoding nucleic acid tags to the cells, thereby preserving the identity of the compounds through the encoding nucleic acid tags bound to the cells.

[0091] In yet another aspect, the disclosure features a method of perturbing cells, encoding the cells with the identity of the perturbation, and measuring the response of the cells to the perturbation. The method includes: (a) contacting the cells in a first defined volume with a library encoded by DNA bound to beads, where the library encoded by DNA bound to the beads 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 bound to the beads and the encoding nucleic acids encode the identity of the compounds; (b) releasing the compounds in the library from the beads and incubating the compounds in the library with the cells in the first defined volume; (c) optionally releasing the encoding nucleic acid tags from the beads in the first defined volume; (d) capturing the encoding nucleic acid tags on the cell surface of the cells, whereby the cells are exposed to compounds in the library and the identities of the exposed compounds are captured on the cell surface; and (e) releasing the cells from the first defined volume, whereby the encoding nucleic acid tags (f) capturing the previously perturbed, nucleic acid-tagged cells with response detection beads in a second defined volume, wherein the cells are exposed to cell lysis conditions that expose the cellular contents of the cells to the response capture beads, and the response capture beads contain capture probes that capture the cellular contents and the nucleic acid tag encoding the perturbation in the previously perturbed, nucleic acid-tagged cells; (g) incubating the response capture beads with the lysed cells in the second defined volume, thereby capturing both the cellular contents and the nucleic acid tag encoding the perturbation on the response capture beads; (h) optionally converting the cellular response to the perturbation into a nucleic acid signal, wherein the cellular response to the perturbation is not a nucleic acid signal; and (i) sequencing the nucleic acid tag bound to the response capture beads, thereby correlating the identity of the perturbation with the cellular response to the perturbation.

[0092] In yet another aspect, the disclosure provides a method for detecting a cell's cellular response to a perturbation contained in a functionalized perturbation bead, comprising: (a) providing an array of picowells and a library of functionalized perturbation beads, wherein a picowell can accommodate a single cell and a single functionalized perturbation bead, each functionalized perturbation bead comprising a plurality of substantially identical, different releasable compounds and a plurality of nucleotide barcodes encoding the compounds, the nucleotide barcodes being functionalized barcodes capable of capturing the cellular contents of the cell, 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 a compound from the functionalized perturbation bead and interfering with the released compound and the cell. (e) lysing the cells to release the cellular contents; (f) capturing one or more components of the cellular contents onto functionalized oligonucleotides on the functionalized perturbation beads, wherein the capturing includes hybridization and enzymatic extension that combines a nucleotide barcode with a nucleic acid element of the cellular contents, thereby forming a hybrid between the nucleotide barcode and the nucleic acid element of the cellular contents; and (g) releasing the hybrids, collecting the hybrids from the library of functionalized perturbation beads, and sequencing the hybrids, thereby correlating the perturbation with the cellular response to the perturbation.

[0093] A system for screening compounds is provided. Possible embodiments include the following:

[0094] The system may include a picowell array plate containing a plurality of picowells, each picowell having an upper aperture defining an opening at the top of the picowell and a bottom defined by a floor, the upper aperture being separated from the floor by a wall, the wall being between the upper aperture and the floor.

[0095] The system can include a single bead disposed in a picowell, the bead comprising a plurality of substantially identical DNA barcodes bound to the bead and a plurality of substantially identical compounds bound to the bead.

[0096] The beads may comprise bead-bound DNA barcodes in the form of linked DNA barcodes or orthogonal DNA barcodes. When the DNA barcodes are in the form of linked DNA barcodes, the linked DNA barcodes are made by methods using click chemistry or repeat cycles or both.

[0097] The repeat cycle can involve using a splint oligonucleotide (splint oligo) that can hybridize to the partially generated bead-bound DNA barcode. Hybridization is mediated by an annealing site on the splint oligo and a corresponding complementary annealing site on the partially generated bead-bound DNA barcode.

[0098] The annealed splint oligo can be used as a template to extend the partially generated DNA barcode using a DNA polymerase. The splint oligo contains bases complementary to the DNA barcode module that will be polymerized into the partially generated bead-bound DNA barcode. The splint oligo also contains bases complementary to the annealing site that will be polymerized into the partially generated bead-bound DNA barcode.

[0099] 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 the corresponding chemical library monomer, and the term "compound" is used to refer to the completed product comprising one or more chemical library members, the completed DNA barcode identifying the compound.

[0100] The system may further include an oligonucleotide sequencing primer capable of directing sequencing of one or more DNA barcode modules included in the bead-bound DNA barcode, and the system may include a DNA sequencing device, wherein the DNA sequencing device is not a luminescence-based sequencer and is not a pH-based DNA sequencing device.

[0101] The system may further include a plurality of spherical caps, each cap capable of fitting to a circular aperture of a picowell, 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.

[0102] At least one bead disposed in at least one picowell includes at least one response capture element linked to said at least one bead.

[0103] At least one of the beads disposed within the picowell comprises at least one response capture element linked to the at least one bead, the at least one response capture element comprising poly(dT), an exon-targeting RNA probe, an antibody, or an aptamer.

[0104] The DNA barcode may be a concatenated DNA barcode or an orthogonal DNA barcode. The DNA barcode comprises one or more DNA barcode modules. Each of the one or more DNA barcode modules encodes information identifying a chemical library monomer. The concatenated DNA barcode or orthogonal DNA barcode further comprises one or both of one or more functional nucleic acids and one or more nucleic acids encoding information other than the identity of the chemical library monomer.

[0105] The linked DNA barcodes attached to the beads may comprise a first DNA barcode module, or a first DNA barcode module, a first annealing site, and a second DNA barcode module, or 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 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 a first DNA barcode module. The DNA barcode module may include 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 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.

[0106] The bead comprises orthogonal DNA barcodes, the bead comprising an exterior surface comprising a first nucleic acid comprising a first DNA barcode module and an annealing site for a sequencing primer and linked to the bead at a first position, a second nucleic acid comprising a second DNA barcode module and an annealing site for a sequencing primer and linked to the bead at a second position, and a third nucleic acid comprising a third DNA barcode module and an annealing site for a sequencing primer and linked to the bead at a third position, the first, second, and third positions on the bead being located at different positions on the exterior surface of the bead.

[0107] The linked DNA barcodes are produced by both click chemistry and splint oligo-based repeat cycles, by both click chemistry and non-click chemistry methods, by click chemistry alone, or by splint oligo-based repeat cycles alone.

[0108] Each of the plurality of substantially identical bead-bound compounds is linked to the bead by a cleavable linker, or by a cleavable linker that is a photocleavable linker, or by a non-cleavable linker.

[0109] At least one bead comprises a graft copolymer consisting of a lightly cross-linked polystyrene matrix onto which polyethylene glycol (PEG) has been grafted.

[0110] At least one picowell contains at least one cell.

[0111] A plurality of substantially identical bead-bound compounds are attached to at least one bead by a cleavable linker, and cleavage of the cleavable linker releases the bead-bound compounds from the bead to produce released compounds.

[0112] The released compound can contact at least one cell, where the at least one cell is a non-cancer mammalian cell, a mammalian cancer cell, a dead mammalian cell, an apoptotic mammalian cell, a necrotic mammalian cell, a bacterial cell, a plasmodium cell, a metabolically active cell but having a crosslinked genome and incapable of undergoing cell division, or a virally infected mammalian cell.

[0113] Each picowell has a bottom defined by a floor and an upper aperture defining an opening at the top of the picowell, the upper aperture being separated from the floor and having a wall between the upper aperture and the floor, the aperture being circular. The floor is circular. The wall is in the form of a truncated cone. The aperture has a first diameter and the floor has a second diameter. The first diameter is larger than the second diameter.

[0114] Each picowell has a top aperture defining an opening at the top of the picowell and a bottom defined by a floor. The top aperture is separated from the floor, and a wall exists between the top aperture and the floor, and the aperture is circular. The floor is circular. The wall is in the form of a truncated cone. The aperture has a first diameter and the floor has a second diameter. The first diameter is larger than the second diameter.

[0115] The cap fits snugly over the aperture. The aperture is made of a polymer with a larger durometer (harder). The cap is made of a polymer with a smaller durometer (softer). The relative durometers of the cap and aperture allow the cap to reversibly fit snugly over the aperture.

[0116] The caps are: a cap intended only to close the picowell to prevent leakage; a passive cap that can absorb metabolites released by cells in a cell culture medium when the cells are cultured in the picowell; an active cap in the form of beads containing multiple essentially identical compounds, each of which is linked to the bead by a cleavable linker; and an active cap in the form of beads containing multiple identical drugs, each of which is linked to the bead by a cleavable linker.

[0117] The system may include at least one spherical cap.

[0118] The system may include at least one non-spherical cap.

[0119] The DNA barcode does not encode any chemical monomer, but instead comprises one or more nucleic acids that identify the class of compound cleavably attached to the bead and one or more steps in a multi-step pathway of organic synthesis, where the bead-attached nucleic acid corresponds to a given chemical monomer used to make the bead-attached compound. The bead-attached nucleic acid corresponding to a given chemical monomer identifies that chemical monomer, the date the bead-attached compound was synthesized, the disease the bead-attached compound is intended to treat, the cellular event the bead-attached compound is intended to stimulate or inhibit, or the reaction conditions used to attach the given chemical library monomer to the bead.

[0120] There is no headpiece that links any of the bead-bound compounds to any of the bead-bound DNA barcodes.

[0121] The linked DNA barcodes include at least one nucleic acid that is a DNA barcode module, and at least one functional nucleic acid that can be used as an annealing site for a sequencing primer, capable of forming a hairpin structure, the hairpin structure including a sequencing primer, an annealing site for the sequencing primer, and a bend in the hairpin structure, wherein the bend is 5-prime to the sequencing primer, 3-prime to the annealing site for the sequencing primer, or a spacer nucleic acid.

[0122] The orthogonal DNA barcode comprises multiple DNA barcode modules, each of which is linked to a different site on a bead, either directly or via a linker, and each of the multiple DNA barcode modules comprises at least one functional nucleic acid capable of serving as an annealing site for a sequencing primer and capable of forming a hairpin structure. The hairpin structure comprises a sequencing primer, an annealing site for the sequencing primer, and a bend in the hairpin structure. The bend is 5-prime to the sequencing primer, 3-prime to the annealing site for the sequencing primer, or a spacer nucleic acid.

[0123] A method for controlling the concentration of a compound in a solution present in a picowell. The method is applied to a bead-bound compound in the picowell. The picowell contains a solution. The bead-bound compound is linked to the bead by a cleavable linker. The method can include exposing the bead-bound compound to conditions that cause cleavage of the cleavable linker. The conditions can include light that can cleave the cleavable linker.

[0124] The method may include the step of releasing the bead-bound compound from the bead to produce a released compound, and following release, the released compound diffusing or dispersing in solution to provide a substantially uniform concentration of the compound in the solution.

[0125] The method can include adjusting conditions to produce a predetermined concentration of substantially uniform concentration, the predetermined concentration being determined relative to the concentration of emitted fluorescent dye emitted from the bead-bound emission monitor.

[0126] The conditions are adjusted by adjusting one or more of the wavelength of light, the intensity of light, and the time of light exposure, and the concentration of emitted fluorescent dye emitted from the bead-bound emission monitor may be determined simultaneously with the release of the bead-bound compound from the bead to form the released compound, or the concentration of emitted fluorescent dye emitted from the bead-bound emission monitor may be determined at a time substantially prior to the release of the bead-bound compound from the bead to form the released compound.

[0127] A cap in combination with a picowell plate containing a plurality of picowells. The cap can be used with said picowell plate.

[0128] Each of the plurality of picowells can be defined by an aperture, a floor, and a wall. The wall is defined by an aperture at the top and a floor at the bottom. The aperture is circular. The floor is circular. The wall takes the shape of the surface of a truncated cone, with the aperture having a first diameter and the floor having a second diameter. The first diameter is larger than the second diameter. The cap is a spherical cap that can fit snugly over the aperture. The aperture is made of a polymer with a larger durometer (hard). The cap is made of a polymer with a smaller durometer (soft). The relative durometers of the cap and aperture allow the spherical cap to reversibly fit snugly over the aperture. The cap can be a passive cap that can close the picowell to prevent leakage and absorb metabolites released by cells in a cell culture medium when the cells are cultured in the picowell; or an active cap that takes the form of beads containing a plurality of essentially identical compounds, each of the plurality of essentially identical compounds being linked to the bead by a cleavable linker, at least one of the plurality of picowells containing an aqueous medium, and cleavage of the cleavable linker releases at least some of the plurality of essentially identical compounds from the beads into the aqueous medium.

[0129] A system including a picowell array plate including a plurality of picowells, each picowell having an upper, generally flat surface, an upper aperture defining an opening at the top of the picowell, and a bottom defined by a floor. The upper aperture is separated from the floor by a wall. A wall may exist between the upper aperture and the floor, and beads may be disposed in at least one of the plurality of picowells. The beads may include a plurality of substantially identical bead-bound DNA barcodes and a plurality of substantially identical bead-bound compounds, and the picowell array plate may further include a mat capable of tightly covering the openings at the top of at least one or all of the plurality of picowells, or capable of practically tightly covering the openings at the top of at least one or all of the plurality of picowells. The tight covering is reversible. The mat may include one or all of 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, generally flat surface of the picowell array plate, and an adhesive surface capable of maintaining reversible adhesion to the top, generally flat surface of the picowell array plate.

[0130] A method of determining a signal from an assay and a sequencing readout on the beads, thereby identifying one or more compounds of interest from the assay, comprising the steps of: providing a plurality of beads, each bead comprising a plurality of substantially related compounds and a plurality of oligonucleotides bound to the beads, wherein the plurality of oligonucleotides bound to each bead identify the plurality of compounds bound to the same bead; performing an assay involving the plurality of compounds bound to the beads; determining at least one signal reflecting the performance of the compound in the assay in step b; sequencing the plurality of oligonucleotides bound to the beads without removing the oligonucleotides from the beads, thereby determining a sequencing readout for each bead; identifying the compound bound to the bead by the sequencing readout in step d and correlating this with the assay performance contained in the determined signal in step c; and identifying the compound of interest by the bead having the signal and sequencing readout from the assay.

[0131] A method for screening a compound library for compounds having a desired property includes providing a plurality of beads, each bead comprising a plurality of oligonucleotides bound to its surface and a plurality of substantially related compounds bound to its surface, wherein the sequences of the oligonucleotides bound to the beads encode the identities of the plurality of substantially related compounds bound to its surface, incorporating the plurality of beads into an assay for the desired property of the compounds in the compound library and capturing a signal from at least one bead, the signal reflecting the performance of the compound on the bead in the assay, wherein the assay signal is also captured without removing the oligonucleotides from the beads; sequencing the plurality of oligonucleotides bound to at least one bead; and identifying at least one compound from the sequencing readout in step (d) and correlating this with its corresponding assay performance captured in the signal in step (c).

[0132] Each bead contains a different plurality of oligonucleotides and a different plurality of substantially related compounds.

[0133] The plurality of oligonucleotides is a plurality of DNA oligonucleotides.

[0134] Multiple compounds are attached to the bead surface by linking multiple compound building blocks in tandem, all of which together make up the compound.

[0135] Each DNA module and each chemical building block is assembled sequentially and alternatively.

[0136] Each compound in the plurality of identical compounds is attached to the surface of a bead by a cleavable linker.

[0137] The cleavable linker is a photocleavable linker, a protease-cleavable linker, or an acid-cleavable linker.

[0138] The compound is cleaved from the bead surface after step (a) and before step (d).

[0139] The signal that reflects the desired property of the compound is a fluorescent signal.

[0140] The size of each bead ranges from 1 μm to 100 μm.

[0141] The size of each bead is 1 μm to 10 μm.

[0142] Each bead is approximately 3 μm in size.

[0143] The method further includes identifying a candidate target among the plurality of possible targets. Compounds having desirable properties bind to the candidate target.

[0144] Step (b) involves incubating a plurality of beads among a plurality of possible targets.

[0145] Possible targets are proteins or nucleic acids.

[0146] Sequencing is performed by single molecule real-time sequencing, ion semiconductor sequencing, thermal sequencing, 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 with mass spectrometry, microfluidic Sanger sequencing, and oligonucleotide extension sequencing.

[0147] A method for screening a compound library for compounds having a desired property includes providing a plurality of beads, each bead comprising a plurality of oligonucleotides bound to its surface and a plurality of substantially related compounds bound to its surface, wherein the sequences of the oligonucleotides bound to the beads encode the synthetic history of the plurality of substantially related compounds bound to its surface; incorporating the plurality of beads into an assay for the desired property of the compounds in the compound library; capturing a signal from at least one bead, the signal reflecting the performance of the compound on the bead in the assay; sequencing the plurality of oligonucleotides bound to at least one bead, where the assay signal is also captured without removing the oligonucleotides from the bead; and identifying at least one compound from the sequencing readout in step (d) and correlating this with its corresponding assay performance captured in the signal in step (c).

[0148] Assays include binding assays.

[0149] Assays include activity assays.

[0150] Assays include competitive binding assays or competitive inhibition assays.

[0151] The assay involves the interaction of the unbound compounds with other assay reagents. The unbound compounds are compounds that have been released from the bead surface.

[0152] The compound is released by cleaving the cleavable linker connecting the compound to the bead.

[0153] The assay is performed in defined volumes, with nominally one bead dispersed per defined volume.

[0154] The confined volume contains a droplet of water.

[0155] The water droplets are suspended in an oil or hydrophobic liquid medium.

[0156] The limited volume comprises a picowell.

[0157] The picowells are organized in a regular array.

[0158] The multiple confined volumes are organized in a regular array.

[0159] The confined volume contains a layer of adhesive aqueous medium surrounding the beads, which are suspended in a hydrophobic medium.

[0160] The assay reagents are washed away before sequencing the oligonucleotides.

[0161] The sequencing step (d) is carried out before the assaying step (b).

[0162] The oligonucleotides on the beads are removed after the sequencing step but before the assay step.

[0163] Removal of oligonucleotides includes enzymatic digestion, chemical cleavage, thermal decomposition, or physical shearing.

[0164] The binding assay involves binding of RNA molecules to beads.

[0165] The signal from the beads comprises the sequence of the bound RNA molecule.

[0166] Binding assays include fluorescently labeled binding assays, in which the molecule that binds to the compound on the bead contains a fluorescent dye.

[0167] Binding assays include nucleic acid tag binding assays, in which the molecule that binds to the compound on the bead comprises a nucleic acid tag, and further the signal from the assay comprises sequencing of the nucleic acid tag attached to the molecule that binds to the compound on the bead.

[0168] Desired properties include one or more of inhibiting or stimulating the catalytic activity of an enzyme, stimulating a Th1-type immune response that can be measured by a cell-based or in vivo assay, stimulating a Th2-type immune response that can be measured by a cell-based or in vivo assay, inhibiting a Th1-type immune response that can be measured by a cell-based or in vivo assay, inhibiting a Th2-type immune response that can be measured by a cell-based or in vivo assay, stimulating or inhibiting ubiquitin-mediated degradation of a protein that can be measured by purified protein, cell-based assay, or in vivo assay.

[0169] A system for screening a compound library for compounds having a desired activity, comprising: a sample compartment that receives a plurality of beads to which compounds are bound and encoded by oligonucleotides; a plurality of encapsulation compartments within the sample compartment, each encapsulation compartment nominally containing a single bead dispersed in an assay medium, the assay medium further containing reagents whose interaction with the compounds on the beads is assayed to produce a measurable signal; a detector for measuring the signal; a sequencing platform; and a user interface that accepts one or more commands from a user.

[0170] The encapsulated compartment contains a droplet.

[0171] The encapsulation compartment comprises a picowell.

[0172] The encapsulated compartment contains the assay reagents.

[0173] The detector includes an optical detector.

[0174] The sequencer includes an optical detector.

[0175] A method for perturbing a cell, comprising providing a nucleic acid-encoded perturbation, confining a cell within the nucleic acid-encoded perturbation, and contacting the cell with the nucleic acid-encoded perturbation in a confined volume, wherein the onset and dose of the perturbation are controlled, the cell is incubated with the nucleic acid-encoded perturbation for a specified time, and a nucleic acid encoding the nucleic acid-encoded perturbation is delivered to the cell.

[0176] Nucleic acid-encoded perturbations are compounds or drug molecules that are encoded by nucleic acids.

[0177] The nucleic acid-encoded perturbations are DNA-encoded libraries.

[0178] The perturbation and the nucleic acid encoding the perturbation are unbound and free in solution.

[0179] The perturbation and the nucleic acid encoding the perturbation are linked to one another.

[0180] The perturbation and the nucleic acid encoding the perturbation are bound to the same substrate but are not bound to each other.

[0181] The bond of the perturbation to the substrate and the bond of the nucleic acid to the substrate are cleavable bonds.

[0182] The cleavable bond is selected from the group consisting of a photocleavable bond, a temperature cleavable bond, a pH sensitive bond, an acid cleavable bond, a base cleavable bond, an acoustic cleavable bond, a salt cleavable bond, a redox sensitive bond, or a physically cleavable bond.

[0183] Confining cells and perturbations includes droplet encapsulation, emulsion encapsulation, picowell encapsulation, macrowell encapsulation, physical binding, bubble encapsulation, or microfluidic confinement.

[0184] Controlling the perturbation includes controlling light exposure, controlling temperature exposure, controlling pH exposure, controlling exposure time, controlling sound exposure, controlling salt exposure, controlling chemical or physical redox potential, or controlling exposure to mechanical agitation.

[0185] Incubating includes exposing the cell to the perturbation after cleaving the perturbation from the substrate or after cleaving the nucleic acid from the substrate.

[0186] Incubating includes exposing the cell to the perturbation without cleaving the perturbation from the substrate or cleaving the nucleic acid from the perturbation.

[0187] Transferring a nucleic acid encoding a nucleic acid-encoded perturbation into a cell includes binding the nucleic acid to the cell surface of the cell.

[0188] Binding a nucleic acid to the cell surface of a cell includes intercalating the nucleic acid into the cell membrane.

[0189] Binding a nucleic acid to the cell surface of a cell includes binding the nucleic acid to a biomolecule on the cell surface.

[0190] The biomolecule is a protein or a carbohydrate.

[0191] Binding the nucleic acid to the cell surface of the cell includes binding through an optical tag on the nucleic acid.

[0192] A method for perturbing cells with perturbations and encoding the cells with the identity of the perturbations includes preparing a bead-bound DNA-encoded library and confining the cells with the bead-bound DNA-encoded library. The bead-bound DNA-encoded library contains one or more copies of combinatorially synthesized compounds and one or more copies of encoding nucleic acid tags. The compounds and encoding nucleic acids are bound to the beads. The encoding nucleic acids encode the identity of the compounds. The bead-bound DNA-encoded library and the cells are confined in a confined volume, and the compounds are optionally released from the beads, incubated with the cells in the confined volume, and the encoding nucleic acid tags are released from the beads, binding the encoding nucleic acid tags to the cells, thereby preserving the identity of the compounds through the encoding nucleic acid tags bound to the cells.

[0193] A method for perturbing cells, encoding the cells with the identity of the perturbation, and measuring the cell's response to the perturbation includes contacting cells in a first limited volume with a library encoded by DNA bound to beads. The library encoded by DNA bound to beads includes 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 are bound to the beads. The encoding nucleic acids encode the identity of the compounds, and the compounds in the library are released from the beads. The compounds in the library are incubated with cells in the first limited volume, and the encoding nucleic acid tags may be released from the beads in the first limited volume. The encoding nucleic acid tags are captured on the cell surface of the cells, thereby exposing the cells to the compounds in the library and capturing the identity of the exposed compounds on the cell surface, and the cells are released from the first limited volume. The encoding nucleic acid tags are bound to the cells, and the encoding nucleic acid tags encode the identity of the compounds to which the cells will be exposed. The previously perturbed, nucleic acid-tagged cells are captured by response detection beads in a second limited volume. The cells are exposed to lysis conditions that expose the intracellular contents of the cells to response capture beads. The response capture beads contain capture probes that capture cellular components and nucleic acid tags that encode perturbations in previously perturbed and nucleic acid-tagged cells, and the response capture beads are incubated with the lysed cells in a second, limited volume, thereby capturing both the intracellular contents and the nucleic acid tags that encode the perturbations on the response capture beads, and converting the cellular response to the perturbation into a nucleic acid signal. The cellular response to the perturbation is not a nucleic acid signal, but rather the nucleic acid tag bound to the response capture beads is sequenced, thereby correlating the identity of the perturbation with the cellular response to the perturbation.

[0194] A method for perturbing cells and capturing the cellular response to the perturbation includes preparing an array of picowells and a library of functionalized perturbation beads. The picowell can accommodate a single cell and a single functionalized perturbation bead, each functionalized perturbation bead containing a plurality of substantially identical, different releasable compounds and a plurality of nucleotide barcodes encoding the compounds. The nucleotide barcodes are functionalized barcodes capable of capturing the cellular contents of the cell. The cellular contents of the cell include the cellular response to the perturbation contained in the functionalized perturbation bead. A single cell is captured in each picowell of the picowell array, a single functionalized perturbation bead is captured in the picowell containing the single cell, a compound is released from the functionalized perturbation bead, and the cell is incubated with the released compound. The compound between the picowells has minimal diffusion, lysing the cell and releasing the cellular contents, and capturing one or more components of the cellular contents to functionalized oligonucleotides on the functionalized perturbation bead. The capturing includes hybridization and enzymatic extension that combines the nucleotide barcode with a nucleic acid element of the cellular contents, thereby forming a hybrid between the nucleotide barcode and the nucleic acid element of the cellular contents, releasing the hybrid, collecting the hybrid from a library of functionalized perturbed beads, and sequencing the hybrid, thereby correlating the perturbation with the cellular response to the perturbation.

[0195] A system for screening compounds for their ability to modulate the biological activity of a cell or a cellular component is provided. The system may include an assay device including multiple wells, each well separated from the other wells. The system may include multiple beads, each a single bead disposed in a single well. Each bead may include multiple substantially identical compounds bound to the bead. The bead-bound compounds are covalently linked to the bead by a cleavable linker, allowing the compounds to be released from the bead in a dose-dependent manner that is measurable as part of the assay.

[0196] Also provided is a method for screening compounds for their ability to modulate the biological activity of a cell or a component of a cell. A system is also provided in conjunction with the method. The system may include an assay device including a plurality of wells, each well separated from the other wells. The system may include a plurality of beads, each suitable for placement in a respective well, each bead comprising a plurality of substantially identical compounds bound to the bead, the bead-bound compounds being covalently linked to the bead by a cleavable linker, such that the compounds can be released from the bead in a dose-dependent manner that is measurable as part of the assay.

[0197] The beads further comprise a plurality of substantially identical bead-bound DNA barcodes linked to the beads by (i) a cleavable linker or (ii) a non-cleavable linker, where if the DNA barcodes are linked to the beads by a cleavable linker, the cleavable linker is orthogonal to the cleavable linker used to link the bead-bound compound to the bead, and the compound is identified by the DNA barcode.

[0198] The system further includes transferring beads to the wells using a transfer dispenser that allows only a single bead to be contained in a single well. In one embodiment, these devices have multiple cavities, each cavity having a single opening diameter, that may be configured to hold only a single bead of the plurality of beads. The assay device and transfer dispenser may be configured to fit or mate with each other, and a gap may exist between the assay device and the transfer device. When the assay device and transfer dispenser fit or mate with each other, the cavity of the transfer dispenser may align with the well of the device.

[0199] Each well may have an opening diameter larger than the opening diameter of each of the cavities, such that when each cavity is placed over a respective well, a confined space including the cavity and the well may be formed.

[0200] A single bead is released from the cavity through the containment space and deposited into the well.

[0201] The beads may be non-magnetic beads.

[0202] The gaps, if present, may have a size (e.g., diameter) smaller than the size (e.g., diameter) of the beads to be transported into the device, thereby preventing movement of the beads through the gaps.

[0203] In conjunction with the method, a device is provided, the device including at least one processor and a memory storing at least one program for execution by the at least one processor, the at least one program including instructions that, when executed by the at least one processor, cause the at least one processor to perform operations.

[0204] Manipulation may involve moving the assay device and the transfer dispenser so that they fit or mate with each other, and there may be a gap between said assay device and said transfer device.

[0205] The manipulation may include aligning a cavity of the transfer dispenser with a well of the device.

[0206] The manipulation may involve fitting or mating the assay device and the transfer dispenser together to form a confined space.

[0207] The manipulation may include expelling a single bead from the cavity through said confining space.

[0208] Further provided is a non-transitory computer-readable storage medium storing at least one program for screening compounds for the ability to modulate the biological activity of a cell or a component of a cell. A system is provided in conjunction with the non-transitory computer-readable storage medium. The system includes an assay device including a plurality of wells, each well separated from the other wells. The system includes a plurality of beads, each suitable for placement in a respective well, each bead comprising a plurality of substantially identical bead-bound compounds, the bead-bound compounds covalently linked to the bead by a cleavable linker, such that the compounds are releasable from the bead in a dose-dependent manner that is measurable as part of the assay.

[0209] The beads may further comprise a plurality of substantially identical bead-bound DNA barcodes linked to the beads by (i) a cleavable linker or (ii) a non-cleavable linker, where if the DNA barcodes are linked to the beads by a cleavable linker, the cleavable linker is orthogonal to the cleavable linker used to link the bead-bound compound to the bead, and the compound is identified by the DNA barcode.

[0210] The system, along with the non-transitory computer-readable storage medium, may further include a transfer dispenser having a plurality of cavities, each cavity having a single opening diameter and configured to hold only a single bead of the plurality of beads. The assay device and the transfer dispenser may be configured to fit or mate with each other, and a gap may exist between the assay device and the transfer device. When the assay device and the transfer dispenser fit or mate with each other, the cavities of the transfer dispenser may align with the wells of the device.

[0211] Each well may have an opening diameter larger than the opening diameter of each of the cavities, such that when each cavity is placed over a respective well, a confined space including the cavity and the well may be formed.

[0212] A single bead is released from the cavity through the containment space and deposited into the well.

[0213] The beads may be non-magnetic beads.

[0214] The gaps, if present, may have a size (eg, diameter) smaller than the size (eg, diameter) of the beads to be transported into the device.

[0215] The at least one program may be for execution by at least one processor and a memory storing the at least one program, the at least one program including instructions that, when executed by the at least one processor, cause the at least one processor to perform an operation. The operation may include moving the assay device and the transfer dispenser to fit or mate with each other, and there may be a gap between the assay device and the transfer device. The operation may include moving a cavity of the transfer dispenser and a well of the device to align. The operation may include moving the assay device and the transfer dispenser to fit or mate with each other to form a containment space. The operation may include moving a single bead from a cavity through the containment space to be released. The operation may include moving a single bead to be deposited into the well.

[0216] The beads may further comprise a plurality of substantially identical bead-bound DNA barcodes linked to the beads by (i) a cleavable linker or (ii) a non-cleavable linker. When the DNA barcodes are linked to the beads by a cleavable linker, the cleavable linker is orthogonal to the cleavable linker used to link the bead-bound compound to the bead, and the compound is identified by the DNA barcode.

[0217] The system may further include a transfer dispenser having multiple cavities, each cavity having a single opening diameter and configured to hold only a single bead of the multiple beads. The assay device and transfer dispenser may be configured to fit or mate with each other, and there may be a gap between the assay device and the transfer device. When the assay device and transfer dispenser fit or mate with each other, the cavities of the transfer dispenser may align with the wells of the device.

[0218] Each well may have an opening diameter larger than the opening diameter of each of the cavities, such that when each cavity is placed over a respective well, a confined space including the cavity and the well may be formed.

[0219] A single bead is released from the cavity through the containment space and deposited into the well.

[0220] The beads may be non-magnetic beads.

[0221] The gaps, if present, may have a size (eg, diameter) smaller than the size (eg, diameter) of the beads to be transported into the device.

[0222] Each of the above-referenced systems, methods, and non-transitory computer-readable storage media may include one or more of each of the features listed below, in any suitable combination.

[0223] If the bead-bound compound is released from the bead, the bead-bound DNA barcode may not be released from the bead.

[0224] Each of the confined volumes may contain a single bead.

[0225] The limited volume may be a picowell or a droplet.

[0226] Cleavable covalent linkers can be cleaved by light, temperature change, pH change, sound, salt, or oxidative change.

[0227] The cleavable linker can be cleaved by light.

[0228] The light may be ultraviolet light.

[0229] The cellular components may be lipids, proteins, carbohydrates, or nucleic acids.

[0230] The cellular component may be nucleic acid.

[0231] The nucleic acid may be mRNA.

[0232] The cellular component may be a cytokine, an antigen, or an enzyme.

[0233] The amount of compound released from the beads in a measurable dose-dependent manner for testing is measured by a change in signal generated from a signal-generating compound that is bound to the beads and is distinct from the compound being tested, the signal-generating compound may generate no signal or an attenuated signal on the beads and generate an increased signal once released from the beads, the increase in signal correlates with the amount of compound to be tested released from the beads, and the compound released from the beads and the signal-generating compound released from the beads may be released under the same conditions.

[0234] The compound and the signal-generating compound may be bound to the same bead.

[0235] The signal-generating compound can be a fluorescent compound whose fluorescence is quenched by the quencher when the signal-generating compound and the quencher are bound to the same bead.

[0236] At least one of the confined volumes may contain beads with bead-bound DNA barcodes and bead-bound compounds that may be different from the bead-bound DNA barcodes and bead-bound compounds on the beads in another of the confined volumes.

[0237] The DNA barcode is linked to the bead by a cleavable linker, which may be different from the cleavable linker that links the bead-binding compound to the bead.

[0238] The DNA barcode may be linked to the bead by a non-cleavable linker.

[0239] The DNA barcode may be linked to the bead by a cleavable linker, which may be cleaved by a different mechanism than the cleavable linker that links the bead-bound compound to the bead.

[0240] The transfer dispenser may further include a locking mechanism that locks the transfer dispenser and the assay device in proper alignment.

[0241] The diameter of each of the cavities may be reversibly expandable to accommodate the beads.

[0242] The diameter of each of the cavities may be at least 100.1% of the diameter of the bead in its original configuration or in its expanded configuration to accommodate the bead.

[0243] The depth of each of the cavities may be at least 50% of the size (eg, diameter) of the bead to about 125% of the size (eg, diameter) of the bead.

[0244] The transfer dispenser may be configured to incorporate a containment cap for transferring beads to multiple cavities of the transfer dispenser beneath the assay device.

[0245] A single bead is a bead that is excluded by size (eg, diameter) such that two beads would fit into a single cavity.

[0246] The beads may have a size (eg, diameter) of about 0.5 to about 100 microns.

[0247] The transfer dispenser is configured to fit onto the assay device such that the transfer dispenser is positioned below and aligned with the assay device in an inverted position, such that each cavity in the transfer dispenser may align with a single well in the assay device, provided that the transfer dispenser does not contain more than one bead in a given cavity.

[0248] The transfer dispenser is configured to fit onto the assay device, and the assay device is aligned with the transfer dispenser below it, so that each well in the assay device contains a single bead, and a single bead is transferred from the transfer dispenser, the transfer requiring a single fitting of the assay device to the transfer dispenser.

[0249] Each cavity of the multiple cavities of the transfer dispenser may include a bottom surface opposite an open end of the cavity, the bottom surface including a sub-cavity recessed therein, the sub-cavity configured to hold a single bead.

[0250] Each cavity of the multiple cavities of the transfer dispenser may include an open bottom surface and an open top surface, the size of the open bottom surface being smaller than the size of the open top surface, and the cavity may be configured such that a single bead resides at a midpoint between the open top surface and the open bottom surface.

[0251] Each cavity of the multiple cavities of the transfer dispenser may include a bottom surface facing the open end of the cavity, the bottom surface having a magnet thereon or thereon, the magnet being configured to hold a single bead.

[0252] Each cavity of the multiple cavities of the transfer dispenser may include an opening having an interior dimension, and the opening may be closed by a closure structure, which may include at least one of the group consisting of a mesh of a solid material having a sub-opening therein, a porous fibrous product, and a sub-structure having at least one sub-opening that is smaller than the interior dimension of the opening and larger than the maximum dimension of a single bead.

[0253] A system for screening compounds for their ability to modulate the biological activity of a cell or a cellular component is provided. The system may include an assay device including a number of wells, including at least 50,000 wells, each well separated from the other wells. The system may include a plurality of beads, each bead suitable for placement into a respective well. Each bead includes a plurality of substantially identical bead-bound compounds. The bead-bound compounds are covalently linked to the bead by a cleavable linker, allowing the compounds to be released from the bead in a dose-dependent manner that is measurable as part of the assay.

[0254] In other embodiments, the system includes an assay device comprising a number of wells, each well separated from the other wells, and a plurality of beads suitable for placement into a single well, each bead comprising a plurality of substantially identical bead-bound compounds, wherein the bead-bound compounds are covalently linked to the beads by cleavable linkers such that the compounds are releasable from the beads in a dose-dependent manner that is measurable as part of the assay, the beads further comprising a plurality of substantially identical bead-bound DNA barcodes linked to the beads by (i) a cleavable linker or (ii) a non-cleavable linker, wherein if the DNA barcodes are linked to the beads by a cleavable linker, the cleavable linker is orthogonal to the cleavable linker used to link the bead-bound compounds to the beads, and the compounds are identified by the DNA barcodes, and each bead comprises at least about 10,000 substantially identical DNA barcodes.

[0255] The beads may further comprise a plurality of substantially identical bead-bound DNA barcodes linked to the beads by (i) a cleavable linker or (ii) a non-cleavable linker. When the DNA barcodes are linked to the beads by a cleavable linker, the cleavable linker may be orthogonal to the cleavable linker used to link the bead-bound compound to the bead. The compound is identified by the DNA barcode.

[0256] The system may further include a transfer dispenser capable of dispensing a single bead into a single well.

[0257] The transfer device may further include at least one pipette capable of transferring a single bead, the at least one pipette including a flexible tip. The flexible tip of the pipette may include polyimide. The flexible tip may extend along 20% ​​or less of the total length of the pipette. The flexible tip may extend along 10% or less of the total length of the pipette.

[0258] Methods for screening compounds for their ability to modulate the biological activity of a cell or a component of a cell are provided, which may be performed by the systems referenced above.

[0259] The method may include moving an assay device and a transfer dispenser to fit or mate with each other, and there may be a gap between the assay device and the transfer device. The method may include aligning a cavity of a transfer dispenser with a well of a device. The method may include fitting or mating the assay device and the transfer dispenser with each other to form a containment space. The method may include releasing a single bead from a cavity through the containment space. The method may include depositing a single bead into the well.

[0260] The transfer device may be operated by a robot, by a human, or by a combination of robotic and manual processes.

[0261] The transfer device may employ magnetic attraction, electrostatic attraction, or engineering principles based on size and gravity to deposit a single bead into a single well.

[0262] The transfer dispenser may include multiple cavities.

[0263] Each cavity of the multiple cavities of the transfer dispenser may include a bottom surface opposite an open end of the cavity. The bottom surface may include a sub-cavity recessed therein. The sub-cavity may be configured to hold a single bead.

[0264] Each cavity of the multiple cavities of the transfer dispenser may include an open bottom surface and an open top surface. The size of the open bottom surface may be smaller than the size of the open top surface. The cavity may be configured such that a single bead resides midway between the open top surface and the open bottom surface.

[0265] Each cavity of the multiple cavities of the transfer dispenser can include a bottom surface facing the open end of the cavity. The bottom surface can have a magnet thereon or thereon. The magnet can be configured to hold a single bead.

[0266] Each cavity of the multiple cavities of the transfer dispenser can include an opening having an interior dimension. The opening can be closed by a closure structure. The closure structure can include at least one of the group consisting of a mesh of a solid material having a sub-opening therein, a porous fibrous product, and a sub-structure having at least one sub-opening that is smaller than the interior dimension of the opening and larger than the maximum dimension of a single bead.

[0267] A method for identifying transcriptomic changes in a cell induced by a compound, wherein the compound is included in an assay of a combinatorial library. The method may be performed by the system referenced above.

[0268] The method may include generating an assay array. The assay array includes a plurality of wells, each well being separated from the other wells, each well containing at least one cell of interest, with the assay array including more than 50,000 wells. The assay array may include a plurality of beads, each containing a unique compound from the combinatorial library, each bead containing a plurality of identical bead-bound compounds, such that each compound in the library is selected as a potential drug candidate. The assay array may include a plurality of functionalized oligonucleotides. The functionalized oligonucleotides may include an oligonucleotide portion encoding the structure of a unique compound or a synthetic step used to create the unique compound, and an RNA capture element. A single bead may be placed in a single well.

[0269] The method may include contacting cells in each defined volume with compounds released into the defined volume from beads and maintaining said contact for a time sufficient to result in transcriptomic changes in RNA expressed by the cells in response to said contact.

[0270] The method can include capturing RNA from cells in each well by lysing the cells and contacting the RNA with RNA capture elements on the beads.

[0271] The method can include identifying captured RNA from at least some of the plurality of beads and assessing transcriptomic changes, if any, in the captured RNA.

[0272] The method can include identifying the structure of the compound that caused the alteration of the transcriptome.

[0273] Beads may be added to the wells of the assay array using a transfer device capable of dispensing a single bead into a single well.

[0274] These and other capabilities of the disclosed subject matter will be more fully understood after review of the following drawings, detailed description, and claims.

[0275] These and other features will become more readily apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0276] [Figure 1] Linked beads. In linked beads, the DNA barcode takes the form of all the DNA barcode modules connected to each other in a single strand, along with any other nucleic acids that have functions such as primer annealing sites as spacers, or information about the manufacturing date. The numbers on this drawing are not structure numbers; they refer to the sequence of the "DNA barcode modules" in the DNA barcode. [Figure 2] Orthogonal beads. In orthogonal beads, the DNA barcode takes the form of all DNA barcode modules where the DNA barcode modules are not present together in a single strand, but instead are present separately linked at different locations on the bead. The numbers on this drawing are not structure numbers; they refer to the sequence of the "DNA barcode modules" in the DNA barcode. [Figure 3-1] Cleavable linkers, conditions for cleavage (UV light or chemicals), 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 alphabetic letters to the left of each linker are from this reference. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] Exemplary amino acid derivatives for 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. [Figure 5] The photographs demonstrate increasing degradation of the fusion protein inside HeLa cells with increasing concentrations of added lenalidomide. Top: Expression of IKZF1 / GFP fusion protein. Bottom: Expression of mScarlett® control. Lenalidomide was added at 0, 0.1, 1.0, or 10 micromolar concentrations. [Figure 6] The photographs demonstrate increasing degradation of the fusion protein inside HeLa cells with increasing concentrations of added lenalidomide. Top: Expression of IKZF3 / GFP fusion protein. Bottom: Expression of mScarlett® control. Lenalidomide was added at 0, 0.1, 1.0, or 10 micromolar concentrations. [Figure 7] Methods and reagents for creating bead-bound DNA barcodes. The most accurate description of a "DNA barcode" is the sum of all information contained in the sum of all DNA barcode modules. However, for convenience, the term "DNA barcode" as used herein refers to the sum of all information in all DNA barcode modules, as well as any additional nucleic acids that provide information such as the number of steps, or the general types of chemical monomers that make up the bead-bound compound, and any additional nucleic acids that serve functions such as linkers, sequencing primer binding sites, hairpins with sequencing primer binding sites, or spacers. If at least a portion of the DNA barcode is produced by click chemistry, the DNA barcode may include residual chemical groups from the click chemistry reaction. [Figure 8] Structure of Alexa Fluor® 488. The goal of this drawing is to identify the compound without having to resort to using trade names. [Figure 9]

[0023] Figure 1 shows a simplified diagram of a bead-bound release monitor. The release monitor provides the user with a measurement of the concentration of a soluble compound after UV-induced release of the compound from the bead. In a preferred embodiment, one type of bead is dedicated to the release monitor, i.e., it does not contain a bead-bound compound, nor does it contain a bead-bound DNA library. "PCL" is a photocleavable linker. [Figure 10] Detailed diagram of the bead release monitor. [Figure 11-1] Chemical synthesis of 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 for chemical synthesis of first type of carboxyl group-modified lenalidomide. [Figure 14] Steps for chemical synthesis of a second type of carboxyl-modified lenalidomide. [Figure 15] Steps for the chemical synthesis of a third type of carboxyl-modified lenalidomide. [Figure 16A] Figures 16A, 16B, and 16C show lenalidomide analogs. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 17-1] Steps for the chemical synthesis of deoxycytidine analogs suitable for click chemistry synthesis of DNA barcodes. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 17-4] Same as above. [Figure 18A] Figures 18A, 18B, and 18C show caps for placing on and sealing picowells. Figure 18A shows an active cap in which a compound is releasable via a cleavable linker. Figure 18B shows another type of active cap to which a reagent, such as an antibody, is attached. The attached reagent may be permanently linked, it may be linked via a cleavable linker, or it may be attached via hydrogen bonding and be released only upon exposure to the solution in the picowell, after which it may diffuse from the active cap into this solution. Figure 18C shows a passive cap that can be used to absorb, adsorb, collect, or capture metabolites from the solution in the picowell. The absorbed metabolites can then be analyzed. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19A]Figure 19A shows a picowell plate without caps on the picowells. Figure 19B shows a picowell plate with caps on each picowell. Figure 19C shows a polyacrylamide solution being poured onto a picowell plate with one cap firmly secured over each picowell. The polyacrylamide then penetrates the porous caps, solidifies, and forms a stable bond to each cap. Figure 19D shows the solidified polyacrylamide "roof" being peeled off the picowell plate along with each cap. Metabolites transferred from the picowell solution and absorbed into each cap can then be analyzed. Preferably, the solution poured onto the picowell plate and onto the beads is a hydrogel, and preferably, the beads are made from a hydrogel. 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 onto the plate and onto the capped well. Also excluded may be those in which a liquid polymer polymerizes to form a solid polymer that adheres to the respective cap. Also excluded may be methods and resulting compositions in which the solid polymer is torn, removing the adhered cap along with it. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above. [Figure 20]

[0039] Figure 1 shows 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 cell genome. The plasmid is IKZF3 mNeon-p2a-mScarlet-w3-2FB (9051 bp). IKZF3 encodes the Aiolos protein. [Figure 22-1] Chemical monomers (compounds 1–6) and their DNA barcodes. [Figure 22-2] Same as above. [Figure 23] Chemical monomers (compounds 7–10) and their DNA barcodes. [Figure 24-1] Chemical monomers (compounds 11–16) and their DNA barcodes. [Figure 24-2] Same as above. [Figure 25-1] Chemical monomers (compounds 17–21) and their DNA barcodes. [Figure 25-2] Same as above. [Figure 26-1] Chemical monomers (compounds 22–16) and their DNA barcodes. [Figure 26-2] Same as above. [Figure 27-1] Chemical monomers (compounds 27–30) and their DNA barcodes. [Figure 27-2] Same as above. [Figure 28] Sequencing of bead-bound DNA barcodes. This figure discloses the intensity of the fluorescent signal for each of the five consecutive bases that are part of the bead-bound DNA barcode. [Figure 29] Stepwise Picowell. [Figure 30] Time course of fluorophore release from beads. This shows the operation of the bead-bound release monitor, acquiring fluorescence data at t=0 s, t=1 s, t=11 s, and t=71 s. [Figure 31A] Emission data obtained after the catalytic action of aspartyl proteases on quencher-fluorophore substrates. [Figure 31B] Same as above. [Figure 32]1A-1C are cross-sectional views of a picowell illustrating various steps. [Figure 33A] Titration data showing how increasing UV dose results in more cleavage of fluorophores from beads. In layman's terms, this shows how more powerful swings of the axe affect shredding of fluorophores from beads (UV dose power is measured in joules per square centimeter). The "Exposure" designation refers only to the parameter at which the photograph was taken; it is simply the exposure time at which the photograph was taken (it does not refer to the exposure time to the cleaving light or to the excitation light). [Figure 33B] Same as above. [Figure 33C] Same as above. [Figure 33D] Same as above. [Figure 33E] Same as above. [Figure 33F] Same as above. [Figure 34] TAMRA concentration vs. luminous flux. The concentration of free TAMRA is shown after release following exposure to UV light at 365 nm. [Figure 35-1] A hand-drawn diagram of the quencher-fluorophore substrate and the cleavage of this substrate by the enzyme, along with the resulting inhibition of the enzyme, is provided. Also shown are the molecular structures of bead-bound pepstatin-A and bead-bound Fmoc-valine (negative control). [Figure 35-2] Same as above. [Figure 36-1] A step in the preparation of beads for use in the eventual capture of mRNA from lysed cells, along with subsequent production of a cDNA library. This drawing is also found in one of the provisional applications (Compositions and Methods for Screening Compound Libraries on Single Cells), from which priority is claimed by the present application. [Figure 36-2] Same as above. [Figure 37-1]Tagging cells with DNA barcodes, where the tagging is by lipids embedded in the cell membrane. This drawing is also in one of the provisional applications (Compositions and methods for screening compound libraries on single cells), to which priority is claimed by this application. [Figure 37-2] Same as above. [Figure 38] FIG. 38 is a schematic diagram of a computing device or system for perturbing cells and capturing the cellular response to the perturbation, according to an exemplary embodiment, including at least one processor and a memory storing at least one program for execution by the at least one processor. [Figure 39] FIG. 39 is a side cross-sectional view of a dispenser having at least one cavity and substructure within the cavity, according to an exemplary embodiment. [Figure 40] FIG. 40 is a side cross-sectional view of a dispenser having at least one cavity, tapered sidewalls, an open top end, and an open bottom end, according to an exemplary embodiment. [Figure 41] FIG. 41 is a side cross-sectional view of a dispenser having at least one cavity and a magnet disposed at the bottom of each cavity, according to an exemplary embodiment. [Figure 42] FIG. 42 is a side cross-sectional view of a pipette with an optional mesh insert, according to an exemplary embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0277] It is understood that the drawings are not necessarily to scale. The drawings are intended to depict only typical aspects of the subject matter disclosed herein and, therefore, should not be considered as limiting the scope of the present disclosure. Those skilled in the art will understand that the structures, systems, devices, and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments, and that the scope of the present invention is defined solely by the claims.

[0278] As used in this specification, including the appended claims, singular terms such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise. All references cited herein, as well as individual patents and published patent applications, figures, drawings, sequence listings, compact discs, etc., are each specifically incorporated by reference and are incorporated by reference to the same extent as if individually indicated and individually indicated.

[0279] Abbreviation

[0280] Table 1 provides abbreviations and non-limiting definitions. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0281] Reagents, kits, enzymes, buffers, live cells, equipment, etc. are available from 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.

[0282] Barcoded gel beads, non-barcoded gel beads, and microfluidic tips are available from 1CellBio, Cambridge, Mass. Guidance and equipment is available for flow cytometry (e.g., FACSCalibur®, BD Biosciences, San Jose, Calif., see BD FACSAria II® User's Guide, part no. 643245, Rev. A, December 2007, 344 pages).

[0283] 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, 125These include I, stable isotopes, epitope tags, fluorescent dyes, Raman tags, electron-dense reagents, substrates, or enzymes, such as those used in enzyme-linked immunoassays or fluorescein (Rozinov and Nolan (1998) Chem. Biol. 5:713-728).

[0284] Table of contents for detailed description (I) Beads (II) One bead one compound (OBOC) (III) Linking nucleic acids to beads (IV) DNA barcode (V) Linking the compound to the beads (VI) Linking chemical monomers together to form compounds (VII) Split-pool synthesis and parallel synthesis (VIII) Manufacturing Picowell (IX) Depositing beads into picowells (X) Sequencing bead-bound nucleic acids in picowells (XI) Releasing bead-bound compounds from beads (XII) Biochemical assay of compounds (XIII) Cell-based assay for compounds (XIV) Cellular perturbation-response analysis

[0285] (I) Beads The disclosed methods and compositions use beads such as monosized TentaGel® M NH2 beads (10, 20, 30 micrometers, etc. in diameter), standard TentaGel® amino resins (90, 130 micrometers, etc. in diameter), and TentaGel Macrobeads® (280-320 micrometers in diameter) (all 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® resin is a graft copolymer consisting of a low-crosslinked polystyrene matrix to which polyethylene glycol (PEG) is grafted. Thus, the present disclosure provides beads or resins modified to contain one or both of DNA barcodes and compounds, where unmodified beads take the form of a graft copolymer consisting of a low-crosslinked polystyrene matrix to which polyethylene glycol (PEG) is grafted.

[0286] TentaGel® is characterized by "PEG chains of up to 20 kilodaltons immobilized on functionalized, cross-linked polystyrene." Graft copolymers with PEG chains of approximately 2000-3000 daltons have proven optimal in terms of kinetic speed, mobility, swelling, and resin capacity (Rapp Polymere, Germany). Accordingly, the present disclosure provides beads or resins in the form of graft copolymers with PEG chains of approximately 2000-3000 daltons. Regarding swelling, Comellas et al. provide guidance for measuring the swelling capacity of beads when immersed in, for example, DCM, DMF, methyl alcohol, water, or buffers used in enzyme assays (Comellas et al. (2009) PLoS ONE. 4:e6222 (12 pages)). Swelling is measured in milliliters per gram of beads.

[0287] 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, and the resin is microspherical and monosized (TentaGel® M resin).

[0288] In a further alternative bead embodiment, the present disclosure uses a resin with a PEG spacer attached to a polystyrene backbone via an alkyl linkage, and the resin type exists in two bifunctional species: the first, a surface-modified resin in which reactive sites on the outer surface of the bead are orthogonally protected relative to reactive sites in the inner volume of the bead; and the second, a hybrid resin in which cleavable and non-cleavable ligands are present on this support and developed for sequential cleavage (TentaGel® B resin).

[0289] In yet another embodiment, the present disclosure utilizes a resin in which a PEG spacer is attached to a polystyrene backbone via an alkyl linkage, resulting in a macrobead resin exhibiting very large particle size and high capacity (TentaGel® MB Resin). The present disclosure also utilizes a resin in which a PEG spacer is attached to a polystyrene backbone via a benzyl ether linkage. This resin can be used in immunization procedures or in the synthesis of PEG-modified derivatives (PEG-conjugated PEG-modified compounds) (TentaGel® PAP Resin).

[0290] Additionally, the beads can be HypoGel® 200 resin, which is a composite of oligoethylene glycol (MW 200) grafted to a low-crosslinked polystyrene matrix (Fluka Chemie GmbH, CH-9471 Buchs, Switzerland).

[0291] In some embodiments, amino-functionalized polystyrene beads without PEG linkers may be used, for example, mono-sized polystyrene M NH2 microbeads (5, 10, 20 micrometers in diameter, etc., also from Rapp Polymere, 72072 Tubingen, Germany).

[0292] In some embodiments, compounds may be encapsulated within pores, 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 temperatures may be used to expand the beads and load the compounds within the beads. In some embodiments, high pressure may be used to force the compounds into the beads. In some embodiments, compounds may be loaded into the beads using a solvent that swells the beads. In some embodiments, 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.

[0293] In such embodiments in which the compound is loaded onto the beads without covalent attachment, the compound may be removed from the beads by diffusion. In some embodiments, the compound may be removed from such beads using, without limitation, temperature, pressure, solvent, pH, salt, buffer, or surfactant, or a combination of such conditions. In some embodiments, the physical integrity of the beads, for example, due to non-crosslinked polymerized beads, may be used to release the compound contained within such beads.

[0294] In exclusive embodiments, the present disclosure can exclude any bead and bead-compound complex, or any method, including one of the beads described above.

[0295] Beads of the present disclosure also include the following: Merrifield resin (chloromethyl polystyrene); PAM resin (4-hydroxymethylphenylacetamidomethyl polystyrene); MBHA resin (4-methylbenzhydrylamine); Brominated Wangresin (alpha-bromopriopiophenone); 4-nitrobenzophenone oxime (Kaiser) resin; Wangresin (4-hydroxymethylphenoxymethyl polystyrene); PHB resin (p-hydroxybenzyl alcohol); HMPA resin (4-hydroxymethylphenoxyacetic acid); HMPB resin (4-hydroxymethyl-3-methoxyphenoxybutanoic acid); 2-chlorotrityl resin; 4-carboxytrityl resin; Rink acid resin (4-[(2,4-dimethoxyphenyl)phenyl] (hydroxymethyl)phenoxymethyl); Rink Amide (RAM) resin "Knorr" resin (4-((2,4-dimethylphenyl)(Fmox-amino)methyl)phenoxyalkyl); PAL resin (5-[4-(Fmoc-amino)methyl-3,5-dimethoxyphenoxy]valeramidomethyl polystyrene); Sieberamide resin (9-Fmox-amino-xanthan-3-yl-oxymethyl); HMBA resin (hydroxymethylbenzoic acid); 4-sulfamoylbenzoyl resin "Kenner Safety Apparatus" resin (N-(4-sulfamoylbenzoyl)aminomethyl-polystyrene); FMP-resin (4-(4-formyl-3-methoxyphenoxy)-ethyl) (see ChemFiles Resins for Solid-Phase Peptide Synthesis Vol. 3 (32 pages) (Fluka Chemie GmbH, CH-9471 Buchs, Switzerland)).

[0296] Beads of the present disclosure further include those beads described above used as passive encapsulants for compounds (which passively hold 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 (polymethyl methacrylate) 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.

[0297] In addition to beads, vesicles or droplets can also be used as vehicles for delivering compounds for some embodiments of the present disclosure. Lipids, non-block copolymers, triblock copolymers, or other membrane-forming materials can be used to form internal volumes in which compounds can be loaded. Compounds can be released from these encapsulated volumes by adding surfactants, mechanical agitation, temperature, salt, pH, or other means. Water-in-oil or oil-in-water emulsions are yet another means of passively encapsulating compounds that can be delivered to the assay volume.

[0298] In all embodiments where passive encapsulation is used to deliver compounds, the DNA tag may additionally be passively loaded, or alternatively, the DNA tag may be covalently attached to a bead, vesicle, or droplet.

[0299] In an exclusive embodiment, the present disclosure may exclude beads or resins made with any of the above chemicals, or derivatives of any one of the above chemicals.

[0300] In embodiments, the beads may be spherical and may have a diameter of about 0.1 to 1 micrometer, about 1 to 5 micrometer, 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.

[0301] Non-spheroid beads that can be defined with respect to the above values ​​and ranges are also provided. For example, one of the axes, or one of the primary dimensions (e.g., side), or one of the secondary dimensions (e.g., diagonal) can include a value in the above range. 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 within one or more of the above values ​​or ranges.

[0302] Bead chains. In one embodiment, provided are a plurality of bead dimers, the bead dimers taking the form of two beads attached to one another, one bead comprising a plurality of attached nucleic acid barcodes (either orthogonal nucleic acid modules or linked nucleic acid modules) and the other bead comprising a plurality of attached compounds, all of which are substantially related to one another (or all of which are substantially identical in chemical structure to one another). The bead dimers can be synthesized by preparing a first bead with attached compounds and a second bead with attached nucleic acid barcodes, separately, and then linking the two beads. In one embodiment, the beads are attached to one another by a reversible linker, and in another embodiment, the beads are attached to one another by a non-reversible linker.

[0303] Bead Permeability. In embodiments, the present disclosure provides beads having various ranges or degrees of permeability. Permeability can be measured as the percentage of the bead's volume that is accessible by solvent, where the unit of measurement is the percentage of the bead's surface that assumes a form or pore, or the unit of measurement is the percentage of the bead's interior that assumes a form of a channel, network, or chamber that is in fluid communication with the bead's surface (and the external medium). The present disclosure can include, or alternatively, can exclude, porous beads.

[0304] U.S. Patent No. 9,062,304 to Rothberg discloses beads having an exterior and interior region. They show an "internal surface (pore surface)," "appropriate pores that will exclude larger molecules," and optionally "differential functionalization of the interior and exterior surfaces," as well as various pore sizes and polymers such as poly(styrene sulfonate) and polystyrene. Figure 1 to Rothberg provides photographs of the surface of the beads and the pores in the beads. U.S. Patent No. 9,745,438 to Bedre provides transmission electron microscope images of porous beads. U.S. Patent No. 5,888,930 to Smith provides scanning electron microscope photographs of the cross-sections of porous beads. Spherical beads with small pores on the surface and larger pores in the interior are shown, and the beads are made from, for example, polystyrene, polyacrylonitrile, polycarbonate, cellulose, or polyurethane. U.S. Patent No. 5,047,437 to Cooke discloses the pore morphology of spherical poly(acrylonitrile) copolymers with skinless surfaces (FIG. 1) and beads with an outer skin on the surface (FIG. 5). U.S. Patent No. 4,090,022 to Tsao discloses the porous openings and interior spaces of cellulose beads.

[0305] Each of the above-identified patents, including all figures, is incorporated herein in its entirety, as if each were individually incorporated by reference in its entirety.

[0306] Without implying any limitation, the outer surface of a bead or microparticle can be determined by tightly wrapping the entire bead or microparticle in an elastic film. The bead or microparticle can be wrapped via a thought experiment, or the wrapped bead can be depicted in a diagram or photograph, or the bead can be actually wrapped. Without implying any limitation, the outer surface of the bead is the portion of the bead that physically contacts the wrapping.

[0307] For example, the present 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%, or at least 40% of the surface area. The present 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%, or at least 80% of the total volume of the beads, and the internal channels or networks are in fluid communication with the outer surface (and external medium) of the beads.

[0308] Additionally, the present disclosure provides beads having pores that occupy less than 1%, 2%, 5%, 10%, 15%, 20%, 30%, or 40% of the surface area. The present disclosure also provides beads in which the volume of the internal channels or networks occupies less than 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total volume of the beads, and the internal channels or networks are in fluid communication with the outer surface (and external medium) of the beads.

[0309] Iron-core beads. The present disclosure encompasses iron-core or magnetic beads. These beads can be manipulated with 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).

[0310] In exclusive embodiments, the present disclosure can exclude any bead or any population of beads, where the bead or population meets one of the above values ​​or ranges.

[0311] Loading of compounds onto beads In many experiments, it is advantageous to load pre-synthesized compounds onto beads, which can be used as a vehicle for delivering 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).

[0312] In embodiments where presynthesized compounds are loaded onto beads, the compounds may be held in conventional 96-, 385-, or 1536-well microtiter plates. Beads may be added to these plates, and the compounds are loaded by diffusion or other active loading methods. In preferred embodiments, the beads selected for impregnation have a pore size or permeability geometry that prevents rapid compound depletion upon removal from the mother liquor. Diffusion out of the beads may be enhanced, if necessary, by heat, pressure, additives, or other stimulants. In some embodiments, compound-loaded beads may be capped in a manner that prevents leakage of the internal contents until triggered by an external impact. One method for capping the exterior of porous beads is to add lipids or amphiphilic molecules to the bead compound solution so that cavities exposed on the surface of the beads are sealed by a bilayer formed by the amphiphilic molecules. In some embodiments, preformed vesicles may be mixed with drug-loaded beads, so that upon agitation, the vesicles rupture and membranes reform on the surfaces of the drug-loaded beads, thereby sealing them. A method for sealing such beads is described (see Tanuj Sapra et al (2012) Nature Scientific Reports volume 2, Article No.: 848).Further experimental protocols for sealing silica beads are available in a report published by Sandia Laboratories by 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 by Hui Zheng et al (bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles) in J. Gen. Physiol. (2016) 147: 77-93.

[0313] In some embodiments utilizing pre-synthesized compounds, beads are generated from the compounds by adding appropriate reagents, such as lipids or diblock copolymers, followed by agitation, thereby forming vesicles containing the compound within their interior or bilayer membranes. In some embodiments, the compounds are extruded through a microfluidic T-junction to create aqueous-phase droplets in an oil phase, with the compound contained within the aqueous phase or at the interface between the aqueous and oil phases. In some embodiments, the formed droplets are further polymerized to create hydrogels, which are more robust and stable to handle 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 can also be utilized to encapsulate compounds within beads. The formation of sol-gel beads is described in Sol-gel Encapsulation of Biomolecules and Cells for Medicinal Applications, Xiaolin Wang et al (2015) Current Topics in Medicinal Chemistry. 15: 223.

[0314] 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 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). An advantage of synthesizing compounds via bead-bound synthesis is that compounds can be made rapidly by a "split-pool" method.

[0315] OBOC combined with coding strategy. Another feature of OBOC is that each bead contains not only a compound but also a coding strategy. When bead-bound nucleic acids are used to code compounds 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.

[0316] A dramatic variation on the use of bead-bound compounds and beads with bead-bound nucleic acids, where the nucleic acid encodes the relevant compound, is as follows: A dramatic variation is to produce a library of conjugates, each member of the library being in the form of a conjugate of a small molecule and a DNA moiety, where the DNA moiety encodes the small molecule. The conjugates are soluble and not bead-bound. After screening with cells or purified proteins, the conjugates remain bound to the cells or purified proteins, allowing for isolation of the conjugates and sequencing of the conjugated nucleic acids to ultimately identify the compound (see Satz et al (2015) Bioconjugate Chemistry. 26:1623-1632).

[0317] 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 represent specific known structures of compounds that bind to them.

[0318] Instead of using coding strategies such as the use of DNA barcodes, beads that are screened for positives (thereby indicating compounds that screen for positives) 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).

[0319] One subtle point in performing split-pool synthesis of combinatorial libraries is that compounds can be produced so that all compounds share a common motif. This strategy has been described as "generating a library of motifs rather than a library of compounds" (Sepetov et al (1995) Proc. Natl. Acad. Sci. 92:5426-5430, Lam et al, supra, at 418).

[0320] To provide a typical example of a large bead, the bead is 0.1 mm in diameter and contains approximately 10% of the same compound. 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 assay can be an "on-bead" assay, or alternatively, the compounds can be detached from the beads and used in a solution-phase assay (Lam et al., supra).

[0321] 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 identity of the attachment site on the bead for attaching each compound, whether a spacer such as polyethylene glycol is used to provide some separation of each compound from the surface of the bead, and the internal volume of the bead.

[0322] 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 group is at the end of the polyoxyethylene chain, and therefore far from the hydrophobic polystyrene. Beads with water-soluble linkers include TentaGel and polydimethylacrylamide beads (PepSyn® gel, Cambridge Research Biochemicals, Northwich, UK).

[0323] The internal volume parameter 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 take advantage of this advantage, beads can be manufactured so that the DNA barcode is located inside the bead, while the compound to be screened is attached to the surface of the bead (Lam et al., supra, pp. 438-439). This advantage of internal volume may be irrelevant if the bead-bound compound is attached by a cleavable linker and compound assays are performed only on the compound that has been cleaved and released.

[0324] Appell et al. provide a non-limiting example of a split-pool method for synthesizing a chemical library and subsequently screening to detect active compounds (Appell et al. (1996) J. Biomolecular Screening. 1:27-31). Library beads are placed, one in each well of 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. Next, "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).

[0325] In a cell-based screening assay for screening bead-bound compounds, Shih et al. (Shih et al. (2017) Mol. Cancer Ther. 16:1212-1223) presents a new type of bead. This new type of bead contains a bead-bound compound that is a member of a library of "ovarian cancer-directed synthetic death ligands." The bead is further decorated with biotin, and two additional chemicals are added to create a sandwich that maintains cell-to-bead adhesion. The sandwich contains streptavidin and a biotin-LXY30 conjugate. This sandwich connects the bead to the receptor for LXY30, which happens to be a well-known protein on the cell surface, namely, integrin. The method of Shih et al. (ibid.) led to the discovery of a new molecule ("LLS2") that can kill cancer cells. The above method uses a bead-bound compound, and the compound binds to the cell (even though the compound is still bead-bound). Cho et al. created 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.

[0326] Linking nucleic acids to beads (orthogonal; linked) One way to understand the topics of concatenated and orthogonal barcoding is to note the significance of one over the other. The advantages of orthogonal barcoding over concatenated barcoding are as follows: With the attachment of each monomer of a growing compound, a DNA barcode module is attached in parallel. In concatenated barcoding, if the attachment of a given module is incomplete (i.e., if not all attachment sites are successfully ligated with the required module), the sequence of the completed barcode will be inaccurate. The term "inaccurate" refers to incomplete ligation, meaning that a chunk may be missing from what was assumed to be the completed, correct DNA barcode. Here, because attachment of all modules failed, the completed barcode sequence contains an error. In contrast, with orthogonal barcoding, each individual module is covalently attached to its own unique attachment site on the bead. Also, once a module is attached to a predetermined site on the bead, no additional modules are connected to the modules already attached.

[0327] 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. Prior to attachment to the growing bead-bound DNA barcode, 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. After completion of synthesis of the dsDNA-form DNA barcode, the dsDNA is converted to ssDNA. Conversion of dsDNA to ssDNA is effective when one of the DNA strands contains a uracil (U) residue, and cleavage of the DNA at the uracil residue is catalyzed by uracil-N-glycosidase (see Figure 5 of Serial No. 62 / 562,905, filed September 25, 2017, which is incorporated herein by reference in its entirety). The above refers to damage inflicted on the growing DNA barcode by the reagents used to create the bead-bound compounds.

[0328] Another method for reducing damage to bead-bound DNA barcodes and partially synthesized DNA barcodes is to synthesize the DNA barcodes in the form of double-stranded DNA, where each of the attached DNA barcode modules 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. This refers to 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).

[0329] 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 first prong of the hairpin anneals to the second prong of the hairpin.

[0330] If the synthesized DNA barcode is in the form of double-stranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can denature the DNA barcode. The methods and reagents described above can prevent denaturation.

[0331] 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.

[0332] 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.

[0333] In an alternative embodiment, the method can use an intermediate between "concatenated DNA barcoding" and "orthogonal DNA barcoding," which involves blocks of DNA barcodes, i.e., each block containing two DNA modules, or three DNA modules, or four DNA modules, or five DNA modules, etc. (but not all the DNA modules that specify the full-length compound).

[0334] Figure 1 discloses an exemplary and non-limiting diagram of linked structured beads. The beads contain multiple DNA barcodes (each made from a DNA barcode module) and multiple chemical compounds (each made from a chemical library monomer). For simplicity, the term "DNA barcode" can be used to refer to all nucleic acids that are "DNA barcode modules" and to 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. Figure 1 further illustrates bead-bound compounds, each made from several chemical library members, each represented by a square, circle, or triangle. Figure 1 shows that each DNA barcode module is numbered sequentially from 1 to 8, corresponding to each of the eight shapes (square, circle, triangle). For clarity, nucleic acids that perform a function (and do not represent or "encode" any particular chemical unit) are not shown in the diagram.

[0335] Figure 2 discloses an exemplary, non-limiting embodiment of an orthogonal structured bead. The bead contains multiple DNA barcodes (each made up of a DNA barcode module), with each DNA barcode module attached to a separate linkage site on the bead. The entire DNA barcode consists of eight DNA barcode modules, numbered 1 through 8 in the figure. If information from a particular DNA barcode is to be read and used to identify a compound bound to the same bead, DNA sequencing must be performed on each of the separately attached DNA barcode modules. In Figure 2, the bead further contains multiple attached compounds, each with eight units, as indicated by the eight shapes (circle, square, and triangle).

[0336] For clarity, the functional nucleic acids attached to each DNA barcode module are not shown in Figure 2. Of course, each DNA barcode module requires a nucleic acid that identifies the position of the chemical library monomer in the completed full-length compound. In the example shown in Figure 2, the position must be 1, 2, 3, 4, 5, 6, 7, or 8.

[0337] 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. Also, organic synthesis procedures may be followed, 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 beads in parallel with the attachment of several blocks of DNA barcode modules.

[0338] In exclusive embodiments, what 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.

[0339] This relates to nucleic acids that may be present in a bead-bound polynucleotide, including nucleic acids that "encode" or serve to identify monomers of a bead-bound compound. In an exclusive embodiment, the present disclosure may exclude nucleic acids that encode "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 encode or specify a particular step in the chemical synthesis of a compound, such as step 1, step 2, step 3, or step 4.

[0340] Furthermore, the present disclosure may include nucleic acids that function as spacers. For example, a spacer can create distance along a polynucleotide chain between a first site that is a sequencing primer annealing site and a second site that specifies a chemical monomer. The present disclosure may also use nucleic acids that repeat or confirm information provided by another nucleic acid. The present disclosure may also use nucleic acids that encode PCR primer binding sites. A polynucleotide having PCR primer binding sites has two PCR primer binding sites, both of which 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 sites can be distinguished from the sequencing primers.

[0341] In exclusive embodiments, the present disclosure can exclude nucleic acids that function as spacers or as sole spacers. Also, the present disclosure can 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.

[0342] Additionally, the disclosure can specify the date 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.

[0343] Providing sequencing primers for specific DNA barcode modules. The present disclosure provides DNA barcodes including 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, two, three, four, five, six, seven, eight, nine, ten, 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.

[0344] Below we describe the situation where each DNA barcode module has its own dedicated sequencing primer binding site. The present disclosure provides bead-bound linked barcodes 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 a first DNA barcode module, a second DNA barcode module, a third DNA barcode module, a fourth DNA barcode module, a fifth DNA barcode module, and a sixth DNA barcode module, wherein the primer binding site is located at the 3 prime of a first DNA barcode module with no other DNA barcode modules between the first DNA barcode module and the primer binding site, the 3 prime of a second DNA barcode module with no other DNA barcode modules between, the 3 prime of a third DNA barcode module with no other DNA barcode modules between, the 3 prime of a fourth DNA barcode module with no other DNA barcode modules between, the 3 prime of a fifth DNA barcode module with no other DNA barcode modules between, or the 3 prime of a sixth DNA barcode module with no other DNA barcode modules between.

[0345] Coding sequences and sequences complementary to the 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 exclusive embodiments, it can exclude any one, any combination, or all of the coding sequences disclosed above or elsewhere in this document. It can also include or exclude double-stranded nucleic acids encoding any one, any combination, or all of the coding sequences described above or elsewhere in this document.

[0346] 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 a bead, resulting in the entire DNA barcode being 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 unique bead attachment site dedicated to that particular DNA barcode module.

[0347] A nucleic acid that specifies the synthesis step number of each DNA barcode module. In an embodiment, an orthogonal DNA barcode comprises a short nucleic acid that specifies the first step of 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 this complex of two nucleic acids: a short nucleic acid that designates "step 1" and is 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 may optionally be attached to the first DNA barcode module via a spacer nucleic acid.

[0348] 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 in compound synthesis. In this embodiment, the parallel attachment of the second chemical monomer and the second DNA barcode module results in the second DNA barcode module actually taking 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 in this complex are in-frame with each other and can be read in a sequencing assay, although the second short nucleic acid may optionally be attached to the second DNA barcode module via a spacer nucleic acid.

[0349] The above-described method is repeated for any given bead up to the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and final DNA barcode module and chemical monomer. The above method can be used when split-pool synthesis is used to create bead-bound DNA barcodes and compounds.

[0350] Orthogonal structures offer the following advantages over ligated structures: In the case of ligation synthesis (where all DNA barcode modules are attached to each other in one continuous polymer), failure to complete any of the intermediate ligation steps can compromise the meaning of the final, completed ligated DNA barcode. In contrast, in orthogonal synthesis (where each and every DNA barcode module is attached to a dedicated site on a bead), failure to attach any of the DNA barcode modules simply results in an empty attachment site on the bead, without compromising the meaning of any of the other attached DNA barcode modules. In a preferred embodiment, each attached DNA barcode module includes an attached second nucleic acid, which identifies a step (during the parallel synthesis of DNA barcodes and compounds).

[0351] For orthogonal synthesis, it is acceptable to use up all attachment sites on the beads (sites for attaching growing chemical library members). However, for orthogonal synthesis, the chemical reaction must be designed so that the first attachment of many DNA barcode modules results in only partial utilization of the entire population of attachment sites on the beads. The following provides an optional limit on using up sites during chemical synthesis of orthogonal barcodes. For unmodified beads, the total number of sites available for attaching DNA barcode modules is 100%.

[0352] The extent to which synthesis of orthogonal constituent beads (with respect to the first DNA barcode) uses up attachment sites on a given bead. The following relates to attaching a first DNA barcode module. In embodiments, attachment of the first DNA barcode module uses up 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 used up. In still other embodiments, attachment of the first DNA barcode module uses up 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%, or between 10-40% of the DNA barcode attachment sites.

[0353] Regarding limitations, attachment of the last DNA barcode module that constitutes a particular DNA barcode uses up 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.

[0354] 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.

[0355] 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 serving as a primer annealing site or spacer, and other nucleic acids have an informational function, and are DNA barcodes. In an exclusive embodiment, the present disclosure may exclude DNA barcodes comprising a DNA crosslinker, such as psoralen. Also excluded may be DNA barcodes having a primer binding region with a higher (or lower) melting temperature than the DNA barcode module. This temperature may simply be "higher" or "lower," or it may be at least 2°C higher, at least 4°C higher, at least 6°C higher, at least 8°C higher, or at least 2°C lower, at least 4°C lower, at least 6°C lower, or at least 8°C lower.

[0356] Also excluded are methods of creating DNA barcodes using DNA ligase. Also excluded are DNA barcodes and methods that comprise hairpins (ssDNA bent into a loop, with one portion of the ssDNA hybridizing to another portion of the same ssDNA). Furthermore, excluded are compositions comprising nucleic acid hairpins, where the nucleic acid hairpin is covalently closed, for example, with a chemical linker. Additionally, excluded are DNA barcodes that are covalently linked to a "headpiece" either directly or indirectly (via a covalent bond to one or more chemicals present between the DNA barcode and the headpiece) to a "headpiece."

[0357] In other, exclusive embodiments, which can 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).

[0358] The extent to which synthesis of orthogonal configuration beads (with respect to the second DNA barcode) uses up attachment sites on a given bead. The following relates to attaching a second DNA barcode module. In embodiments, attachment of the second DNA barcode module (in the case of creating orthogonal configuration beads) uses up 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 used up. In still other embodiments, attachment of the first DNA barcode module uses up 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%, or between 10-40% of the remaining free DNA barcode attachment sites.

[0359] 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.

[0360] The above embodiments, as well as the above-mentioned exclusionary embodiments, can also be applied to methods involving attaching a third DNA module barcode, or attaching a fourth DNA module barcode, or attaching a fifth DNA barcode module, etc.

[0361] 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).

[0362] Synthesis of bead-bound tethered DNA barcodes. The present disclosure provides bead-bound tethered DNA barcodes, wherein a bead comprises a plurality of tethered DNA barcodes, most or nearly all of which have essentially the same structure. The tethered DNA barcode can comprise one or more DNA barcode modules, and the order of these DNA barcode modules (from the bead-attached end to the distal end) along the entire DNA barcode is the same as the time the bead-bound tethered DNA barcode is synthesized. Also, the order of these DNA barcode modules along the entire DNA barcode is the same as the time the corresponding chemical library monomers are tethered to the growing bead-bound compounds.

[0363] The concatenated DNA barcode can include a linker that is used to link the entire concatenated DNA barcode to a bead, and 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.

[0364] One order of sequencing primer hybridizing sites in bead-bound DNA barcodes. In an embodiment of 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.

[0365] Alternative ordering of sequencing primer hybridizing sites occurring within bead-bound DNA barcodes. In alternative sequencing primer hybridizing site embodiments, the concatenated DNA barcode can include, in this order: linker, first DNA barcode module, first sequencing primer binding site, first annealing site, second DNA barcode module, second sequencing primer binding site, second annealing site, third DNA barcode module, third sequencing primer binding site, third annealing site, etc.

[0366] 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. Therefore, for purposes of this description, it is not important that both annealing sites have the same name. In other words, it is not important that a second annealing site on a splint oligo is disclosed as hybridizing to a second annealing site on the growing bead-bound DNA barcode.

[0367] Block-wise synthesis. In alternative embodiments, the growing compound and growing DNA barcode module sequences can be synthesized in blocks. For example, a block comprised of two chemical library units can be attached to a bead in parallel with the attachment of a block comprised of a corresponding two-DNA barcode module. Similarly, a block comprised of three chemical library units can be attached to a bead in parallel with the attachment of a block comprised of a corresponding three-DNA barcode. Block syntheses involving four blocks, five blocks, six blocks, seven blocks, eight blocks, nine blocks, ten blocks, etc. are also provided. Each of these block transfer embodiments can also be excluded by the present disclosure. The block-wise transfer of DNA barcode monomers can be performed orthogonally, with a unique attachment point for receiving each successive block of DNA barcode monomer. Alternatively, block-wise transfer of DNA barcode monomers can be performed to generate a concatemeric structure (all DNA barcode modules occur only as one continuous linear polymer).

[0368] Additionally, during parallel split-pool synthesis of bead-bound DNA barcodes and bead-bound compounds, 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.

[0369] 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 produced by the "sprint oligo" method. Alternatively, concatenated DNA barcodes can be produced by click chemistry. The "sprint oligo" method and click chemistry can also be used in combination. Split-pool synthesis can be performed in a 96-well plate, with each well containing a 0.25 micrometer filter bed. 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 96 wells. This suction method is used when beads are exposed to a first set of reagents, when the first set of reagents needs to be washed away, or when the first set of reagents needs to be replaced with a second set of reagents. A manifold was used to hold the 96-well plates (Resprep VM-96 manifold), and a pump was used to draw fluid from the bottom of all filters (BUCHI Vac V-500 pump). The 96-well plates with filter bottoms were AcroPrep Advance 96-well, 350 μL, 0.45 μm, REF 8048 (Pall Corp., Multi-Well Plates, Ann Arbor, MI).

[0370] Distance from the primer annealing site to the DNA barcode module. For the purpose of sequencing the bead-bound DNA barcode, i.e., for the purpose of sequencing all of the DNA barcode modules forming the DNA barcode, a polynucleotide comprising a first nucleic acid that is the annealing site for the sequencing primer and a second nucleic acid that is the DNA barcode module, the first nucleic acid can be immediately upstream of the second nucleic acid. Alternatively, the first nucleic acid can be upstream of the second nucleic acid, and the first and second nucleic acids can be 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 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 name of a class of compound, or a disease that may be treated with the bead-bound compound, or a date, or a lot number.

[0371] Synthesis of bead-bound concatenated DNA barcodes using click chemistry Click chemistry can be used for the stepwise synthesis of DNA barcodes, where the first DNA barcode module can be directly attached to a bead or attached to a bead-attached linker.

[0372] Also linkable 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.

[0373] To provide another example, what can be linked is a second DNA barcode module directly linked to a bead-bound first DNA barcode module. Alternatively, what can be linked 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 read-through to the first DNA barcode module, what can be determined is the sequence of both of these DNA barcode modules.

[0374] To provide yet another example, a polynucleotide can be linked 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 multi-stage parallel synthesis of a compound. Additionally or alternatively, the second nucleic acid can identify a general class of compound to be produced by split-pool synthesis. Additionally or alternatively, the second nucleic acid can identify a disease that is treated by the compound being screened. Furthermore, the second nucleic acid can identify a date, the name of a chemist, etc.

[0375] A preferred method for synthesizing DNA barcodes is presented below, using the same reaction cycle to incrementally attach each DNA barcode module.

[0376] Step 1. Provide beads with TCO groups attached. In practice, the beads have hundreds or thousands of similarly attached TCO groups, with each TCO group attached to a different site on the bead. In practice, a split-pool method is used to simultaneously modify a large number of beads by click chemistry.

[0377] Step 2. [Tetrazine]-[first DNA barcode module]-[azide] is added to the beads, and the TCO groups are condensed with the tetrazine groups. 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 instead contains the condensation product that is produced when TCO condenses with tetrazine.

[0378] Step 3. Optional washing.

[0379] Step 4. Add DBCO-TCO to cap the azide and create a TCO terminus. The result is the following structure: BEAD-TCO-tetrazine-first DNA barcode module-azide-DBCO-TCO

[0380] Step 5. Optional washing.

[0381] Step 6. Add the following reagents to attach the second DNA barcode module to the distal end of the growing DNA barcode. The reagents are: [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-first DNA barcode module-azide-DBCO-TCO-[tetrazine]-[second DNA barcode module]-[azide]

[0382] The above scheme involves a series of steps for incrementally adding more DNA barcode modules, these additions being in parallel with the addition of more chemical monomers. As noted elsewhere, this "parallel" synthesis can involve attaching a chemical monomer followed by a DNA barcode module that specifies that monomer, or alternatively, attaching a DNA barcode module followed by a chemical monomer specified by that specific chemical monomer.

[0383] Compounds for click chemistry synthesis of DNA barcodes Figure 17 discloses the chemical synthesis of a compound suitable for connecting deoxycytidine residues (dC) during the synthesis of DNA barcode modules and ultimately entire DNA barcodes. 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 organic synthesis pathway 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 residual chemicals (never naturally occurring in nucleic acids) are generated as "trace" from the click chemistry being performed. Available DNA polymerases can be used for sequencing-by-synthesis of DNA barcodes created by click chemistry; the DNA polymerase can migrate across the trace, and the trace does not introduce sequencing errors. TBAI is tetrabutylammonium iodide.

[0384] Synthesis of concatenated DNA barcodes In the following description, DNA barcode modules are assembled in a row to create a DNA barcode. However, in the figures in the text below, the term "DNA barcode" is used instead of "DNA barcode module" to fit the figures in the text to the page. Figure 7 shows the same process as 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.

[0385] An option is to create a DNA barcode containing a terminal nucleic acid encoding a DNA hairpin. This involves a DNA barcode containing a nucleic acid at its 3-prime end that has a sequencing primer annealing site, a bend in the form of approximately four unpaired bases, and a sequencing primer that can bend around and base-pair around the sequencing primer annealing site. Again, the sequencing primer anneals to the sequencing primer annealing site, and the actual sequencing reaction begins at the 3' end of the annealed sequencing primer.

[0386] When performing the final step of synthesizing the DNA barcode and ligating the final DNA barcode module to the growing bead-bound DNA barcode, the "sprint oligo" can comprise a sequence encompassing a DNA hairpin (the DNA hairpin includes, in this order, an annealing site for the sequencing primer, several nucleotides that do not base-pair with each other or any nearby bases, 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, using the splint oligo as a template to polymerize, 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 each other, and (3) the sequencing primer.

[0387] Reversible terminator group at the 3' end of a hairpin sequencing primer. The present disclosure provides reagents, compositions, and methods for attaching a preformed nucleotide / reversible terminator group complex to the 3' end of an annealed sequencing primer. The reversible terminator group is an optional component of the hairpin sequencing primer and is part of a bead-bound DNA barcode.

[0388] Step 1. First, beads are placed in picowells, each having a polynucleotide attached thereto, the 5' end of which is optionally linked to the bead using a linker. Figure 7 shows that the bead-bound polynucleotide comprises a first DNA barcode and a first annealing site. The linker can be made from nucleic acid, or it can be chemically made from some other material. Preferably, the linker is hydrophobic, and preferably, the linker separates the bead-bound grown DNA barcode from the hydrophobic polystyrene bead, e.g., TentaGel® bead.

[0389] For convenience of notation, 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" are both referred to as the "first annealing site," even though they do not have the same sequence of bases (instead, their 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 serving as a template for DNA polymerase, which extends the bead-bound DNA barcode by copying what is on the splint oligo).

[0390] Also, for ease of notation, 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).

[0391] The bead-bound grown DNA barcode from the 5' end to the 3' end may comprise nucleic acids in the following order: Beads / first DNA barcode / first annealing site /

[0392] Alternatively, the bead-bound grown DNA barcode from the 5' end to the 3' end can comprise nucleic acids encoding the number of steps, 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 /

[0393] 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 /

[0394] Not shown in the figures in these texts is any linker that mediates the attachment 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.

[0395] Step 2. A soluble splint oligonucleotide (splint oligo) is added, which is composed of a first annealing site, a second DNA barcode module, and a second annealing site.

[0396] Figure 7 also shows the step where the hybridized splint oligo is used as a template, and a DNA polymerase catalyzes the attachment of a second DNA barcode module and a second annealing site to the bead-bound growing DNA barcode. Figure 7 shows the enzymatic product of the DNA polymerase using the splint oligo as a template to catalyze the growth of the bead-bound DNA barcode (growth by covalent attachment of a second DNA barcode and a second annealing site) a little longer. Shown immediately below the text is the complex of the splint oligo hybridized to the bead-bound growing DNA barcode: Beads / first DNA barcode / first annealing site / First annealing site / second DNA barcode / second annealing site

[0397] To repeat some of the information shown in Figure 7, shown immediately below is the splint oligo: " First annealing site / second DNA barcode / second annealing site "

[0398] Step 3. Add DNA polymerase and dNTPs to extend the bead-bound DNA barcode. Below is the bead-bound DNA barcode, and the splint oligo is still hybridized, with the "second DNA barcode module" and "second annealing site" nucleic acids attached, making the bead-bound growing barcode longer than before. Figure 7 further illustrates this step. The splint oligo is displayed below the bead-bound growing barcode: Bead / first DNA barcode / first annealing site / second DNA barcode / second annealing site First annealing site / second DNA barcode / second annealing site

[0399] Step 4. Washing the splint oligos. The splint oligos can be induced 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, by adding dilute NaOH to the picowell array to neutralize it.

[0400] 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): Second annealing site / Third DNA barcode / Third annealing site /

[0401] 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 following complement: "third DNA barcode / third annealing site / "

[0402] Step 7. Wash the second splint oligo.

[0403] Step 4. Add the following splint oligos (this specific addition is not shown in Figure 7): Third annealing site / Fourth DNA barcode / Fourth annealing site /

[0404] This soluble oligonucleotide contains a nucleic acid capable of annealing to the "third annealing site" of the bead-bound oligonucleotide. Upon annealing, a DNA polymerase with four dNTPs is utilized to extend the bead-bound oligonucleotide, which is used to encode yet another DNA barcode module (the fourth DNA barcode). This cycle of steps is repeated throughout the split-pool procedure, which creates a library of compounds and associated DNA barcodes in parallel, each associated with a given compound (each DNA barcode informs the chemical synthesis history of the associated compound). When the chemical synthesis of the compound library is complete, the cycle of steps is stopped. With the completed bead-bound DNA barcode chemical library in hand, the beads can then be dispensed into picowells of a picowell array.

[0405] The DNA barcode of each bead also constitutes a DNA barcode associated with each picowell. The DNA barcode can enable identification of the bead-bound compound. The sequencing method of the present disclosure is performed within the picowell while the bead is still within the picowell. In exclusive embodiments, the present disclosure can exclude any sequencing method, can exclude any reagents used for sequencing, and sequencing is not performed on bead-bound DNA templates, or sequencing is not performed on bead-bound DNA templates disposed within the picowell.

[0406] Sequencing primer annealing site. In one embodiment, each DNA barcode module within the completed DNA barcode is operably linked to, and in-frame with, 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).

[0407] 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. Figure 7 does not show the sequencing primer annealing site.

[0408] Nucleic acid linked to beads via the 3' end of the nucleic acid Although various embodiments disclosed herein relate to linking DNA to beads via the 5' end of the DNA, in other embodiments, DNA, such as DNA barcodes or DNA tags, can be linked to beads via their 3' end. The 3' hydroxyl group of DNA can react under certain chemical synthesis conditions (e.g., Mitsunobu conversion), damaging the 3' end and making it unable to participate in elongation, ligation, or other processes. Therefore, DNA tags can be attached to beads via their 3' end to prevent unwanted chemical reactions and damage to the DNA barcode.

[0409] Exclusionary embodiments of the bead-bound DNA barcodes of the present disclosure can exclude any bead, microparticle, microsphere, resin, or polymer composition in which the linked DNA barcode is linked to the bead via a photocleavable or cleavable linker.

[0410] What can be excluded is any bead, microparticle, microsphere, resin, or polymer composition that does not contain both: (1) a linked DNA barcode linked to a first location on a bead; and (2) a compound linked to a second location on a bead, where the first location is not the same as the second location. In a preferred embodiment, this "compound" is made of multiple chemical library monomers.

[0411] Excluded can be any bead, microparticle, microsphere, resin, or polymer composition that does not have an outer surface (or outer surfaces) and an inner surface (or inner surfaces, or inner regions), and the bead does not include at least 10,000 substantially identical linked DNA barcodes linked to the bead, and at least 90% of the at least 10,000 substantially identical linked DNA barcodes are linked to the outer surface. In other words, excluded can be any bead in which at least 90% of the linked DNA barcodes are not linked to the outer surface.

[0412] What may be excluded is any bead, microparticle, microsphere, resin, or polymer composition made substantially of or including any polyacrylamide.

[0413] What may be excluded is any bead, microparticle, microsphere, hydrogel, resin, or polymer composition that includes a promoter, such as the T7 promoter, or that includes a polyA region, or that contains a promoter and also a polyA region.

[0414] A method using only one cycle of annealing / polymerization to generate bead-bound DNA barcodes with two DNA barcode modules. The present disclosure encompasses systems, reagents, and methods in which the bead-bound DNA barcodes involve only one annealing / polymerization step. This embodiment is represented by the following diagram, where the first diagram shows annealing of the splint oligo and the second diagram shows filling using DNA polymerase. The end result is a bead-bound DNA barcode containing two DNA barcode modules. In this particular procedure, the bead-bound starting material can optionally include a linker (but preferably not any cleavable linker), optionally a nucleic acid encoding information other than the compound's identity, and optionally a functional nucleic acid such as a sequencing primer or DNA hairpin. Two diagrams are shown in the text (see immediately below):

[0415] Beads / first DNA barcode / first annealing site / First annealing site / second DNA barcode / second annealing site Bead / first DNA barcode / first annealing site / second DNA barcode / second annealing site First annealing site / second DNA barcode / second annealing site

[0416] A method using two cycles of annealing / polymerization to generate bead-bound DNA barcodes with three DNA barcode modules. The present disclosure encompasses bead-binding 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 comprises the structure: first annealing site / second DNA barcode / second annealing site, and the second splint oligo comprises the structure: second annealing site / third DNA barcode / third annealing site.

[0417] A method using three cycles of annealing / polymerization to generate bead-bound DNA barcodes with four DNA barcode modules. The present disclosure encompasses bead-binding compositions, systems, and methods in which three different split oligos are used (first splint oligo; second splint oligo; third splint oligo). In this context, the first splint oligo includes the structure: first annealing site / second DNA barcode / second annealing site, the second splint oligo includes the structure: second annealing site / third DNA barcode / third annealing site, and the third splint oligo includes the structure: third annealing site / fourth DNA barcode / fourth annealing site.

[0418] A method using four cycles of annealing / polymerization to generate bead-bound DNA barcodes with five DNA barcode modules. The present disclosure encompasses bead-binding 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 context, the first splint oligo includes the structure: first annealing site / second DNA barcode / second annealing site, the second splint oligo includes the structure: second annealing site / third DNA barcode / third annealing site, the third splint oligo includes the structure: third annealing site / fourth DNA barcode / fourth annealing site, and the fourth splint oligo includes the structure: fourth annealing site / fifth DNA barcode / fifth annealing site.

[0419] Embodiments using multiple annealing / polymerization steps to generate bead-bound DNA barcodes with multiple DNA barcode modules. The present disclosure encompasses bead-bound compositions, systems, and methods involving concatenated barcodes, using only one splint oligo (creating a two-module DNA barcode), using only two splint oligos (creating a three-module DNA barcode), using only three splint oligos (creating a four-module DNA barcode), using only four splint oligos (creating a five-module DNA barcode), using only five splint oligos (creating a six-module DNA barcode), using only six splint oligos (creating a seven-module DNA barcode), etc.

[0420] Included are bead-binding compositions, systems, and methods that use at least 1 splint oligo, at least 2 splint oligos, at least 3 splint oligos, at least 4 splint oligos, at least 5 splint oligos, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 20 splint oligos, or fewer than 20, fewer than 15, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 3, fewer than 2 splint oligos. These numbers refer to the splint oligos themselves, as well as the number of steps of adding splint oligos and the numbering of DNA modules added to the growing bead-bound DNA barcode.

[0421] Reduce damage to DNA barcodes Use orthogonal DNA barcodes to reduce damage (instead of concatenated DNA barcodes). One way to apply the topics 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.

[0422] In ligated barcoding, if the attachment of any given module is incomplete (i.e., not all attachment sites are successfully ligated with the required module), the sequence of the completed barcode will be inaccurate. The term "inaccurate" means that incomplete ligation results in missing chunks, and the completed product was assumed by the user to be a complete, correct DNA barcode. Here, the completed DNA barcode sequence will contain errors because attachment of all DNA modules failed. In contrast, orthogonal barcoding covalently binds each individual DNA module to its own unique attachment site on the bead. Furthermore, once a DNA module is attached to a predetermined site on the bead, there is no need to ligate additional DNA modules to the DNA modules already ligated to the bead.

[0423] Damage is reduced by using a crosslinking agent. The present disclosure provides reagents and methods for reducing damage to bead-bound DNA barcodes and 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 dsDNA form of the DNA barcode, the dsDNA is converted to ssDNA. Conversion of dsDNA to ssDNA is effective when one of the DNA strands has a uracil (U) residue, and cleavage of the DNA at the uracil residue is catalyzed by uracil-N-glycosidase (see Figure 5 of Serial No. 62 / 562,905, filed September 25, 2017, which is incorporated herein by reference in its entirety). The above refers to damage inflicted on the growing DNA barcode by the reagents used to create the bead-bound compounds.

[0424] Using double-stranded DNA (dsDNA) to create DNA barcodes reduces damage. Another method for reducing damage to bead-bound DNA barcodes and partially synthesized DNA barcodes is to synthesize the DNA barcodes in the form of double-stranded DNA, where each of the attached DNA barcode modules 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 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).

[0425] 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 first prong of the hairpin anneals to the second prong of the hairpin.

[0426] If the synthesized DNA barcode is in the form of double-stranded DNA (dsDNA), solvents such as DCM, DMF, and DMA can denature the DNA barcode. The methods and reagents described above can prevent denaturation.

[0427] 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.

[0428] Damage is reduced by avoiding proteinaceous solvents, avoiding strong acids and bases, and avoiding strong reducing and oxidizing agents. The types of chemistries compatible with the presence of deoxyribonucleic acid (DNA), whether bead-bound or non-bead-bound, may require the absence of proteinaceous solvents, avoiding strongly acidic conditions, avoiding strong bases such as t-butyllithium, 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 (Luk and Sats (2014) DNA-Compatible Chemistry (Chapter 4) in A Handbook for DNA-Encoded Chemistry, 1 st ed. John Wiley and Sons, Inc.).

[0429] 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 a compound.

[0430] The use of DNA-compatible chemicals reduces damage to nucleic acids. Satz et al. disclose various chemicals 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 description in Satz et al. (supra) is directed to chemical reactions performed on DNA / chemical library member conjugates, the types of DNA-compatible chemicals described are also relevant, and organic chemistry should be performed on beads containing bead-bound compounds and bead-bound DNA.

[0431] DNA-compatible reactions for the formation of benzimidazole compounds, imidazolidinone compounds, quinazolinone compounds, isoindolinone compounds, thiazole compounds, and imidazopyridine compounds are disclosed (see Satz et al., Table 1, entries 1-6).

[0432] 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).

[0433] Additionally, methods for linking reagents to DNA have been disclosed, where the linkage occurs at a functional group already attached to the DNA. These methods include the Suzuki ligation, an optimized procedure for the Sonogashira ligation between an alkyne and an aryl halide; the conversion of aldehydes to alkynes using dimethyl-1-diazo-2-oxopropylphosphonate; a new method for the cycloaddition of triazoles directly from purified alkynes; and an improved method for the reaction of isocyanate building blocks with amine-functionalized DNA, where improved reactions occur using isocyanate reagents in pH 9.4 buffer (see Satz et al., Table 1, entries 12-15).

[0434] Additional methods for linking reagents to DNA have been disclosed, where linkage occurs at functional groups already attached to the DNA. These include methods for conjugating primary amines to DNA, optimized procedures for forming DNA-conjugated thioureas, methods for alkylating secondary amines and bis-alkylating aliphatic primary amines using hetaryl halides as building blocks that can react with amine-functionalized DNA conjugates, methods for mono-alkylating primary amine-DNA conjugates, and methods for the Wittig reaction (see Satz et al., Table 1, entries 16-20).

[0435] Damaged DNA is reduced through DNA repair enzymes. Various proteins, including enzymes, DNA damage-binding proteins, and helicases, are available for DNA damage repair. Commercially available DNA repair proteins 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 (which do not cleave ssDNA or dsDNA) that remove damaged bases 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 apyrimidinic sites. EndoVIII is available for repairing oxidized pyrimidines and apyrimidinic sites. EndoV is available for mismatch repair. HaaG is a glycosylase that can repair alkylated purines. When DNA repair enzymes leave gaps, or when double-stranded DNA is missing one or more consecutive deoxyribonucleotides on one of the strands, various DNA polymerases are available to fill the gaps (see Catalog (2018) New England BioLabs, Ipswich, MA).

[0436] Various DNA repair enzymes and DNA repair systems have been isolated from mammals, yeast, and bacteria. These include those that mediate nucleotide excision repair (NER), direct repair, base excision repair, transcription-coupled DNA repair, and recombinational repair. Interstrand DNA crosslinks can be repaired using a combination of NER and homologous recombination. Direct repair involves the repair of cyclobutane pyrimidine dimers and 6-4 products via photolyase enzymes. Direct repair involves the repair of O-methyltransferases via DNA methyltransferases. 6 -O from methylguanine 6This also 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.

[0437] 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.

[0438] By linking DNA to beads via their 3'-end, damage is reduced. Certain chemical conversions can damage the exposed 3'-hydroxyl group of nucleic acids. For example, the Mitsunobu reaction allows the conversion of primary and secondary alcohols into esters, phenyl ethers, thioethers, and various other compounds, which makes the exposed 3'-end unresponsive to subsequent processing steps or potentially allows the now-modified 3'-end to participate in further chemical reactions. In some embodiments, DNA tags can be attached to beads via their 3'-end, so that only the 5'-end is exposed to solution.

[0439] The disclosed reagents, systems, and methods encompass bead-bound nucleic acids, such as bead-bound DNA or bead-bound DNA tags, where the linkage to the bead involves the 3' terminus (or 3' end) of the DNA. When ssDNA containing a DNA barcode is linked 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 linked ssDNA. Instead of using only one sequencing primer, multiple sequencing primers can be used, each hybridizing upstream to a specific DNA barcode module. For example, if a given DNA barcode contains five DNA barcode modules, the DNA can be linked via its 3' end to a bead, and the DNA barcode can include five different primer annealing sites, each located just upstream or immediately upstream of a given DNA barcode module.

[0440] In another embodiment, the 5'-linking embodiment with dsDNA is dsDNA, and the 5'-end of only one strand of the dsDNA is linked to the bead.

[0441] (V) Compounds and linkage 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 or enzymes such as proteases, (4) non-cleavable linkers, (5) bifunctional linkers, (6) multifunctional linkers, and (7) beads used in linking. For example, available linkers include 4-hydroxymethylbenzoic acid (HMBA) linkers and 4-hydroxymethylphenylacetic acid linkers (see Camperi, Marani, Cascone (2005) Tetrahedron Letters. 46:1561-1564).

[0442] 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. 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.

[0443] 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; and yet another tine can be linked to a metal ion.

[0444] 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, and the enzyme is used to assay the activity of the attached chemical library member.

[0445] (VI) Linking the monomers together to make the 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 indicates 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.

[0446] 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 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 can be used to identify each of the chemical monomers used to build the compounds in that library.

[0447] Ligation reaction. The following describes the ligation of chemical monomers to beads and to each other, i.e., the first step is to directly ligate 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 disclosed below are DNA compatible.

[0448] We describe a method for preparing three amino acid compounds on Tentagel® beads. Fmoc-protected resin (1 mg, Rapp Polymere GmbH, 10 μm, TentaGel M-NH2, 0.23 mmol / g) modified with Fmoc-Photo-Linker, 4-{4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butanoic acid, or another suitable linker with Fmoc protection, was suspended in each well of a DMA (150 μL) reaction plate (Merck Millipore Ltd, 0.45 μm hydrophobic PTFE). The 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 μL of a mixture of 5% piperazine, 2% DBU in DMF. The plate was sealed with an Excel Scientific Alumna Seal and shaken at 40°C for 15 minutes. The solvent was removed by applying a vacuum, and the deprotection procedure was repeated for 5 minutes. After filtration, each well was washed with 150 μL each of 2XDMA, 3XDCM, and 1XDMA, with the solvent removed by applying a vacuum between each wash. Next, each well of the resin was acylated with the appropriate amino acid by adding 150 μL of a preactivation mixture of 60 mM Fmoc-amino acid, 80 mM Oxyma, 200 mM DIC, and 80 mM 2,4,6-trimethylpyridine, which had been left at room temperature for 2 minutes. The plate was resealed and shaken at 40°C for 1 hour. After filtration, each well was washed with 150 μL each of 2XDMA and 3XDCM. The beads in each well were resuspended in 150 μl of DCM, and the contents of each well were combined into one container via pipetting. The combined beads were thoroughly mixed and redistributed across the plate by pipetting equal volumes into the appropriate wells (1 mg / well). The solvent is removed by applying a vacuum, and each well is ready for the next appropriate step. For each additional amino acid attachment, the Fmoc deprotection step is repeated, followed by the attachment of the desired amino acid. If a split pool is required, the coupling and redistribution method is repeated.

[0449] We describe a method for preparing 3-mer amino acids by the split-pool method on beads. Fmoc-protected resin (1 mg, Rapp Polymere GmbH, 10 μm, TentaGel M-NH2, 0.23 mmol / g) modified with Fmoc-Photo-Linker, 4-{4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butanoic acid, or any other suitable linker, was suspended in each well of a DMA (150 μL) reaction plate (Merck Millipore Ltd, 0.45 μm hydrophobic PTFE). The 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 μL of a mixture of 5% piperazine, 2% DBU in DMF. The plate was sealed with an Excel Scientific Alumna Seal and shaken at 40°C for 15 minutes. The solvent was removed by applying a vacuum, and the deprotection procedure was repeated for 5 minutes. After filtration, each well was washed with 150 μL each of 2XDMA, 3XDCM, and 1XDMA, with the solvent removed by applying a vacuum between each wash. Next, each well of the resin was acylated with the appropriate AA by adding 150 μL of a pre-activation mixture of 60 mM Fmoc-amino acid, 80 mM Oxyma, 200 mM DIC, and 80 mM 2,4,6-trimethylpyridine, which had been left at room temperature for 2 minutes. The plate was resealed and shaken at 40°C for 1 hour. After filtration, each well was washed with 150 μL each of 2XDMA, 3XDCM, and 1XDMA. For each additional AA conjugation, the Fmoc deprotection step was repeated first, followed by the conjugation step with the desired AA. To analyze each successive conjugation, a 1 mg portion of beads was suspended in 100 μL of DMSO and exposed to the full power of a 365 nm LED for 2 hours. The resin is filtered and the filtrate is injected into an Agilent 1100 Series LCMS equipped with an Agilent Poroshell SB-C-18, 3.0 × 50 mm, 2.7 μm column using a gradient from 5% CH3CN in 0.1% TFA in water to 100% CH3CN in 0.1% TFA over 4 min at a flow rate of 1.2 mL / min and monitoring at 220 nm.

[0450] This experiment involved the creation of non-amino acid pendants with lenalidomide (Revlimid®), which would be attached to the final amino acid after deprotection. This was also performed during spinning. Each well of resin was acylated with 150 μL of a 5-minute pre-treatment mixture of 40 mM chloroacetic acid, 40 mM Oxyma, 80 mM DIC, and 40 mM TMP in DMA (after Fmoc deprotection). The plate was sealed and shaken at 40°C for 1 hour. Each well was washed with 150 μL each of 3×DMA, 3×DCM, and 2×DMA. The resin was then resuspended in a suspension of 100 mM K2CO3 and 100 mM Rev in DMA. The plate was sealed and shaken at room temperature for 3 hours. The resin was washed with 150 μL each of 2×50 / 50 DMA / water, 3×DMA, 3×DCM, and 2×DMA.

[0451] Defines the degree of fidelity of synthesis of a compound attached to a given bead. This 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 characterizes the compound attached to a given bead. This given bead can be the product of a split-pool synthesis of a library of compounds, where each bead has a unique compound.

[0452] 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 the following two chemical groups: the first chemical group is N-alpha-9-fluorenyl-methyloxycarbonyl (Fmoc, base-labile); the second chemical group 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).

[0453] Preferably, at least 99.5%, at least 99.0%, at least 95%, at least 90%, at least 85%, or at least 80% of the chemical library members bound to a given bead have the exact same chemical structure after synthesis is complete. Incomplete ligation, which may occur at one or more steps in the multi-step synthesis of chemical library members, is possible. For this reason, the compositions of the present disclosure may be characterized or limited by one of the following limitations or ranges:

[0454] 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 monomer that may occur during solid-phase synthesis).

[0455] In exclusive embodiments, the present disclosure can exclude any method or reagent that does not meet one of the above cutoff values ​​for "exactly the same structure."

[0456] 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 are similar and contain the same compound in the population of beads (not considering any errors in incorporation of chemical monomers during solid phase synthesis, not considering any chemical damage that occurs to the chemical monomers during organic synthesis).

[0457] Introduction to Click Chemistry. Jewett et al. define "click reactions as...selective, high-yielding, and have favorable reaction rates." A subclass of click reactions in which the components are inert to the surrounding biological environment is called biolsogonal (Jewett and Bertozzi (2010) Chem. Soc. Rev. 39:1272-1279). "Click chemistry" can be used to join 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 in combinatorial chemistry. Click chemistry reactions are characterized by high yields, irreversibility, and insensitivity to oxygen or water. Classes of chemical reactions used in "click chemistry" include (1) cycloaddition reactions, particularly 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. In contrast, non-click chemistry reactions 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).

[0458] 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).

[0459] Hartwig-Buchwald amination. The Hartwig-Buchwald amination reaction can be used in solid-phase pharmaceutical synthesis. This amination reaction is used to synthesize carbon-nitrogen bonds. The reaction involves palladium-catalyzed aryl halide and amine (R1-NH-R2), generating an aryl product in which the amine replaces the halide, directly attaching the nitrogen of the amino group 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 Hartwig-Buchwald amination is compatible with a variety of amines, making it well suited for combinatorial chemistry (Zimmermann and Brase (2007) J. Comb. Chem. 9:1114-1137).

[0460] 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 copper catalyst accelerates the rate of the Huisgen cycloaddition reaction. The Huisgen reaction functions via "click chemistry" or "click reaction." When catalyzed by copper, the Huisgen reaction can generate a 1,2,3-triazole nucleus suitable for creating small molecule drugs. The Huisgen reaction is compatible with the presence of amino acid side chains, at least in protected form. Molecules created with 1,2,3-triazoles can have bonds similar to the amide bonds of polypeptides, and therefore, these molecules can replace peptide bonds (Angell and Burgess (2007) Chem. Soc. Rev. 36:1674-1689).

[0461] Peptide Nucleic Acids (PNAs). The present disclosure provides split-pool, combinatorial, 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).

[0462] The present disclosure encompasses bead-bound compounds, wherein the compound is in the form of only one monomer. For example, the bead-bound compound 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 in which the amino group is modified with a small chemical moiety having a carboxylic acid group, or the compound is a lenalidomide analog that is a stereoisomer or enantiomer of lenalidomide.

[0463] (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 a later point, disclosed below is the attachment of non-amino acids, and the preparation of polyethylene glycol (PEG)-modified beads.

[0464] The present disclosure provides split-pool synthesis for generating chemical libraries. In one embodiment, this method involves (a) dividing beads into different containers and (b) adding different components to each container. For example, if three containers are used, species A is added to the first containing container, species B is added to the second containing container, and species C is added to the third containing container, and the species are reacted, covalently binding to the attachment sites of the beads in the containers; (c) pooling all beads into one container; (d) dividing the beads into three containers; and (e) adding different components to each container, species A is added to the first containing container, species B is added to the second containing container, and species C is added to the third containing container, and the species are covalently bound to the previously attached first species (see Stockwell (2000) Trends Biotechnol. 18:449-455).

[0465] The split pool synthesis of the present disclosure includes a DNA barcode ligation step either before or after each chemical ligation step (which creates a chemical library member), where the DNA barcode identifies the chemical being ligated in that step.

[0466] In exclusive embodiments, the present disclosure may exclude methods and reagents in which a barcode is attached before attaching a chemical for a given step of a parallel synthesis. Conversely, the present disclosure may exclude methods and reagents in which a chemical is attached before attaching a barcode for a given step of a parallel synthesis.

[0467] One of the characteristics of bead-bound chemical libraries prepared by split-pooling is that only one compound is attached to each bead. Some heterogeneity can arise if ligation is incomplete, e.g., if only 4,000 of the 5,000 attachment sites in a given split-pooling step are successfully ligated to the desired chemical species.

[0468] 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, the bead receives one or more chemical units (chemical monomers) followed by a DNA barcode module encoding that particular chemical unit. The term "parallel" refers to the fact that as the polymer of chemical library monomers grows, the polymer of DNA barcode modules also grows. When all DNA barcode modules are attached to the bead and form a linked or orthogonal structure, the full-length DNA barcode (not just the DNA barcode modules) is referred to as a "DNA barcode."

[0469] Ratio of the number of externally attached DNA barcodes to the total number of attached chemical library members. This relates to the external and internal 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 external and internal 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 an exclusive embodiment, the present disclosure may exclude any bead, or any population of beads, that meets one of the above values.

[0470] Uniformity of DNA barcodes across typical beads, uniformity of chemical library members across typical beads The present disclosure provides for "chemical library homogeneity" for any given bead (or any population of beads) to be 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.

[0471] 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%.

[0472] Similarly, the present disclosure provides the above cutoff values ​​for assessing the uniformity of barcodes, such as DNA barcodes.

[0473] The uniformity of the DNA barcodes and the uniformity of the chemical library members can be defined as the percentage of the total population that matches the exact sequence as desired, as planned in the methods section of a laboratory manual or notebook.

[0474] In exclusive embodiments, the present disclosure may exclude any reagent, composition, or method that does not meet one or more of the above cutoff values.

[0475] 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.

[0476] In exclusive embodiments, the present disclosure can exclude any bead, or any population of beads, where the DNA barcode uniformity 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 chemical library member uniformity 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.

[0477] Ratio of internally attached DNA barcodes to externally attached DNA barcodes In some embodiments of the present disclosure, it may be desirable to manufacture and use beads in which DNA barcodes are primarily attached to the outer surface. One reason for not manufacturing and using beads with internal DNA barcodes is the low penetration of DNA oligomers into the inner space and the low penetration of DNA ligase (a ligase for connecting DNA modules to each other to create a completed DNA barcode) into the inner space. For sequencing purposes, a reason for not manufacturing and using internal DNA barcodes is the low penetration of the enzymes required to amplify the DNA required for final sequencing of the barcode. Yet another reason for not manufacturing and using beads with internal DNA barcodes is the cost of the inner space for attaching chemical library members.

[0478] The present disclosure provides beads having DNA barcodes, wherein 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.

[0479] The present disclosure also provides beads having DNA barcodes, wherein 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.

[0480] A population of beads in aqueous suspension can be contacted with a substrate, such as a picowell array, so that the beads enter and occupy the picowells. The ratio of the number of beads in the suspension to the number of picowells in the substrate can be adjusted to achieve a desired occupancy 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 empty of beads, and most picowells containing beads will contain only one bead. A small percentage of occupied picowells will contain two beads.

[0481] In valuable embodiments, the ratio of the number of beads in the suspension to the number of picowells can be about 0.2:100, about 0.4:100, about 0.6:100, about 0.8:100, about 1:100, about 2:100, about 4:100, about 6:100, about 8:100, about 10:100, about 20:100, about 30:100, about 40:100, about 50:100, about 60:100, 80:100, about 100:100 (which is the same as 1:1), about 2:1, about 4:1, about 6:1, about 8:1, about 10:1, etc.

[0482] In exclusive embodiments, the present disclosure may exclude any method or system that falls within one of the above values ​​or ranges.

[0483] In embodiments of the range, the ratio of the number of beads in the suspension to the number of picowells 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 0 to about 20:100, about 20:100 to about 30:100, about 30:100 to about 40:100, about 40:100 to about 50:100, about 50:100 to about 60:100, about 60:100 to 80:100, about 80:100 to about 100:100 (equivalent to 1:1), about 100:100 (equivalent to 1:1) to about 2:1, about 2:1 to about 4:1, about 4:1 to about 6:1, about 6:1 to about 8:1, about 8:1 to about 10:1, etc.

[0484] In exclusive embodiments, the present disclosure may exclude any method or system that falls within one of the above values ​​or ranges.

[0485] (VIII) Manufacturing Picowell Combining UV light, photomasks, and photoresist to create picowell array plates. Plates containing a large number of microwells, or picowells, can be fabricated for use in this disclosure as follows. Briefly, a three-layer sandwich is assembled. The top layer is photoresist. The middle layer is a glass wafer. The bottom layer is a photomask. Picowells are cut from the photoresist using UV light. When picowells are cut from a flat sheet of photoresist, the photoresist resembles a typical metal dish containing muffin cups, with slanted sides used to hold the muffin batter inside the dish. UV light acts as a "non-crosslinker" because it breaks down the photoresist polymer. After UV treatment, a solvent is added to wash away the UV-treated photoresist, leaving behind clean-looking picowells.

[0486] Tilting and rotating creates the slanted walls. Picowells with slanted walls are fabricated as follows: A photomask has multiple openings, each corresponding to the desired bottom dimensions of the picowell. The bottom dimensions can include circumference, diameter, and shape, i.e., a circular shape. The top dimensions of the well are created by directing a tilted UV light beam through the openings in the photomask while rotating the light source or the stage holding the sandwich (photomask / glass wafer / photoresist sandwich). In the rotation, the light source is not at a 90-degree angle relative to the photomask / wafer / photoresist sandwich; instead, it is tilted slightly from the 90-degree position to carve out the slanted 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 for inexpensively fabricating multiple picowell array plates.

[0487] Han et al. describe equipment and reagents for fabricating microwell plates with tilted 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-side 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°C hotplate for 10 minutes, followed by a 95°C hotplate for 30 minutes. The resulting photoresist / wafer sandwich is then contacted with a UV mask using a contact stage. The term "tilt and rotate UV lithography" refers to a method for fabricating microwell array plates or picowell array plates, each with tilted 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). A mask is contacted with photoresist, where each opening in the mask is circular. Figure 8 of 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 truncated cones. A soft material such as PDMS (polydimethylsiloxane) is poured over the conical array and cured, causing the PDMS layer to peel off and form the conical wells.

[0488] A mold is created for mass production of picowell array plates. If a picowell array plate is 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 attached array of picoprotrusions is removed (picoprotrusions are the inverse of the desired picowells). The solid platform with picoprotrusions is a reusable mold that can be used to manufacture many picowell array plates.

[0489] The procedure for creating replicas from an epoxy mold (or a cone array mold made of 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 pico-protrusions is pressed uniformly into the heat-softened material. After the pico-protrusions are transferred to the substrate material as pico-indentations, the mold can be separated from the substrate. This disclosure preferably discloses pico-cones and pico-wells as patterns on the mold and substrate, respectively.

[0490] 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).

[0491] Other microwell plate embodiments. Plastic microwell arrays can be fabricated via thermoforming using a silicon mold containing an array of microwells, e.g., 800,000 microwells. Tapered shapes, smooth sidewalls, and a high degree of control, resulting in submicron tolerances, can be fabricated 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 an inability to control lateral dimensions during etching.

[0492] A non-pulsed dry etching process is used to fabricate plastic arrays by thermoforming plastic onto a silicon master that has been fabricated by a non-pulsed isotropic dry etching process using a chrome mask. The process uses three gases: Ar, SF6, and C4F8. The process is performed at 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 varied is the ratio of SF6 to C4F8, and the result of varying the ratio is tapered walls of molds (silicon pillars) that exist at slopes of, for example, 18 degrees (highly sloped walls), 9 degrees (slightly sloped walls), or 2 degrees (walls nearly perpendicular to the substrate) (see Perry, Henley, and Ramsey (Oct. 26-30, 2014) Development of Plastic Microwell Arrays for Improved Replication Fidelity. 18th Int. Conference on Miniaturized Systems for Chemistry and Life Sciences. San Antonio, TX (pages 1700-1703)).

[0493] In embodiments, the present disclosure provides substrates, arrays, grids, microfluidic devices, etc., comprising 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.

[0494] Additionally, the volume can take the form of a range between any two adjacent values ​​listed above, such as a range between about 40 femtoliters and about 60 femtoliters, or a range between any two values ​​listed above that are not directly adjacent to each other in the list above.

[0495] Furthermore, 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 immediately adjacent to each other in the above list.

[0496] In exclusive embodiments, the present 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 a range of any of the two values ​​above that are adjacent to each other, or by a range of any of the two values ​​above that are not adjacent to each other in the list.

[0497] Spherical plugs (also known as capped beads) on picowells. The present disclosure provides spherical plugs, or alternatively, porous spherical plugs for each well or substantially all wells of a picowell array. The purpose of the plugs is to retain the drug, drug candidate, cellular contents, and metabolites within the well. The plugs also serve to isolate the contents of the picowells from each other. The spherical plugs may not need to be perfectly spherical, as long as they can serve the purpose of covering the top (or opening or mouth) of the picowell. The wells can have a top diameter and a bottom diameter. The diameter of the spherical plugs before capping the wells 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 plugs can be added to cover the picowells simply by flowing the plugs across the picowell array. Centrifugation, pressure, agitation, or other methods can be used to pack the beads into the top (or mouth or opening) of the picowell and create a tight seal. In some embodiments, a solvent can be used to expand and / or resize the capped beads. In some embodiments, the capped beads are loaded into a solvent that shrinks the beads, and when replaced with assay buffer or a different solvent, the capped beads return to their original size or expand, thereby tightly sealing the picowell. In some embodiments, temperature can be used to expand or shred the capped beads to obtain a better seal at the mouth of the picowell. Optionally, the capped beads can be held in place and prevented from falling further into the picowell by one of the steps in a stepped picowell array.

[0498] The capped beads may be the same type of bead carrying the disclosed compounds, or they may be different types of beads. In some embodiments, the capped beads may actually be the compounds themselves. The capped beads may function as passive caps that prevent or slow the diffusion of molecules from the picowell, or the beads may be active beads that capture reagents from the picowell using functional moieties attached to the capped beads. In some embodiments, porous capped beads may passively capture metabolites released from cell-based assays performed in the picowell. In some embodiments, capped beads may nonspecifically capture cellular materials such as lipids, proteins, carbohydrates, and nucleic acids. In some embodiments, capped beads may be functionalized with antibodies to specifically capture proteins released from healthy, diseased, lysed, or fixed cells. In some embodiments, capped beads may be functionalized with DNA or RNA oligonucleotides that specifically capture cellular nucleic acids. In some embodiments, DNA- or RNA-functionalized capped beads may be used to capture microRNA released from cells in the capped picowell. 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 bearing a compound. In some embodiments, the capped bead captures material released from the compound bead. In some embodiments, the capped bead captures a sample of the compound released from the compound bead. In some embodiments, the capped bead captures DNA barcodes released from the compound bead. In some embodiments, the capped beads capture different types of analytes released from within the picowells they cap.

[0499] Relative Hardness of Caps and Picowells. A preferred device is a microtiter plate, with each microtiter plate containing thousands of picowells in its base. The ability of a cap to properly seat or seal each picowell can be a function of the hardness of the plastic that makes up the picowell opening and interior walls relative to the hardness of the cap.

[0500] The hardness of plastics can be defined in terms of a "durometer" value. Hardness is defined and tested as the resistance of a material to indentation. The hardness of a spherical plug and the hardness of a picowell wall can be defined in terms of its "durometer." 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 (see Silicon Design Manual, 6th ed., Albright Technologies, Inc., Leominster, MA).

[0501] 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 discrete step from the top diameter to the bottom diameter (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 discrete 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, the small beads can be deposited at the bottom of the stepped picowell and the capped beads can be deposited at the top opening of the stepped picowell. In some embodiments, the picowell can contain more than two beads.

[0502] Method for making a stepped picowell. Figure 29 discloses a stepped picowell. The embodiment shown has three compartments and two steps. The top compartment is the widest and is configured to accept a cap when the picowell is capped, with the cap occupying most of the top compartment. The middle compartment is configured to be occupied primarily by reagents or reagents alone. The reagents may 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 to which both a DNA library and a releasable compound are linked. In addition to carrying the DNA barcode and the releasable compound, the same beads can also carry a "response capture element." One of the steps in the stepped picowell holds the capped beads in place and prevents them from falling further into the picowell. In Figure 29, Structure 1 is the cap, Structure 2 is the bead, and Structure 3 is the upper region located just above the first step. Structure 4 is the central region 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.

[0503] For the space in the lowest compartment occupied by a 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.

[0504] Furthermore, for the space occupied by the beads (assuming there is only one bead in a picowell), about 50% of the beads are in the lowest section, and about 50% of the same beads are in the middle section; 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. For these calculations, the space occupied by the beads is assumed (hypothetically) 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.

[0505] Similar to conical and cylindrical picowells, the use of a molding system is one preferred embodiment for fabricating stepped picowells. For this purpose, a mold containing an array of multilayer pillars is desired, which, when stamped into a thermoplastic or other curable polymer substrate, can form the imprint of a stepped picowell. Layered pillar arrays with multiple steps, each step of a different diameter (smaller at the top), can be formed in a multilayer lithography process. Briefly, to crosslink the first layer of the micropillar array, a first layer of photoresist is exposed through a first mask. A second layer of photoresist can be deposited directly onto the first layer (previously exposed), and a second photomask can be used to later crosslink a second pattern of the second photoresist. At the end of the multilayer patterning, the resist stack can be developed to wash away the uncrosslinked areas, leaving behind an array of multilayer pillars. A detailed protocol for fabricating multilayer pillar arrays is described in Francisco Perdigones et al., (January 8 th, 2011). Microsystem Technologies for Biomedical Applications, Biomedical Engineering, Trends in Electronics Anthony N. Laskovski, 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.

[0506] Removing the capped beads. In many embodiments, it is advantageous to harvest the capped beads to study their reaction to chemical perturbations, analytes, or cellular responses within the picowells. In some embodiments, the capped beads can be removed from the picowell openings by inverting the picowell array and using mechanical agitation. In some embodiments, a solvent can be used to shrink the picowells, allowing them to be easily removed from the picowell openings. 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.

[0507] 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. In some embodiments, a crosslinked gel can be poured over the capped picowells, and the crosslinked gel crosslinks to the capped beads and itself, causing the capped beads to become embedded in the crosslinked sheet, which can be peeled off.

[0508] The relative positions of the picowells are maintained in the peeled layer. It should be understood that in embodiments where the capped beads are interlocked with a peelable gel layer, the relative positions of the capped beads relative to each other and to the picowells are maintained in the peeled layer. This allows for direct connectivity between the picowells, the assays in the picowells, the beads within the picowells, and any material captured in the capped beads.

[0509] In some embodiments, fiducial markers can be used to orient the relative features of the picowell array to the capped beads of the exfoliated layer.

[0510] Fiducial markers that allow for alignment and registration of the picowells. Arranging the picowells in an irregular array allows for easy identification of shifts and drifts during imaging of the picowell array. In some embodiments, the picowells are arranged in an irregular order to facilitate detection of optical and mechanical drift 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 drift during imaging. In some embodiments, external markers, such as fluorescent beads, may be misted onto the picowell array to provide a fiducial pattern.

[0511] 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 picowells in a given picowell array. Alternatively, the mat can be sized to cover a predetermined section of the picowells in the array. The mat can be secured to the top of the picowell plate, covering the picowells and also covering the generally flat top surface of the picowell plate between the picowells. Secure contact can be achieved by one or more of: (i) maintaining 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 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 so 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.

[0512] Membranes are available for use as mats to contact the substantially flat surface of picowell plates and for 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, WA. These include membranes made from 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 cutoff include 150 Da, 200 Da, 300 Da, 500 Da, 900 Da, 600 Da, 1,000 Da, 2,000 Da, 3,000 Da, 5,000 Da, 10,000 Da, 50,000 Da, 20,000 Da, 30,000 Da, 70,000 Da, 100,000 Da, 200,000 Da, 300,000 Da, 400,000 Da, 500,000 Da, 800,000 Da, 3500 Da, 0.005 micrometer, 0.030 micrometer, 0.05 micrometer, 0.10 micrometer, 0.20 micrometer, 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 while excluding 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.

[0513] Reversible adhesion is mediated by "molecular Velcro," e.g., metalloporphyrin- 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, and 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.

[0514] 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®, and Agarose®, as well as ion-exchange beads made of DEAE cellulose, carboxymethyl cellulose, and phosphocellulose, or any combination of the above, all combined into a single absorbent mat. Absorbent ligands include those used in high-performance liquid chromatography (HPLC) (see BioRad catalog, Hercules, CA). Specific absorbents include responsive capture elements such as poly(dT), which can capture mRNA by hybridizing with polyA tails. Response capture elements also include exon-targeting RNA probes, antibodies, and aptamers. Any or any combination of these can be covalently attached to the mat to create an absorbent mat that, when contacted with the top surface of a picowell, can capture aqueous assay medium or aqueous cell culture medium that may be present inside the picowell.

[0515] (IX) Depositing beads into picowells Plates with picowells can take the form of 96-well plates, each containing thousands of picowells. Alternatively, plates with picowells can take the form of 24-well plates, each containing thousands of picowells. For 96-well plates, each well can be filled using 0.1–0.2 mL of a suspension of beads in water or an aqueous solution. For 24-well plates, each well can be filled using approximately 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 vary, with approximately one-third of the picowells containing only one bead, approximately one-third containing two beads, and approximately one-third containing either no beads or more than two beads. The number of beads in the suspension may also be 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.

[0516] After the beads have settled, any excess liquid can be removed by touching the pipette tip to the wall of each well of a 96-well plate or the wall of each well of a 24-well plate to remove excess liquid.

[0517] Regarding assay reagents, when picowells are used to perform reactions, such as DNA sequencing, biochemical assays, or assays on cultured cells, assay reagents can be added to picowells already containing precipitated beads. As described above for the initial addition of the bead suspension, assay reagents are added 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 each of the 24 wells of a 24-well plate, can be removed with the pipette tip touching the wall of each of the 96 wells of a 96-well plate, or each of the 24 wells of a 24-well plate.

[0518] Picowell array flow cell embodiments. The picowell array can 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 embodiments, beads, cells, and other assay materials of the present disclosure can flow in through the inlet and out through the outlet. Gravity or centrifugal force can be used to retain beads in the picowells as they flow through the flow cell.

[0519] (X) Sequencing bead-bound nucleic acids in picowells The bead-bound nucleic acids can be sequenced while still attached to the beads. Alternatively, or additionally, the bead-bound nucleic acids can be sequenced after cleavage of the DNA barcodes from the beads.

[0520] Cleavage of DNA barcodes from beads prior to sequencing. In some embodiments, the present disclosure can encompass methods in which 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-specific technique, such as hybridization with a nucleic acid probe.

[0521] EXCLUSIVE EMBODIMENTS. In embodiments, the present disclosure may exclude any method, related 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 determination 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, sequencing, or prior to any type of sequence determination technique, such as hybridization with a nucleic acid probe.

[0522] Polymerase chain reaction (PCR); quantitative PCR (qPCR). PCR and qPCR methods rely on a three-step method: (1) denaturing a DNA template at high temperature, annealing primers at reduced temperatures, and finally extending the primers via DNA synthesis catalyzed by DNA polymerase (Gadkar and Filion (2014) Curr. Issues Mol. Biol. 16:1-6). qPCR is also referred to as "real-time PCR" (Kralik and Ricchi (2017) Frontiers Microbiology. 8 (9 pages)).

[0523] Recent modifications or improvements to PCR and qPCR methods include the use of helicase-dependent amplification (HDA), internal amplification standards, locked nucleic acids (LNA), and inhibitor-binding additives (Gadkar and Filion (2014) Curr. Issues Mol. Biol. 16:1-6). Locked nucleic acids offer the advantage of highly precise target recognition and binding.

[0524] qPCR can simultaneously amplify and quantify target DNA molecules. The qPCR method compares the number of amplification cycles required for the response curve to reach a specific fluorescence threshold (Pabinger, Rodiger, Kriegner (2014) Biomolecular Detection Quantification. 1:23-33). Refsland et al. provide a concise description of apparently typical conditions for performing qPCR (Refsland, Stenglein, Harris (2010) Nucleic Acids Res. 38:4274-4284).

[0525] Guidance is available on PCR primer design and validation, as well as variables such as annealing temperature (Ta), melting temperature (Tm), extension temperature, and buffer type (Bustin and Huggett (2017) Biomolecular Detection Quantification. 14:19-28).

[0526] Rolling circle amplification (RCA). DNA can be amplified while still attached to the beads. The amplified form of DNA is easier to sequence than unamplified DNA. In rolling circle amplification, DNA tags (DNA barcodes) are created as single strands. 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' to 3' exonuclease activity), the DNA is catalyzed to extend the splint oligos, ensuring a ligatable junction. 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 for the use of synthetic chemicals that can damage DNA, since any surviving DNA molecules can be thermally amplified to a sufficient amount for easy sequencing. DNA can be made single stranded by exonuclease digestion, nicking and melting at high temperature, or treatment with sodium hydroxide.

[0527] Further details of rolling circle amplification (RCA) will be revealed in the following steps that can be used to perform RCA.

[0528] Step 1: Begin with bead-bound ssDNA. If the bead-bound DNA is initially in double-stranded form (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).

[0529] Step 2: Add a "splint oligo" to the bead-bound ssDNA. The splint oligo is designed to hybridize to approximately 10-20 base pairs at the end (5' end) of the ssDNA that will be covalently linked to the bead, and also hybridizes 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 with the free end of the bead-bound ssDNA; all that is required is for the far ends of the bead-bound ssDNA sequences to be joined together to form a large loop.

[0530] Step 3: Sulfolobus DNA polymerase IV is added; the polymerase uses the large loop of ssDNA as a template to create a complementary large loop that is covalently attached at one end to the splint oligo.

[0531] Step 4: DNA ligase is used to covalently close the complementary giant loop, resulting in a circular ssDNA. It is this closed ssDNA ring that performs the "rolling" during RCA.

[0532] Step 5: A DNA polymerase with strand displacement activity is added, followed by the addition of dNTPs. The added DNA polymerase covalently attaches the dNTPs to the bead-bound ssDNA and extends the distal end of the bead-bound ssDNA to create a complementary copy of what is on the "rolling circle," which then extends further to create yet another complementary copy of what is on the "rolling circle," which then extends further to create 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 its continued activity.

[0533] 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 (7 pages)). 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).

[0534] 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.

[0535] In one embodiment of the present disclosure, the RCA amplified ssDNA is cleaved from the beads prior to PCR amplification to generate PCR products. In another embodiment of the present disclosure, the PCR amplification to generate PCR products can be performed without cleaving the RCA amplified ssDNA from the beads.

[0536] As described by Baner et al., "RCA reactions 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 a suitable enzyme due to its strand displacement activity and high processivity. RCA has been similarly characterized by Li et al. as follows: "In RCA, a circular template is isothermally amplified by the DNA polymerase phi29, which has strand displacement properties. The long single-stranded DNA product contains thousands of sequence repeats" (Li and Zhong (2007) Anal. Chem. 79:9030-9038).

[0537] The DNA barcodes of the present disclosure can be sequenced using the method 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 Sanger sequencing, or by methods using "next generation sequencing."

[0538] Illumina DNA Sequencing. The Illumina method for DNA sequencing is as follows: DNA can be fragmented into sizes ranging from 100 to 400 base pairs (bp) by sonication (Hughes, Magrini, Demeter (2014) PLoS Genet. 10:e1004462). In the Illumina method, a DNA library is created by modifying fragments of DNA from cells or cells 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 then associated with another adapter, which 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 in the sandwich. In effect, the lawn contains two types of adapters, one of which binds (hybridizes) to one of the adapters in the complex, noncovalently tethering the complex to the plate. These can be referred to as the "first lawn-bound adapter" and the "second lawn-bound adapter." The first task of the DNA polymerase is to create a daughter strand using the tethered (but noncovalently bound) DNA as a template. As DNA polymerization occurs, the daughter strand becomes covalently attached to the "first lawn-bound adapter." This covalent linkage is generated by the catalytic action of the DNA polymerase. After the daughter strand is fully synthesized, its distal end (the end that protrudes into the media) contains a DNA sequence complementary to the second adapter 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 adapter." Described above is how both adapters in a sandwich are used, as well as how both a "first lawn-binding adapter" and a "second lawn-binding adapter" are used.

[0539] The reaction is then cycled multiple times, resulting in a cluster of amplified versions of the original dsDNA. In effect, the cluster takes the form of covalently attached (tethered) ssDNA molecules, each of which corresponds to only one strand of the original dsDNA (the dsDNA isolated from the living cell or tissue). This cluster of tethered ssDNA molecules is called a "polony." Polony generation is achieved by a technique called "bridge amplification." Finally, after bridge amplification and polony creation, the reverse strand, covalently attached to the solid surface, is cleaved from its tether, washed away, and discarded, leaving only the forward strand.

[0540] 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).

[0541] 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 bead-attached fragments have 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 a slide.

[0542] The primer then hybridizes to the P1 adapter sequence within the library template. A set of four fluorescently labeled dinucleotide probes competes for ligation to the sequencing primer. Specificity of the dinucleotide probes is achieved by interrogating every first and second base in each ligation reaction. Multiple cycles of ligation, detection, and cleavage are performed, with the number of cycles determining the final read length. 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 for the second ligation cycle (see Wu et al. (2010) Nature Methods. 7:336-337).

[0543] 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 adding deoxynucleoside triphosphates (dNTPs) and washing. The DNA templates are attached to a solid support as a clonal population of templates. Each such incorporation releases a hydrogen ion, resulting in the assembly of 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). The present disclosure may exclude pH-based DNA sequencing.

[0544] 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, due to the fact that each DNA barcode module is attached to a unique site on the bead, each DNA barcode module that makes up the orthogonal DNA barcode requires its own dedicated sequencing primer.

[0545] EXCLUSIVE EMBODIMENTS: In embodiments, the present disclosure may exclude any systems, devices, device combinations, and methods involving microfluidics, aqueous droplets present in an oil medium, aqueous droplets created by joining a first channel containing an aqueous reagent with a second channel containing oil, generating aqueous droplets that travel through the oil medium via a third channel originating from the junction 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).

[0546] In other, exclusive embodiments, any reagent, composition, nucleic acid, or bead that constitutes 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 in which beads are functionalized with azide DNA headpiece moieties.

[0547] Additional Exclusionary Embodiments Related to Sequencing Methods and Sequencing Reagents. In embodiments, the present disclosure may exclude reagents, systems, or methods that do not involve the use of "reversible terminators" in DNA sequencing. Also excluded may be any reagents, systems, or methods that do not include methoxy protecting groups. Furthermore, any reagents, systems, or methods that involve DNA sequencing, but in which the DNA being sequenced is not covalently attached to beads at the time information about the polynucleotide order is detected and collected, may be excluded. Furthermore, any reagents, systems, or methods that amplify the DNA template before performing the sequencing reaction, e.g., by PCR or rolling circle techniques, may be excluded. In embodiments, any method of barcoding, e.g., nucleic acid barcoding that is linked (all information about the synthesis of a member of a chemical library is present on one single nucleic acid), may be excluded. In another aspect, any method of barcoding, e.g., nucleic acid barcoding that is orthogonal (information about the synthesis of a given monomer of a compound library is distributed across multiple attachment locations on a bead), may be excluded. In exclusive embodiments relating to DNA ligase, the present disclosure may exclude any reagent, system, or method that uses DNA ligase to connect modules of nucleic acid barcodes.

[0548] Fluorophores, quenchers, and FRET-based assays. The present disclosure provides fluorophores and quenchers for screening members of chemical libraries or characterizing isolated members of chemical libraries. FRET is Förster resonance energy transfer.

[0549] Assays can be performed on bead-bound chemical library members. Assays can also 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. Additionally, assays can be performed on soluble chemical library members that have never been attached to any beads or that have been cleaved from beads and then purified.

[0550] 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), and the like. Biochemicals, Urbana, IL), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, tetramethylrhodamine (TMR), Texas Red, tetrachlorofluorescein (TET), hexachlorofluorescein (HEX), and Joe dye (4'-5'-dichloro-2',7'-dimethoxy-6-carboxyfluorescein), SYBR Green I (absorption 497 nm, emission 520 nm), 6-carboxyfluorescein (6-FAM) (absorption 492 nm, emission 518 nm), 5-carboxyfluorescein (5-FAM) (absorption 492 nm, emission 518 nm), FITC, and rhodamine. Quenchers include TAMRA quencher, black hole quencher-1 (BHQ1), black hole quencher-2 (BHQ2), and DABCYL quencher. Note that, as disclosed elsewhere in this patent document, TAMRA can be both a fluorophore and a quencher.

[0551] Guidance is available for reagents for FRET-based assays, which 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 involves measuring the activity of signal peptidase (SpsB) using a "SceD peptide" substrate. 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 botulinum toxin assays. SNAP-25 activity can be measured using a substrate of BoNT-A. In FRET-based assays, 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).

[0552] 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 biodegradation of a given protein. These reagents, compositions, and methods can use FRET-based assays, or alternatively, can use assays other than FRET-based assays.

[0553] Molecular beacons have been described (see Baruch, Jefferey, 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 can 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.

[0554] (XI) Releasing the bead-bound compound Cleavable linkers. Provided are linkers that cannot be cleaved. 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, as cited by Gordon et al (1999) J. Chem. Technology Biotechnology. 74:835-851). Cleavable linkers also include acylsulfonamide linkers that 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 C-terminal aldehydes upon cleavage, where cleavage is by periodate oxidation (see Paulick et al (2006) J. Comb. Chem. 8:417-426).

[0555] Figure 3 discloses various cleavable linkers suitable for the compositions and methods of the present disclosure. Figure 3 is reproduced from Table 1 in Yinliang Yang's (2014) Design of Cleavable Linkers and Applications in Chemical Proteomics. Technische Universitat Munchen Lehrstuhl fur Chemie der Biopolymere. From Figure 3, preferred cleavable linkers for the present disclosure are linkers a, c, d, p, q, r, and t. Linker p was used in the experimental results disclosed herein. These cleavage conditions are DTT (linker a), Na2SO4 (linker c), Na2SO4 (linker d), UV light (linker p), UV light (linker q), UV light (linker r), and TEV protease (linker t). These specific cleavage conditions are mild and are not expected to damage the beads, the bead-bound compounds, or the chemical library members (units) of the bead-bound compounds.

[0556] 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 connected to R2, which is para to the azo moiety.

[0557] Photolabile Cleavable Linkers. The present disclosure encompasses photocleavable linkers containing an o-nitrobenzyl group. This group can be cleaved by irradiation at 330-370 nm (see Saran and Burke (2007) Bioconjugate Chem. 18:275-279, Mikkelsen, Grier, Mortensen (2018) ACS Combinatorial Science. DOI:10.1021). A linker with a shorter photolysis time than the o-nitrobenzyl linker is the 2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC) linker. A variant of the o-nitrobenzyl linker is the o-nitrobenzylamino linker. When attached to a peptide chain and subsequently cleaved, this linker releases an amide. Linkers ...

Claims

1. 1. A system for screening compounds for their ability to modulate the biological activity of a cell or a component of a cell, said system comprising: an assay device comprising a plurality of over 50,000 wells, each well separated from the other wells, each well comprising an upper aperture defining an opening at the top of the respective well, a bottom defined by a floor, and a wall existing between the upper aperture and the floor; a plurality of beads, each a single bead suitable for placement into a single well, each bead comprising a plurality of substantially identical bead-bound compounds covalently linked to said bead by a cleavable linker such that said compounds are releasable from said bead in a dose-dependent manner that is measurable as part of the assay; a plurality of beads, the beads further comprising a plurality of substantially identical bead-bound DNA barcodes linked to the beads by (i) a cleavable linker or (ii) a non-cleavable linker, wherein if the DNA barcodes are linked to the beads by a cleavable linker, the cleavable linker is orthogonal to the cleavable linker used to link the bead-bound compounds to the beads, and the compounds are identified by the DNA barcodes; and a transfer dispenser that allows only a single bead to be dispensed into a single well, the transfer dispenser comprising multiple cavities, each cavity having a single opening diameter, each cavity comprising an open bottom surface, an open top surface, and a sidewall between the open bottom surface and the open top surface, the size of the open bottom surface being smaller than the size of the open top surface, and when each cavity is aligned over a respective corresponding well of an assay device, a closed chamber is formed between each cavity and each corresponding well, and the transfer dispenser comprises a fixing mechanism configured to align and fix the transfer dispenser and the assay device; Including, the system.

2. 10. The system of claim 1, wherein the transfer dispenser is operated by a robot, by a human, or by a combination of robotic and manual processes.

3. 10. The system of claim 1, wherein the transfer dispenser employs magnetic attraction, electrostatic attraction, or magnitude and gravity-based engineering principles to deposit a single bead into a single well.

4. The system of claim 1 , wherein the transfer dispenser further comprises at least one pipette capable of delivering the single bead, the at least one pipette comprising a flexible tip.

5. The system of claim 4 , wherein the flexible tip of the pipette comprises polyimide.

6. 5. The system of claim 4, wherein the flexible tip extends along no more than 20% of the total length of the pipette.

7. 5. The system of claim 4, wherein the flexible tip extends along no more than 10% of the total length of the pipette.

8. 1. A method for identifying transcriptomic changes in a cell induced by a compound, wherein the compound is included in a combinatorial library assay, the method comprising: generating an assay array, said assay array comprising: i) a plurality of wells, each well separated from the other wells, each well containing at least one cell of interest, the assay array comprising over 50,000 wells, each well comprising an upper aperture defining an opening at the top of each well, a bottom defined by a floor, and a wall existing between the upper aperture and the floor; ii) a plurality of beads, each bead comprising a unique compound from the combinatorial library, each bead comprising a plurality of identical bead-bound compounds such that each compound in the library is selected as a potential drug candidate, each bead comprising an oligonucleotide portion encoding the structure of the unique compound or the synthetic steps used to make the unique compound, and a plurality of functionalized oligonucleotides comprising an RNA capture element; wherein a single bead is disposed in a single well; aligning the wells with a transfer dispenser that allows only a single bead to be dispensed into a single well to form a confined space, the transfer dispenser including multiple cavities, each cavity having a single opening diameter, each cavity including an open bottom surface, an open top surface, and a sidewall between the open bottom surface and the open top surface, the size of the open bottom surface being smaller than the size of the open top surface, such that when each cavity is aligned over a respective corresponding well of the assay device, a closed chamber is formed between each cavity and each corresponding well; securing the transfer dispenser using a securing mechanism to align and secure the transfer dispenser and the assay device; releasing a single bead into each well via a transfer dispenser; contacting the cells in each defined volume with the compounds released from the beads into the defined volume and maintaining the contact for a time sufficient to result in transcriptomic changes in RNA expressed by the cells in response to the contact; capturing the RNA from the cells in each well by lysing the cells and contacting the RNA with the RNA capture elements on the beads; identifying the captured RNA from at least some of the plurality of beads and assessing transcriptomic changes, if any, in the captured RNA; and Identifying the structure of the compound that caused the change in the transcriptome. A method comprising:

9. 10. The method of claim 8, wherein the transfer dispenser is operated by a robot, by a human, or by a combination of robotic and manual processes.

10. 10. The method of claim 8, wherein the transfer dispenser employs magnetic attraction, electrostatic attraction, or size and gravity based engineering principles to deposit a single bead into a single well.

11. 9. The method of claim 8, wherein the transfer dispenser further comprises at least one pipette capable of delivering the single bead, the at least one pipette comprising a flexible tip.

12. The method of claim 11 , wherein the flexible tip of the pipette comprises polyimide.

13. 12. The method of claim 11, wherein the flexible tip extends along no more than 20% of the total length of the pipette.

14. 12. The method of claim 11, wherein the flexible tip extends along no more than 10% of the total length of the pipette.

15. 1. A system for screening compounds for their ability to modulate the biological activity of a cell or a component of a cell, said system comprising: an assay device comprising a plurality of wells, each well separated from the other wells, each well comprising an upper aperture defining an opening at the top of the respective well, a bottom defined by a floor, and a wall existing between the upper aperture and the floor; a plurality of beads, each a single bead suitable for placement into a single well, each bead comprising a plurality of substantially identical bead-bound compounds covalently linked to said bead by a cleavable linker, such that said compounds are releasable from said bead in a dose-dependent manner that is measurable as part of an assay; a plurality of beads, the beads further comprising a plurality of substantially identical bead-bound DNA barcodes linked to the beads by (i) a cleavable linker or (ii) a non-cleavable linker, wherein if the DNA barcodes are linked to the beads by a cleavable linker, the cleavable linker is orthogonal to the cleavable linker used to link the bead-bound compounds to the beads, the compounds being identified by the DNA barcodes, each bead being at least about 10,000 substantially identical DNA barcodes; and a transfer dispenser that allows only a single bead to be dispensed into a single well, the transfer dispenser comprising multiple cavities, each cavity having a single opening diameter, each cavity comprising an open bottom surface, an open top surface, and a sidewall between the open bottom surface and the open top surface, the size of the open bottom surface being smaller than the size of the open top surface, and when each cavity is aligned over a respective corresponding well of an assay device, a closed chamber is formed between each cavity and each corresponding well, and the transfer dispenser comprises a fixing mechanism configured to align and fix the transfer dispenser and the assay device; Including, the system.

16. 16. The system of claim 15, further comprising a transfer dispenser capable of dispensing a single bead into a single well.

17. 16. The system of claim 15, wherein the transfer dispenser is operated by a robot, by a human, or by a combination of robotic and manual processes.

18. 17. The system of claim 16, wherein the transfer dispenser employs magnetic attraction, electrostatic attraction, or magnitude and gravity-based engineering principles to deposit a single bead into a single well.

19. 17. The system of claim 16, wherein the transfer dispenser further comprises at least one pipette capable of delivering the single bead, the at least one pipette comprising a flexible tip.

20. 20. The system of claim 19, wherein the flexible tip of the pipette comprises polyimide.

21. 20. The system of claim 19, wherein the flexible tip extends along no more than 20% of the total length of the pipette.

22. 20. The system of claim 19, wherein the flexible tip extends along no more than 10% of the total length of the pipette.

23. 1. A method for identifying transcriptomic changes in a cell induced by a compound, wherein the compound is included in a combinatorial library assay, the method comprising: generating an assay array, said assay array comprising: i) a plurality of wells, each well separated from the other wells, each well containing at least one cell of interest, each well including an upper aperture defining an opening at the top of the respective well, a bottom defined by a floor, and a wall existing between the upper aperture and the floor; ii) a plurality of beads, each bead comprising a unique compound from the combinatorial library, each bead comprising a plurality of identical bead-bound compounds such that each compound in the library is selected as a potential drug candidate, each bead comprising a DNA barcode encoding the structure of the unique compound or the synthetic steps used to make the unique compound, and a plurality of functionalized oligonucleotides comprising an RNA capture element; wherein a single bead is disposed in a single well, the compounds are identified by the DNA barcodes, and each bead comprises at least about 10,000 substantially identical DNA barcodes; aligning the wells with a transfer dispenser that allows only a single bead to be dispensed into a single well to form a confined space, the transfer dispenser including multiple cavities, each cavity having a single opening diameter, each cavity including an open bottom surface, an open top surface, and a sidewall between the open bottom surface and the open top surface, the size of the open bottom surface being smaller than the size of the open top surface, such that when each cavity is aligned over a respective corresponding well of the assay device, a closed chamber is formed between each cavity and each corresponding well; securing the transfer dispenser using a securing mechanism to align and secure the transfer dispenser and the assay device; releasing a single bead into each well via a transfer dispenser; contacting the cells in each defined volume with the compounds released from the beads into the defined volume and maintaining the contact for a time sufficient to result in transcriptomic changes in RNA expressed by the cells in response to the contact; capturing the RNA from the cells in each well by lysing the cells and contacting the RNA with the RNA capture elements on the beads; identifying the captured RNA from at least some of the plurality of beads and assessing transcriptomic changes, if any, in the captured RNA; and Identifying the structure of the compound that caused the change in the transcriptome. A method comprising:

24. 24. The method of claim 23, wherein the transfer dispenser is operated by a robot, by a human, or by a combination of robotic and manual processes.

25. 24. The method of claim 23, wherein the transfer dispenser employs magnetic attraction, electrostatic attraction, or engineering principles based on size and gravity to deposit a single bead into a single well.

26. 24. The method of claim 23, wherein the transfer dispenser further comprises at least one pipette capable of delivering the single bead, the at least one pipette comprising a flexible tip.

27. 27. The method of claim 26, wherein the flexible tip of the pipette comprises polyimide.

28. 27. The method of claim 26, wherein the flexible tip extends along no more than 20% of the total length of the pipette.

29. The system of claim 1 , further comprising a computer system configured to program one or more robotic components for aligning the transfer dispenser and the assay device.

30. The method of claim 8 , wherein aligning comprises inverting the transfer dispenser and the assay device.

31. The system of claim 1 , wherein the transfer dispenser includes a tapered sidewall.

32. The system of claim 1 , wherein the bottom surface includes a sub-cavity recessed into the bottom surface, the sub-cavity configured to hold a single bead.

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