Magnetic Hydrogel Particles and Methods of Use

Abstract

The present invention relates to particles comprising a magnetic core coated within a polymer gel, methods for making such particles, and methods for using such particles, for example, to facilitate both chemical and biological synthesis.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 442,396, filed January 31, 2023, and U.S. Provisional Application No. 63 / 504,191, filed May 24, 2023, the entire contents of which are incorporated herein by reference.

[0002] Federal Rights Statement This invention was made with government support under Grant No. R35GM140890 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Electronic Sequence Listing Reference The contents of the electronic sequence listing (146392063540SEQLIST.xml; size: 24,966 bytes; created: January 12, 2024) are incorporated herein by reference in their entirety.

[0004] FIELD OF THE INVENTION The present invention relates to particles comprising a magnetic core coated within a polymer gel, methods for making such particles, and methods for using such particles, for example, to facilitate both chemical and biological synthesis. [Background technology]

[0005] Background of the Invention Fractional and pooled libraries of small molecules and biopolymers can be prepared by solid-phase chemistry and used in drug discovery campaigns. Each bead in these libraries is a spatially isolated clonal population of a single library member that can be interrogated individually. Initial studies using these libraries were limited to affinity-based interactions with bead-tethered compounds. More recent studies (Cochrane et al. ACS Comb. Sci. 2019, 21, 5, 425-435; Price et al. Anal. Chem. 2016, 88, 5, 2904-2911; Cochrane et al. Anal. Chem. 2017, 89, 24, 13227-13234; Hackler et al. Anal. Chem. 2016, 88, 5, 2904-2911) have established that these particles can be used to deliver compounds for both affinity and functional assays. While affinity-driven selection can be performed in bulk reactions, functional screening requires the compartmentalization of individual particles in volumes small enough to result in high local concentrations, for example in microfluidic droplets.

[0006] While microfluidics can generate highly monodisperse droplets and particles, these systems present a high barrier to entry due to the highly specialized fabrication, components, and operation techniques. Conversely, bulk emulsification is available in every laboratory with minimal equipment and does not require specialized training. However, bulk emulsification (e.g., via vortexing or shaking) generally results in droplet sizes spanning several orders of magnitude. Particle-templated emulsions (Hatori et al. Anal. Chem. 2018, 90, 16, 9813-9820) introduce monosized particles into the emulsion volume, which serves as a support for droplet generation, resulting in a shell of aqueous mixture around the particle support. This results in monodisperse droplets surrounding the solid support. However, the final emulsion still contains a population of particle-free "satellite droplets."

[0007] Therefore, there is a need for particles that can facilitate chemical reactions during library synthesis and can be used for library screening. Production methods must be scalable to large numbers of beads (ideally at or above the scale of library synthesis) while still yielding uniform particles. Summary of the Invention

[0008] overview Provided herein are compositions comprising particles including a magnetic core coated within a polymer gel. In some embodiments, the polymer gel comprises polyacrylamide. In some embodiments, the polymer gel comprises about 4% w / v to about 10% w / v polyacrylamide. In some embodiments, the polymer gel comprises about 10:1 to about 40:1 acrylamide to bisacrylamide. In some embodiments, the polymer gel comprises 19:1 or 37.5:1 acrylamide to bisacrylamide.

[0009] In some embodiments, the particles further comprise an additive that inhibits phase separation, aggregate formation, and / or coacervation. In some embodiments, the additive comprises albumin. In some embodiments, the albumin is modified with one or more reactive groups.

[0010] In some embodiments, about 0.001 mM to about 20 mM of the polymer gel is functionalized. In some embodiments, the particles are fluorescently labeled. In some embodiments, the polymer gel has a pore size of about 20 nm to about 200 nm. In some embodiments, the magnetic core is a magnetic bead. In some embodiments, the magnetic bead has a diameter of about 0.5 μm to about 10 μm. In some embodiments, the magnetic bead has a diameter of about 1.0 μm, 2.8 μm, or 10.0 μm. In some embodiments, the total particle diameter is less than about 40 μm, optionally, the total particle diameter is about 6 μm to about 12 μm. In some embodiments, the composition comprises particles having a uniform diameter distribution. In some embodiments, the composition comprises particles having a particle diameter coefficient of variation of about 3% to about 50%. In some embodiments, the average total diameter of the particles is about 5 μm to about 10 μm. In some embodiments, the average total diameter of the particles is about 7 μm. In some embodiments, at least 95% of the total particles in the composition comprise a magnetic core.

[0011] In some embodiments, provided herein are methods for producing a composition comprising particles including a magnetic core encapsulated in a polymer gel, the method comprising emulsifying an aqueous solution comprising a monomer and magnetic beads with a solution comprising a polymerization initiator to cause polymerization of the monomer, thereby producing a composition comprising a magnetic core encapsulated in a polymer gel. In some embodiments, the polymer gel comprises polyacrylamide. In some embodiments, the method comprises emulsifying a solution comprising 4% w / v to 10% acrylamide monomer and magnetic beads with a solution comprising an initiator. In some embodiments, the monomer-containing solution comprises acrylamide monomer and bisacrylamide monomer. In some embodiments, the solution comprising the monomer and magnetic beads is an aqueous solution. In some embodiments, the initiator is in an oil-based solution, and optionally, the initiator is TEMED. In some embodiments, the aqueous solution comprises ammonium persulfate. In some embodiments, emulsifying the solution comprises vortexing, homogenizing, mixing, stirring, and / or shaking. In some embodiments, the initiator causes polymerization of acrylamide to coat the magnetic beads in the polyacrylamide gel.

[0012] In some embodiments, the method further comprises combining a solution comprising acrylamide and bisacrylamide monomers and magnetic beads with a solution comprising a polymerization initiator to produce a combined composition prior to emulsification. In some embodiments, the combined composition comprises an oil phase and an aqueous phase. In some embodiments, the method further comprises sparging the combined composition with an inert gas. In some embodiments, the inert gas is argon.

[0013] In some embodiments, the method further comprises applying a magnetic field to the composition to separate the particles. In some embodiments, the method further comprises removing a particle-free supernatant. In some embodiments, the method further comprises washing the particles. In some embodiments, the method further comprises resuspending the particles. In some embodiments, the method further comprises functionalizing a portion of the gel.

[0014] Provided herein are particles produced by the methods provided herein.

[0015] Provided herein are libraries of particles produced by the methods provided herein.

[0016] In some embodiments, provided herein is a kit comprising a composition comprising an aqueous solution comprising magnetic beads and acrylamide, and a composition comprising an oily solution comprising a polymerization initiator. Instructions for use of the kit are provided in accordance with the methods provided herein.

[0017] Provided herein are kits that include the particles provided herein, and instructions for use according to the methods provided herein. [Brief explanation of the drawings]

[0018] [Figures 1A-1E]Various gel particle functionalizations by copolymerization are shown. Figure 1A shows blank gel beads. Gel particles were copolymerized with 5'-methacrylamide oligonucleotides (Figure 1B), methacrylamide-modified hairpin headpiece DNA (HDNA) (Figure 1C), propargyl methacrylate (PMA) (Figure 1D), or primary amines using 3-(aminopropyl)methacrylamide (APMA) (Figure 1E). Gel functionalization was detected by chemical reactions with fluorescent dye-labeled complementary functional groups, including complementary oligonucleotide hybridization (Figure 1B), enzymatic ligation of double-stranded (dsDNA) modules used in DEL synthesis (Figure 1C), Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with Alexa Fluor 488 azide (488AF-N3) (Figure 1D), or amine acylation with fluorescein succinimidyl ester (FAM-OSu) (Figure 1E). Gel labeling was detected by flow cytometry and compared to blank gel beads (Figures 1A-1E, right panels).

[0019] [Figure 2] Structure and characterization of methacrylamide-modified HDNA (ac-HDNA) structures and characterization are shown. Product formation was confirmed by MALDI-TOF MS analysis of HPLC fractions. MS labels indicate the theoretical exact mass (top) and observed mass (bottom) of [M+H]+.

[0020] [Figure 3A-3B] Gel particles prepared with various concentrations of APMA, acylated with FAM-OSu, analyzed by flow cytometry (Figure 3A), and quantified (gray borders indicate standard error) are shown (Figure 3B).

[0021] [Figure 4A]DNA-templated beads prepared by PCR using DNA oligonucleotide primer P1 (black dashed line, single-sided arrow) functionalized magnetic beads (black circle) (P1 sequence shown in SEQ ID NO:1) and DNA oligonucleotide primer P2 (black single-sided arrow) (P2 sequence shown in SEQ ID NO:3) are shown. The DNA template contained a T7 RNA polymerase promoter element (T7 prom, black arrow). Non-templated negative control magnetic beads were labeled with Alexa Fluor 647 (647AF, black hollow circle with black dot). Each bead set was encapsulated in a P2 copolymer hydrogel. [Figure 4B] In vitro transcription of gel-encapsulated DNA-templated and non-templated beads is shown in the presence of a FAM-labeled DNA oligonucleotide probe (P3, black single arrow) of the 5' region of the RNA transcript; the P3 sequence is shown in SEQ ID NO: 5. The P3 probe detected the presence of the RNA transcript (black) hybridized via P2 in the gel. [Figure 4C] Two-dimensional flow cytometry analysis showing that the majority of particles exhibited either exclusively red fluorescence (660 nm, non-templated negative control in Q1) or green fluorescence (520 nm, templated and RNA-loaded beads in Q3).

[0022] [Figure 5A]DNA-templated beads encapsulated in polyacrylamide hydrogels copolymerized with oligonucleotide primer P1 (black dashed line, single-sided arrow) are shown. The transcribed mRNA was hybridized to gel-linked P1 and a 3' puromycin-modified DNA oligonucleotide P4 (black "puro"); the P4 sequence is shown in SEQ ID NO:6. The mRNA (black strand) encoded a ribosome binding site (RBS), an epitope tag (gray), a glycine-serine linker (G4S, gray) (SEQ ID NO:19), a HiBiT luciferase complementation tag (black), and a stop codon (*). Ribosomal protein synthesis was terminated by incorporation of a 3' puromycin into the nascent strand, tethering the translated peptide to P4. The epitope was detected by immunofluorescence, and HiBiT was quantified by luciferase complementation with LgBiT. [Figure 5B] Figure 5C shows in-gel translation of FLAG-templated particles probed with 647AF-labeled anti-FLAG antibody, visualized, and compared to untranslated particles in Figure 5C in confocal imaging of antibody fluorescence (λex / λem = 650 / 720 nm) and magnetic bead autofluorescence (λex / λem = 490 / 560 nm). Scale = 50 μm. [Figure 5D] IVTT reactions programmed with FLAG, HA, or V5-templated particles (FLAG coding sequence shown in SEQ ID NO: 13, HA coding sequence shown in SEQ ID NO: 12, and V5 coding sequence shown in SEQ ID NO: 11) and translated epitopes detected by flow cytometry with 647AF-anti-FLAG, APC-anti-HA, or CF488A-anti-V5 immunofluorescence are shown (FLAG and HA: λex / λem=640 / 660 nm; V5: λex / λem=488 / 530 nm; translated particles are black, untranslated particles are gray). [Figure 5E] In-gel capture of translated FLAG, HA, and V5 epitopes with and without 10 μM P4 as quantified by HiBiT complementation is shown (black, gray). Error bars reflect the standard deviation of the mean.

[0023] [Figure 6]Sorting of hydrogel magnetic particles using fluorescence-activated cell sorting (FACS) is shown. HA- and V5-templated particles were spiked (1% each) into a background of library particles (NNK5). The NNK5 library template sequence is shown in SEQ ID NO: 16. Particles were then translated in a bulk IVT reaction and incubated with APC-labeled anti-HA and CF488A-labeled anti-V5 antibodies prior to FACS. After sorting, each population was PCR amplified and sequenced by next-generation sequencing (NGS).

[0024] [Figure 7] Flow cytometry analysis of magnetic bead templates of different diameters for gel particle formation is shown. Magnetic beads were encapsulated in gels copolymerized with ac-P1 (20 μM). The ac-P1 sequence is shown in SEQ ID NO: 2. Populations of 1 μm, 2.8 μm, and 10 μm diameter magnetic beads encapsulated in hydrogels were gated by forward and side scatter correlation (top), and encapsulation was confirmed by comparing the gated population fluorescence intensity (bottom, λex / λem = 490 / 530 nm) of unprobe (gray) and FAM-P1'-probe (black) samples.

[0025] [Figure 8] A standard curve for the HiBiT quantitation assay is shown. Various concentrations of HiBiT peptide (0.1-1000 nM) were assayed for chemiluminescence upon addition of excess LgBiT complementation reagent.

[0026] [Figure 9] Figure 1 shows hydrogel particle diameter as a function of templating magnetic bead diameter. The box plot shows the mean, standard deviation, upper and lower quartiles (box), and 1.5 x interquartile range (whiskers).

[0027] [Figure 10A] Figure 1 shows the uniformity of hydrogel particle size analyzed by confocal fluorescence microscopy. Scale = 100 μm. [Figure 10B] Measured hydrogel particle diameters for 32k particles (median = 7.3 μm) are shown. [Figure 10C] Flow cytometry (λex / λem=488 / 520 nm) used to analyze probed (black) and unprobed (gray) gel particles is shown.

[0028] [Figure 11] A schematic of the particle synthesis approach is shown: (i) magnetic beads are suspended in a monomer premix, a layer of oil-containing initiator is added, and the suspension is emulsified; (ii) the droplets are cured to form hydrogel droplets; and (iii) the bead-containing gel particles are magnetically isolated.

[0029] [Figure 12] The enrichment rates of various control tags by sequencing selected hit particles are shown. Reads were pattern-matched to epitope tag sequences or degenerate NNK5 library sequences. The HA-positive hit pool was approximately 99% HA-coding sequence, and the V5-hit pool was approximately 50% V5-coding sequence, resulting in approximately 100-fold and 50-fold enrichment from the library starting material.

[0030] [Figure 13] Figure 1 shows hydrogels prepared by bulk emulsification scale with templated magnetic bead sizes (e.g., 1.0 μm, 2.8 μm, or 10.0 μm). The degree of particle dispersion is inversely proportional to the diameter of the magnetic beads.

[0031] [Figure 14] We demonstrate the translation and detection of non-canonical amino acid-containing gel particle library beads. NNU library particles (3 x 106) were subjected to an engineered IVT reaction to recode them, introducing an azido-lysine (AzK) at the CUG codon. The translated particles were washed and treated with AF647-alkyne in a CuAAC reaction prior to analysis by flow cytometry. Particles were sorted to isolate the top 2% of the AF647 population, and sequences were compared to the starting library. A 6-fold enrichment was observed after a single round of screening.

[0032] [Figure 15] NNU library particles (3 x 10) were prepared by emPCR and subjected to an engineered IVT reaction recoded to introduce an azido-lysine (AzK) at the CUG codon. The translated particles were washed and incubated with AF647-alkyne in a CuAAC reaction before analysis by flow cytometry. Particles were sorted by gating on the top 2% of AF647 signal.

[0033] [Figures 16A-16B] qPCR analysis of emPCR library preparation is shown. Aliquots of 100 beads were quantified to obtain the average loading (dotted trace), and limiting dilutions of beads were sampled into 77 wells (gray trace) to obtain single-bead quantification of DNA-templated beads (Figure 16A). Traces for the standard (light gray) and negative template control (black) are also shown in Figure 16A. Quantification of 100 beads and DNA-templated single beads is shown in box plots (Figure 16B). The average particle loading of the 100-bead aliquot was 4,200 DNA molecules per bead (Figure 16B). The average particle loading of single DNA-templated beads was 42,000 DNA molecules per bead (Figure 16B).

[0034] [Figure 17A] Figure 1 shows time-dependent quantification of trypsin activity by hydrogel particles loaded with a fluorogenic (turn-on) green fluorescent probe. [Figure 17B] Time-dependent quantitation of hydrogel particles loaded with an N-terminally labeled tryptic peptide, the N-terminal label being the red fluorescent dye Cy5. Upon trypsin digestion, Cy5 is released from the gel, resulting in a decrease in the fluorescent signal over time (extinction).

[0035] [Figure 18]Schematics of various copolymerized functionalities are shown, including but not limited to crosslinkers, cell adhesion promoters (e.g., alkylamines), affinity capture tags (e.g., halotags for chloroalkanes), enzyme capture, probe capture, and oligonucleotides for hybridization, the latter two of which are useful for labeling and characterization.

[0036] [Figure 19] 1 shows gel particles copolymerized with oligonucleotides and then hybridized with a trypsin activity-based probe conjugated to a complementary oligonucleotide, and a schematic showing that the probe-hybridized gel particles were digested with trypsin, dequenching the activity-based probe and yielding fluorescent gel particles. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention In some embodiments, provided herein are versatile hydrogel-based particles that facilitate both chemical and biological synthesis. Particle preparation via the bulk emulsification method disclosed herein is simple, scales well, and results in a relatively uniform size distribution. The hydrogel layer of the particles can exhibit a range of functionalities commonly used in both traditional split-and-pool combinatorial chemical synthesis or templated enzymatic biosynthesis. (Gartner et al.Science,2004,305,5690,1601-1605;Gartner et al.J.Am.Chem.Soc.2001,123,28,6961-6963;Halpin et al.DNA Display I.Sequence-Encoded Routing of DNA Populations,PLoS Biol,2004,2(7),e173;Halpin et al.DNA Display II.Genetic Manipulation of Combinatorial Chemistry Libraries for Small-Molecule Evolution,PLoS Biol,2004,2(7),e174;Halpin et al.DNA Display III.Solid-Phase Organic Synthesis on Unprotected DNA,PLoS Biol, 2004, 2(7), e175.) Furthermore, templated library preparation using this particle system by in vitro translation can be particularly advantageous because the RNA transcripts and subsequently translated peptides are localized to their host particles, generating monoclonal beads for screening without further emulsification or other compartmentalization.

[0038] In some embodiments, the particles provided herein can be used for library screening using particle systems in combination with fluorescence-activated cell sorting (FACS) instruments. This new particle format introduces important handling advantages that can enable the automation of encoded library synthesis while unlocking functional screening capabilities through multivalent encoded library member display. (MacConnell et al. ACS Comb. Sci. 2017, 19, 3, 181-192; Komnatnyy et al. Chem. Commun. 2018, 54, 6759-6771.)

[0039] We have found that magnetic bead-templated emulsion polymerization yields remarkably uniform particles that do not require microfluidics for either preparation or analysis, and that exhibit excellent handling and biocompatibility.

[0040] As used herein, "building block" refers to a chemical building block that is attached to or can be attached to other chemical building blocks. Building blocks are diverse chemical structures characterized by one or more functional groups that are variously used to couple via chemical synthesis and potentially interact with a target of interest.

[0041] As used herein, a "functionalization site" or "functional group" refers to a chemical group that can participate in a reaction and produce a bond between two moieties. Examples of functional groups include, but are not limited to, -NH, -SH, -OH, -COH, halides, -N, -CONH, etc.

[0042] As used herein, "particle" refers to a discrete composite comprising a magnetic core coated with a polymer.

[0043] "A" or "an" means one (one) and more than one. For example, "a particle" includes one, two, three, or more particles.

[0044] "Library" means a collection of molecules or chemical entities.

[0045] "Oligonucleotide" means a polymer of nucleotides having one or more nucleotides at the 5' end, the 3' end, and at an internal position between the 5' and 3' ends. Oligonucleotides may comprise DNA, RNA, or any derivative thereof known in the art.

[0046] "Tag" or "oligonucleotide tag" refers to an oligonucleotide portion of a library, at least a portion of which contains information for identifying the particle and / or library. For example, an oligonucleotide tag may contain information that allows the identification of an associated functional group. In some embodiments, an oligonucleotide tag is used as a barcode.

[0047] I. Particles In some embodiments, provided herein are particles comprising a magnetic core coated within a polymer gel. In some embodiments, the particles comprise a hydrogel coating the magnetic core. In some embodiments, hydrogel refers to a material formed when organic polymers (natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules to form a gel. In some embodiments, the hydrogel may be a biocompatible hydrogel, which refers to a polymer that forms a gel that is not toxic to living cells and allows sufficient diffusion of oxygen and nutrients to the trapped cells to maintain their viability.

[0048] In some embodiments, provided herein are particles containing acrylamide droplets surrounding magnetic particles. In some embodiments, the particles are prepared by bulk emulsification to obtain uniform hydrogel compartments that are easily polymerized and magnetically isolated. Acrylamide provides a direct polymerization mechanism already used in many chemistry and molecular biology laboratories, and the polyacrylamide product is generally inert to chemical and biochemical reactions. This radical-mediated polymerization easily incorporates various functionalities into the polymer matrix by simply including molecules that have both the desired functionality and a vinyl group for polymerization. In some embodiments, the inventors have prepared hydrogels with both chemical (e.g., amine, azide) and biochemical (oligonucleotide) groups and then demonstrated that these groups can participate in various reactions in the gel matrix (see below). Thus, in some embodiments, the particles comprise a polyacrylamide gel coating a magnetic core.

[0049] In some embodiments, provided herein are particles comprising a magnetic core coated with a hydrogel layer. In some embodiments, hydrogel layers comprising polyacrylamide can be easily synthesized, preventing aggregation and inhibiting adhesion to tube walls. Polyacrylamide is also inert and therefore compatible with a variety of different chemical and biochemical reactivities. Gel particles comprising polyacrylamide can be functionalized in various ways by adding different reagents to the acrylamide monomer solution, such as propargyl methacrylate (which can couple with molecules containing "azide" functionality) for "alkyne" functionality, N-(3-aminopropyl) methacrylamide for "amine" functionality, methacrylamide (ac)-modified oligonucleotide P1 for "reverse primer" functionality, or ac headpiece DNA (used in DNA-encoded library synthesis) for HDNA functionality. In some embodiments, the hydrogel polymer layer comprises polyacrylamide, acrylamide, and / or bisacrylamide. In some embodiments, the hydrogel polymer layer comprises polyacrylamide and / or acrylamide. In some embodiments, the hydrogel polymer layer comprises polyacrylamide.

[0050] In some embodiments, the hydrogel or polymer layer is made of polyacrylamide, polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PVSA), poly(L-aspartic acid), poly(L -glutamic acid), polylysine, agar, agarose, alginic acid, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or a combination thereof.

[0051] In some embodiments, gel particles can be variously functionalized by adding different reagents to the respective monomer solutions, including the aforementioned polymers or hydrogels surrounding magnetic beads, including molecules with both the desired functionality and vinyl groups for polymerization. In some embodiments, hydrogel layers around magnetic beads can be created that have both chemical (e.g., alkyne, amine, azide) and biochemical (oligonucleotide) functional groups. In some embodiments, the functionalized hydrogel or polymer layer is selected from the group consisting of polyacrylamide, polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PVSA), poly(L-aspartic acid), poly( L-glutamic acid), polylysine, agar, agarose, alginic acid, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or a combination thereof.

[0052] In some embodiments, hydrogel particles with magnetic cores are prepared by emulsifying a hydrogel monomer solution and magnetic microbeads. In some embodiments, polyacrylamide hydrogel particles with magnetic cores can be prepared by suspending magnetic beads in an aqueous monomer solution containing acrylamide and bisacrylamide, and an initiator such as TEMED can be added to the oil phase. The aqueous and oil phases can then be mixed together to form an emulsified suspension. In some embodiments, the magnetic beads are carboxylic acid-functionalized magnetic beads (M-270 carboxylic acid Dynabeads, 5×10 7 , ThermoFisher Scientific). In some embodiments, the ratio of acrylamide:bis-acrylamide in the acrylamide monomer solution is 19:1 or 37.5:1, or any ratio between about 10:1 and about 40:1. In some embodiments, the oil / aqueous sample may be emulsified using a bead mill homogenizer (65 seconds, 2500 rpm, BeadBug, Benchmark Scientific, Sayreville, NJ), vortexing, or stirring. In some embodiments, the emulsified suspension may be polymerized on ice, and the hydrogel particles with magnetic cores can be isolated on a magnet after polymerization.

[0053] In some embodiments, the ratio of acrylamide:bisacrylamide in the acrylamide monomer solution used to prepare hydrogel particles having a magnetic core is about 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, 20:1, 20.5:1, 21:1, 21.5:1, 22:1, 22.5:1, 23:1, 23. 5:1, 24:1, 24.5:1, 25:1, 25.5:1, 26:1, 26.5:1, 27:1, 27.5:1, 28:1, 28.5:1, 29:1, 29.5:1, 30:1, 30.5:1, 31:1, 31.5:1, 32:1, 32.5:1, 33:1, 33.5:1, 34:1, 34.5:1, 35:1, 35.5:1, 36:1, 36.5:1, 37:1, 37.5:1, 38:1, 38.5:1, 39:1, 39.5:1, or 40:1, or a ratio within a range defined by any two of the foregoing ratios.

[0054] In some embodiments, the particles and / or compositions comprising the particles include an additive for inhibiting phase separation, aggregate formation, and / or coacervation of the hydrogel layer of the hydrogel particles having a magnetic core. In some embodiments, the additive includes albumin, such as bovine serum albumin (BSA) and acrylamide-modified BSA. In other embodiments, the albumin is modified with one or more reactive groups, such as vinyl-modified albumin and acrylamide-modified BSA. In some embodiments, the particles include bovine serum albumin (BSA), modified BSA, human serum albumin (HSA), and / or modified HSA.

[0055] In some embodiments, the hydrogel polymer layer comprises about 70-96% weight / volume (w / v) fluid (e.g., water) and about 4-30% w / v polymer. Percentages are weight / volume unless otherwise specified. In some embodiments, the hydrogel layer of the hydrogel particle having a magnetic core comprises about 70% fluid and 30% polymer, 72% fluid and 28% polymer, 74% fluid and 26% polymer, 76% fluid and 24% polymer, 78% fluid and 22% polymer, 80% fluid and 20% polymer, 82% fluid and 18% polymer, 84% fluid and 16% polymer, 86% fluid and 14% polymer, 88% fluid and 12% polymer, 90% fluid and 10% polymer, 92% fluid and 8% polymer, 94% fluid and 6% polymer, or 96% fluid and 4% polymer, or a percentage of fluid or polymer within a range defined by any two of the foregoing percentages.In some embodiments, the polymer is polyacrylamide, polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamine), or the like. acid), polylysine, agar, agarose, alginic acid, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or a combination thereof.

[0056] In certain embodiments, the fluid in the hydrogel polymer layer of the hydrogel particle having a magnetic core comprises water or any other solvent compatible with the hydrogel. In some embodiments, the water content of the hydrogel layer is about 90%, 91%, 92%, 93%, 94%, 95%, or 96%, or a percentage within a range defined by any two of the foregoing percentages. In some embodiments, the hydrogel polymer layer of the hydrogel particle having a magnetic core comprises about 10% polymer, 9% polymer, 8% polymer, 7% polymer, 6% polymer, 5% polymer, or 4% polymer, or a percentage within a range defined by any two of the foregoing percentages. In some embodiments, the hydrogel polymer layer of the hydrogel particles having a magnetic core comprises about 10% polyacrylamide, 9% polyacrylamide, 8% polyacrylamide, 7% polyacrylamide, 6% polyacrylamide, 5% polyacrylamide, or 4% polyacrylamide, or a percentage within a range defined by any two of the foregoing percentages.

[0057] In some embodiments, the particles comprise pores in the polymer that allow diffusion out of the gel. In some embodiments, the pores are of a size selected to retain reagents, substrates, reactants, and / or enzymes within the polymer. In some embodiments, the pore size is selected to produce a high local concentration of reactants, substrates, or enzymes within the hydrogel to facilitate proximity-driven synthesis. In some embodiments, the pore size is about 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, or 200 nm, or a size within a range defined by any two of the foregoing sizes. In some embodiments, the hydrogel polymer forms a hydrogel matrix having pores (e.g., a porous hydrogel matrix). These pores can retain sufficiently large genetic material or peptides within the hydrogel matrix coated on the magnetic beads, while allowing smaller materials, such as reagents, to pass through the pores and thereby enter and exit the hydrogel matrix. In some embodiments, the pore size is determined by the ratio of the concentration of polymer to the concentration of crosslinker. In some embodiments, the polymer to crosslinker ratio is 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, or 1:30, or a ratio within a range defined by any two of the foregoing ratios.

[0058] In some embodiments, the magnetic core of the hydrogel comprises magnetic beads. In some embodiments, the magnetic core responds to an external magnetic field but can be demagnetized when the magnetic field is removed. Thus, paramagnetic microparticles can be efficiently separated from solution using a magnet but easily resuspended without magnetically induced aggregation. In some embodiments, the magnetic beads comprise a magnetite-rich core (e.g., iron oxide) encapsulated by a pure polymer shell. In one embodiment, suitable magnetic beads comprise a magnetite / encapsulation ratio of approximately 20-35%. For example, magnetic beads comprising a magnetite / encapsulation ratio of approximately 23%, 25%, 28%, 30%, 32%, or 34% are suitable for use in the present invention.

[0059] In some embodiments, the magnetic beads within the hydrogel particles have a diameter of about 0.5 μm to about 10 μm, such as 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or a diameter within a range defined by any two of the foregoing values. In some embodiments, the magnetic beads within the hydrogel particles have a diameter of about 2.8 μm.

[0060] In some embodiments, the magnetic beads used herein can have a functional group-coated surface. Methods for directly or indirectly coating magnetic beads with functional groups are known in the art. For example, functional groups (e.g., COOH) can be coated on magnetic beads during their formation. See, for example, U.S. Pat. No. 5,648,124. Furthermore, magnetic beads can be coated with functional groups by covalently coupling one or more functional groups to one or more COOH groups on the magnetic beads. A specific example of a functional group-coated surface is a surface coated with moieties each having a free functional group attached to the amino group of the aminosilane on the magnetic beads. As a result, the surface of the magnetic beads is coated with functional group-containing moieties. In particular, carboxylic acid-coated magnetic beads are commercially available. Other functional groups can be coated on magnetic beads, including, but not limited to, amino-coated, carboxyl-coated, and encapsulated carboxyl-coated magnetic beads. In some embodiments, other functional groups can be coupled to magnetic beads via carbodiimide coupling to carboxy groups on the surface of the magnetic beads. Other functional groups include, but are not limited to, amine groups, carboxyl groups, encapsulated carboxyl groups, silica (SiOH), and diethylaminoethyl (DEAE).

[0061] In some embodiments, the particles are about 5 μm to about 10 μm in diameter. In some embodiments, the particles are of a size sufficient to allow multiple synthesis reactions to occur within the polymer coating. In some embodiments, the overall diameter of the hydrogel particles is measured using confocal fluorescence microscopy. In some embodiments, the median diameter is 7±2 μm. The overall hydrogel particle diameter is less than about 40 μm. In some embodiments, the total particle diameter is less than about 40 μm, optionally, the total particle diameter is about 6 μm to about 12 μm. In some embodiments, the overall diameter of the hydrogel particles having a magnetic core is about 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45 μm, 46 μm, 47 μm, 48 μm, 49 μm, 50 μm, 51 μm, 52 μm, 53 μm, 54 μm, 55 μm, 56 μm, 57 μm, 58 μm, 59 μm, 60 μm, 61 μm, 62 μm, 63 μm, 64 μm, 65 μm, 66 μm, 67 μm, 68 μm, 69 μm, 70 μm, 71 μm, 72 μm, 73 μm, 74 μm, 75 μm, 76 μm, 77 μm, 78 μm, 79 μm, 80 μm, 81 μm, 82 μm, 83 μm, 84 μm, 85 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, or a diameter within a range defined by any two of the foregoing values, e.g., from about 2 μm to about 30 μm, from about 3 μm to about 20 μm, from about 4 μm to about 10 μm, or from about 5 μm to about 9 μm.

[0062] In some embodiments, hydrogel particles with magnetic cores have a wide variety of functionalities embedded in the hydrogel layer or hydrogel matrix. In some embodiments, gel particles can be variously functionalized by adding the following reagents to an acrylamide monomer solution containing propargyl methacrylate (PMA) or a propargyl methacrylate analog or other alkyne-derivatized or containing compound known to those skilled in the art for "alkyne" functionality: N-(3-aminopropyl) methacrylamide (APMA) or an N-(3-aminopropyl) methacrylamide analog, or other amine-derivatized or containing compound known to those of skill in the art for an "amine" functionality, 5'-methacrylamide modified DNA oligonucleotide P1 (acrydite) or acrydite-P1 (ac-P1) or an acrydite-P1 analog, or other acrydite-derivatized compound known to those of skill in the art for a "reverse primer" functionality, or methacrylamide DNA headpiece or ac-hairpin headpiece DNA (HDNA) or analogs thereof for an "HDNA" functionality, or aminoethyldithio or sulfhydryl-derivatized compound or analogs thereof known to those of skill in the art for a "sulfhydryl" functionality, or any combination thereof.

[0063] In some embodiments, hydrogel particles with magnetic cores have a wide variety of functionalities embedded in the hydrogel layer or hydrogel matrix. In some embodiments, gel particles can be variously functionalized by adding the following reagents to the monomer solution of a particular gel, including propargyl methacrylate or propargyl methacrylate analogs or other alkyne-derivatized or containing compounds known to those skilled in the art for "alkyne" functionality: N-(3-aminopropyl) methacrylamide or an N-(3-aminopropyl) methacrylamide analog, or other amine-derivatized or containing compound known to those of skill in the art for an "amine" functionality, a 5'-methacrylamide modified DNA oligonucleotide P1 (acrydite) or acrydite-P1 or acrydite-P1 analog, or other acrydite-derivatized compound known to those of skill in the art for a "reverse primer" functionality, or a methacrylamide DNA headpiece or ac-hairpin headpiece DNA (HDNA) or analog thereof for an "HDNA" functionality, or an aminoethyldithio or sulfhydryl-derivatized compound or analog known to those of skill in the art for a "sulfhydryl" functionality, or any combination thereof.

[0064] In some embodiments, the hydrogel or polymer layer comprises one or more reagents including propargyl methacrylate (PMA) or a propargyl methacrylate analog, or other alkyne-derivatized or containing compounds known to those skilled in the art; N-(3-aminopropyl) methacrylamide (APMA) or an N-(3-aminopropyl) methacrylamide analog, or other amine-derivatized or containing compounds known to those skilled in the art; 5'-methacrylamide-modified DNA oligonucleotide P1 (acrydite) or acrydite-P1 or an acrydite-P1 analog, or other acrydite-derivatized compounds; methacrylamide DNA headpiece or ac-hairpin headpiece DNA (HDNA) or analogs thereof; or aminoethyldithio or sulfhydryl-derivatized compounds or analogs thereof known to those skilled in the art. In some embodiments, the hydrogel or polymer layer of the hydrogel particle is labeled with a different fluorescent label, such as a suitable fluorescent dye-labeled substrate containing a complementary oligonucleotide, a double-stranded oligonucleotide ligation module, an azide, or a succinimidyl ester.

[0065] In some embodiments, functionalization of hydrogel particles can be detected by flow cytometry by adding appropriate fluorescent dye-labeled substrates, including complementary oligonucleotides, double-stranded oligonucleotide ligation modules, azides, or succinimidyl esters. In some embodiments, hybridization and amine acylation result in the largest shifts, but all functionalization reactions result in baseline separation of product and starting material by flow cytometry. In some embodiments, hydrogels with increasing amine loading capacities can be prepared by copolymerizing increasing concentrations of APMA.In some embodiments, the APMA concentration is about 0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.10 mM, 0.12 mM, 0.14 mM, 0.16 mM, 0.18 mM, 0.20 mM, 0.22 mM, 0.24 mM, 0.26 mM, 0.28 mM, 0.30 mM, 0.32 mM, 0.34 mM, 0.36 mM, 0.38 mM, 0.40 mM, 0.42 mM, 0.44 mM, 0.46 mM, 0.48 mM, 0.50 mM, 0.55 mM, 0.60 mM, 0.65 mM, 0.70 mM, 0.75 mM, 0.80 mM, 0.85 mM , 0.90mM, 0.95mM, 1.0mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7m M, 1.8mM, 1.9mM, 2.0mM, 2.1mM, 2.2mM, 2.3mM, 2.4mM, 2.5mM, 2.6mM, 2.7mM , 2.8mM, 2.9mM, 3.0mM, 3.1mM, 3.2mM, 3.3mM, 3.4mM, 3.5mM, 3.6mM, 3.7mM, 3.8mM, 3.9mM, 4.0mM, 4.1mM, 4.2mM, 4.3mM, 4.4mM, 4.5mM, 4.6mM, 4.7mM, 4. 8mM, 4.9mM, 5.0mM, 5.1mM, 5.2mM, 5.3mM, 5.4mM, 5.5mM, 5.6mM, 5.7mM, 5.8 mM, 5.9mM, 6.0mM, 6.1mM, 6.2mM, 6.3mM, 6.4mM, 6.5mM, 6.6mM, 6.7mM, 6.8m M, 6.9mM, 7.0mM, 7.1mM, 7.2mM, 7.3mM, 7.4mM, 7.5mM, 7.6mM, 7.7mM, 7.8mM , 7.9mM, 8.0mM, 8.1mM, 8.2mM, 8.3mM, 8.4mM, 8.5mM, 8.6mM, 8.7mM, 8.8mM, 8 0.9mM, 9.0mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7mM, 9.8mM, 9.9mM, 10.0mM, 10.5mM, 11.0mM, 11.5mM, 12.0mM, 12.5mM, 13.0mM, 13.5mM, 14.0mM, 14.5mM, 15.0mM, 15.5mM, 16.0mM, 16.5mM, 17.0mM, 17.5mM, 18.0mM, 18.5mM, 19.0mM, 19.5mM, 20.0mM, or a concentration within a range defined by any two of the foregoing values.In some embodiments, gel particles can be prepared at various APMA concentrations, acylated with FAM-OSu, and analyzed and quantified by flow cytometry, as shown in Figures 3A and 3B. In some embodiments, acylation with FAM-OSu resulted in a log-linear increase in gel particle fluorescence over a four-order of magnitude range.

[0066] In some embodiments, hydrogel particles with magnetic cores have a wide variety of functionalities embedded in the hydrogel layer or hydrogel matrix. In some embodiments, gel particles can be variously functionalized by adding the following reagents to the monomer solution of a particular gel, including propargyl methacrylate or propargyl methacrylate analogs or other alkyne-derivatized or containing compounds known to those skilled in the art for "alkyne" functionality: N-(3-aminopropyl) methacrylamide or an N-(3-aminopropyl) methacrylamide analog, or other amine-derivatized or containing compounds known to those skilled in the art for "amine" functionality, 5'-methacrylamide-modified (acrydite) DNA oligonucleotide P1 (ac-P1) or an acrydite-P1 analog, or other acrydite-derivatized compounds known to those skilled in the art for "reverse primer" functionality, or methacrylamide DNA headpiece or ac-hairpin headpiece DNA (HDNA) or analogs thereof for "HDNA" functionality, or aminoethyldithio or sulfhydryl-derivatized compounds or analogs known to those skilled in the art for "sulfhydryl" functionality, or any combination thereof. In some embodiments, about 0.001 mM to about 20 mM of the hydrogel or polymer gel is functionalized. In some embodiments, about 0.01 mM to about 20 mM of the hydrogel or polymer gel is functionalized. In some embodiments, about 0.1 mM to about 20 mM of the hydrogel or polymer gel is functionalized. In some embodiments, about 1 mM to about 20 mM of the hydrogel or polymer gel is functionalized. In some embodiments, about 10 mM to about 20 mM of the hydrogel or polymer gel is functionalized.

[0067] In some embodiments, the hydrogel or polymer layer of the hydrogel particle comprises a protein, an enzyme, a polynucleotide, an oligonucleotide, a polysaccharide, a fluorophore, a lipid, or a supramolecular assembly.

[0068] In some embodiments, the concentration of the functionalized hydrogel or polymer is about 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02 mM, 0.025 mM, 0.03 mM, 0.035 mM, 0.04 mM, 0.045 mM, 0.05 mM, 0.055 mM, 0.06 mM, 0.065 mM, 0.07 mM, 0.075 mM, 0.08 mM, 0.085 mM, 0.09 mM, 0.095 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, or a concentration within a range defined by any two of the foregoing values.

[0069] In some embodiments, hydrogels can be functionalized in various ways by including a substoichiometric methacrylamide-modified additive in the acrylamide:bisacrylamide monomer solution for copolymerization. For example, the incorporation of synthetic hairpin headpiece DNA (HDNA) and subsequent enzymatic oligonucleotide ligation demonstrates the feasibility of a standard DEL synthesis workflow using these particles. Commercially available NH2-HDNA can be easily converted to its methacrylamide analog for copolymerization. Amine functionality can be routinely used in solid-phase synthesis and DEL synthesis. In some embodiments, particle loading capacity is quantitative over more than four orders of magnitude. For example, at the highest site density of APMA-functionalized hydrogel particles (i.e., 20 mM), each median particle (approximately 7 μm in diameter) contains approximately 4 fmol of amine sites.

[0070] In some embodiments, beads can be templated with DNA encoding various affinity tag epitopes, such as FLAG, HA, or V5, fused to a HiBiT luciferase complementary tag and encapsulated in a P1-functionalized gel. The gel particles are subjected to an mRNA-display in vitro transcription / translation reaction incorporating a puromycin-modified peptide capture oligonucleotide, P4, complementary to the RNA immediately 3′ of the stop codon (Figure 5A). The translated beads can be analyzed by imaging microscopy and flow cytometry to visualize specific epitope translation. Translated gel particles exhibit homogenous antibody-derived fluorescence throughout the gel periphery (Figure 5B); only magnetic bead autofluorescence is detected in the same particles prior to translation (Figure 5C). The fluorescence of translated gel particles by flow cytometry is baseline resolved compared to untranslated particles for all three exemplary epitopes (Figure 5D). In some embodiments, in-gel peptide capture yield can be quantified via HiBiT luminescence. In some embodiments, particles translated in the presence of puromycin capture oligonucleotide P4 retained approximately 100 nM of HiBiT peptide, whereas translations lacking P4 retained less than 1 nM (Figure 5E).

[0071] In some embodiments, gel particles can be variously functionalized by adding different reagents to the respective monomer solutions, including the aforementioned polymers or hydrogels surrounding magnetic beads, including molecules with both the desired functionality and vinyl groups for polymerization. In some embodiments, hydrogel layers around magnetic beads can be created that have both chemical (e.g., alkyne, amine, azide) and biochemical (oligonucleotide) functional groups. In some embodiments, the functionalized hydrogel or polymer layer is selected from the group consisting of polyacrylamide, polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PVSA), poly(L-aspartic acid), poly( L-glutamic acid), polylysine, agar, agarose, alginic acid, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or a combination thereof.In some embodiments, the coefficient of diameter variation of the hydrogel particles is about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or a percentage defined by any two of the foregoing values.

[0072] In some embodiments, gel particles can be variously functionalized by adding different reagents to the respective monomer solutions, including the aforementioned polymers or hydrogels surrounding magnetic beads, including molecules with both the desired functionality and vinyl groups for polymerization. In some embodiments, hydrogel layers around magnetic beads can be created that have both chemical (e.g., alkyne, amine, azide) and biochemical (oligonucleotide) functional groups. In some embodiments, the functionalized hydrogel or polymer layer is selected from the group consisting of polyacrylamide, polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PVSA), poly(L-aspartic acid), poly( L-glutamic acid), polylysine, agar, agarose, alginic acid, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or a combination thereof.In some embodiments, the average total diameter of the hydrogel particles is about 5.1 μm, 5.2 μm, 5.3 μm, 5.4 μm, 5.5 μm, 5.6 μm, 5.7 μm, 5.8 μm, 5.9 μm, 6.0 μm, 6.1 μm, 6.2 μm, 6.3 μm, 6.4 μm, 6.5 μm, 6.6 μm, 6.7 μm, 6.8 μm, 6.9 μm, 7.0 μm, 7.1 μm, 7.2 μm, 7.3 μm, 7.4 μm, 7.5 μm, 7.6 μm, 7.7 μm, 7.8 μm, 7.9 μm, 8.0 μm, 8.1 μm, 8.2 μm, 8.3 μm, 8.4 μm, 8.5 μm, 8.6 μm, 8.7 μm, 8.8 μm, 8.9 μm, 9.0 μm, 9.1 μm, 9.2 μm, 9.3 μm, 9.4 μm, 9.5 μm, 9.6 μm, 9.7 μm, 9.8 μm, 9.9 μm, 9.10 μm, 9.11 μm, 9.12 μm, 9.13 μm, 9.14 μm, 9.15 μm, 9.16 μm, 9.17 μm, 9.18 μm, 9.19 μm, 9.20 μm, 9.21 μm, 9.22 μm, 9.23 μm, 9.24 μm, 9.25 μm, 9.26 μm, 9.27 μm, 9.28 μm, In some embodiments, the average diameter of the hydrogel particles is about 7 μm.

[0073] In some embodiments, gel particles can be variously functionalized by adding different reagents to the respective monomer solutions, including the aforementioned polymers or hydrogels surrounding magnetic beads, including molecules with both the desired functionality and vinyl groups for polymerization. In some embodiments, hydrogel layers around magnetic beads can be created that have both chemical (e.g., alkyne, amine, azide) and biochemical (oligonucleotide) functional groups. In some embodiments, the functionalized hydrogel or polymer layer is selected from the group consisting of polyacrylamide, polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PVSA), poly(L-aspartic acid), poly( L-glutamic acid), polylysine, agar, agarose, alginic acid, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or a combination thereof. In some embodiments, at least 95% of the total hydrogel particles in the composition comprise a magnetic core. In some embodiments, the percentage of total hydrogel particles in the composition comprising the magnetic core is 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or a percentage defined by any two of the foregoing values.

[0074] In some embodiments, the library of hydrogel particles having a magnetic core comprises about 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11, 9×10 11 , 1×10 12 particles, or a number defined by any two of the aforementioned values.

[0075] In some embodiments, the library of hydrogel particles having a magnetic core comprises about 1×10 6 ~1×10 12 particles, 1×10 6 ~1×10 11 particles, 1×10 6 ~1×10 10 particles, 1×10 6 ~1×10 9 particles, 1×10 6 ~1×10 8 particles, 1×10 6 ~1×10 7 particles, 1×10 7 ~1×10 12 particles, 1×10 7 ~1×10 11 particles, 1×10 7 ~1×10 10 particles, 1×10 7 ~1×10 8 particles, 1×10 8 ~1×10 12 particles, 1×10 8 ~1×10 11 particles, 1×10 8 ~1×10 10 particles, 1×10 8 ~1×10 9 particles, 1×10 9 ~1×10 12 particles, 1×10 9 ~1×10 11 particles, 1×10 9 ~1×10 10 particles, 1×10 10 ~1×10 12 particles, or 1×10 11 ~1×10 12 Contains particles.

[0076] II. Particle Preparation Method In some embodiments, provided herein are methods for producing compositions comprising particles comprising a magnetic core encapsulated within a polymer gel. In some embodiments, the methods provided herein are advantageous in that they produce gel-encapsulated nanoparticles in a single step by emulsifying an aqueous solution and an oily solution, and do not require complex assembly techniques or machinery. In some embodiments, the methods provided herein are suitable for producing gel-encapsulated nanoparticles in a high-throughput manner and / or for producing gel-encapsulated nanoparticles in bulk.

[0077] In some embodiments, the methods provided herein include emulsifying an aqueous solution containing a monomer and magnetic beads with a solution containing a polymerization initiator to cause polymerization of the monomer. In some embodiments, the monomer is a monomer unit of a polymer. In some embodiments, the aqueous solution includes an acrylamide monomer. In some embodiments, the aqueous solution includes about 4% to about 10%, about 5% to about 9%, about 6% to about 8%, or about 7% to about 10% acrylamide monomer.

[0078] In some embodiments, the aqueous solution comprises bisacrylamide. In some embodiments, the aqueous solution comprises a mixture of bisacrylamide and acrylamide monomers. In some embodiments, the ratio of bisacrylamide to acrylamide is about 10:1 to about 40:1, e.g., about 15:1 to about 20:1, about 30:1 to about 40:1, about 18:1 to about 38:1, or about 35:1 to about 40:1. In some embodiments, the ratio of bisacrylamide to acrylamide is about 17:1, about 18:1, about 19:1, about 20:1, or about 21:1. In some embodiments, the ratio of bisacrylamide to acrylamide is about 36:1, about 37:1, about 37.5:1, about 38:1, about 39:1, or about 40:1.

[0079] In some embodiments, the aqueous solution further comprises a second polymerization initiator that initiates polymerization when mixed with the solution containing the polymerization inhibitor. In embodiments, the initiator is ammonium persulfate (APS) or TEMED. In some embodiments, the aqueous solution comprises APS and the oil solution comprises TEMED. In some embodiments, the aqueous solution comprises TEMED and the oil solution comprises TEMED. In some embodiments, APS and TEMED cause the polymerization of acrylamide and bisacrylamide to encapsulate the magnetic beads. In other embodiments, initiation of polymerization can be achieved using alternative radical sources (e.g., azobisisobutyronitrile, AIBN) or stimulus-responsive species (e.g., photosensitive photoinitiators such as dimethoxyphenylacetophenone, DMPA).

[0080] In some embodiments, the method includes combining two solutions, such as an aqueous solution and an oily solution. In some embodiments, the aqueous solution includes acrylamide and / or bisacrylamide, and the oily solution includes a polymerization initiator. In some embodiments, the two solutions are combined by mixing, emulsifying, vortexing, homogenizing, stirring, and / or shaking. In some embodiments, the method includes combining the solutions using a mechanical system. In some embodiments, the method includes combining the solutions using a vortexer, a rotary mixer, or a stir bar. In some embodiments, the method includes manually shaking a container containing the two solutions.

[0081] In some embodiments, the solutions are mixed for a period of time. In some embodiments, the solutions are mixed for a time sufficient to combine the solutions. In some embodiments, the solutions are mixed for a time sufficient to allow polymerization of the monomers and encapsulation of the beads. In some embodiments, the solutions are combined, mixed, vortexed, emulsified, homogenized, stirred, and / or shaken for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes. In some embodiments, the solutions are combined, mixed, vortexed, emulsified, homogenized, stirred, and / or shaken for 1 to 60 minutes, 1 to 45 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, or 1 to 5 minutes. In some embodiments, the solutions are combined, mixed, vortexed, emulsified, homogenized, stirred, and / or shaken for 10-60 minutes, 20-60 minutes, 30-60 minutes, or 45-60 minutes.

[0082] In some embodiments, the solutions are mixed at room temperature. In some embodiments, the solutions are mixed at a temperature above room temperature. In some embodiments, the solutions are mixed below room temperature. In some embodiments, the solutions are mixed at about 15°C to about 25°C, about 17°C to about 23°C, or about 20°C to about 22°C.

[0083] In some embodiments, the method further comprises sparging a gas through the combined composition. In some embodiments, the combined composition is sparged with an inert gas. In some embodiments, the combined composition is sparged with nitrogen, argon, or helium. In some embodiments, the combined composition is sparged with an inert gas for at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes. In some embodiments, the combined composition is sparged for 1 to 60 minutes, 1 to 45 minutes, 1 to 30 minutes, 1 to 20 minutes, 1 to 10 minutes, or 1 to 5 minutes. In some embodiments, the combined composition is sparged for 10 to 60 minutes, 20 to 60 minutes, 30 to 60 minutes, or 45 to 60 minutes.

[0084] In some embodiments, the method further comprises applying a magnetic field to the combined composition to purify or isolate particles comprising one or more magnetic beads encapsulated in the hydrogel. In some embodiments, applying a magnetic field to the combined mixture uses a magnet to localize the beads while removing or exchanging the solution. In some embodiments, applying a magnetic field is used to separate particles comprising one or more magnetic beads encapsulated in the hydrogel from particles that do not contain one or more magnetic beads. In some embodiments, particles having two or more, three or more, or four or more magnetic beads are separated from particles or gels that do not contain magnetic beads. In some embodiments, applying a magnetic field allows for the isolation of a homogenous population of particles containing one or more different reagents. In some embodiments, applying a magnetic field produces a library of particles each containing a different reagent.

[0085] In some embodiments, after applying a magnetic field to the combined composition, particles, including magnetic beads, are removed from the solution. In some embodiments, particles, including magnetic beads, form a pellet and / or localize to a portion of the tube or vessel containing the combined solution. In some embodiments, the magnetic field causes precipitation of particles, including magnetic beads. In some embodiments, a supernatant, free of particles, including magnetic beads, is aspirated and / or removed from the combined composition. In some embodiments, aspirating the supernatant results in purification of particles, including magnetic beads.

[0086] In some embodiments, after application of the magnetic field, particles comprising one or more magnetic beads are substantially purified from particles that do not contain magnetic beads. In some embodiments, after application of the magnetic field, the ratio of particles that contain magnetic beads to particles that do not contain magnetic beads is at least 100:1, at least 1,000:1, or at least 10,000:1. In some embodiments, after application of the magnetic field, particles that contain a single magnetic bead are separated from particles that contain multiple magnetic beads.

[0087] In some embodiments, after aspirating the supernatant, the remaining particles, including one or more magnetic beads, are washed one or more times. In some embodiments, the washing removes particles that do not contain magnetic beads. In some embodiments, the wash solution comprises a neutral buffer solution. In some embodiments, the particles are resuspended using an excess of buffer solution, a magnetic field is applied, the supernatant is aspirated one or more times, and the particles are washed. In some embodiments, after washing, the ratio of particles containing magnetic beads to particles not containing magnetic beads is at least 100:1, at least 1,000:1, or at least 10,000:1. In some embodiments, the method comprises washing the beads one, two, three, four, five, or more times.

[0088] In some embodiments, after application of the magnetic field, the particles are resuspended. In some embodiments, the particles are resuspended in a buffer solution. In some embodiments, the particles are resuspended in a buffer solution suitable for a chemical or biochemical reaction.

[0089] In some embodiments, the method includes producing a functionalized gel or a functionalized portion of a gel. In some embodiments, the gel is functionalized after particles comprising the polymer gel coating a magnetic core are purified, for example by magnetic separation. In some embodiments, the gel is functionalized before purification of the encapsulated particles. In some embodiments, a portion of the monomers are functionalized before polymerization. In some embodiments, the polymerized gel is functionalized.

[0090] In some embodiments, functionalized gels or particles contain functional groups that are substrates for further reactions or synthetic methods. For example, suitable functional groups include oligonucleotides, synthetic hairpin headpiece DNA, alkynes, and / or primary amines.

[0091] In some embodiments, the particles are functionalized by copolymerization. In some embodiments, the particles are copolymerized with 5' methacrylamide oligonucleotides, methacrylamide-modified HDNA, propargyl methacrylate, or APMA. In some embodiments, the gel is functionalized. In some embodiments, the gel is functionalized before polymerization. In some embodiments, the gel is functionalized simultaneously with polymerization. In some embodiments, the gel is functionalized after polymerization.

[0092] In some embodiments, the magnetic beads are functionalized. In some embodiments, the magnetic beads are functionalized with 5' methacrylamide oligonucleotides, methacrylamide-modified HDNA, propargyl methacrylate, or APMA. In some embodiments, the magnetic beads are functionalized before polymerization of the gel encapsulating the beads. In some embodiments, the magnetic beads are functionalized at the same time as the gel is polymerized. In some embodiments, the magnetic beads are functionalized after the gel is polymerized. In some embodiments, the magnetic beads are functionalized with a carboxylic acid.

[0093] In some embodiments, the gel is functionalized by adding a compound containing a functional group to a monomer solution. In some embodiments, the reagent is added under conditions that allow coupling of the functional group to the gel. In some embodiments, the compound is incubated with the monomer solution so that at least a portion of the monomers are functionalized. In some embodiments, at least 0.01%, at least 0.1%, at least 1%, or at least 10% of the monomers are functionalized.

[0094] In some embodiments, particles or monomers are functionalized using an excess of functional agent. In some embodiments, particles are functionalized by incubation with about 0.0001 mM to about 100 mM (e.g., about 0.0002 mM to about 50 mM, about 0.0002 mM to about 20 mM, about 1 mM to about 100 mM, 5 mM to about 80 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, or about 15 mM to about 30 mM) of a compound containing a functional group. In some embodiments, particles are functionalized by incubation with about 20 mM of a compound containing a functional group. In some embodiments, the compound containing a functional group is propargyl methacrylate.

[0095] In some embodiments, the gel and / or beads are functionalized so that the particles contain one or more functional groups. In some embodiments, a given particle contains multiple copies of the same functional group. In some embodiments, a given particle contains multiple copies of different functional groups. In some embodiments, different particles in a mixture of particles contain different functional groups. In some embodiments, each particle in the mixture contains the same functional group.

[0096] In some embodiments, functionalization of the gel results in particles that include templates for synthesis reactions, e.g., in some embodiments, functionalization of the gel results in particles that include HDDNA or oligonucleotide templates.

[0097] In some embodiments, prior to polymerization, the solution containing the monomers and magnetic beads includes one or more reagents. In some embodiments, the one or more reagents include a protein, an enzyme, a polynucleotide, an oligonucleotide, a polysaccharide, a fluorophore, or a lipid. In some embodiments, the reagent is a synthesizable enzyme. In some embodiments, the reagent includes a polynucleotide or oligonucleotide substrate for a synthesis reaction. In some embodiments, the reagent includes a fluorophore that can be used to detect particles. In some embodiments, the solution includes multiple reagents, such as a fluorophore and an oligonucleotide.

[0098] In some embodiments, one or more reagents are incorporated into the gel by polymerization of monomers. In some embodiments, the reagents are covalently attached to the beads and / or gel. In some embodiments, the reagents are encapsulated in the gel. In some embodiments, the reagents are non-covalently retained within the gel.

[0099] In some embodiments, the method includes polymerizing monomers to produce particles, including gel-coated magnetic beads, and subsequently functionalizing the formed particles. In some embodiments, the particles are functionalized using an excess of functional agent. In some embodiments, the particles are functionalized by incubation with about 0.0001 mM to about 100 mM (e.g., about 0.0002 mM to about 50 mM, about 0.0002 mM to about 20 mM, about 1 mM to about 100 mM, 5 mM to about 80 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, or about 15 mM to about 30 mM) of a compound comprising a functional group. In some embodiments, the particles are functionalized by incubation with about 20 mM of a compound comprising a functional group. In some embodiments, the compound comprising a functional group is propargyl methacrylate.

[0100] In some embodiments, the method further comprises detecting the functionalization of the gel and / or beads. In some embodiments, the detection is by chemical reaction with a fluorescent dye-labeled functional group. In some embodiments, the detection is by complementary oligonucleotide hybridization, enzymatic ligation of dsDNA molecules, CuAAC using AlexaFluor 488 azide (488A-FN3), or amine acylation using fluorescein succinimidyl ester (FAM-OSu).

[0101] In some embodiments, the methods provided herein result in a library of particles. In some embodiments, the library of particles comprises a plurality of particles having different functional groups. In some embodiments, the library of particles comprises a plurality of particles having the same functional group.

[0102] In some embodiments, the methods provided herein are compatible with different magnetic bead sizes, such as about 1.0 μm, 2.8 μm, or 10.0 μm in diameter, and in some embodiments, the methods provided herein result in gel particles having magnetic core sizes of about 1 μm, 2.8 μm, or 10 μm in diameter after encapsulation.

[0103] In some embodiments, increasing the magnetic bead size results in a larger, more uniform gel particle distribution using the methods provided herein. In some embodiments, for hydrogels prepared by bulk emulsification with different templated magnetic bead sizes, such as approximately 1.0 μm, 2.8 μm, or 10.0 μm in diameter, the resulting overall hydrogel particle size dispersity is inversely proportional to the magnetic bead diameter. For example, in some embodiments, when the templated magnetic bead size is approximately 1 μm in diameter, the mean diameter of the hydrogel particles after magnetic bead encapsulation is approximately 4.9 μm in diameter, with a standard deviation of approximately 1.8 μm and a coefficient of variation of approximately 36% based on a count of 447 particles. In some other embodiments, when the templated magnetic bead size is approximately 2.8 μm in diameter, the mean diameter of the hydrogel particles after magnetic bead encapsulation is approximately 7.5 μm in diameter, with a standard deviation of approximately 1.9 μm and a coefficient of variation of 25% based on a count of 390 particles. In some other embodiments, when the templated magnetic bead size is about 10 μm in diameter, the mean diameter of the hydrogel particles after encapsulation of the magnetic beads is about 12 μm in diameter, with a standard deviation of about 2.0 μm and a coefficient of variation of 16% based on counting 66 particles.

[0104] In some embodiments, hydrogel particles having a 1 μm diameter magnetic core prepared by the methods provided herein have a hydrogel volume of about 88 fL. In some embodiments, hydrogel particles having a 2.8 μm diameter magnetic core prepared by the methods provided herein have a hydrogel volume of about 260 fL. In some embodiments, hydrogel particles having a 10 μm diameter magnetic core prepared by the methods provided herein have a hydrogel volume of about 570 fL.

[0105] III. Methods for detecting synthetic products Also provided herein are methods for detecting synthesis products of reactions in particles. In some embodiments, the particles and methods provided herein advantageously result in concentration of reactants and retention of synthesis products within the particles. In some embodiments, the methods include synthesizing the synthesis products in the particles. In some embodiments, the particles include a gel coating a magnetic core. In some embodiments, the particles include a synthesis template. In some embodiments, the gel is a polymer gel.

[0106] In some embodiments, the method includes detecting a synthesis product of a reaction in particles comprising a polyacrylamide gel coating magnetic beads. In some embodiments, the polymer gel facilitates proximity-driven synthesis by an enzyme. In some embodiments, the polymer gel increases the local concentration of the substrate, enzyme, and template, resulting in production of the synthesis product. In some embodiments, one or more of the enzyme, substrate, and template are attached to the polymer gel or the bead. In some embodiments, one or more of the enzyme, substrate, and template are entrapped within the polymer gel. In some embodiments, the pore size of the polymer gel is such that the enzyme, substrate, and / or template are retained.

[0107] In some embodiments, the method includes providing a synthetic enzyme and a synthetic substrate within a polymer gel. In some embodiments, the synthetic substrate is a nucleic acid or an amino acid. In some embodiments, the synthetic substrate is a nucleotide. In some embodiments, the synthetic substrate is a tRNA. In some embodiments, the synthetic substrate is a naturally occurring tRNA or a synthetic tRNA. In some embodiments, the synthetic substrate is a tRNA for any of the naturally occurring amino acids.

[0108] In some embodiments, the enzyme incorporates the substrate into a synthetic product. In some embodiments, the substrate is incorporated into a polymer product. In some embodiments, the substrate is incorporated into a nucleic acid or protein product. In some embodiments, the sequence of the synthetic product is determined by sequencing the template. For example, in some embodiments, the template is mRNA and the synthetic product is a protein containing the corresponding amino acid sequence. In some embodiments, the template is a DNA or RNA sequence and the synthetic product is a complementary DNA or RNA sequence.

[0109] In some embodiments, the functionalized particles are further modified to produce particles comprising a template. In some embodiments, templated particles are produced. In some embodiments, the particles are functionalized with a primer. In some embodiments, the primer is incubated with the template DNA and oligonucleotides under PCR conditions to produce templated particles comprising double-stranded DNA. In some embodiments, the particles are templated after polymerization of the gel and purification of the gel-encapsulated particles. In some embodiments, magnetic beads are functionalized and templated. In some embodiments, polymer gels are functionalized and templated. In some embodiments, monomers are functionalized before or after polymerization.

[0110] In some embodiments, the number or percentage of templated particles is determined. In some embodiments, the templated particles are detected by qPCR using primers that hybridize to DNA synthesized during templating. In some embodiments, the templated particles are detected by binding of a fluorescent probe to the template. In some embodiments, the templated particles are detected by binding of an antibody to the template. In some embodiments, the templated particles are purified from non-templated particles.

[0111] In some embodiments, the template is single-stranded DNA. In some embodiments, the template is double-stranded DNA. In some embodiments, the template comprises a transcription initiation sequence. In some embodiments, the template comprises a transcription termination sequence. In some embodiments, the template comprises a promoter sequence for an RNA polymerase. In some embodiments, the template comprises a promoter sequence for T7 RNA polymerase.

[0112] In some embodiments, the template is mRNA. In some embodiments, the template contains a translation initiation sequence such as AUG. In some embodiments, the template comprises a ribosome binding site. In some embodiments, the template comprises a transcription termination sequence. In some embodiments, the template comprises a puromycin translation termination sequence. In some embodiments, the template comprises a HiBit tag. In some embodiments, the template comprises a sequence encoding an epitope tag. In some embodiments, the template comprises a sequence encoding an amino acid tag, such as a 6HIS or FLAG tag (SEQ ID NO: 21). In some embodiments, the template comprises one or more tags or epitopes separated by a flexible linker.

[0113] In some embodiments, the substrate is a naturally occurring nucleotide. In some embodiments, the substrate is selected from the group consisting of adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, deoxycytidine, inosine, or diaminopurine), base analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), modified bases (e.g., 2'-substituted nucleotides such as 2'-O-methylated bases and 2'-fluoro bases), intercalating bases, modified sugar ... For example, 2'-fluororibose; ribose; 2'-deoxyribose; arabinose; hexose; anhydrohexitol; altritol; mannitol; cyclohexanyl; cyclohexenyl; morpholino, which also has a phosphoramidate backbone; locked nucleic acids (LNA, e.g., the 2'-hydroxyl of ribose is connected to the 4'-carbon of the same ribose sugar by a Ci-β alkylene bridge or a Ci-6 heteroalkylene bridge; exemplary bridges included methylene bridges, propylene bridges, ether bridges, or amino bridges); glycol nucleic acids (GNA, e.g., R-GNA or S-GNA, in which the ribose is replaced by a glycol unit attached to a phosphodiester bond); threose nucleic acids (TNA, in which the ribose is replaced by aL-threofuranosyl-(3'→2'));and / or oxygen replacement in ribose (e.g., with S, Se, or alkylene such as methylene or ethylene), modified backbone (e.g., peptide nucleic acid (PNA) in which 2-amino-ethyl-glycine bond replaces ribose and phosphodiester backbone), and / or modified phosphate group (e.g., phosphorothioate, 5'-N-phosphoramidite, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, phosphorodiamidate, alkyl or aryl phosphonate, phosphotriester, bridged phosphoramidate, bridged phosphorothioate, and bridged methylene-phosphonate). Oligonucleotides can have single-stranded (e.g., hairpin), double-stranded, or other secondary or tertiary structures (e.g., stem-loop structure, double helix, triplex, quadruplex, etc.). In some embodiments, particles contain a mixture of oligonucleotide substrates (e.g., A, C, G, and T or U). In some embodiments, the particles comprise a mixture of oligonucleotide substrates in approximately equal amounts;

[0114] In some embodiments, the enzyme is a DNA or RNA polymerase. In some embodiments, the enzyme is DNA polymerase I, T7 DNA polymerase, DNA polymerase II, DNA polymerase VI, Taq DNA polymerase, T4 DNA polymerase, RNA polymerase I, DNA polymerase III, RNA polymerase II, RNA polymerase III, T7 RNA polymerase, or reverse transcriptase. In some embodiments, the enzyme is a ribosome. In some embodiments, the enzyme is derived from a microorganism such as a bacterium, a virus, or a yeast.

[0115] In some embodiments, the synthetic product is a peptide of 50 amino acids or less in length. In some embodiments, the synthetic product is a polypeptide of more than 50 amino acids in length. In some embodiments, the synthetic product comprises one or more amino acids linked by peptide bonds. In some embodiments, the synthetic product is about 2 to about 1000 amino acids in length. In some embodiments, the synthetic product is about 2 to about 50, about 2 to about 40, about 2 to about 30, about 2 to about 20, or about 2 to about 10 amino acids in length. In some embodiments, the synthetic product is a naturally occurring peptide or a synthetic peptide.

[0116] In some embodiments, the product of synthesis is an oligonucleotide, e.g., a polynucleotide. In some embodiments, the product of synthesis is double-stranded DNA. In some embodiments, the product of synthesis is single-stranded DNA or RNA. In some embodiments, the product of synthesis comprises two or more nucleotides linked by phosphodiester bonds. In some embodiments, the product of synthesis comprises two or more nucleotides linked by a synthetic backbone. In some embodiments, the product of synthesis is an oligonucleotide about 2 to about 100 nucleotides in length. In some embodiments, the product of synthesis is about 2 to about 100, about 2 to about 80, about 2 to about 60, about 2 to about 40, or about 2 to about 20 nucleotides in length. In some embodiments, the product of synthesis is a naturally occurring oligonucleotide or a synthetic oligonucleotide.

[0117] In some embodiments, the synthetic product is a drug candidate. In some embodiments, the synthetic product is a drug target. In some embodiments, the synthetic product is part of a larger molecule. For example, in some embodiments, the synthetic protein is part of a protein.

[0118] In some embodiments, the template is conjugated to a magnetic bead. In some embodiments, the template is covalently bound to the magnetic bead. In some embodiments, the template is conjugated to the magnetic bead before polymerization of the gel around the beads. In some embodiments, the template is conjugated to the beads after polymerization. In some embodiments, the template is conjugated directly to the beads. In some embodiments, the template is conjugated to the beads by a linker. In some embodiments, the linker is a small molecule, peptide, or oligonucleotide linker.

[0119] In some embodiments, the template is conjugated to the polymer gel. In some embodiments, the template is covalently bound to the polymer gel. In some embodiments, the template is non-covalently held within the polymer gel. In some embodiments, the template is conjugated to the monomer prior to polymerization of the gel around the beads. In some embodiments, the template is conjugated to the polymer after polymerization. In some embodiments, the template is conjugated directly to the polymer. In some embodiments, the template is conjugated to the polymer by a linker. In some embodiments, the linker is a small molecule, peptide, or oligonucleotide linker.

[0120] In some embodiments, the synthesis product remains associated with the particle after synthesis. In some embodiments, the synthesis product does not diffuse into the solution surrounding the particle. In some embodiments, the synthesis product is non-covalently retained in the particle. In some embodiments, the pore size of the polymer gel particle is such that the synthesis product cannot escape from the polymer gel. In some embodiments, the synthesis product non-covalently interacts with the template or enzyme, resulting in its retention within the particle. In some embodiments, ribosome stalling results in the retention of the synthesis product. In some embodiments, the synthesis product contains 3' puromycin, which results in tethering the product to the template.

[0121] In some embodiments, the method further comprises detecting the activity of the enzyme. In some embodiments, the presence or amount of a synthesis product is detected. In some embodiments, a detection or selection assay is performed. In some embodiments, the enzyme activity is measured using flow cytometry, a cell sorting device, density measurement, affinity tag detection, or DNA sequencing. In some embodiments, the activity is measured by binding a fluorescently labeled protein, e.g., an antibody. In some embodiments, the activity is measured by binding a fluorescently labeled oligonucleotide. In some embodiments, the labeled oligonucleotide is complementary to the synthesis product or a portion of the synthesis product. In some embodiments, the labeled oligonucleotide is not complementary to the template.

[0122] In some embodiments, the method further comprises detecting the synthesis product. For example, in some embodiments, the method comprises detecting the synthesis product using a fluorescent label and performing flow cytometry or cell sorting to detect the product. In some embodiments, the synthesis product is a polynucleotide, and the polynucleotide product is detected using sequencing.

[0123] IV. Methods for Producing DNA-Encoded Libraries Also provided herein are methods for producing DNA-encoded libraries (DELs). In some embodiments, the DELs comprise particles, each particle comprising a different DNA sequence. In some embodiments, the DNA sequences are associated with building blocks. In some embodiments, the DNA or oligonucleotide sequences associated with the building blocks are "tags." In some embodiments, the DNA sequences are associated with chemical building blocks, such as functional groups. In some embodiments, the DNA is used as a barcode or tag to identify small molecules associated with the particles.

[0124] In some embodiments, the method involves incubating particles comprising a magnetic core coated in a polyacrylamide gel containing functionalized sites and synthetic hairpin headpiece DNA (HDNA) under conditions that couple the building blocks to the functionalized sites. Previous publications provide examples of DNA headpieces in which beads are functionalized with azide DNA headpiece moieties (MacConnell et al. ACS Comb. Sci. 2015, 17, 9, 518-534). In certain embodiments, the headpiece comprises an oligonucleotide selected from the group consisting of a double-stranded oligonucleotide, a single-stranded oligonucleotide, or a hairpin oligonucleotide. In some embodiments, the headpiece comprises a primer binding region.

[0125] In some embodiments, the library comprises a plurality of headpieces, each headpiece of the plurality of headpieces comprising the same sequence region (e.g., a primer binding region) and a different coding region (e.g., a first tag encoding a first component or oligonucleotide sequence that facilitates the use of the library, the identity of the library, linkage, spacer, or hybridization, amplification, or sequencing techniques).

[0126] Typically, the headpiece contains a non-self-complementary sequence at the 5' or 3' end that allows for the attachment of an oligonucleotide tag via polymerization, enzymatic ligation, or chemical reaction. The headpiece can allow for ligation of the oligonucleotide tag and optional purification and phosphorylation steps. After the final tag is added, an additional adapter sequence can be added to the 5' end of the final tag. Exemplary adapter sequences include primer binding sequences or sequences containing a label (e.g., biotin). If many building blocks and corresponding tags are used (e.g., 100), a mix-and-split strategy can be used during the oligonucleotide synthesis process to create the required number of tags. Such mix-and-split strategies for DNA synthesis are known in the art. The resulting library members can be amplified by PCR after selection of binding entities for the target of interest.

[0127] In some embodiments, the headpiece comprises one or more primer binding sequences. For example, the headpiece comprises a sequence within the loop region of a hairpin that functions as a primer binding region for amplification, and the primer binding region has a higher melting temperature for its complementary primer (e.g., it can include an adjacent identifier region) than the sequence within the headpiece. In other embodiments, the complex comprises two primer binding sequences (e.g., to enable a PCR reaction) on either side of one or more tags encoding one or more building blocks. Alternatively, the headpiece may contain one primer binding sequence at the 5' or 3' end. In some embodiments, the headpiece is a hairpin, and the loop region forms a primer binding site, or a primer binding site is introduced by hybridization of an oligonucleotide to the headpiece on the 3' side of the loop. A primer oligonucleotide containing a homologous region at the 3' end of the headpiece and carrying a primer binding region at its 5' end (e.g., to enable a PCR reaction) can hybridize to the headpiece and contain a tag encoding a building block or the addition of a building block. The primer oligonucleotide may contain additional information, such as a region of randomized nucleotides, e.g., 2-16 nucleotides in length, that is included in the bioinformatics analysis.

[0128] The headpiece can optionally include a hairpin structure, which can be achieved by any useful method. For example, the headpiece can include complementary bases that form intermolecular base-pairing partners through Watson-Crick DNA base pairing and / or wobble base pairing, etc. In another example, the headpiece can include modified or substituted nucleotides that can form duplexes with higher affinity than unmodified nucleotides, and such modified or substituted nucleotides are known in the art. In some embodiments, the headpiece includes one or more crosslinking bases to form a hairpin structure. In some embodiments, bases within a single strand or bases in different double strands can be crosslinked, for example, by using psoralen.

[0129] In some embodiments, the headpiece or tag comprises one or more labels that allow detection. For example, the headpiece, one or more oligonucleotide tags, and / or one or more primer sequences comprise an isotope, a radiocontrast agent, a marker, a tracer, a fluorescent label (e.g., rhodamine or fluorescein), a chemiluminescent label, a quantum dot, and / or an affinity tag such as biotin or a his tag.

[0130] In some embodiments, the headpiece or tag is modified to increase solubility in semi-aqueous, reduced aqueous, or non-aqueous (e.g., organic) conditions. In some embodiments, the C5 position of a T or C base is modified with an aliphatic chain without significantly disrupting their ability to hydrogen bond to their complementary base. Exemplary modified or substituted nucleotides are 5'-dimethoxytrityl-N4-diisobutylaminomethylidene-5-(l-propynyl)-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 5'-dimethoxytrityl-5-(l-propynyl)-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 5'-dimethoxytrityl-5-fluoro-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite; and 5'-dimethoxytrityl-5-(pyren-l-yl-ethynyl)-2'-deoxyuridine, or 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

[0131] Additionally, headpieces and / or oligonucleotide tags can be interspersed with modifications that promote solubility in organic solvents. For example, azobenzene phosphoramidites can introduce hydrophobic moieties into the headpiece design. Such insertion of hydrophobic amidites into the headpiece can occur anywhere within the molecule. Thus, adding hydrophobic residues to the headpiece design allows for improved solubility in semi-aqueous or non-aqueous (e.g., organic) conditions while making the headpiece suitable for oligonucleotide tagging. Furthermore, DNA tags subsequently introduced into the library can also be modified at the C5 position of a T or C base, thereby making the library more hydrophobic and soluble in organic solvents for subsequent steps in library synthesis.

[0132] Various ligation techniques can be used to add scaffolds, building blocks, spacers, linkers, tags, and / or headpieces to produce complexes. Thus, any of the binding steps described herein can include any useful ligation technique, such as enzymatic ligation and / or chemical ligation. These binding steps can include adding one or more tags to the headpiece or complex; adding spacers to the headpiece; and adding one or more scaffolds or building blocks to the headpiece or complex. In certain embodiments, the ligation technique used for any of the oligonucleotides provides a resulting product that can be transcribed and / or reverse transcribed to enable library decoding or template-dependent polymerization by one or more DNA or RNA polymerases.

[0133] Generally, enzymatic ligation produces oligonucleotides with native phosphodiester bonds that can be transcribed and / or reverse transcribed. Exemplary methods of enzymatic ligation are provided herein and involve the use of one or more RNA or DNA ligases, such as T4 RNA ligase, T4 DNA ligase, CircLigase™ ssDNA ligase, CircLigase™ II ssDNA ligase, and ThermoPhage™ ssDNA ligase (Prokazyme Ltd., Reykjavik, Iceland). In some embodiments, ligation involves the use of an RNA ligase or a combination of an RNA ligase and a DNA ligase. Ligation can further include one or more soluble polyvalent cations in combination with one or more ligases.

[0134] Chemical ligation can also be used to produce oligonucleotides capable of transcription or reverse transcription. It may be necessary to test the effectiveness of chemical ligation techniques to provide oligonucleotides capable of transcription or reverse transcription. This effectiveness can be tested by any useful method, such as liquid chromatography-mass spectrometry, RT-PCR analysis, and / or PCR analysis. In certain embodiments, chemical ligation involves the use of one or more chemically reactive pairs to provide spacing moieties that can be transcribed or reverse transcribed. In particular, reactions suitable for chemically reactive pairs are preferred candidates for the ligation process (Kolb et al., Angew. Chem. Int. Ed., 40:2004-2021 (2001); Van der Eycken et al., QSAR Comb. Sci., 26:1115-1326 (2007)). In one embodiment, the ligated oligonucleotide contains a linkage that reduces the ability of polymerases to read or transpose, for example, through a "non-readable" linkage.

[0135] In some embodiments, the methods described herein include reaction conditions that promote enzymatic or chemical ligation between a headpiece and a tag or between two tags. These reaction conditions include using modified nucleotides in the tag, as described herein; using donor and acceptor tags of different lengths and varying the concentration of the tags; using different types of ligases and combinations thereof (e.g., CircLigase™ DNA ligase and / or T4 RNA ligase) and varying their concentrations; using polyethylene glycols (PEGs) of different molecular weights and varying their concentrations; using non-PEG aggregating agents (e.g., betaine or bovine serum albumin); varying the temperature and duration for ligation; varying the concentrations of various agents, including ATP, Co(NH3)6Ch, and yeast inorganic pyrophosphate; using enzymatically or chemically 20 phosphorylated oligonucleotide tags; using 3'-protecting tags; and using pre-adenylated tags. These reaction conditions also include chemical ligation.

[0136] The headpiece and / or tag can contain one or more modified or substituted nucleotides. In preferred embodiments, the headpiece and / or tag contains one or more modified or substituted nucleotides that promote enzymatic ligation, such as 2'-O-methyl nucleotides (e.g., 2'-O-methylguanine or 2'-O-methyluracil), 2'-fluoro 25 nucleotides, or any other modified nucleotides that serve as substrates for ligation. In some embodiments, the headpiece and / or tag is modified to contain one or more chemically reactive groups to promote chemical ligation (e.g., an optionally substituted alkynyl group and an optionally substituted azide group). In some embodiments, the tag oligonucleotide is functionalized at both ends with a chemically reactive group, and optionally, one of these ends can be protected to reduce side reactions (e.g., to reduce polymerization side reactions).

[0137] In some embodiments, the complex is purified before or after ligation. In some embodiments, the complex is purified to remove unreacted headpieces or tags that may cause cross-reaction. In some embodiments, the complex is purified to remove any reagents or unreacted starting materials that may inhibit or reduce the ligation activity of the ligase. For example, the presence of phosphate may reduce ligation activity. In some embodiments, entities introduced into the chemical or ligation step may need to be removed to enable subsequent chemical or ligation steps.

[0138] Enzymatic and chemical ligations can include polyethylene glycol having an average molecular weight greater than 300 daltons (e.g., greater than 600 daltons, greater than 3,000 daltons, greater than 4,000 daltons, or greater than 4,500 daltons). In some embodiments, the polyethylene glycol has an average molecular weight of about 3,000 daltons to 9,000 daltons. In some embodiments, the polyethylene glycol can be present in any useful amount, for example, about 25% (w / v) to about 35% (w / v), e.g., 30% (w / v).

[0139] In some embodiments, the tag is introduced by ligation of a single-stranded oligonucleotide to the single-stranded oligonucleotide.

[0140] Branched oligomeric or polymeric compounds can also be synthesized, so long as at least one building block contains three functional groups that are reactive with other building blocks. The libraries of the present disclosure can include linear molecules, branched molecules, or combinations thereof.

[0141] In some embodiments, a building block refers to a chemical building block that is attached to or can be attached to other chemical building blocks. In some embodiments, a building block is part of an organic small molecule. When the functional moiety is a polymer or oligomer, the structural unit is a monomeric unit of the multimer or oligomer. A structural unit can also include a backbone structure (e.g., a backbone structural unit) that is connected or can be connected to one or more additional structures (e.g., peripheral structural units). In some embodiments, the building blocks are complementary (i.e., the building blocks must be able to react together to form a structure comprising two or more building blocks). In some embodiments, a building block has at least two reactive groups. In some embodiments, a building block has only one reactive group. In some embodiments, the reactive groups on two different building blocks are complementary, i.e., can react together to form a covalent bond. Examples of building blocks are provided in U.S. Patent Application Publication No. 2007 / 0224607, incorporated herein by reference.

[0142] Furthermore, chemically reactive pairs (or functional groups) can be easily incorporated into the synthesis scheme to facilitate efficient chemical ligation of oligonucleotides. Furthermore, the resulting ligated oligonucleotides can serve as templates for template-dependent polymerization by one or more polymerases. In some embodiments, any of the binding steps described herein for tagging encoded libraries can be modified to include enzymatic ligation and / or chemical ligation techniques.

[0143] In some embodiments, the method includes providing building blocks and associated oligonucleotides under conditions that allow the building blocks to be attached to the particles and the oligonucleotides to be attached to the headpiece DNA or the oligonucleotides attached to the headpiece DNA. In some embodiments, the conditions are ligation conditions. In some embodiments, the method includes incubation with a DNA ligase. In some embodiments, the oligonucleotides are attached to the HDNA or the oligonucleotides attached to the HDNA by polymerization. In some embodiments, the oligonucleotides are attached by chemical reaction. In some embodiments, the oligonucleotides are ligated using a chemically reactive pair (e.g., a pair comprising an optionally substituted alkynyl functional group and an azide functional group) and / or by enzymatic ligation, for example, using one or more RNA ligases and / or DNA ligases.

[0144] In another aspect, the present invention features a library comprising one or more types of particles described herein. In some embodiments, the library comprises a plurality of headpieces. In other embodiments, each headpiece of the plurality of headpieces comprises an identical sequence region (e.g., a primer binding region) and a different coding region (e.g., a tag encoding the identity of the library). In certain embodiments, the library comprises about 10 2 ~10 20 (e.g., about 10 2 ~10 3 , 10 2 ~10 4 , 10 2 ~10 5 , 10 2 ~10 6 , 10 2 ~10 7 , 10 2 ~10 8 , 10 2 ~10 9 , 10 2 ~10 10 , 10 2 ~10 11 , 10 2~10 12 ,10 2 ~10 13 ,10 2 ~10 14 ,10 2 ~10 15 ,10 2 ~10 16 ,10 2 ~10 17 ,10 2 ~10 18 ,10 2 ~10 19 ,10 4 ~10 s ,10 4 ~10 6 ,10 4 ~10 7 ,10 4 ~10 8 ,10 4 ~10 9 ,10 4 ~10 10 ,10 4 ~10,10 4 ~10 12 ,10 4 ~10 13 ,10 4 ~10 14 ,10 4 ~10 15 ,10 4 ~10 16 ,10 4 ~10 17 ,10 4 ~10 18 ,10 4 ~10 19 ,10 4 ~IO 20 ,10 5 ~10 6 ,10 5 ~10 7 ,10 5 ~10 8 ,10 5 ~35 10 9 ,10 5 ~10 10 ,10 5 ~10 11 ,10 5 ~10 12 ,10 5 ~10 13 ,105 ~10 14 , 10 5 ~10 15 , 10 5 ~10 16 , 10 5 ~10 17 , 10 5 ~10 18 , 10 5 ~10 19 , or 10 5 ~10 20 ) particles. In some embodiments, each particle is different.

[0145] In any of the above embodiments, the method further comprises one or more steps for diversifying the library or for examining library members, as described herein. In some embodiments, the method further comprises identifying small, drug-like library members that bind to or inactivate a protein of therapeutic interest. In other embodiments, the method further comprises contacting members of the library with a biological target under conditions suitable for at least one member of the library to bind to the target, removing one or more library members that do not bind to the target, and analyzing one or more oligonucleotide tags associated with the target.

[0146] In any of the above embodiments, the coded information is provided by one or more tags or a combination of two or more tags. In some embodiments, the coded information is represented by two or more tags (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more tags). In some embodiments, the coded information is represented by two or more tags, and all of the coded tags are included in the coding sequence (e.g., by using a particular tag combination to code the information). In some embodiments, the coded information is represented by two or more tags, and less than all of the coded tags are included in the coding sequence (e.g., by using one tag from a set of two or more individual tags to code within an individual coding sequence).

[0147] In any of the above embodiments, the headpiece and / or oligonucleotide tag comprises about 5 to about 300 nucleotides. In some embodiments, the headpiece and / or oligonucleotide tag comprises 5 to 250 nucleotides, 5 to 200 nucleotides, 5 to 150 nucleotides, 5 to 100 nucleotides, 5 to 90 nucleotides, 5 to 80 nucleotides, 5 to 70 nucleotides, 5 to 60 nucleotides, 5 to 50 nucleotides, 5 to 40 nucleotides, 5 to 30 nucleotides, 5 to 20 nucleotides, 5 to 10 nucleotides, 10 to 300 nucleotides, 10 to 250 nucleotides, 10 to 200 nucleotides, 10 to 150 nucleotides. nucleotides, 10-100 nucleotides, 10-50 nucleotides, 10-25 nucleotides, 20-300 nucleotides, 20-250 nucleotides, 20-200 nucleotides, 20-150 nucleotides, 20-100 nucleotides, 20-50 nucleotides, 30-300 nucleotides, 30-250 nucleotides, 30-200 nucleotides, 30-150 nucleotides, 30-100 nucleotides, 30-50 nucleotides, 40-300 nucleotides, 40-250 nucleotides, 40-200 nucleotides nucleotides, 40 to 150 nucleotides, 40 to 100 nucleotides, 40 to 75 nucleotides, 40 to 50 nucleotides, 50 to 300 nucleotides, 50 to 250 nucleotides, 50 to 200 nucleotides, 50 to 150 nucleotides, 50 to 100 nucleotides, 50 to 75 nucleotides, 60 to 300 nucleotides, 60 to 250 nucleotides, 60 to 200 nucleotides, 60 to 150 nucleotides, 60 to 100 nucleotides, 60 to 75 nucleotides, 70 to 300 nucleotides, or 70 to 250 nucleotides nucleotides, 70 to 200 nucleotides, 70 to 150 nucleotides, 70 to 100 nucleotides, 80 to 300 nucleotides, 80 to 250 nucleotides, 80 to 200 nucleotides, 80 to 150 nucleotides, 80 to 100 nucleotides, 90 to 300 nucleotides, 90 to 250 nucleotides, 90 to 200 nucleotides, 90 to 150 nucleotides, or 90 to 100 nucleotides, 100 to 300 nucleotides, 100 to 250 nucleotides, 100 to 200 nucleotides, 100 to 150 nucleotides,The length may be 100 to 120 nucleotides, 150 to 300 nucleotides, 150 to 250 nucleotides, 150 to 200 nucleotides, or 200 to 300 nucleotides, 200 to 250 nucleotides, and 250 to 300 nucleotides.

[0148] In some embodiments, the HDNA is conjugated to a polymer gel. In some embodiments, the HDNA is conjugated to polyacrylamide. In some embodiments, the HDNA is conjugated to a magnetic core.

[0149] In some embodiments, the method includes coupling a building block to a functionalization site located on or within the particle. In some embodiments, the functionalization site is located on the magnetic core. In some embodiments, the functionalization site is located in the polymer gel. In some embodiments, the functionalization site is reactive with one or more reactive groups of the building block. In some embodiments, the functionalization site includes a boronate, amine, isocyanate, carboxylic acid, or aryl halide reactive group.

[0150] In some embodiments, the method for producing a DNA-encoded library includes multiple rounds of incubation, each round including incubating particles with building blocks attached to functional groups in the particles and headpiece DNA or oligonucleotide tags attached to previous oligonucleotide tags. In some embodiments, one or more cleanup or purification steps are performed during or between rounds. In some embodiments, the method results in particles comprising multiple building blocks and polynucleotide sequences containing associated tags for each building block. Thus, in some embodiments, the method includes incubating particles with a second oligonucleotide tag and a second building block, a third oligonucleotide tag and a third building block, etc.

[0151] In some embodiments, the oligonucleotide sequence is associated with additional information, such as the concentration of functionalized moieties or different batches of particles.

[0152] In some embodiments, the method further comprises amplifying the oligonucleotides. In some embodiments, the oligonucleotides are amplified to produce a library. In some embodiments, the oligonucleotides are sequenced to determine the building blocks present on the particles. In some embodiments, the sequencing determines additional information, such as the concentration of functionalization sites or different batches of particles.

[0153] In yet another aspect, the invention features a method of screening a plurality of chemical entities, the method including: (a) contacting a target with any of the particles described herein and / or libraries described herein; and (b) selecting one or more particles having predetermined characteristics relative to the target compared to a control, thereby screening the chemical entities.

[0154] To create a large number of chemical entities in a library, a solution containing headpieces can be divided into multiple aliquots and then placed into a large number of physically separate compartments, such as the wells of a multi-well plate. Generally, this is a "splitting" process. Within each compartment or well, successive chemical reactions and ligation steps are performed using the single-stranded tags in each aliquot. The relationship between the chemical reaction conditions and the sequence of the single-stranded tag is recorded. The reaction and ligation steps can be performed in any order. The reacted and ligated aliquots are then combined, or "pooled," at which point purification can optionally occur. These splitting and pooling steps can be optionally repeated.

[0155] The library can then be tested and / or selected for specific characteristics or functions as described herein. For example, a mixture of tagged chemical entities can be separated into at least two populations, with the first population binding to a specific biological target and the second population not (e.g., by negative or positive selection). The first population can then be selectively captured (e.g., by eluting with a column that provides the target of interest or by incubating an aliquot with the target of interest), and optionally further analyzed or tested, optionally using any washing, purification, negative selection, positive selection, or separation steps, etc.

[0156] In some embodiments, the library is contacted with a biological target under conditions suitable for at least one member of the library to bind to the target, and then the library members that do not bind to the target are removed, and one or more oligonucleotide tags associated with the target are analyzed.This method can optionally include amplifying the tag by methods known in the art.Exemplary biological targets include enzymes (e.g., kinases, phosphatases, methylases, demethylases, proteases, and DNA repair enzymes), proteins involved in protein-protein interactions (e.g., receptor ligands), receptor targets (e.g., GPCRs and RTKs), ion channels, bacteria, viruses, parasites, DNA, RNA, prions, and carbohydrates.

[0157] In another embodiment, the chemical entities that bind to the target are not subjected to amplification and are directly analyzed. Exemplary analytical methods include evanescent resonant photonic crystal analysis; bead-based methods for deconvoluting tags; label-free photonic crystal biosensor analysis; or microarray analysis, including hybridization-based approaches.

[0158] These methods can be used, for example, in a selection process, to identify and discover any number of chemical entities with a particular characteristic or function. The desired characteristic or function can be used as the basis for partitioning the library into at least two portions, with at least one concomitant enrichment of members or related members in the library with the desired function. In certain embodiments, the method involves identifying small, drug-like library members that bind to or inactivate a protein of therapeutic interest. In any of these cases, oligonucleotide tags encode the chemical history of the library members, and in each case, a set of chemical possibilities can be represented by any particular tag combination. In some embodiments, a series of chemical reactions is designed, with a set of building blocks selected such that reaction of the selected building blocks under defined chemical conditions generates a combinatorial plurality (or library of molecules), one or more of which may be useful as therapeutic agents for a particular disease.

[0159] V. In Vitro Translation Methods Also provided herein are in vitro translation methods that utilize the particles provided herein. In some embodiments, the method comprises incubating particles comprising a magnetic core coated with a polyacrylamide gel containing an RNA template, ribosomes, and amino acids under conditions for translating the RNA into a polypeptide.

[0160] In some embodiments, the RNA template is contained in or on a particle. In some embodiments, the RNA template is conjugated to a magnetic core. In some embodiments, the RNA template is conjugated to a polyacrylamide gel. In some embodiments, the transcribed RNA is captured by hybridization to the gel periphery of the magnetic beads. In some embodiments, the RNA template is conjugated before polyacrylamide polymerization. In some embodiments, the RNA template is conjugated to a particle after polyacrylamide polymerization.

[0161] In some embodiments, the RNA template is a template for a protein or peptide. In some embodiments, the RNA template is a template for a therapeutic protein or therapeutic candidate. In some embodiments, the RNA template is a template for a therapeutic target. In some embodiments, the RNA template is for an antibody, e.g., a single-chain antibody. In some embodiments, the RNA template comprises a sequence complementary to a gel-binding oligonucleotide.

[0162] In some embodiments, the methods include a translation system created by isolating, refining, and mixing factors involved in the translational synthesis of a protein or peptide, such as ribosomes, translation factors, tRNAs, amino acids, and energy sources including ATP and GTP.

[0163] An example of natural tRNA is a mixture of purified tRNA fractions obtained by collecting and disrupting E. coli, which is also commercially available. Some A, U, C, and G residues in natural tRNA are chemically modified by enzymes. Alternatively, tRNAs transcribed in vitro but with naturally occurring sequences can be used. In contrast, artificial tRNAs, which are the transcription products of tRNAs, are preferably used as orthogonal tRNAs instead of natural tRNAs. Artificial tRNAs can be prepared by in vitro transcription reactions using template DNA and an appropriate RNA polymerase. Such artificial tRNAs do not contain any chemical modifications.

[0164] In some embodiments, the translation system is the E. coli S30 translation system (Promega, Madison, Wisconsin) for efficient in vitro translation. The E. coli S30 translation system advantageously provides highly efficient translation of various mRNA templates compared to other in vitro translation systems (e.g., wheat germ extract, rabbit reticulocyte lysate). Furthermore, the S30 system for in vitro translation is well characterized and highly suitable for preparing very large reaction mixtures, thus facilitating the construction of very large libraries according to the methods of the present invention. The S30 system is also suitable for the incorporation of unnatural amino acids using tRNA molecules charged with the unnatural amino acid. See PCT Patent Publication No. 90 / 05785, incorporated herein by reference. In some embodiments, the translation system is a synthetic mixture of recombinantly expressed and purified proteins involved in translation (e.g., PURExpress). These systems are advantageous because they allow for the elimination of different components to drive the construction of specific types of libraries (e.g., elimination of release factors).

[0165] In some embodiments, the polypeptide encoded by the RNA comprises a detection tag. In some embodiments, the detection tag is an epitope recognized by an antibody or a sequence that can be bound by another protein. In some embodiments, the polypeptide comprises HiBit, 6His, FLAG, HA, V5, a streptavidin tag, a GST tag, or an MBP tag. In some embodiments, binding of the detection tag to the detection agent produces a detectable signal (e.g., fluorescence).

[0166] In some embodiments, the translated peptide or polypeptide is retained within the particle. In some embodiments, the translated peptide or polypeptide does not diffuse from the particle. In some embodiments, the translated peptide or polypeptide is non-covalently bound to the particle or a molecule associated with the particle. In some embodiments, the peptide or polypeptide is covalently bound to the particle or a molecule associated with the particle. In some embodiments, the mRNA variously displays a puromycin tag, which results in either covalent or non-covalent attachment of the peptide to the mRNA.

[0167] In some embodiments, RNA template encodes a peptide or polypeptide that comprises multiple domains.For example, in some embodiments, RNA template encodes a peptide or polypeptide of interest and a detection tag.In some embodiments, RNA template encodes multiple detection tags.

[0168] In some embodiments, the domains are separated by a linker sequence. In some embodiments, the domains are separated by a flexible linker. In some embodiments, the linker sequence comprises glycine and serine. In some embodiments, the linker is an amino acid sequence (G n S) X where n is 1 to 4 and x is 1 to 5. In some embodiments, the linker comprises the amino acid sequence G4S (SEQ ID NO: 19).

[0169] In some embodiments, the RNA template comprises one or more modifications. In some embodiments, the RNA template comprises a 3' modification. In some embodiments, the RNA template comprises a 3' puromycin-modified nucleotide.

[0170] In some embodiments, the translated peptide or polypeptide is about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, or about 5 to about 10 amino acids in length.

[0171] In some embodiments, the method further comprises detecting and / or isolating particles having desired properties. In some embodiments, the particles are isolated using flow cytometry, cell sorting devices, density measurements, affinity tag detection, and / or DNA sequence detection. In some embodiments, fluorescence microscopy and flow cytometry are used to visualize specific epitope translation within the particles.

[0172] In some embodiments, translated gel particles exhibit homogenous antibody-derived fluorescence throughout the gel periphery, while only magnetic bead autofluorescence is detected in the same particles prior to translation. In some embodiments, the fluorescence of translated gel particles by flow cytometry is baseline resolved compared to untranslated particles for selected epitopes such as FLAG, HA, or V5.

[0173] In some embodiments, translation of gel-immobilized RNA and subsequent immobilization of the translation product in gel particles is verified by orthogonal detection of multiple epitope tags. In some embodiments, the translation product immobilized in the gel particles is a peptide. In some embodiments, the translation product immobilized in the gel particles is a therapeutic agent. In some embodiments, immunofluorescence measurements are used to confirm the presence of each epitope in the immobilized or captured translation product in the gel particles by specific epitope tagging and in-gel protein binding assays. In some embodiments, in-gel peptide capture yields are quantified by an in vitro translation efficiency assay based on HiBiT luminescence.

[0174] In some embodiments, gel particle libraries prepared by the methods provided herein are screened by FACS. For example, in some embodiments, the methods provided herein result in gel particle libraries of peptide templates with degenerate codons fused to HiBiT tags that are used to template magnetic beads via limiting dilution emulsion PCR (emPCR). In some embodiments, single-bead qPCR analysis of templated library beads shows greater than 4000 DNA templates / bead, with approximately 30% of the beads being templated. In some embodiments, library beads (5×10 6 ) are combined with control epitope beads (e.g., 1% HA or V5), translated in the presence of puromycin oligonucleotide P4, probed with dye-labeled antibodies (e.g., anti-HA-647AF; anti-V5-CF488A), and sorted by FACS into two populations: high V5 and high HA. The selected hit particles are sequenced to determine the enrichment of various control tags. Reads are pattern-matched to the sequences of the epitope tags or degenerate sequences of the library. In some embodiments, the HA-positive hit pool is 99% HA-encoding sequences, and the V5-hit pool is 50% V5-encoding sequences, representing 100-fold and 50-fold enrichment from the library starting material.

[0175] In some embodiments, the library-scale synthesis and selection provided herein are compatible with engineered genetic codes. For example, in some embodiments, beads are functionalized with a DNA template encoding a single codon (e.g., the natural Gln codon) fused with a HiBiT tag and encapsulated in a P1-functionalized gel. A CUGtRNA codon lacking glutamine and glutamine-tRNA synthetase (GlnRS) is synthesized using dFx flexizyme. Asn GGCAn engineered in vitro transcription and translation (IVTT) mixture containing the non-canonical amino acid azidolysine (AzK) loaded onto the IVTT mix is ​​used to exhibit azide functionality via IVTT. In some embodiments, HiBiT quantitation yields similar levels of captured peptide (10±7 nM) to the total native peptide epitopes captured in several other embodiments and examples provided herein. In some embodiments, to generate an engineered IVTT mix, the methods described herein are used to prepare a gel particle library of peptide templates in which engineered codons are fused to HiBiT tags and exhibit AzK functionality.

[0176] In some embodiments, a peptide library template with 5NNT degenerate codons (excluding the GAC codon) was used to template magnetic beads by emPCR, yielding an average of 2 x 10 4 After gel encapsulation, the NNT library beads (3 × 10 6 The 647AF-rich population (approximately the top 2%) is then sorted by FACS. After sequencing and decoding, a roughly six-fold enrichment of GAC-templated sequences is observed in the sorted population compared to the starting library pool.

[0177] In some embodiments, provided herein are methods that result in the translation and detection of non-canonical amino acid-containing gel particle library beads. For example, in some embodiments, NNU library particles (3×10 6(1000) are subjected to an engineered in vitro translation (IVT) reaction to introduce an azido-lysine (AzK) at the CUG codon. The translated particles are washed and treated with AF647-alkyne in a CuAAC reaction, followed by analysis by flow cytometry. The resulting particles are sorted to isolate the top 2% of the AF647 population, and the sequences are compared to the starting library. In some embodiments, a 6-fold enrichment is observed after one round of screening, where the percentage of AzK sequences increases from approximately 0.09% in the original NNU library particles to approximately 0.58% after sorting and isolating the top 2% of the AF647 population using FACS.

[0178] In some embodiments, a standard PCR protocol for library generation is provided herein, where magnetic beads were templated with several control epitope-encoding sequences, doped into a conventional library as background, and successfully selected as hits, showing substantial enrichment compared to the library input.

[0179] In some embodiments, provided herein is coupled transcription / translation of mixed library / epitope particles without additional compartmentalization, resulting in sufficiently pure in-gel epitope display for immunofluorescence-based screening. Translation products, like RNA transcripts, are concentrated in the gel matrix of DNA-templated magnetic beads, demonstrating that all enzymatic steps are proximity-driven.

[0180] VI. Kits and Libraries In some embodiments, provided herein are kits for preparing particles comprising a polymer coating and a magnetic core. In some embodiments, the kits comprise a monomer. In some embodiments, the kits comprise acrylamide and bisacrylamide. In some embodiments, the kits comprise one or more polymerization initiators. In some embodiments, the kits comprise an oily solution and an aqueous solution. In some embodiments, the kits comprise an oily solution comprising a polymerization initiator and an aqueous solution comprising a monomer. In some embodiments, the aqueous solution comprises magnetic beads. In some embodiments, the aqueous solution comprises an initiator. In some embodiments, the aqueous solution comprises TEMED. In some embodiments, the oily solution is in one container and the aqueous solution is in another container. In some embodiments, emulsification of the oily solution with the aqueous solution results in polymerization of the monomer.

[0181] In some embodiments, the kit further comprises instructions for producing particles according to the methods provided herein, hi some embodiments, the kit comprises instructions for producing a library of particles provided herein.

[0182] Also provided herein are libraries of particles. In some embodiments, the library includes particles with different oligonucleotide tags. In some embodiments, the library includes particles with different building blocks. In some embodiments, the library includes particles with different small molecules. In some embodiments, the library includes particles with different mRNA sequences. In some embodiments, the library includes particles with different peptides or polypeptides. In certain embodiments, the library includes particles with about 10 2 ~10 10 (e.g., about 10 2 ~10 3 , 10 2 ~10 4 , 10 2 ~10 5 , 10 2 ~10 6 , 10 2~10 7 、10 2 ~10 8 、10 2 ~10 9 、10 3 ~10 4 、10 3 ~10 5 、10 3 ~10 6 、10 3 ~10 7 、10 3 ~10 8 、10 3 ~10 9 、10 3 ~10 10 、10 4 ~10 5 、10 4 ~10 6 、10 4 ~10 7 、10 4 ~10 8 、10 4 ~10 9 、10 4 ~10 10 、10 5 ~10 6 、10 5 ~10 7 、10 5 ~10 8 、10 5 ~10 9 、10 5 ~10 10 、10 6 ~10 7 、10 6 ~10 8 、10 6 ~10 9 、10 6 ~10 10 、10 7 ~10 8 、10 7 ~10 9 、10 7 ~10 10 、10 8 ~I0 9 、10 8 ~10 10 、10 9 ~10 10) particles. In some embodiments, each particle comprises a different set of oligonucleotides, peptides, small molecules, polypeptides, or building blocks. In some embodiments, multiple particles in the library comprise the same set of oligonucleotides, peptides, small molecules, polypeptides, or building blocks. In some embodiments, each particle in the library comprises an identifying sequence that can be used to identify the set of building blocks, peptides, small molecules, or polypeptides. [Example]

[0183] Example 1 - Versatile functionalization of polyacrylamide hydrogel magnetic particles by copolymerization This example demonstrated that diverse functionalization of polyacrylamide hydrogel-coated magnetic particles can be achieved by copolymerization. Radical-mediated polymerization created polyacrylamide hydrogels incorporating various functionalities into the polymer matrix by including molecules with both the desired functionality and vinyl groups for copolymerization. Various copolymerized functionalities were used, including but not limited to crosslinkers, cell adhesion promoters (e.g., alkylamines), affinity capture tags (e.g., halotags for chloroalkanes), enzyme capture, probe capture, and oligonucleotides for hybridization (Figure 18). The latter two are useful for labeling and characterization.

[0184] Using this methodology, we synthesized polyacrylamide hydrogels bearing both chemical (e.g., amine, azide) and biochemical (e.g., oligonucleotide) groups. We also demonstrated that these groups can participate in reactions within the hydrogel matrix of particle systems.

[0185] method Azidooligonucleotide preparation: 5'-NH2P1 DNA oligonucleotide primer was azide-modified with EDC, NHS, and 5-azidopentanoic acid to give 5'-N3P1, followed by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) coupling of 5'-N3P1 to propargyl-modified magnetic microbeads (e.g., Dynabeads, 1.0 μm, 2.8 μm, or 10.0 μm diameter) as previously reported (Malone et al. ACS Comb. Sci. 2017, 19, 1, 9-14).

[0186] Methacrylamide DNA headpiece (ac-HDNA) functionalization: Methacrylic acid (100 μmol) was added to NHS (100 μmol) and EDC (100 μmol) in DMF, and the esterification reaction was incubated at room temperature for 5 min. A methacrylic acid NHS ester solution (1 M, 15 μL) was added to HDNA (100 nmol) in phosphate buffer (0.4 M, pH 7.9), and the reaction was incubated at room temperature for 1.5 h. It was then quenched by the addition of Tris buffer (20 μL, 1 M). Methacrylamide-HDNA (ac-HDNA) was purified by reverse-phase HPLC purification (Waters XTerra C18, 2.5 µm, 10 mm x 50 mm) using gradient elution (mobile phase A: 97.5% 50 mM TEAA pH 8; mobile phase B: ACN; 2.5% B 1 min, 2.5–12.5% ​​B 20 min, 4 mL / min) and absorbance detection (260 nm). Fractions containing the desired product were pooled, dried, and reconstituted in HPLC-grade water for quantification at A260. Fractions of interest were spotted (1 μL) onto a MALDI-TOF MS target plate, dried, and covered with THAP matrix solution (1 μL, 18 mg / mL THAP, 7 mg / mL ammonium citrate dibasic in 1:1 ACN:HO), dried, and analyzed by MALDI-TOF MS (Microflex, Bruker Daltonics, Inc., Billerica, MA).

[0187] Preparation of hydrogel particles: Aliquots of carboxylic acid functionalized magnetic beads (M-270 carboxylic acid Dynabeads, 5 × 10 7Aqueous solution (ThermoFisher Scientific, ThermoFisher Scientific) was added to a 1.5 mL microcentrifuge tube and magnetically isolated (Figure 11). The supernatant was removed, and the beads were suspended in acrylamide monomer solution (300 μL, 0.5 M 19:1 mono:bisacrylamide, 0.6% APS). Oil (900 μL, 4 / 20 / 76 w / w, KF-6038 / mineral oil / DMF-A-6cs) was layered onto the bead suspension, and the separated oil / aqueous sample was sparged with Ar (10 min). TEMED (1 μL) was added to the oil layer, the headspace in the tube was filled with Ar, and the sample was emulsified using a bead mill homogenizer (65 s, 2500 rpm, BeadBug, Benchmark Scientific, Sayreville, NJ) and then incubated on ice (2 h, 4 °C). The polymerized gel particles were magnetically isolated, the supernatant removed, and the particles washed with disruption buffer (4 × 1 mL) until no traces of oil remained. The gel particles were suspended in bead buffer (1 mL) for storage. The gel particles were variously functionalized by adding the following reagents to the acrylamide monomer solution: propargyl methacrylate (20 μM, "alkyne" functionality), N-(3-aminopropyl)methacrylamide (0.002–20 mM, "amine" functionality), ac-P1 (20 μM, "reverse primer" functionality), or ac-HDNA (20 μM, HDNA functionality).

[0188] In-gel oligonucleotide hybridization: Load gel particles with FAM-P1' (1 µM, 20 pmol / 1 x 10 in 2 x SSC, 0.5% SDS). 6 The gel particles were combined with the FAM-P1' beads (the FAM-P1' sequence is shown in SEQ ID NO: 4) and incubated for 2 minutes at room temperature. The gel particles were magnetically isolated, washed (2 x 200 μL 2 x SSC, 0.5% SDS), and suspended in bead buffer (250 μL) for storage and analysis.

[0189] In-gel hybridization of activity-based probes: Gel particles were hybridized with trypsin activity-based probes copolymerized with oligonucleotides and subsequently conjugated to complementary oligonucleotides. The probe-hybridized gel beads were digested with trypsin to dequench the activity-based probes, resulting in fluorescent gel particles (Figure 19).

[0190] Intragel amine acylation: gel particles (1 × 10 7 ) was suspended in phosphate buffer (0.2 M, pH 7.5, 100 μL), combined with NHS-647AF (1 nmol), and incubated at room temperature for 1 h. The gel particles were magnetically isolated, washed with bead buffer (2 × 200 μL), and suspended in bead buffer (200 μL) for storage and analysis.

[0191] In-gel CuAAC (Cu(I)-catalyzed azide-alkyne cycloaddition): gel particles (1 × 10 7 ) was suspended in reaction buffer (1 M TEAA pH 7, 0.5% Tween-20, 100 μL) and combined with N3-647AF (5 nmol). Catalyst mixture (50 nmol CuSO4, 250 nmol ascorbic acid, 60 nmol THPTA) was added, and the reaction was incubated at room temperature for 1 h. Gel particles were magnetically isolated, washed with bead buffer (2 × 200 μL), and suspended in bead buffer (200 μL) for storage and analysis.

[0192] In-gel DNA ligation: ac-H DNA (20 μM) and copolymerized gel particles (2 × 10 6 The gel particles were suspended in T4 ligase buffer (20 μL, NEB) containing enzymatic ligation substrate DNA oligonucleotides (L(+) and L(-), 50 μM each), heated (2 min, 95 °C), and cooled to room temperature. The heat-treated reaction mixture was divided in half, T4 ligase (20 U, NEB) was added to one aliquot, and the sample was incubated at room temperature for 1 h. The gel particles were magnetically isolated, washed with bead buffer (2 × 200 μL), and suspended in bead buffer (200 μL) for storage and analysis.

[0193] Confocal fluorescence imaging (particle analysis): Gel particles hybridized with FAM-P10' were imaged by a confocal fluorescence microscope (Stellaris 8, Leica). Particles (approximately 10 6 ) was loaded into the imaging well and allowed to settle (15 min). Particle fluorescence (l ex / l em = 488 / 520 nm) was obtained and used to calculate the object diameter (LAS).

[0194] Flow cytometry (particle analysis): Gel particles were analyzed by flow cytometry (NovoCyte, Agilent). For analysis, beads in buffer (1–5 × 10 4 Gel particles were suspended in 100 μL of PBS (beads / μL). Particles were gated based on forward scatter (FSC) and side scatter (SSC) to isolate single particle populations (Figure 7). Population fluorescence was measured in various channels (FAM λ, FAM λ) based on the dye label used to probe the gel particles. ex / λ em = 488 / 530 nm; TMR λ ex / λ em = 561 / 580 nm; APC λ ex / λ em = 640 / 660 nm).

[0195] result 2.8 µm magnetic beads were encapsulated in hydrogels exhibiting diverse functionalities (Figure 1A-E). Specifically, oligonucleotides with commercially available 5'-acryldiite modifications or amino oligonucleotides modified with methacrylic acid (Figure 2), alkynes with propargyl methacrylate (PMA) (Figure 1D), or primary amines with 3-(aminopropyl)methacrylamide (APMA) (Figure 1E) were produced. Gel particles were copolymerized with 5'-methacrylamide oligonucleotides (Figure 1B), methacrylamide-modified hairpin headpiece DNA (HDNA) (Figure 1C), propargyl methacrylate (PMA) (Figure 1D), or 3-(aminopropyl)methacrylamide (APMA) (20 µM additive) (Figure 1E). Gel functionalization was detected by chemical reactions with fluorescent dye-labeled complementary functional groups, including complementary oligonucleotide hybridization (Figure 1B), enzymatic ligation of the dsDNA modules used for DEL synthesis (Figure 1C), CuAAC with Alexa Fluor 488 azide (488AF-N3) (Figure 1D), or amine acylation with fluorescein succinimidyl ester (FAM-OSu) (Figure 1E). Gel labeling was detected by flow cytometry and compared to blank gel beads (Figure 1, right).

[0196] Copolymerization of 5'-methacrylamide-modified DNA oligonucleotide P1 (acrydite) yielded P1-functionalized gel particles, which were hybridized with a 5'-fluorescein (FAM)-labeled DNA oligonucleotide complementary to P1 (FAM-P1') and analyzed by confocal fluorescence imaging (Figure 10A). Across nine preparations (32,000 sampled particles), a median diameter of 7 ± 2 µm (Figure 10B) was observed. Flow cytometry analysis of gel particles probed with FAM-P1' yielded a distinct signal population with a 100-fold increase in fluorescence intensity compared to unprobed gel particles, indicating that >95% of the magnetic beads were encapsulated in the hydrogel (Figure 10C).

[0197] Increasing magnetic bead size produced larger, more uniform gel particle distributions (Figure 13) by imaging microscopy (Figure 9), and all bead sizes (e.g., 1.0 μm, 2.8 μm, and 10.0 μm diameter magnetic beads encapsulated in hydrogels, respectively) produced populations that were easily gated by forward and side scatter in flow cytometry for comparison of hydrogel fluorescence upon probing (Figure 7).

[0198] Functionalization of hydrogel particles was detected and characterized by flow cytometry by the addition of appropriate fluorescent dye-labeled substrates, including complementary oligonucleotides, double-stranded oligonucleotide ligation modules, azides, or succinimidyl esters, respectively. Depending on the reactivity, relative fluorescence increased 5- to 25-fold compared to naive gel particles (Figure 1, right). Hybridization (Figure 1B, right) and amine acylation (Figure 1E, right) resulted in the largest shifts; all functionalization reactions resulted in baseline separation of product and starting material by flow cytometry (Figure 1, right).

[0199] Hydrogel particles were also tested for enzyme activity-based assays. For example, hydrogel particles were first copolymerized with oligonucleotides and then hybridized with a trypsin activity-based probe conjugated to a complementary oligonucleotide. The probe-hybridized gel beads were then digested with trypsin, dequenching the activity-based probe and yielding fluorescent gel particles. Figure 17A shows time-dependent quantification of hydrogel particles loaded with a fluorogenic (turn-on) green fluorescent probe for trypsin activity. Figure 17B shows time-dependent quantification of hydrogel particles loaded with an N-terminally labeled tryptic peptide, which is N-terminally labeled with the red fluorescent dye Cy5. Upon trypsin digestion, Cy5 is released from the gel, resulting in a decrease in the fluorescent signal over time (turn-off).

[0200] Hydrogel particles with increasing amine loading capacity were prepared by copolymerizing increasing concentrations of APMA (0.02–20 mM). Acylation with FAM-OSu resulted in a log-linear increase in gel particle fluorescence over four orders of magnitude (Figure 3A,B).

[0201] Hydrogel particles were variably functionalized by including substoichiometric methacrylamide-modified additives in the acrylamide:bisacrylamide monomer solution for copolymerization. Incorporation of synthetic hairpin headpiece DNA (HDNA) and subsequent proof-of-concept enzymatic oligonucleotide ligation reactions demonstrated the feasibility of standard DEL synthesis workflows using these particles.

[0202] The particle loading capacity of amine functionality was demonstrated to be quantitative over four orders of magnitude: at the highest site density (20 mM), each median particle (7 μm diameter) had approximately 4 fmol of amine sites.

[0203] conclusion In this example, magnetic core hydrogel particles successfully facilitated diverse functionalization via copolymerization and subsequent chemical and biochemical solid-phase synthesis-like procedures. Radical-mediated polymerization is mild and suitable for incorporating a variety of acryloyl and methacryloyl moieties. While this example demonstrates some of the most common functionalities deployed in chemical library synthesis, the technique can be applied to many other functionalities. This example should not be construed as limiting the scope of the invention.

[0204] Furthermore, time-dependent fluorescence data (Figure 17A,B) demonstrate the utility of the hydrogel scaffold for facilitating biochemical enzyme activity assays. The in-gel assay format can be used, for example, to detect the presence of inhibitor elements (e.g., DNA-encoded library members, aptamers, peptides) that are also synthesized in the gel. In-gel assays can also be used to probe catalytic activity in enzyme engineering experiments, where each particle contains a nucleic acid template for in vitro translation.

[0205] Example 2—In-gel transfer of DNA and subsequent capture of the resulting mRNA by hybridization to the hydrogel periphery of magnetic beads While enzymatic ligation plays an important role in the preparation of DNA-encoded chemical libraries, DNA transcription is central to the preparation of genetically encoded RNA and protein libraries. This example demonstrates that RNA transcribed from a bead-bound DNA template is trapped in the surrounding hydrogel by complementary RNA sequences in the hydrogel.

[0206] method Bead-templated PCR (single template): P1-functionalized magnetic microbeads (1 × 10 8 ) were suspended in PCR mix (0.4 mM dNTPs, 4 μM forward primer P2, 0.1 U / μL Taq DNA polymerase in 1× standard Taq buffer) containing DNA template (1 pmol / μL) and thermocycled ([95°C, 20 s; 60°C, 20 s; 68°C, 20 s] × 25 cycles, C1000 Touch, Bio-Rad). The beads were washed with bead buffer (4 × 1 mL) and suspended in bead buffer (1 mL) for analysis.

[0207] A qPCR mix (0.2 mM each dNTP, 0.5 μM each P1 and P2 primer, 0.25 μM qPCR probe (qPCR probe sequence shown in SEQ ID NO: 7), 0.05 U / μL Taq polymerase, and 1× standard Taq buffer) was prepared and aliquoted into a 96-well PCR plate (20 μL each). Dilutions of templated bead samples (1 / 100 and 1 / 1000) were prepared in bead buffer. qPCR wells were assembled by adding 1 μL of the diluted suspension. The reactions were thermal cycled ([95°C, 20 s; 60°C, 20 s; 68°C, 20 s] × 40 cycles) and fluorescence was monitored (530 nm, QuantStudio3, Thermo Scientific). A fixed volume (1 µL) was added to each standard reaction (logarithmically ranging from 100 pg / µL to 0.1 fg / µL) to prepare a standard curve using serial dilutions of the template. Beads were counted by hemocytometer to obtain the average per bead-template load.

[0208] In-gel transfer: Non-templated beads labeled with 647AF and beads templated with DNA encoding the FLAG epitope (FLAG-templated beads) were encapsulated in hydrogels copolymerized with P1 (50 μM) in separate emulsion polymerizations. The hardened gel particles (1 × 10 7 Each non-templated and FLAG-templated sample was suspended in a T7 RNAP reaction mixture (0.5 mM NTP, 5 mM DTT, 5 U / μL T7 RNAP, 0.4 U / μL TIPP, 1 μM P3 in 1× NEB T7 buffer) and incubated (1 h, 37°C). The particles were washed with disruption buffer (2× 500 μL) and suspended in bead buffer (400 μL) for storage and analysis.

[0209] Flow cytometry (particle analysis): Gel particles were analyzed by flow cytometry (NovoCyte, Agilent). For analysis, beads in buffer (1–5 × 10 4Gel particles were suspended in 100 μL of PBS (beads / μL). Particles were gated based on forward scatter (FSC) and side scatter (SSC) to isolate single particle populations (Figure 7). Population fluorescence was measured in various channels (FAM λ, FAM λ) based on the dye label used to probe the gel particles. ex / λ em = 488 / 530 nm; TMR λ ex / λ em = 561 / 580 nm; APC λ ex / λ em = 640 / 660 nm).

[0210] result DNA-templated 2.8 μm magnetic beads were mixed with non-templated dye-labeled (Alexa Fluor 647, 647AF) beads as a negative control (Figure 4A). RNA transcripts were detected by including a FAM-labeled DNA oligonucleotide probe of the 5' region (P3) of the RNA transcript (Figure 4B). After in vitro transcription of the bead mixture, flow cytometry analysis showed the presence of three bead populations: 38% high 647AF fluorescence (i.e., non-templated negative control beads), 59% high FAM fluorescence (i.e., DNA-templated beads), and 2% fluorescence in both channels (Figure 4C).

[0211] DNA-templated beads were prepared by PCR using DNA oligonucleotide primer P1 (Figure 4A) functionalized magnetic beads (Figure 4A) and DNA oligonucleotide primer P2 (Figure 4A). The DNA template contained a T7 RNA polymerase promoter element (T7 prom, Figure 4A). Non-templated negative control beads were labeled with Alexa Fluor 647 (647AF, Figure 4A). Each bead set was encapsulated in a P2 copolymer hydrogel. In vitro transcription of the gel-encapsulated DNA-templated and non-templated beads in the presence of a FAM-labeled DNA oligonucleotide probe of the 5' region of the RNA transcript (Figure 4B, P3) detected the presence of the RNA transcript hybridized via P2 in the gel. Two-dimensional flow cytometry analysis showed that the majority of particles exhibited either exclusively red fluorescence (38%) (non-templated negative control in Figure 4C, 660 nm, Q1) or green fluorescence (59%) (templated and RNA-loaded beads in Figure 4C, 520 nm, Q3).

[0212] conclusion Flow cytometry results showed that RNA was captured by hybridization at the hydrogel periphery of the magnetic beads. The results also showed that transcript migration to non-template beads was not only rare but also occurred as a distinct population. Therefore, the low level of transcript migration makes this method suitable for producing libraries with a large number of different templates.

[0213] Example 3—In-gel translation of peptides and capture of translated peptides in the hydrogel periphery of magnetic beads This example demonstrated in-gel translation of peptides and subsequent capture or immobilization of the peptide products in the hydrogel periphery of magnetic beads.

[0214] method In-gel translation (biogenic amino acids): DNA-templated beads were encapsulated in hydrogels copolymerized with ac-P1 (50 μM) and acrylamide-modified BSA (50 μM). BSA (2 μmol) and N-acryloxysuccinimide (20 μmol) were combined in buffer (20 mM phosphate pH 7.5, 20 μL) and incubated at room temperature for 1 h. Hydrogel particles (1 × 10 5 particles / µL, final volume 10–70 µL) in NEB PURExpress containing puromycin capture oligonucleotide P4 (10 µM) (登録商標) The reaction mixture was incubated (3 h, 37 °C), washed with disruption buffer (0.5 mL), washed with PBST (0.5 mL), and then washed with PBST (10 mL). 4 The suspension was suspended in 0.1 mL of 0.1% ethanol (particles / μL).

[0215] In-gel translation (unnatural amino acids): Reagents and protocols for in vitro translation of non-canonical amino acids were adapted from previously described methods (Adaligil et al. ACS Chem. Biol. 2021, 16, 6, 1011-1018, Murakami et al. Nature Methods 2006, 3, 357-359).

[0216] In-gel immunofluorescence detection: Translated particles were suspended in detection antibody solution (4 × 10 particles / μL, 10 ng / μL APC anti-HA, 50 ng / μL CF488 anti-V5, 10% non-fat milk in PBST), incubated (16 h, 4 °C), washed with PBST (0.5 mL), and suspended in PBST (1 mL) for flow cytometry.

[0217] Flow cytometry (particle analysis): Gel particles were analyzed by flow cytometry (NovoCyte, Agilent). For analysis, gel particles were suspended in bead buffer (1–5 × 104 beads / µL). Particles were gated based on forward scatter (FSC) and side scatter (SSC) to isolate single particle populations (Figure S2). Population fluorescence was measured in various channels (FAM λ) based on the dye label used for the gel particle probe. ex / λem = 488 / 530 nm; TMR λ ex / λ em = 561 / 580 nm; APC λ ex / λ em = 640 / 660 nm).

[0218] HiBiT assay: Sample (5 µL of solution in 5 µL PBS or 2.5 x 10 4 The LgBiT / substrate mixture (5 µL, 2x LgBiT / substrate in PBS) was combined with the LgBiT / substrate mixture (5 µL, 2x LgBiT / substrate in PBS) in a black, 384-well microtiter plate and incubated for 15 min at 37 °C. A HiBiT peptide standard solution (0.1–1000 nM HiBiT peptide) was similarly assembled and incubated. Luminescence was analyzed using a plate reader (CLARIOstar Plus, BMG LABTECH), and unknown concentrations were determined based on the HiBiT standard analysis (Figure 8).

[0219] result Magnetic beads were templated with DNA encoding various affinity tag epitopes (FLAG, HA, or V5) fused to a HiBiT luciferase complementary tag and encapsulated in P1-functionalized gels. The gel particles were subjected to mRNA-display in vitro transcription / translation reactions incorporating a puromycin-modified peptide capture oligonucleotide, P4, complementary to the RNA immediately 3′ of the stop codon (Figure 5A). The translated beads were analyzed by imaging microscopy and flow cytometry to visualize specific epitope translation. Translated gel particles displayed homogeneous antibody-derived fluorescence throughout the gel periphery (Figure 5B); only magnetic bead autofluorescence was detected in the same particles prior to translation (Figure 5C). Flow cytometric fluorescence of translated gel particles was baseline-separated compared to untranslated particles for all three exemplary epitopes (Figure 5D). In-gel peptide capture yield was quantified via HiBiT luminescence. Particles translated in the presence of puromycin capture oligonucleotide P4 retained approximately 100 nM of HiBiT peptide, whereas translations lacking P4 retained less than 1 nM ( Figure 5E ).

[0220] conclusion Orthogonal detection of multiple epitope tags in this example successfully demonstrated translation of gel-immobilized RNA and subsequent capture or immobilization of the translation products in gel particles. Immunofluorescence measurements confirmed the presence of each epitope via specific epitope tagging, demonstrating proof-of-concept for performing in-gel protein binding assays. HiBiT detection further confirmed the presence of the captured peptides and enabled sensitive quantification of the captured peptides. The in-gel peptide capture yield (approximately 100 nM) was sufficient for robust detection in both the immunofluorescence and HiBiT assays.

[0221] Example 4—Compatibility of Polyacrylamide Hydrogel Magnetic Particles with FACS Analysis as a High-Throughput Screening Strategy This example demonstrated the utility of the novel gel particle format by showing its compatibility with FACS analysis as a high-throughput screening strategy. Furthermore, the data also demonstrated that library-scale synthesis and selection are compatible with engineered genetic codes.

[0222] method Bead-templated PCR (single template): P1-functionalized magnetic microbeads (1 × 10 8 ) were suspended in PCR mix (0.4 mM dNTPs, 4 μM forward primer P2, 0.1 U / μL Taq DNA polymerase in 1× standard Taq buffer) containing DNA template (1 pmol / μL) and thermocycled ([95°C, 20 s; 60°C, 20 s; 68°C, 20 s] × 25 cycles, C1000 Touch, Bio-Rad). The beads were washed with bead buffer (4 × 1 mL) and suspended in bead buffer (1 mL) for analysis.

[0223] A qPCR mix (0.2 mM each dNTP, 0.5 μM each P1 and P2 primer, 0.25 μM qPCR probe, 0.05 U / μL Taq polymerase, 1x standard Taq buffer) was prepared and aliquoted into a 96-well PCR plate (20 μL each). Dilutions of templated bead samples (1 / 100 and 1 / 1000) were prepared in bead buffer. qPCR wells were assembled by adding 1 μL of the diluted suspension. The reactions were thermal cycled (95°C, 20 s; 60°C, 20 s; 68°C, 20 s) for 40 cycles, and fluorescence was monitored (530 nm, QuantStudio3, Thermo Scientific). A fixed volume (1 μL) was added to each standard reaction (logarithmically ranging from 100 pg / μL to 0.1 fg / μL) to prepare a standard curve using serial dilutions of the template. Beads were counted by hemocytometer to obtain an average per bead template load.

[0224] Bead-templated emPCR (NNK library template): P1-functionalized magnetic microbeads (1 × 10 8 ) was suspended in PCR mix (0.2 mM each dNTP, 8 μM forward primer P2, 0.02% w / v KF-6102, 0.3 U / μL Taq DNA polymerase in 1× standard Taq buffer) containing NNK5 DNA template (1.2 fg / μL). Oil (900 μL, 4 / 20 / 76, KF-6038 / mineral oil / DMF-A-6CS, w / w / w) was added to the top of each aqueous reaction mixture. Reactions were emulsified (65 s, 2500 rpm) using a bead mill homogenizer (BeadBug, Benchmark Scientific). Aliquots (50 μL) were transferred to a 96-well PCR plate using a wide-bore pipette tip, and the samples were thermocycled (35 cycles of [95°C, 20 s; 60°C, 20 s; 68°C, 30 s], 68°C, 5 min). The plate was placed on a magnetic stand and incubated (30 min), the supernatant was removed, and the isolated beads were transferred to a clean 1.5 mL tube in disruption buffer and washed with disruption buffer (4 x 1 mL). The washed beads were isolated and suspended in bead buffer (1 mL) for analysis.

[0225] A qPCR mix (0.2 mM each dNTP, 0.5 μM each P1 and P2 primer, 0.25 μM qPCR probe, 0.05 U / μL Taq polymerase, 1× standard Taq buffer) was prepared and aliquoted into a 96-well PCR plate (20 μL each). Beads were counted using a hemocytometer to prepare suspensions in bead buffer of known densities (100 beads / μL and 1 bead / μL). Three 100-bead and 77 single-bead qPCR wells were assembled by adding the appropriate suspension (1 μL). The reactions were thermocycled ([95°C, 20 s; 60°C, 20 s; 68°C, 20 s] × 40 cycles) and fluorescence was monitored (530 nm, QuantStudio3). A fixed volume (1 μL) was added to each standard reaction (logarithmically ranging from 100 pg / μL to 0.1 fg / μL) to prepare a standard curve using serial dilutions of the template.

[0226] Flow cytometry (library screening): Gel particles were screened by FACS (FACSAria III, BD Biosciences). Single particle populations (gated by FSC / SSC correlation as described above) were analyzed in various channels (FAM λ, λ λ) based on the dye label used for the gel particle probe. ex / λ em = 488 / 530 nm; APC λ ex / λ em Multispectral fluorescence at 640 / 660 nm was analyzed.

[0227] In-gel immunofluorescence detection: Translated particles were suspended in detection antibody solution (4 × 10 particles / μL, 10 ng / μL APC anti-HA, 50 ng / μL CF488 anti-V5, 10% non-fat milk in PBST), incubated (16 h, 4 °C), washed with PBST (0.5 mL), and suspended in PBST (1 mL) for flow cytometry.

[0228] NGS library preparation and analysis: Bead aliquots (approximately 1,000 beads) from each flow cytometry-sorted population were amplified using primers P1 and P2 and gel-purified to isolate amplicons within the template range (180–220 bp for all samples). Illumina sequencing libraries were constructed using the Bioo Scientific NEXTflex Rapid DNA-Seq kit and NEXTflex unique dual-index DNA barcodes (Bioo Scientific Corporation, Austin, TX). Five nanograms of amplicon samples were subjected to end repair, adenylation, and adapter ligation reactions. The adapter ligation products were cleaned using Agencourt AMPure XP beads (Beckman Coulter, Inc., Brea, CA) without library size selection. The cleaned ligated DNA was amplified for 10 PCR cycles to enrich for adapter ligation products. Amplified PCR products were cleaned with AMPure XP beads and quantified by Kapa qPCR (Kapa Biosystems, Inc., Wilmington, MA). Libraries were denatured and diluted to 12 pM for clustering on a MiSeq (Illumina Inc., San Diego, CA) using v2 MicroSR 300-cycle chemistry and dual indexing. Analysis was performed by aligning each sequence to a control epitope or library sequence and counting the percentage of matches (up to three mismatches) for each aligned sequence.

[0229] Aminoacyl-azidolysine-CUG-tRNA AsnSynthesis of: The aminoacylation reaction mixture (20 μL in 0.1 M bicine, pH 9.0, 20 μM tRNA, and 20 μM dFx Flexizyme) was heated (95°C, 3 min) and cooled to room temperature over 5 min. 20 mM MgCl2 was added, and the mixture was cooled on ice (5 min). The reaction was initiated by the addition of azidolysine 3,5-dinitrobenzyl ester (25 mM in DMSO) and incubated on ice for 2 h. After the acylation reaction, aminoacyl-Ne-azidolysine-CUG-tRNA was obtained. Asn The pellet was then precipitated (0.3 M NaOAc, pH 5.2, 100% EtOH, 10,000 g, 15 m). The pellet was rinsed (0.1 M NaOAc, pH 5.2, 70% EtOH) and dried.

[0230] In vitro translation of AzK-containing peptides: Ne-azidolysine-containing peptides were translated in a genetically reprogrammed in vitro translation system from recombinant E. coli, omitting glutamine and GlnRS to reprogram the Ne-azidolysine to a GAC ​​codon. Briefly, the in vitro translation reaction contained 50 mM HEPES pH 7.6, Mg(OAc)2, 100 mM KOAc, 1 mM DTT, 2 mM spermidine, 20 mM creatine phosphate, 2 mM ATP, 2 mM GTP, 1 mM CTP, 1 mM UTP, 0.2 mM each amino acid, and 1.5 mg / mL E. coli total tRNA, with a final protein concentration of 0.03 μM ArgRS, 0.09 μM GlyRS, 0.02 μM HisRS, 0.4 μM IleRS, 0.02 μM LeuRS, 0.11 μM LysRS, 0.68 μM PheRS, 0.04 μM SerRS, and 0.02 μM ValRS, 0.6 μM MTF, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 0.26 μM The hydrogel particles (10 μM) contained EF-G, 10 μM EF-Tu / Ts, 5 μM EF-P, 0.25 μM RF2, 0.17 μM RF3, 0.5 μM RRF, 1 μM T7 RNA polymerase, 3 μg / mL MK, 4 μg / mL creatine kinase, and 1.2 μM ribosomes. 5particles / µL, final volume 10–30 µL) with puromycin capture oligonucleotide P4 (10 µM) and Ne-azidolysine-CUG-tRNA Asn The reaction was incubated (3 h, 37 °C), washed with disruption buffer (0.5 mL), washed with PBST (0.5 mL), and then suspended in PBST (10 mL) for analysis. 4 The suspension was suspended in 0.1 mL of 0.1% ethanol (particles / μL).

[0231] Library-scale synthesis and selection using an engineered genetic code: Beads were functionalized with a DNA template encoding a single GAC codon (naturally encoding Gln) along with a HiBiT tag and encapsulated in a P1-functionalized gel. Using an engineered IVT mix to exclude glutamine, GlnRS was loaded with the non-canonical amino acid azidolysine (AzK) into CUGtRNAAsn using dFx flexizyme, allowing for specific display of the azide functionality using in vitro translation.

[0232] result A peptide library template with 5 NNK degenerate codons fused to a HiBiT tag was used to template magnetic beads by limiting dilution emPCR. Single-bead qPCR analysis of the templated library beads showed that approximately 30% of the beads were templated, with 4.2 kDa DNA template / bead. Library beads (5 x 10 6 ) were combined with control epitope beads (1% HA or V5), translated in the presence of puromycin oligonucleotide P4, probed with dye-labeled antibodies (anti-HA-647AF; anti-V5-CF488), and sorted by FACS into two populations (high V5 and high HA; Figure 6 ).

[0233] Selected hit particles were sequenced to determine the enrichment of various control tags. Reads were pattern-matched to epitope tag sequences or degenerate NNK5 library sequences. The HA-positive hit pool was approximately 99% HA-encoding sequence, and the V5-hit pool was approximately 50% V5-encoding sequence, representing approximately 100-fold and 50-fold enrichments from the library starting material (Figure 12).

[0234] The data also demonstrated that library-scale synthesis and selection were compatible with the engineered genetic code. HiBit quantification yielded similar levels of capture peptide (10 ± 7 nM) to all previously tested natural peptide epitopes (Figure 5E). We designed peptide library templates with 5 NNT degenerate codons (to eliminate the GAC codon) and generated encapsulated magnetic beads (Beads QC) bearing these sequences. NNT library beads (3 × 10 6 ) was combined with a small amount of our AzK-templated beads (0.1% of beads) (the azidolysine (AzK) peptide-coding sequence is shown in SEQ ID NO: 18), and translation was performed using an engineered IVT mix in the presence of P4. After translation, in-gel CuAAC was performed on the entire library mixture with 647AF-alkyne to label any AzK-displaying particles, and the 647AF-rich population (top 2%) was sorted by FACS (Figure 15). After high-throughput sequencing and decoding, we observed an approximately 6-fold enrichment of CUG-templated sequences in the sorted population compared to the starting library pool (Table 2) and (Figure 14).

[0235] conclusion These experiments in this example established the feasibility of FACS-based high-throughput screening of gel particle libraries. Using standard PCR protocols for library generation, magnetic beads were templated with several control epitope-encoding sequences and doped with a conventional library as background. Hits were successfully selected, demonstrating substantial enrichment compared to the library input. Importantly, coupled transcription / translation of mixed library / epitope particles without additional compartmentalization resulted in sufficiently pure in-gel epitope display for immunofluorescence-based screening. Translation products, such as RNA transcripts, were enriched in the gel periphery of DNA-templated magnetic beads. [Table 1] TIFF2026508113000002.tif211170TIFF2026508113000003.tif127170 [Table 2] Values ​​are the % of all sequences that match either the epitope or the NNU5 library reference.

Claims

1. A composition comprising particles comprising a magnetic core coated within a polymer gel.

2. The composition of claim 1 , wherein the polymer gel comprises polyacrylamide.

3. The composition of any one of claims 1 to 2, wherein the polymer gel comprises about 4% w / v to about 10% w / v polyacrylamide.

4. The composition of any one of claims 1 to 3, wherein the polymer gel comprises from about 10:1 to about 40:1 acrylamide to bisacrylamide.

5. 5. The composition of claim 4, wherein the polymer gel comprises 19:1 or 37.5:1 acrylamide to bisacrylamide.

6. The composition of any one of claims 1 to 5, wherein the particles further comprise an additive that inhibits phase separation, condensate formation, and / or coacervation.

7. The composition of claim 6 , wherein the additive comprises albumin.

8. The composition of claim 7, wherein the albumin is modified with one or more reactive groups.

9. The composition of any one of claims 1 to 8, wherein about 0.001 mM to about 20 mM of the polymer gel is functionalized.

10. The composition of any one of claims 1 to 9, wherein the particles are fluorescently labeled.

11. The composition of any one of claims 1 to 10, wherein the polymer gel has a pore size of about 20 nm to about 200 nm.

12. The composition of any one of claims 1 to 11, wherein the magnetic core is a magnetic bead.

13. The composition of claim 12, wherein the magnetic beads have a diameter of about 0.5 μm to about 10 μm.

14. 14. The composition of claim 13, wherein the magnetic beads have a diameter of about 1.0 μm, 2.8 μm, or 10 μm.

15. The composition of any one of claims 1 to 14, having a total particle diameter of less than about 40 μm, optionally having a total particle diameter of from about 6 μm to about 12 μm.

16. The composition of any one of claims 1 to 15, wherein the composition comprises particles having a uniform diameter distribution.

17. The composition of any one of claims 1 to 16, wherein the composition comprises particles having a particle diameter coefficient of variation of about 3% to about 50%.

18. 18. The composition of any one of claims 16 to 17, wherein the particles have an average total diameter of from about 5 μm to about 10 μm.

19. 19. The composition of any one of claims 16 to 18, wherein the particles have an average total diameter of about 7 μm.

20. 20. The composition of any one of claims 16 to 19, wherein at least 95% of the total particles in the composition comprise a magnetic core.

21. 1. A method for producing a composition comprising particles comprising a magnetic core encapsulated within a polymer gel, comprising: emulsifying an aqueous solution containing a monomer and magnetic beads with a solution containing a polymerization initiator to cause polymerization of the monomer, thereby producing a composition comprising a magnetic core encapsulated in the polymer gel.

22. 22. The method of claim 21, wherein the polymer gel comprises polyacrylamide.

23. 23. The method of claim 21 or claim 22, comprising emulsifying a solution comprising 4% w / v to 10% acrylamide monomer and magnetic beads with said solution comprising an initiator.

24. The method of any one of claims 21 to 23, wherein the solution comprising the monomers comprises acrylamide monomers and bisacrylamide monomers.

25. The method according to any one of claims 21 to 24, wherein the solution containing the monomer and magnetic beads is an aqueous solution.

26. 26. The method of any one of claims 21 to 25, wherein the initiator is in an oily solution, and optionally, the initiator is TEMED.

27. 27. The method of any one of claims 21 to 26, wherein the aqueous solution comprises ammonium persulfate.

28. 28. The method of any one of claims 21 to 27, wherein emulsifying the solution comprises vortexing, homogenizing, mixing, stirring, and / or shaking.

29. 29. The method of any one of claims 21 to 28, wherein the initiator causes polymerization of the acrylamide to coat the magnetic beads in the polyacrylamide gel.

30. 30. The method of any one of claims 21-29, further comprising combining the solution comprising acrylamide and bisacrylamide monomers and magnetic beads with the solution comprising a polymerization initiator to produce a combined composition prior to emulsification.

31. The method of any one of claims 21 to 30, wherein the combined composition comprises an oil phase and an aqueous phase.

32. 32. The method of any one of claims 21 to 31, further comprising sparging the combined composition with an inert gas.

33. 33. The method of claim 32, wherein the inert gas is argon.

34. 34. The method of any one of claims 21 to 33, further comprising applying a magnetic field to the composition to separate the particles.

35. 35. The method of any one of claims 21 to 34, further comprising removing the particle-free supernatant.

36. The method of any one of claims 21 to 35, further comprising washing the particles.

37. 37. The method of any one of claims 21 to 36, further comprising resuspending the particles.

38. The method of any one of claims 21 to 37, further comprising functionalizing a portion of the gel.

39. A particle produced by the method of any one of claims 21 to 38.

40. A library of particles produced by the method of any one of claims 21 to 39.

41. A kit comprising a composition comprising an aqueous solution containing magnetic beads and acrylamide, and a composition comprising an oily solution containing a polymerization initiator.

42. A kit according to claim 41 and instructions for use according to the method of any one of claims 21 to 38.

43. A kit comprising the composition of any one of claims 1 to 20 and instructions for use.

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