Magnetic hydrogel particles and methods of use

Abstract

The present invention relates to: a particle comprising a magnetic core coated in a polymeric gel; methods of making such particles; and methods of using such particles, e.g., for supporting both chemical synthesis and biosynthesis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 442,396, filed on January 31, 2023, and U.S. Provisional Application Serial No. 63 / 504,191, filed on May 24, 2023, which are hereby incorporated by reference in their entirety.

[0003] Statement on Federal Government Rights

[0004] This invention was made with government support awarded by the National Institutes of Health under Grant No. R35GM140890. The government has certain rights in this invention.

[0005] References to electronic sequence listings

[0006] The contents of the electronic sequence listing (146392063540SEQLIST.xml; size: 24,966 bytes; and creation date: January 12, 2024) are incorporated herein by reference in their entirety. Technical Field

[0007] The present invention relates to particles comprising a magnetic core encapsulated in a polymeric gel; methods of making such particles; and methods of using such particles, for example, to support both chemical and biological synthesis. Background Art

[0008] Split and pooled libraries of small molecules and biopolymers can be prepared via solid-phase chemistry and used in drug discovery activities. Each bead in these libraries is a spatially isolated clonal population of a single library member that can be queried individually. Initial work using these libraries has been limited to affinity-based interactions of compounds tethered to the beads. 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 determined that these particles can be used to deliver compounds for affinity and functional assays. While affinity-driven selection can be performed in batch reactions, functional screening requires compartmentalizing individual particles into a volume small enough to yield high local concentrations, such as in microfluidic droplets.

[0009] Although microfluidics can produce highly monodisperse droplets and particles, these systems have a high barrier to entry due to the high degree of specialization in manufacturing, assembly and operating skills. In contrast, every laboratory only needs minimal equipment to perform batch emulsification, and no specialized training is required. However, batch emulsification (e.g., via vortexing or shaking) typically results in droplet size variations exceeding several orders of magnitude. Particle templated emulsions (Hatori et al. Anal. Chem. 2018, 90, 16, 9813–9820) introduce single-sized particles into the emulsion volume, which serve as supports for droplet generation, obtaining a shell of an aqueous mixture around the particle support. This results in monodisperse droplets surrounding the solid support. But the final emulsion still contains a large number of “satellite droplets” that do not contain particles.

[0010] 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 on the scale of library synthesis or larger) while still resulting in uniform particles. Summary of the Invention

[0011] Provided herein is a composition comprising particles comprising a magnetic core encapsulated in a polymer gel. In some embodiments, the polymer gel comprises polyacrylamide. In some embodiments, the polymer gel comprises from about 4% w / v to about 10% w / v polyacrylamide. In some embodiments, the polymer gel comprises acrylamide to bisacrylamide in a ratio of from about 10:1 to about 40:1. In some embodiments, the polymer gel comprises acrylamide to bisacrylamide in a ratio of 19:1 or 37.5:1.

[0012] In some embodiments, the particles further comprise an additive that inhibits phase separation, aggregate formation, and / or aggregation. In some embodiments, the additive comprises albumin. In some embodiments, the albumin is modified with one or more reactive groups.

[0013] In some embodiments, the polymer gel of about 0.001 mM to about 20 mM 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 when 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 coefficient of variation in particle diameter 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.

[0014] In some embodiments, the present invention provides a method for producing a composition comprising particles comprising 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, and thereby producing a composition comprising a magnetic core encapsulated in the 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 solution comprising monomer comprises acrylamide monomer and bisacrylamide monomer. In some embodiments, the solution comprising monomer and magnetic beads is an aqueous solution. In some embodiments, the initiator is in an oil solution, optionally wherein 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 encapsulate the magnetic beads in the polyacrylamide gel.

[0015] In some embodiments, the method further comprises, prior to emulsification, combining the solution comprising acrylamide and bisacrylamide monomers and magnetic beads with a solution comprising a polymerization initiator to produce a combined composition. In some embodiments, the combined composition comprises an oil phase and an aqueous phase. In some embodiments, the method further comprises bubbling the combined composition with an inert gas. In some embodiments, the inert gas is argon.

[0016] 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 the supernatant that does not contain the particles. 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.

[0017] Provided herein is a particle produced by a method provided herein.

[0018] Provided herein is a particle library produced by the methods provided herein.

[0019] In some embodiments, the present invention provides a kit comprising: a composition comprising an aqueous solution comprising magnetic beads and acrylamide; and a composition comprising an oil solution comprising a polymerization initiator. Instructions for use of the kit are provided according to the methods provided herein.

[0020] Provided herein is a kit comprising particles provided herein and instructions for use according to the methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figures 1A to 1E Various gel particle functionalizations via copolymerization are shown. Figure 1A Blank gel beads are shown. Gel beads were mixed with 5'-methacrylamide oligonucleotides ( Figure 1B ), and methacrylamide-modified hairpin DNA (HDNA) ( Figure 1C ), and propargyl methacrylate (PMA) ( Figure 1D ), or with primary amines using 3-(aminopropyl)methacrylamide (APMA) ( Figure 1E ) for copolymerization. Gel functionalization is detected by chemical reactions involving complementary functional groups labeled with fluorescent dyes, including hybridization of complementary oligonucleotides ( Figure 1B ), enzymatic ligation of double-stranded (dsDNA) modules for DEL synthesis ( Figure 1C ), Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) using Alexa Fluor 488 azide (488AF-N3) ( Figure 1D ), or acylation with fluorescein succinimidyl ester (FAM-OSu) ( Figure 1E Gel markers were detected by flow cytometry and compared with blank gel beads ( Figures 1A to 1E , right).

[0022] Figure 2The structure and characterization of methacrylamide-modified HDNA (ac-HDNA) are shown. Product formation was confirmed by MALDI-TOF MS analysis of HPLC fractions. MS labeling indicates [M+H] + Theoretical accurate mass (top) and measured mass (bottom) of .

[0023] Figures 3A to 3B Shown are samples prepared with different concentrations of APMA, acylated with FAM-OSu, and analyzed by flow cytometry ( Figure 3A ) and were quantified (gray boundaries indicate standard errors) ( Figure 3B ) of gel particles.

[0024] Figure 4A Shown are DNA-templated beads prepared by PCR using magnetic beads (black circles) functionalized with DNA oligonucleotide primer P1 (black dashed line, unidirectional arrow), wherein the P1 sequence is shown in SEQ ID NO: 1; and DNA oligonucleotide primer P2 (black unidirectional arrow), wherein the P2 sequence is shown in SEQ ID NO: 3. The DNA template contains a T7 RNA polymerase promoter element (T7 prom, black arrow). Negative control magnetic beads that were not templated were labeled with AlexaFluor 647 (647AF, black open circle with black dots). Each bead set was encapsulated in a P2 copolymer hydrogel. Figure 4B Shown are in vitro transcription of gel-encapsulated DNA templates and untemplated beads in the presence of a FAM-labeled DNA oligonucleotide probe to the 5' region of the RNA transcript (P3, black single-pointing arrow), where the P3 sequence is shown in SEQ ID NO: 5. The P3 probe detects the presence of RNA transcripts (black) in the gel that hybridize via P2. Figure 4C Two-dimensional flow cytometry analysis is shown, which indicates that the majority of particles exhibited exclusively red fluorescence (660 nm, untemplated negative control in Q1) or green fluorescence (520 nm, templated and RNA-loaded beads in Q3).

[0025] Figure 5AShown is the DNA template bead encapsulated in the polyacrylamide hydrogel copolymerized with oligonucleotide primer P1 (black dotted line, unidirectional arrow).The P1 and 3' puromycin modified DNA oligonucleotide P4 (black " puro ") hybridization of transcribed mRNA and gel connection, wherein P4 sequence is shown in SEQ ID NO:6.MRNA (black chain) encoding ribosome binding site (RBS), epitope tag (grey), glycine-serine linker (G4S, grey) (SEQ ID NO:19), HiBiT luciferase complementary tag (black) and stop codon (*).Ribosomal protein synthesis terminates along with 3' puromycin incorporation into nascent chain, so that translated peptide is tethered to P4.Epitope is detected via immunofluorescence, and HiBiT is quantitatively detected via the complementation of luciferase and LgBiT. Figure 5B In-gel translation of FLAG-templated particles is shown, which was probed with 647AF-labeled anti-FLAG antibody, visualized, and imaged confocally with antibody fluorescence (λ ex / λ em =650 / 720 nm) and magnetic bead autofluorescence (λ ex / λ em =490 / 560nm) Figure 5C Untranslated particles were used for comparison. Scale bar = 50 μm. Figure 5D IVTT reactions programmed with particles templated with FLAG, HA, or V5 are shown, wherein the FLAG coding sequence is shown in SEQ ID NO: 13, the HA coding sequence is shown in SEQ ID NO: 12, and the V5 coding sequence is shown in SEQ ID NO: 11, and the translated epitopes (FLAG and HA: λ) were detected by flow cytometry with 647AF-anti-FLAG, APC-anti-HA, or CF488A-anti-V5 immunofluorescence. ex / λ em =640 / 660nm; V5:λ ex / λ em = 488 / 530 nm; translated particles are black, untranslated particles are gray). Figure 5E Shown are in-gel capture of translated FLAG, HA and V5 epitopes with and without 10 μM P4 (black, grey), which were quantified via HiBiT complementation. Error bars reflect standard deviation of the mean.

[0026] Figure 6Shown is the sorting of hydrogel magnetic particles using fluorescence-activated cell sorting (FACS). HA- and V5-templated particles (1% each) were spiked into a background of library particles (NNK5), where the NNK5 library template sequence is shown in SEQ ID NO: 16. The particles were then translated in a bulk IVT reaction, incubated with APC-labeled anti-HA and CF488A-labeled anti-V5 antibodies, and then subjected to FACS. After sorting, each population was PCR amplified and sequenced by next-generation sequencing (NGS).

[0027] Figure 7 Figure 2 shows flow cytometric analysis of magnetic bead templates of different diameters used for gel particle formation. Magnetic beads were encapsulated in a gel copolymerized with ac-P1 (20 μM), wherein the ac-P1 sequence is shown in SEQ ID NO: 2. Magnetic bead populations of 1, 2.8, and 10 μm diameter encapsulated in the hydrogel were gated via forward and side scatter correlation (top), and the gated population fluorescence intensity (bottom, λ) of the unprobed sample (grey) and the FAM-P1' probed sample (black) was plotted. ex / λ em =490 / 530 nm) were compared to confirm encapsulation.

[0028] Figure 8 The HiBiT quantification assay calibration curve is shown. After adding an excess of LgBiT complementary reagent, chemiluminescence was measured for different concentrations of HiBiT peptide (0.1 to 1000 nM).

[0029] Figure 9 Shown are the changes in hydrogel particle diameter as a function of templated magnetic bead diameter. Box and whisker plots indicate the mean, standard deviation, upper and lower quartiles (boxes), and 1.5X interquartile range (whiskers).

[0030] Figure 10A Shown is the uniformity of hydrogel particle diameter analyzed by confocal fluorescence microscopy. Scale bar = 100 μm. Figure 10B Shown are the diameters of the hydrogel particles measured for 32k particles (median = 7.3 μm). Figure 10C Flow cytometry (λex / λem=488 / 520 nm) for analysis of probed (black) and unprobed (grey) gel particles is shown.

[0031] Figure 11 Schematic diagram of the particle synthesis process. (i) Magnetic beads are suspended in a monomer premix, a layer of oil containing an initiator is added, and the suspension is emulsified. (ii) The droplets solidify to form hydrogel droplets. (iii) The gel particles containing the beads are magnetically separated.

[0032] Figure 12 The enrichment rate of various control tags obtained by sequencing sorted hit particles is shown. The read pattern matches the epitope tag sequence or the degenerate sequence of the NNK5 library. The HA positive hit pool is approximately 99% HA coding sequence, and the V5 hit pool is approximately 50% V5 coding sequence, which are enriched by approximately 100-fold and approximately 50-fold compared to the library starting material, respectively.

[0033] Figure 13 Shown are hydrogels prepared by batch emulsion scale using templated magnetic bead sizes (e.g., 1.0 μm, 2.8 μm, or 10.0 μm). The particle dispersion is inversely proportional to the magnetic bead diameter.

[0034] Figure 14 The translation and detection of gel particle library beads containing non-canonical amino acids are shown. NNU library particles (3 x 10 6 ) was subjected to an engineered IVT reaction recoded to install azidolysine (AzK) at CUG codons. The translated particles were washed and treated with AF647-alkyne in a CuAAC reaction and then analyzed by flow cytometry. The particles were sorted to isolate the top 2% of the AF647 population and sequences compared to the starting library. A 6-fold enrichment was observed after one round of screening.

[0035] Figure 15 NNU library particles (3 x 10 6 ) that underwent an engineered IVT reaction recoded to install azidolysine (AzK) at the CUG codon. The translated particles were washed and incubated with AF647-alkyne in a CuAAC reaction and then analyzed by flow cytometry. Particles were sorted by gating on the top 2% of AF647 signal.

[0036] Figure 16A and Figure 16B qPCR analysis of emPCR library preparation is shown. Aliquots of 100 beads were quantified to obtain the average loading (dashed trace), and limiting dilutions of beads were sampled in 77 wells (grey trace) to obtain single bead quantification results for DNA template ( Figure 16A ). The traces for the standards (light grey) and negative template control (black) are also shown. Figure 16A The box plots show the quantification of 100 beads and DNA-templated single beads ( Figure 16B The average particle loading of 100 bead aliquots was 4,200 DNA molecules / bead ( Figure 16BThe average particle loading of a single DNA-templated bead was 42,000 DNA molecules / bead ( Figure 16B ).

[0037] Figure 17A Shown is the time-dependent quantification of hydrogel particles loaded with a fluorescent (on) green fluorescent probe with trypsin activity. Figure 17B Shown is the time-dependent quantification of hydrogel particles loaded with N-terminally labeled tryptic peptides with the red fluorescent dye Cy5. Following trypsin digestion, Cy5 is released from the gel, resulting in a decrease (off) in the fluorescence signal over time.

[0038] Figure 18 Schematic diagrams showing various copolymerization functionalities including, but not limited to, cross-linkers, cell adhesion promoters (e.g., alkylamines), affinity capture tags (e.g., chloroalkyl HaloTags), enzyme capture, probe capture, and oligonucleotides for hybridization. The latter two can be used for labeling and characterization.

[0039] Figure 19 Shown are gel particles copolymerized with oligonucleotides and subsequently hybridized with trypsin-active probes conjugated to complementary oligonucleotides, schematically illustrating digestion of the probe-hybridized gel particles with trypsin, dequenching the activity-based probes and yielding fluorescent gel particles. DETAILED DESCRIPTION

[0040] In some embodiments, a versatile hydrogel-based particle is provided herein that supports both chemical and biological synthesis. The batch emulsification method disclosed herein allows for simple, scalable particle preparation and a relatively uniform size distribution. The hydrogel layer of the particle can exhibit a range of functionalities commonly used in 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 III. Solid-Phase Organic Synthesis on Unprotected DNA, PLoS Biol, 2004, 2(7), e175.) Furthermore, the use of this particle system is particularly advantageous for templated library preparation via in vitro translation, because RNA transcripts and subsequently translated peptides are localized to their host particles, eliminating the need for further emulsification or other partitioning to generate monoclonal beads for screening.

[0041] In some embodiments, the particles provided herein can be used for library screening using a particle system combined with a fluorescence activated cell sorting (FACS) instrument. This new particle format introduces important processing advantages, which can realize the automation of encoded library synthesis and unlock functional screening capabilities through the display of multivalent encoded library members. (MacConnell et al. ACS Comb. Sci. 2017, 19, 3, 181–192; Komnatnyy et al. Chem. Commun., 2018, 54, 6759–6771.)

[0042] The present inventors have discovered that magnetic bead-templated emulsion polymerization yields unexpectedly uniform particles that do not require microfluidics for preparation or analysis and that exhibit excellent handling and biocompatibility.

[0043] As used herein, "building block" refers to a chemical building block that is attached to or potentially attached to other chemical building blocks. Building blocks are diverse chemical structures that have one or more functional groups that are used in various ways for coupling via chemical synthesis and that may interact with a target of interest.

[0044] As used herein, "functional site" or "functional group" refers to a chemical group that participates in the reaction and is capable of producing a connection between two moieties. Examples of functional groups include, but are not limited to, -NH2, -SH, -OH, -CO2H, halide, -N3, -CONH2, etc.

[0045] As used herein, "particles" refer to discrete composites comprising a magnetic core coated by a polymer.

[0046] "A" or "an" means one or more than one. For example, "a particle" includes one, two, three or more particles.

[0047] "Library" means a collection of molecular or chemical entities.

[0048] "Oligonucleotide" means a nucleotide polymer having a 5' terminus, a 3' terminus, and one or more nucleotides at internal positions between the 5' terminus and the 3' terminus. An oligonucleotide may comprise DNA, RNA, or any derivative thereof known in the art.

[0049] "Tag" or "oligonucleotide tag" means an oligonucleotide portion of a library, at least a portion of which contains information identifying the particle and / or library. For example, an oligonucleotide tag can contain information that allows identification of a functional group of interest. In some embodiments, an oligonucleotide tag is used as a barcode.

[0050] I. Particles

[0051] In some embodiments, provided herein are particles comprising a magnetic core coated in a polymer gel. In some embodiments, the particles comprise a hydrogel coating the magnetic core. In some embodiments, a hydrogel refers to a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent bonds, ionic bonds, or hydrogen bonds to produce a three-dimensional open lattice structure that captures 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 non-toxic to living cells and allows oxygen and nutrients to diffuse fully to the captured cells to maintain vitality.

[0052] In certain embodiments, provided herein are particles comprising acrylamide droplets around magnetic particles. In certain embodiments, particles are prepared in batch emulsification to obtain uniform hydrogel compartments that are easy to polymerize and magnetically separate. Acrylamide provides a simple and easy polymerization mechanism that has been used in many chemical and molecular biology laboratories, and polyacrylamide products are generally inert to chemical and biochemical reactions. This free radical-mediated polymerization reaction only incorporates molecules with both the desired functionality and the vinyl groups for polymerization, and various functional groups can be easily incorporated into the polymer matrix. In certain 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 (hereinafter) in the gel matrix. Therefore, in certain embodiments, the particle comprises a polyacrylamide gel coating a magnetic core.

[0053] In some embodiments, a particle is provided herein comprising a magnetic core coated with a hydrogel layer. In some embodiments, a hydrogel layer comprising polyacrylamide can be easily synthesized, and it can prevent aggregation and inhibit adhesion to the tube wall. Polyacrylamide is also inert and is therefore compatible with a range of different chemical and biochemical reactivities. The gel particles comprise polyacrylamide, which can be functionalized in various ways by adding different reagents to an acrylamide monomer solution, such as propargyl methacrylate for realizing an "alkyne" function (which can be coupled to a molecule containing an "azide" function), N-(3-aminopropyl) methacrylamide for realizing an "amine" function, methacrylamide (ac)-modified oligonucleotide P1 for realizing a "reverse primer" function, or ac head DNA for realizing an HDNA function (which is used for DNA-encoded library synthesis). In some embodiments, the hydrogel polymer layer comprises polyacrylamide, acrylamide and / or bisacrylamide. In some embodiments, the hydrogel polymer layer includes polyacrylamide and / or acrylamide. In some embodiments, the hydrogel polymer layer includes polyacrylamide.

[0054] In some embodiments, the hydrogel or polymer layer comprises 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), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyvinylsulfonic acid (PVSA), Poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, 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.

[0055] In some embodiments, the gel particles comprise a hydrogel surrounding the aforementioned polymers or magnetic beads, which can be variously functionalized by adding different reagents to the corresponding monomer solution, such as by incorporating molecules with desired functionality and vinyl groups for polymerization. In some embodiments, a hydrogel layer surrounding the magnetic beads can be prepared with both chemical (e.g., alkyne, amine, azide) and biochemical (oligonucleotide) functional groups. In some embodiments, the functionalized hydrogel or polymer layer comprises 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), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyvinylsulfonic acid (PVSA) , poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, 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 some embodiments, hydrogel particles with a magnetic core are prepared by emulsification of a hydrogel monomer solution and magnetic microbeads. In some embodiments, polyacrylamide hydrogel particles with a magnetic core 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. Subsequently, the aqueous phase and the oil phase can 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 to bisacrylamide 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 / water sample can be emulsified using a bead mill homogenizer (65 s, 2500 rpm, BeadBug, Benchmark Scientific, Sayreville, NJ), vortexing, or stirring. In some embodiments, the emulsified suspension can be polymerized on ice, and after polymerization, the hydrogel particles having a magnetic core can be separated on a magnet.

[0057] In some embodiments, the ratio of acrylamide to 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, 38:1, 38.5:1, 39:1, 39.5:1 or 40:1, or a ratio within the range defined by any two of the above ratios.

[0058] In some embodiments, the particles and / or compositions comprising the particles comprise additives to inhibit phase separation, coacervate formation, and / or coagulation of the hydrogel layer of the hydrogel particles having a magnetic core. In some embodiments, the additive comprises albumin, such as bovine serum albumin (BSA) and acrylamide-modified BSA. In another embodiment, the albumin is modified with one or more reactive groups, such as albumin modified with vinyl groups and acrylamide-modified BSA. In some embodiments, the particles comprise bovine serum albumin (BSA), modified BSA, human serum albumin (HSA), and / or modified HSA.

[0059] In some embodiments, the hydrogel polymer layer comprises about 70% to 96% weight / volume (w / v) fluid (such as water) and about 4% to 30% w / v polymer. Unless otherwise stated, percentages are weight / volume. In some embodiments, the hydrogel layer of hydrogel particles with 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 the percentage of fluid or polymer within the range defined by any two of the above percentages. In some embodiments, the polymer comprises 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), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyvinyl sulfonic acid (PVSA), poly(L -aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, 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.

[0060] In certain embodiments, the fluid in the hydrogel polymer layer of hydrogel particles 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 the range defined by any two of the foregoing percentages. In some embodiments, the hydrogel polymer layer of hydrogel particles 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 the range defined by any two of the foregoing percentages. In some embodiments, the hydrogel polymer layer of 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 the range defined by any two of the foregoing percentages.

[0061] In some embodiments, the particles comprise pores in the polymer that allow diffusion into and out of the gel. In some embodiments, the pore size is selected so that reagents, substrates, reactants, and / or enzymes are retained in the polymer. In some embodiments, the pore size is selected so that a high local concentration of reactants, substrates, or enzymes is generated in the hydrogel to promote 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 the range defined by any two of the above sizes. In some embodiments, the hydrogel polymer forms a hydrogel matrix (e.g., a porous hydrogel matrix) having pores. These pores can retain genetic material or peptides large enough to be coated on the magnetic beads within the hydrogel matrix, but allow small substances such as reagents to pass through the pores and thus enter and exit the hydrogel matrix. In some embodiments, the pore size is determined by the ratio of polymer concentration to crosslinker concentration. 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 above ratios.

[0062] 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 may be demagnetized when the magnetic field is removed. Thus, paramagnetic particles can be efficiently separated from solution using a magnet, but may be easily resuspended without magnetically induced aggregation. In some embodiments, the magnetic beads comprise a magnetite-rich core (such as iron oxide) encapsulated by a pure polymer shell. In one embodiment, suitable magnetic beads comprise a magnetite / encapsulation ratio of about 20% to 35%. For example, magnetic beads comprising a magnetite / encapsulation ratio of about 23%, 25%, 28%, 30%, 32% or 34% are suitable for use in the present invention.

[0063] In some embodiments, the magnetic beads within the hydrogel particles have a diameter of about 0.5 μm to about 10 μm, such as, for example, 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.

[0064] In some embodiments, the magnetic beads used herein may have a surface coated with functional groups. Methods for directly or indirectly coating magnetic beads with functional groups are known in the art. For example, functional groups (e.g., COOH) can coat magnetic beads during the formation of magnetic beads. See, for example, U.S. Patent No. 5,648,124. In addition, the magnetic beads can be coated with functional groups by covalently coupling the functional group (one or more) to the COOH group (one or more) on the magnetic beads. A specific example of a surface coated with functional groups is a surface coated with a portion each having a free functional group, which is bound to the amino group of the aminosilane of the magnetic beads; therefore, the surface of the magnetic beads is coated with a portion containing functional groups. In particular, carboxylic acid-coated magnetic beads are commercially available. Other functional groups can be coated on the magnetic beads, such as, but not limited to, amino-coated, carboxyl-coated, and encapsulated carboxyl-coated magnetic beads. In some embodiments, other functional groups can be coupled to the magnetic beads via carbodiimide coupling with carboxyl groups on the surface of the magnetic beads. Other functional groups include, but are not limited to, amine groups, carboxyl groups, coated carboxyl groups, silicon dioxide (SiOH), and diethylaminoethyl (DEAE).

[0065] In some embodiments, the particles have a diameter of about 5 μm to about 10 μm. In some embodiments, the particles are of sufficient size to enable multiple synthesis reactions to occur in the polymer coating. In some embodiments, the total diameter of the hydrogel particles is measured using a confocal fluorescence microscope. In some embodiments, the median diameter is 7 ± 2 μm. The diameter of the total hydrogel particles is less than about 40 μm. In some embodiments, the total particle diameter is less than about 40 μm, optionally when the total particle diameter is about 6 μm to about 12 μm. In some embodiments, the total 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, 26μm, 27μm, 28μm, 29μm, 30μm, 31μm, 32μm, 33μm, 34μm, 35μm, 36μm, 37μm, 38μm, 39μm, or a diameter within the range defined by any two of the above values ​​(e.g., about 2μm to about 30μm, about 3μm to about 20μm, about 4μm to about 10μm, or about 5μm to about 9μm).

[0066] In some embodiments, hydrogel particles having a magnetic core have various functionalities embedded in the hydrogel layer or hydrogel matrix. In some embodiments, the hydrogel particles can be functionalized by adding the following reagents to an acrylamide monomer solution comprising propargyl methacrylate (PMA) or a propargyl methacrylate analog or other alkyne-derived or alkyne-containing compounds known to those skilled in the art for achieving "alkyne" functionality. N-(3-aminopropyl)methacrylamide (APMA) or N-(3-aminopropyl)methacrylamide analogs or other amine-derivatized compounds or amine-containing compounds known to those skilled in the art for achieving "amine" functionality; 5'-methacrylamide-modified DNA oligonucleotide P1 (acrydite) or acrydite-P1 (ac-P1) or acrydite-P1 analogs or other acrylic acid phosphoramidite-derivatized compounds known to those skilled in the art for achieving "reverse primer" functionality; or methacrylamide DNA head segment or ac-hairpin head segment DNA (HDNA) or analogs thereof for achieving "HDNA" functionality; or aminoethyldisulfide or thiol-derivatized compounds or analogs known to those skilled in the art for achieving "thiol" functionality; or any combination thereof.

[0067] In some embodiments, hydrogel particles having a magnetic core have various functionalities embedded in the hydrogel layer or hydrogel matrix. In some embodiments, the hydrogel particles can be functionalized by adding acrylamide monomer solutions containing propargyl methacrylate or propargyl methacrylate analogs or other alkyne-derived or alkyne-containing compounds known to those skilled in the art for achieving "alkyne" functionality to the monomer solution of the particular gel. N-(3-aminopropyl)methacrylamide or N-(3-aminopropyl)methacrylamide analogs or other amine-derived compounds or amine-containing compounds known to those skilled in the art for achieving "amine" functionality; 5'-methacrylamide-modified DNA oligonucleotide P1 (acrydite) or acrydite-P1 or acrydite-P1 analogs or other acrydite-derived compounds known to those skilled in the art for achieving "reverse primer" functionality; or methacrylamide DNA head segment or ac-hairpin head segment DNA (HDNA) or analogs thereof for achieving "HDNA" functionality; or aminoethyldisulfide or thiol-derived compounds or analogs known to those skilled in the art for achieving "thiol" functionality; or any combination thereof.

[0068] In some embodiments, the hydrogel or polymer layer comprises one or more reagents, the one or more reagents comprising: propargyl methacrylate (PMA) or propargyl methacrylate analogs or other alkyne-derivatized or alkyne-containing compounds known to those skilled in the art; N-(3-aminopropyl) methacrylamide (APMA) or N-(3-aminopropyl) methacrylamide analogs or other amine-derivatized or amine-containing compounds known to those skilled in the art; 5'-methacrylamide-modified DNA oligonucleotide P1 (acrydite) or acrydite-P1 or acrydite-P1 analogs or other acrylic acid phosphoramidite acrydite-derivatized compounds known to those skilled in the art; or methacrylamide DNA head segment or ac-hairpin head segment DNA (HDNA) or its analogs; or aminoethyl disulfide or thiol-derivatized compounds or its analogs 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 an appropriate fluorescent dye-labeled substrate (including a complementary oligonucleotide, a double-stranded oligonucleotide connecting module, an azide or a succinimide ester).

[0069] In some embodiments, hydrogel particle functionalization can be detected by flow cytometry with the addition of an appropriate fluorescent dye-labeled substrate (including complementary oligonucleotides, double-stranded oligonucleotide linker modules, azides, or succinimidyl esters). In some embodiments, hybridization and amine acylation resulted in the largest shifts, but all functionalization reactions resulted in baseline separation of product and starting material by flow cytometry. In some embodiments, hydrogels with increasing amine loading capacity 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, 2.7m M, 2.8mM, 2.9mM, 3.0mM, 3.1mM, 3.2mM, 3.3mM, 3.4mM, 3.5mM, 3.6mM, 3.7m M, 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 ,5.8mM, 5.9mM, 6.0mM, 6.1mM, 6.2mM, 6.3mM, 6.4mM, 6.5mM, 6.6mM, 6.7mM , 6.8mM, 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, .5 mM, 19.0 mM, 19.5 mM, 20.0 mM, or a concentration within the range defined by any two of the above values.In some embodiments, gel particles with varying APMA concentrations can be prepared, acylated with FAM-OSu, analyzed by flow cytometry, and quantified, e.g., Figure 3A and Figure 3B In some embodiments, acylation with FAM-OSu results in a log-linear increase in gel particle fluorescence over a four-order-of-magnitude range.

[0070] In some embodiments, hydrogel particles having a magnetic core have various functionalities embedded in the hydrogel layer or hydrogel matrix. In some embodiments, the hydrogel particles can be functionalized by adding acrylamide monomer solutions containing propargyl methacrylate or propargyl methacrylate analogs or other alkyne-derived or alkyne-containing compounds known to those skilled in the art for achieving "alkyne" functionality to the monomer solution of the particular gel. N-(3-aminopropyl)methacrylamide or N-(3-aminopropyl)methacrylamide analogs or other amine-derivatized compounds or amine-containing compounds known to those skilled in the art for achieving "amine" functionality; 5'-methacrylamide-modified (acrydite) DNA oligonucleotide P1 (ac-P1) or acrydite-P1 analogs or other acrydite-derivatized compounds known to those skilled in the art for achieving "reverse primer" functionality; or methacrylamide DNA head or ac-hairpin head DNA (HDNA) or analogs thereof for achieving "HDNA" functionality; or aminoethyldisulfide or thiol-derivatized compounds or analogs known to those skilled in the art for achieving "thiol" functionality; or any combination thereof. In some embodiments, about 0.001 mM to about 20 mM hydrogel or polymer gel is functionalized. In some embodiments, about 0.01 mM to about 20 mM hydrogel or polymer gel is functionalized. In some embodiments, about 0.1 mM to about 20 mM hydrogel or polymer gel is functionalized. In some embodiments, about 1 mM to about 20 mM hydrogels or polymer gels are functionalized. In some embodiments, about 10 mM to about 20 mM hydrogels or polymer gels are functionalized.

[0071] 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.

[0072] 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 the range defined by any two of the above values.

[0073] In some embodiments, the hydrogels can be functionalized in a variety of ways by incorporating substoichiometric amounts of methacrylamide-modified additives into the acrylamide:bisacrylamide monomer solution used for copolymerization. For example, the incorporation of synthetic hairpin DNA (HDNA) and subsequent enzymatic oligonucleotide ligation reactions demonstrated the feasibility of standard DEL synthesis workflows using these particles. Commercially available NH2-HDNA can be readily converted to methacrylamide analogs for copolymerization. Amine functionality can be routinely used for solid phase synthesis as well as DEL synthesis. In some embodiments, the particle loading capacity was quantified over more than 4 orders of magnitude. For example, at the highest site density (i.e., 20 mM) of APMA-functionalized hydrogel particles, each median particle (approximately 7 μm in diameter) carried approximately 4 fmol of amine sites.

[0074] 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 undergo an mRNA display in vitro transcription / translation reaction, incorporating a puromycin-modified peptide capture oligonucleotide P4 ( complementary to the RNA directly 3' of the stop codon). Figure 5A The translated beads can be analyzed by imaging microscopy and flow cytometry to visualize translation of specific epitopes. The translated gel particles exhibit uniform antibody-derived fluorescence throughout the periphery of the gel ( Figure 5B ); before translation, only magnetic bead autofluorescence was detected in the same particles ( Figure 5CFluorescence of the translated gel particles by flow cytometry achieved baseline separation compared to untranslated particles for all three exemplary epitopes ( Figure 5D In some embodiments, the in-gel peptide capture yield can be quantified via HiBiT luminescence. In some embodiments, particles translated in the presence of puromycin capture oligonucleotide P4 retain approximately 100 nM HiBiT peptide, while translations in the absence of P4 retain less than 1 nM peptide ( Figure 5E ).

[0075] In some embodiments, the gel particles comprise a hydrogel surrounding the aforementioned polymers or magnetic beads, which can be variously functionalized by adding different reagents to the corresponding monomer solution, such as by incorporating molecules with desired functionality and vinyl groups for polymerization. In some embodiments, a hydrogel layer surrounding the magnetic beads can be prepared with both chemical (e.g., alkyne, amine, azide) and biochemical (oligonucleotide) functional groups. In some embodiments, the functionalized hydrogel or polymer layer comprises 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), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyvinylsulfonic acid (PVSA) , poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, 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 variation in diameter 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 above values.

[0076] In some embodiments, the gel particles comprise a hydrogel surrounding the aforementioned polymers or magnetic beads, which can be variously functionalized by adding different reagents to the corresponding monomer solution, such as by incorporating molecules with desired functionality and vinyl groups for polymerization. In some embodiments, a hydrogel layer surrounding the magnetic beads can be prepared with both chemical (e.g., alkyne, amine, azide) and biochemical (oligonucleotide) functional groups. In some embodiments, the functionalized hydrogel or polymer layer comprises 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), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyvinylsulfonic acid (PVSA) , poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, 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, In some embodiments, the hydrogel particles have an average diameter of about 7 μm, or about 10 μm, or about 20 μm, or about 30 μm, or about 40 μm, or about 50 μm, or about 60 μm, or about 70 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm, or about 80 μm

[0077] In some embodiments, the gel particles comprise a hydrogel surrounding the aforementioned polymers or magnetic beads, which can be variously functionalized by adding different reagents to the corresponding monomer solution, such as by incorporating molecules with desired functionality and vinyl groups for polymerization. In some embodiments, a hydrogel layer surrounding the magnetic beads can be prepared with both chemical (e.g., alkyne, amine, azide) and biochemical (oligonucleotide) functional groups. In some embodiments, the functionalized hydrogel or polymer layer comprises 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), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyvinylsulfonic acid (PVSA) , poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, 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 hydrogel particles in the composition comprise a magnetic core. In some embodiments, the percentage of total hydrogel particles in the composition comprising a 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.

[0078] In some embodiments, the library of hydrogel particles with a magnetic core comprises about 1 x 10 6 pcs, 2x 10 6 3x 10 6 4x 10 6 5x 10 6 pcs, 6x 10 6 7x 10 6 8x 10 6 9x 10 6 1x 10 7 pcs, 2x 10 7 3x 10 7 4x 10 7 5x107 pcs, 6x 10 7 7x 10 7 8x 10 7 9x 10 7 1x 10 8 pcs, 2x 10 8 3x 10 8 4x 10 8 5x 10 8 pcs, 6x 10 8 7x 10 8 8x 10 8 9x 10 8 1x 10 9 2x10 9 3x 10 9 4x 10 9 5x 10 9 6x10 9 7x 10 9 8x 10 9 9x 10 9 1x 10 10 2x10 10 3x 10 10 4x 10 10 5x 10 10 pcs, 6x 10 10 7x 10 10 8x 10 10 9x 10 10 1x10 11 pcs, 2x 10 11 3x 10 11 4x 10 11 5x 10 11 pcs, 6x 10 11 7x 10 11 8x 10 11 9x10 11 1x 10 12 particles, or a number defined by any two of the above values.

[0079] In some embodiments, the library of hydrogel particles with a magnetic core comprises about 1 x 10 6 to 1x 10 12 particles, 1x 10 6 to 1x 10 11 particles, 1x 10 6to 1x 10 10 particles, 1x 10 6 to 1x 10 9 particles, 1x10 6 to 1x 10 8 particles, 1x 10 6 to 1x 10 7 particles, 1x 10 7 to 1x 10 12 particles, 1x 10 7 to 1x10 11 particles, 1x 10 7 to 1x 10 10 particles, 1x 10 7 to 1x 10 8 particles, 1x 10 8 to 1x10 12 particles, 1x 10 8 to 1x 10 11 particles, 1x 10 8 to 1x 10 10 particles, 1x10 8 to 1x 10 9 particles, 1x 10 9 to 1x 10 12 particles, 1x 10 9 to 1x 10 11 particles, 1x 10 9 to 1x 10 10 particles, 1x 10 10 to 1x10 12 particles, or 1x10 11 to 1x 10 12 particles.

[0080] II. Methods for Preparing Granules

[0081] In certain embodiments, this paper provides a method for producing a composition comprising particles comprising a magnetic core encapsulated in a polymer gel. In certain embodiments, the advantage of the method provided herein is that they produce gel-encapsulated nanoparticles in a single step by emulsifying aqueous solutions and oil solutions, and do not require complicated assembly techniques or machinery. In certain embodiments, the method provided herein is applicable to producing gel-encapsulated nanoparticles and / or producing gel-encapsulated nanoparticles in batches with a high-throughput method.

[0082] In certain embodiments, the method provided herein includes emulsifying an aqueous solution comprising monomer and magnetic beads with a solution comprising a polymerization initiator to cause polymerization of the monomer. In certain embodiments, the monomer is a monomeric unit of a polymer. In certain embodiments, the aqueous solution comprises acrylamide monomer. In certain embodiments, the aqueous solution comprises about 4% to about 10%, about 5% to about 9%, about 6% to about 8%, or about 7% to about 10% acrylamide monomer.

[0083] 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 between about 10:1 and about 40:1, such as 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.

[0084] In some embodiments, the aqueous solution further comprises a second polymerization initiator that initiates polymerization when mixed with a solution comprising a 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 induce polymerization of acrylamide and bisacrylamide to encapsulate the magnetic beads. In other embodiments, alternative free radical sources (e.g., azobisisobutyronitrile, AIBN) or stimuli-responsive substances (e.g., photosensitive photoinitiators such as dimethoxyphenylacetophenone, DMPA) can be used to initiate polymerization.

[0085] In certain embodiments, method comprises combining two solutions (such as an aqueous solution and an oil solution). In certain embodiments, the aqueous solution comprises acrylamide and / or bisacrylamide, and the oil solution comprises a polymerization initiator. In certain embodiments, two solutions are combined by mixing, emulsifying, vortexing, homogenizing, stirring and / or shaking. In certain embodiments, method comprises using a mechanical system to combine solutions. In certain embodiments, method comprises using a vortexer, rotary mixer or stirring rod to combine solutions. In certain embodiments, method comprises manually shaking a container comprising two solutions.

[0086] In certain embodiments, solution is mixed within a time period.In certain embodiments, solution is mixed enough time to make solution combination.In certain embodiments, solution is mixed enough time to realize the polymerization of monomer and the encapsulation of bead.In certain embodiments, solution is combined, mixed, vortex, emulsification, homogenization, stirring and / or jolting for at least one minute, at least two minutes, at least three minutes, at least four 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 certain embodiments, solution is combined, mixed, vortex, emulsification, homogenization, stirring and / or jolting for one minute to 60 minutes, one minute to 45 minutes, one minute to 30 minutes, one minute to 20 minutes, one minute to 10 minutes or one minute to five minutes.In certain embodiments, solution is combined, mixed, vortex, emulsification, homogenization, stirring and / or jolting for 10 minutes to 60 minutes, 20 minutes to 60 minutes, 30 minutes to 60 minutes or 45 minutes to 60 minutes.

[0087] In some embodiments, the solutions are mixed at room temperature. In some embodiments, the solutions are mixed at temperatures above room temperature. In some embodiments, the solutions are mixed at temperatures below room temperature. In some embodiments, the solutions are mixed at temperatures between about 15°C and about 25°C, between about 17°C and about 23°C, or between about 20°C and about 22°C.

[0088] In certain embodiments, method further comprises bubbling the combined composition with a gas.In certain embodiments, the mechanical energy of the combined composition is bubbled with an inert gas.In certain embodiments, the combined composition is bubbled with nitrogen, argon or helium.In certain embodiments, the combined composition is bubbled with an inert gas for at least two minutes, at least three minutes, at least four 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 certain embodiments, the combined composition is bubbled for one minute to 60 minutes, one minute to 45 minutes, one minute to 30 minutes, one minute to 20 minutes, one minute to 10 minutes or one minute to five minutes.In certain embodiments, the combined composition is bubbled for 10 minutes to 60 minutes, 20 minutes to 60 minutes, 30 minutes to 60 minutes or 45 minutes to 60 minutes.

[0089] In certain embodiments, the method further includes applying a magnetic field to the combined composition to purify or separate the particles comprising one or more magnetic beads encapsulated in a hydrogel. In certain embodiments, a magnet is used to apply a magnetic field to the combined mixture to cause the positioning of the beads while removing or exchanging the solution. In certain embodiments, the application of a magnetic field is used to separate the particles comprising one or more magnetic beads encapsulated in a hydrogel from particles that do not comprise one or more magnetic beads. In certain embodiments, particles with two or more, three or more, or four or more magnetic beads are separated from particles or gels that do not comprise magnetic beads. In certain embodiments, the application of a magnetic field allows the separation of a colony of uniform particles containing one or more different reagents. In certain embodiments, the application of a magnetic field produces a library of particles that each comprise different reagents.

[0090] In some embodiments, after applying a magnetic field to the combined composition, the particles comprising the magnetic beads are removed from the solution. In some embodiments, the particles comprising the magnetic beads form a precipitate and / or are positioned to a portion of a tube or container containing the combined solution. In some embodiments, the magnetic field causes the particles comprising the magnetic beads to precipitate. In some embodiments, the supernatant that does not comprise the particles comprising the magnetic beads is aspirated and / or removed from the combined composition. In some embodiments, aspirating the supernatant causes purification of the particles comprising the magnetic beads.

[0091] In some embodiments, after application of the magnetic field, particles comprising one or more magnetic beads are substantially purified from particles that do not comprise magnetic beads. In some embodiments, after application of the magnetic field, the ratio of particles comprising magnetic beads to particles that do not comprise 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 comprising a single magnetic bead are separated from particles comprising a plurality of magnetic beads.

[0092] In some embodiments, after aspirating the supernatant, the remaining particles containing 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 washing solution comprises a neutral buffer solution. In some embodiments, an excess of buffer solution is used to resuspend the particles, a magnetic field is applied, and the supernatant is aspirated one or more times to wash the particles. 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 once, twice, three times, four times, five times, or more.

[0093] In some embodiments, after applying 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.

[0094] In some embodiments, the method comprises producing a functionalized gel or a functionalized portion of a gel. In some embodiments, the gel is functionalized after particles comprising a polymer gel encapsulating a magnetic core are purified by, for example, magnetic separation. In some embodiments, the gel is functionalized prior to purification of the encapsulated particles. In some embodiments, a portion of the monomers is functionalized prior to polymerization. In some embodiments, the polymerized gel is functionalized.

[0095] In some embodiments, the functionalized gel or particle is a gel or particle comprising a functional group that serves as a substrate for further reaction or synthesis. For example, suitable functional groups include oligonucleotides, synthetic hairpin DNA, alkynes, and / or primary amines.

[0096] 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 prior to polymerization. In some embodiments, the gel is functionalized while being polymerized. In some embodiments, the gel is functionalized after polymerization.

[0097] In some embodiments, the magnetic beads are functionalized. In some embodiments, the magnetic beads are functionalized 5' methacrylamide oligonucleotides, methacrylamide-modified HDNA, propargyl methacrylate, or APMA. In some embodiments, the magnetic beads are functionalized prior to gel polymerization of the encapsulated particles. In some embodiments, the magnetic beads are functionalized concurrently with gel polymerization. In some embodiments, the magnetic beads are functionalized after gel polymerization. In some embodiments, the magnetic beads are functionalized with carboxylic acid.

[0098] In some embodiments, the gel is functionalized by adding a compound comprising a functional group to the 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 such 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.

[0099] In some embodiments, the particles or monomers are functionalized using an excess of a functionalizing agent. In some embodiments, the particles are functionalized by incubating with about 0.0001 mM to about 100 mM (such as 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 incubating 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 gel and / or beads are functionalized such that the particles comprise one or more functional groups. In some embodiments, a given particle comprises multiple copies of the same functional group. In some embodiments, a given particle comprises multiple copies of different functional groups. In some embodiments, different particles in a mixture of particles comprise different functional groups. In some embodiments, each particle in the mixture comprises the same functional group.

[0101] In some embodiments, functionalization of the gel results in particles comprising a template for a synthesis reaction. For example, in some embodiments, functionalization of the gel results in particles comprising a HDDNA or oligonucleotide template.

[0102] In certain embodiments, before polymerization, the solution comprising monomers and magnetic beads comprises one or more reagents. In certain embodiments, the one or more reagents comprise proteins, enzymes, polynucleotides, oligonucleotides, polysaccharides, fluorophores, or lipids. In certain embodiments, the reagent is an enzyme capable of synthesis. In certain embodiments, the reagent comprises a polynucleotide or oligonucleotide substrate for synthesis reaction. In certain embodiments, the reagent comprises a fluorophore that can be used for detection of particles. In certain embodiments, the solution comprises a variety of reagents, such as fluorophores and oligonucleotides.

[0103] In certain embodiments, the polymerization of monomers causes one or more reagents to be incorporated into the gel. In certain embodiments, the reagent is covalently linked to beads and / or gel. In certain embodiments, the reagent is encapsulated in the gel. In certain embodiments, the reagent is maintained in the gel in a non-covalent manner.

[0104] In certain embodiments, method comprises making monomer polymerization to produce the particle comprising the magnetic bead coated with gel, and then functionalizing the particle formed.In certain embodiments, use excess functional agent to functionalize the particle.In certain embodiments, by hatching together with about 0.0001mM to about 100mM (such as about 0.0002mM to about 50mM, about 0.0002mM to about 20mM, about 1mM to about 100mM, 5mM to about 80mM, about 10mM to about 50mM, about 10mM to about 40mM or about 15mM to about 30mM) compound comprising functional group so that particle functionalization.In certain embodiments, by hatching together with about 20mM compound comprising functional group so that particle functionalization.In certain embodiments, the compound comprising functional group is propargyl methacrylate.

[0105] In some embodiments, the method further comprises functionalizing the detection gel and / or beads. In some embodiments, detection is a chemical reaction using a functional group labeled with a fluorescent dye. In some embodiments, detection is by hybridization of complementary oligonucleotides, enzymatic ligation of dsDNA molecules, CuAAC using Alexa Fluor 488 azide (488A-FN3), or amine acylation using fluorescein succinimidyl ester (FAM-OSu).

[0106] In some embodiments, the methods provided herein produce a particle library. In some embodiments, the particle library comprises a plurality of particles having different functional groups. In some embodiments, the particle library comprises a plurality of particles having the same functional group.

[0107] In some embodiments, the methods provided herein are compatible with different magnetic bead sizes (such as about 1.0, 2.8, or 10.0 μm in diameter). In some embodiments, the methods provided herein yield gel particles having a magnetic core size of about 1, 2.8, or 10 μm in diameter after encapsulation.

[0108] In certain embodiments, the method provided herein is used to increase the magnetic bead size to form a larger and more uniform distribution of gel particles. In certain embodiments, for the hydrogel prepared by batch emulsification scale with different templated magnetic bead sizes (such as diameters of about 1.0, 2.8 or 10.0 μm), the overall hydrogel particle size dispersity obtained is inversely proportional to the magnetic bead diameter. For example, in certain embodiments, when the diameter of the templated magnetic bead size is about 1 μm, by counting 447 particles, the average diameter of the hydrogel particles after encapsulating the magnetic beads is about 4.9 μm, and its standard deviation is about 1.8 μm and the coefficient of variation is about 36%. In some other embodiments, when the diameter of the templated magnetic bead size is about 2.8 μm, by counting 390 particles, the average diameter of the hydrogel particles after encapsulating the magnetic beads is about 7.5 μm, and its standard deviation is about 1.9 μm and the coefficient of variation is 25%. In some other embodiments, when the templated magnetic bead size is about 10 μm in diameter, the average diameter of the hydrogel particles after encapsulating the magnetic beads is about 12 μm by counting 66 particles, with a standard deviation of about 2.0 μm and a coefficient of variation of 16%.

[0109] In some embodiments, hydrogel particles with a magnetic core having a diameter of 1 μm prepared by the methods provided herein have a hydrogel volume of about 88 fL. In some embodiments, hydrogel particles with a magnetic core having a diameter of 2.8 μm prepared by the methods provided herein have a hydrogel volume of about 260 fL. In some embodiments, hydrogel particles with a magnetic core having a diameter of 10 μm prepared by the methods provided herein have a hydrogel volume of about 570 fL.

[0110] III. Methods for Detecting Synthetic Products

[0111] This paper also provides a method for detecting the reaction synthesis product in the particle. In certain embodiments, advantageously, the particle and method provided herein cause reactants to concentrate in the particle and the synthesis product is retained. In certain embodiments, the method includes synthesizing the synthesis product in the particle. In certain embodiments, the particle comprises a gel coating a magnetic core. In certain embodiments, the particle comprises a synthesis template. In certain embodiments, the gel is a polymeric gel.

[0112] In certain embodiments, the method includes detecting the synthetic product of the reaction in the particles of the polyacrylamide gel comprising the coated magnetic beads. In certain embodiments, the polymeric gel promotes the synthesis of the proximity-driven enzyme. In certain embodiments, the polymeric gel increases the local concentration of the substrate, enzyme, and template to obtain the production of the synthetic product. In certain embodiments, one or more of the enzyme, substrate, and template is attached to the polymeric gel or beads. In certain embodiments, one or more of the enzyme, substrate, and template is trapped in the polymeric gel. In certain embodiments, the pore size of the polymeric gel is such that the enzyme, substrate, and / or template is retained.

[0113] In some embodiments, the method includes providing a synthetase and a synthetic substrate in a polymeric 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 any one of the tRNAs for naturally occurring amino acids.

[0114] In some embodiments, the enzyme incorporates the substrate into the 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 the sequence of the template. For example, in some embodiments, the template is an mRNA and the synthetic product is a protein comprising a 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.

[0115] 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 primers. In some embodiments, primers are incubated with template DNA and oligonucleotides under PCR conditions to produce template particles comprising double-stranded DNA. In some embodiments, the particles are templated after gel polymerization and gel-encapsulated particle purification. In some embodiments, magnetic beads are functionalized and templated. In some embodiments, polymerized gels are functionalized and templated. In some embodiments, monomers are functionalized before or after polymerization.

[0116] In some embodiments, the number or percentage of templated particles is determined. In some embodiments, templated particles are detected by qPCR using primers that hybridize to DNA synthesized during templating. In some embodiments, templated particles are detected by binding of a fluorescent probe to the template. In some embodiments, templated particles are detected by binding of an antibody to the template. In some embodiments, templated particles are purified from non-templated particles.

[0117] 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 RNA polymerase. In some embodiments, the template comprises a promoter sequence for T7 RNA polymerase.

[0118] In some embodiments, the template is mRNA. In some embodiments, the template contains a translation mimicking 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 (e.g., 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.

[0119] In some embodiments, the substrate is a naturally occurring nucleotide. In some embodiments, the substrate is any one of the following: adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, deoxycytidine, inosine or diaminopurine, base analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 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 sugars (e.g., 2'-fluororibose; ribose; 2'-deoxyribose; arabinose; hexose; anhydrohexitol; altritol; mannitol; cyclohexyl; morpholino also having a phosphoramidate backbone); locked nucleic acids (LNA, e.g., wherein ribose the 2'-hydroxyl group of the ribose is connected to the 4'-carbon of the same ribose via a C1-β alkylene or C1-6 heteroalkylene bridge, wherein exemplary bridges include methylene, propylene, ether, or amino bridges); diol nucleic acids (GNA, e.g., R-GNA or S-GNA, wherein the ribose is replaced by a diol unit attached to a phosphodiester bond); threose nucleic acids (TNA, wherein the ribose is replaced by an α-L-threofuranosyl-(3'—>2')); and / or replacement of the oxygen in the ribose (e.g., by S, Se, or alkyl groups such as methylene or vinyl groups); modified backbones (e.g., peptide nucleic acids (PNAs) in which 2-amino-ethyl-glycine linkages replace ribose and phosphodiester backbones); and / or modified phosphate groups (e.g., phosphorothioate, 5'-N-phosphoramidite, phosphoselenate, borophosphate, borophosphate, hydrogen phosphonate, phosphoramide, phosphorodiamidate, alkyl or aryl phosphonates, phosphotriester, bridged phosphoramidate, bridged phosphorothioate, and bridged methylenephosphonate). The oligonucleotides may be single-stranded (e.g., hairpins), double-stranded, or have other secondary or tertiary structures (e.g., stem-loop structures, double helices, triplexes, quadruplexes, etc.). In some embodiments, the particles comprise a mixture of oligonucleotide substrates (e.g., A, C, G, and T or U). In some embodiments, the particles comprise a mixture of approximately equal amounts of oligonucleotide substrates.

[0120] In certain embodiments, the enzyme is a DNA or RNA polymerase. In certain 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 certain embodiments, the enzyme is a ribosome. In certain embodiments, the enzyme is from a microorganism (such as a bacterium, a virus or a yeast).

[0121] In certain embodiments, the synthetic product is a peptide such as 50 amino acids in length or less. In certain embodiments, the synthetic product is a polypeptide having a length of more than 50 amino acids. In certain embodiments, the synthetic product comprises one or more amino acids connected by peptide bonds. In certain embodiments, the length of the synthetic product is about 2 to about 1000 amino acids. In certain embodiments, the length of 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 certain embodiments, the synthetic product is a naturally occurring or synthetic peptide.

[0122] In certain embodiments, the synthetic product is an oligonucleotide, such as a polynucleotide. In certain embodiments, the synthetic product is a double-stranded DNA. In certain embodiments, the synthetic product is a single-stranded DNA or RNA. In certain embodiments, the synthetic product comprises two or more nucleotides connected by a phosphodiester bond. In certain embodiments, the synthetic product comprises two or more nucleotides connected by a synthetic backbone. In certain embodiments, the synthetic product is an oligonucleotide having a length of about 2 to about 100 nucleotides. In certain embodiments, the length of the synthetic product 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 certain embodiments, the synthetic product is a naturally occurring or synthetic oligonucleotide.

[0123] 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 a portion of a larger molecule. For example, in some embodiments, a synthetic protein is a portion of a protein.

[0124] In some embodiments, the template is conjugated to magnetic beads. In some embodiments, the template is covalently linked to the magnetic beads. In some embodiments, the template is conjugated to the magnetic beads before the gel is polymerized around the magnetic beads. In some embodiments, the template is conjugated to the beads after polymerization. In some embodiments, the template is directly conjugated to the beads. In some embodiments, the template is conjugated to the beads via a linker. In some embodiments, the linker is a small molecule, peptide, or oligonucleotide linker.

[0125] In some embodiments, the template is conjugated to a polymer gel. In some embodiments, the template is covalently linked to the polymer gel. In some embodiments, the template is retained in the polymer gel in a non-covalent manner. In some embodiments, the template is conjugated to a monomer before the gel is polymerized around the magnetic 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 via a linker. In some embodiments, the linker is a small molecule, peptide, or oligonucleotide linker.

[0126] 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 within the particle. In some embodiments, the particle pore size of the polymer gel is such that the synthesis product cannot escape 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 arrest results in the retention of the synthesis product. In some embodiments, the synthesis product contains 3' puromycin, resulting in tethering of the product to the template.

[0127] In certain embodiments, the method further includes detecting the activity of the enzyme. In certain embodiments, the presence or amount of the synthetic product is detected. In certain embodiments, detection or selection is performed. In certain embodiments, the enzyme activity is measured using flow cytometry, cell sorting equipment, density measurement, affinity tag detection or DNA sequencing. In certain embodiments, the activity is measured by binding fluorescently labeled protein (e.g., antibody). In certain embodiments, the activity is measured by binding fluorescently labeled oligonucleotides. In certain embodiments, the labeled oligonucleotide is complementary to the synthetic product or a part of the synthetic product. In certain embodiments, the labeled oligonucleotide is not complementary to the template.

[0128] In some embodiments, the method further includes detecting the synthetic product. For example, in some embodiments, the method includes using fluorescent markers to detect the synthetic product and performing flow cytometry or cell sorting to detect the product. In some embodiments, the synthetic product is a polynucleotide and sequencing is used to detect the polynucleotide product.

[0129] IV. Methods for Generating DNA-Encoded Libraries

[0130] Also provided herein are methods for generating DNA-encoded libraries (DELs). In some embodiments, the DELs comprise particles, wherein each particle comprises a different DNA sequence. In some embodiments, the DNA sequence is associated with a building block. In some embodiments, the DNA or oligonucleotide sequence associated with the building block is a "tag." In some embodiments, the DNA sequence is associated with a chemical building block (such as a functional group). In some embodiments, the DNA is used as a barcode or tag to identify a small molecule associated with the particle.

[0131] In some embodiments, the method includes incubating particles comprising a magnetic core coated in a polyacrylamide gel under conditions that allow the building blocks to couple to the functional sites, the polyacrylamide gel comprising the functional sites and a synthetic hairpin header DNA (HDNA). Previous publications provide examples of DNA headers in which beads are functionalized with azido DNA header moieties (MacConnell et al. ACS Comb. Sci. 2015, 17, 9, 518–534). In particular embodiments, the header 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 header comprises a primer binding region.

[0132] In some embodiments, the library comprises a plurality of header segments. In some embodiments, each of the plurality of header segments comprises the same sequence region (e.g., a primer binding region) and a different coding region (e.g., a first tag encoding an oligonucleotide sequence for use in the library, a property of the library, a junction, a spacer, or adding a first component or facilitating hybridization, amplification, or sequencing techniques).

[0133] In general, the head segment comprises a non-self-complementary sequence at the 5'-end or 3'-end, which allows the oligonucleotide tag to be bound by polymerization, enzymatic connection or chemical reaction. The head segment allows the connection of oligonucleotide tags and optional purification and phosphorylation steps. After adding the last tag, an additional adapter sequence can be added to the 5' end of the last tag. Exemplary adapter sequences include primer binding sequences or sequences with labels (e.g., biotin). In the case where many structural units and corresponding tags (e.g., 100) are used, a mix-and-split strategy can be used during the oligonucleotide synthesis step to create the necessary number of tags. Such mix-and-split strategies for DNA synthesis are known in the art. After selecting the binding entity and the target of interest, the resulting library members can be amplified by PCR.

[0134] In certain embodiments, the header comprises one or more primer binding sequences.For example, the header comprises a sequence in a hairpin loop region, which is used as a primer binding region for amplification, wherein the primer binding region has a higher melting temperature than the sequence in the header for its complementary primer (for example, it may include a flanking identifier region). In other embodiments, the complex is included in two primer binding sequences on either side of one or more tags encoding one or more structural units (for example, so as to be able to perform a PCR reaction). Alternatively, the header can include a primer binding sequence at the 5' end or 3' end. In certain embodiments, the header is a hairpin, and the loop region forms a primer binding site, or the primer binding site is introduced by hybridizing an oligonucleotide with the header on the ring 3' side. The primer oligonucleotide contains a region homologous to the header 3' end and carries a primer binding region (for example, so as to be able to perform a PCR reaction) on its 5' end, can hybridize with the header and can contain a coding structural unit or a tag that adds a structural unit. Primer oligonucleotides may contain additional information, such as a randomized nucleotide region, for example 2 to 16 nucleotides in length, which is included to facilitate bioinformatics analysis.

[0135] The header may optionally include a hairpin structure, wherein the structure can be achieved by any useful method. For example, the header may include complementary bases that form an intermolecular base pairing partner, such as formed by Watson-Crick DNA base pairing and / or by wobble base pairing. In another example, the header may include modified or substituted nucleotides that can form a duplex formation with higher affinity than unmodified nucleotides, such modified or substituted nucleotides being known in the art. In some embodiments, the header includes one or more cross-linked bases to form a hairpin structure. In some embodiments, bases within a single strand or bases in different duplexes can be cross-linked, for example, by cross-linking using psoralen.

[0136] In some embodiments, the header or tag comprises one or more labels that allow detection. For example, the header, one or more oligonucleotide tags, and / or one or more primer sequences comprise an isotope, a radioactive imaging 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).

[0137] In some embodiments, the header or tag is modified to increase solubility under semi-aqueous, reducing, or non-aqueous (e.g., organic) conditions. In some embodiments, the C5 position of the T or C base is modified with an aliphatic chain without significantly disrupting their ability to form hydrogen bonds with their complementary bases. Exemplary modified or substituted nucleotides are 5'-dimethoxytrityl-N4-diisobutylaminomethylene-5-(1-propynyl)-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 5'-dimethoxytrityl-5-(1-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-1-yl-ethynyl)-2'-deoxyuridine or 3'-[(2-cyanoethyl)-(N,N15 diisopropyl)]-phosphoramidite.

[0138] In addition, the header and / or oligonucleotide tags may be interspersed with modifications that promote solubility in organic solvents. For example, azobenzene phosphoramidites can introduce hydrophobic moieties in the header design. Such hydrophobic amidites are inserted into the header and can occur at any position in the molecule. Therefore, adding hydrophobic residues to the header design allows for improved solubility under semi-aqueous or non-aqueous (e.g., organic) conditions while enabling the header to be oligonucleotide labeled. In addition, the DNA tags subsequently introduced into the library may also be modified at the C5 position of the T or C base so that they also make the library more hydrophobic and soluble in the organic solvents used for subsequent library synthesis steps.

[0139] Various ligation techniques can be used to add scaffolds, building blocks, spacers, linkages, tags, and / or headers to create 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 header or complex; adding spacers to the header; and adding one or more scaffolds or building blocks to the header or complex. In certain embodiments, the ligation technique for any oligonucleotide provides a resulting product that can be transcribed and / or reverse transcribed to allow decoding of the library or for template-dependent polymerization with one or more DNA or RNA polymerases.

[0140] In general, enzymatic ligation produces oligonucleotides with natural phosphodiester bonds that can be transcribed and / or reverse transcribed. Exemplary methods of enzymatic ligation are provided herein and include the use of one or more RNA or DNA ligases, such as T4 RNA ligase, T4 DNA ligase, CircLigase, or a combination thereof.TM ssDNA ligase, CircLigase TM II ssDNA ligase and ThermoPhage TM ssDNA ligase (Prokazyme Ltd., Reykjavik, Iceland). In some embodiments, ligation comprises the use of RNA ligase, or a combination of RNA ligase and DNA ligase. Ligation may further comprise one or more soluble multivalent cations in combination with one or more ligases.

[0141] Chemical ligation can also be used to produce oligonucleotides that can be transcribed or reverse transcribed. It may be necessary to test the efficacy of chemical ligation technology to provide oligonucleotides that can be transcribed or reverse transcribed. This efficacy can be tested by any useful method (such as liquid chromatography-mass spectrometry, RT-PCR analysis and / or PCR analysis). In a particular embodiment, chemical 5 connection includes using one or more chemical reactivity pairs to provide a spacer that can be transcribed or reverse transcribed. In particular, reactions suitable for chemical reaction pairs are preferred candidates for the connection process (Kolb et al., Angew. Chem. Int. Editing, 40: 2004-2021 (2001); Van der Eycken et al., QSAR Comb. Sci., 26: 1115-1326 (2007)). In one embodiment, the oligonucleotides connected contain connections, and the polymerase has a reduced ability to read or shift through the connection, such as "unreadable" connections.

[0142] In some embodiments, the methods described herein include reaction conditions that promote enzymatic or chemical ligation between a header and a tag or between two tags. These reaction conditions include using modified nucleotides within the tags 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 TM DNA ligase and / or T4 RNA ligase), and varying their concentrations; using polyethylene glycol (PEG) with different molecular weights, and varying their concentrations; using non-PEG crowding agents (e.g., betaine or bovine serum albumin); varying the temperature and duration for ligation; varying the concentrations of various reagents, including ATP, Co(NH3)6Ch, and yeast inorganic pyrophosphate; using enzymatically or chemically phosphorylated oligonucleotide tags; using 3' protected tags; and using pre-adenylated tags. These reaction conditions also include chemical ligation.

[0143] The header and / or tag may include one or more modified or substituted nucleotides. In a preferred embodiment, the header and / or tag include 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 used as substrates for ligation. In some embodiments, the header and / or tag are modified to include one or more chemically reactive groups to support chemical ligation (e.g., optionally substituted alkynyl groups and optionally substituted azido groups). In some embodiments, the tag oligonucleotide is functionalized with chemically reactive groups at both ends, and optionally, one of these ends is protected so that side reactions can be reduced (e.g., reducing polymerization side reactions).

[0144] In some embodiments, the complex is purified before or after ligation. In some embodiments, the complex is purified to remove unreacted headers or tags that may cause cross-reactions. 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 phosphates may cause reduced ligation activity. In some embodiments, it may be necessary to remove entities introduced into a chemical or ligation step in order to proceed to a subsequent chemical or ligation step.

[0145] Enzymatic and chemical linkages can include polyethylene glycol having an average molecular weight greater than 300 daltons (e.g., greater than 600 daltons, 3,000 daltons, 4,000 daltons, or 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, such as about 25% (w / v) to about 35% (w / v), such as 30% (w / v).

[0146] In some embodiments, the tag is installed by ligating a single-stranded oligonucleotide to a single-stranded oligonucleotide.

[0147] Branched oligomers or polymers can also be synthesized, provided that at least one building block comprises three functional groups that are reactive with other building blocks.The libraries of the present disclosure can comprise linear molecules, branched molecules, or a combination thereof.

[0148] In certain embodiments, structural unit refers to the chemical structural unit that is attached to other chemical structural units or may be attached to other such structural units.In certain embodiments, structural unit is a part for organic micromolecule.When functional part is polymeric part or oligomeric part, structural unit is the monomeric unit of polymer or oligomer.Structural unit can also include skeleton structure (for example, skeleton structural unit), which is connected to or can be connected to one or more other structures (for example, peripheral structural unit).In certain embodiments, structural unit is complementary (that is, structural unit must be able to react together to form a structure comprising two or more structural units).In certain embodiments, structural unit has at least two reactive groups.In certain embodiments, this structural unit only has one reactive group.In certain embodiments, the reactive groups on two different structural units are complementary, i.e. can react together to form a covalent bond.The example of structural unit is provided in U.S. Patent Application Publication No. 2007 / 0224607, which is incorporated herein by reference.

[0149] In addition, chemically reactive pairs (or functional groups) can be easily incorporated into synthetic protocols and will support efficient chemical ligation of oligonucleotides. Furthermore, the resulting ligated oligonucleotides can be used as templates for template-dependent polymerization with one or more polymerases. In some embodiments, any of the binding steps described herein for labeling encoded libraries can be modified to incorporate enzymatic ligation and / or chemical ligation techniques.

[0150] In certain embodiments, method is included in allowing structural unit to be attached to particle and oligonucleotide is attached to head section DNA or is attached to the oligonucleotide attached to head section DNA and provides structural unit and associated oligonucleotide under the condition of.In certain embodiments, condition is connection condition.In certain embodiments, method includes hatching together with DNA ligase.In certain embodiments, oligonucleotide is attached to HDNA or is the oligonucleotide that is attached to HDNA by polymerization.In certain embodiments, oligonucleotide is attached by chemical reaction.In certain embodiments, oligonucleotide is by utilizing chemical reactivity to (for example, including optionally substituted alkynyl and azido functional group pair) and / or enzyme connection (such as using one in multiple RNA ligase and / or DNA ligase) be connected.

[0151] In another aspect, the invention features a library comprising one or more particles described herein. In some embodiments, the library comprises a plurality of header segments. In other embodiments, each of the plurality of header segments comprises the same sequence region (e.g., a primer binding region) and a different coding region (e.g., a tag encoding a characteristic of the library). In a specific embodiment, the library comprises about 10 2 to 10 20 Particles between (e.g., about 10 2to 10 3 10 2 to 10 4 10 2 to 10 5 10 2 to 10 6 10 2 to 10 7 10 2 to 10 8 10 2 to 10 9 10 2 to 10 10 10 2 to 10 11 10 2 to 10 12 10 2 to 10 13 10 2 to 10 14 10 2 to 10 15 10 2 to 10 16 10 2 to 10 17 10 2 to 10 18 10 2 to 10 19 10 4 to 10 s 10 4 to 10 6 10 4 to 10 7 10 4 to 10 8 10 4 to 10 9 10 4 to 10 10 10 4 to 10, 10 4 to 10 12 10 4 to 10 13 10 4 to 10 14 10 4 to 10 15 10 4 to 10 16 10 4 to 10 17 104 to 10 18 10 4 to 10 19 10 4 to 10 20 10 5 to 10 6 10 5 to 10 7 10 5 to 10 8 10 5 to 35 10 9 10 5 to 10 10 10 5 to 10 11 10 5 to 10 12 10 5 to 10 13 10 5 to 10 14 10 5 to 10 15 10 5 to 10 16 10 5 to 10 17 10 5 to 10 18 10 5 to 10 19 , or 10 5 to 10 20 In some embodiments, each particle is different.

[0152] In any of the above embodiments, the method further comprises one or more steps to diversify the library or query the members of the library, as described herein. In some embodiments, the method further comprises identifying small drug-like library members that bind to or inactivate proteins of therapeutic interest. In other embodiments, the method further comprises contacting the members of the library with a biological target under conditions suitable for binding of at least one member of the library 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.

[0153] In any of the above embodiments, the encoded information is provided as one or more tags or in a combination of multiple tags. In some embodiments, the encoded information is represented by more than one tag (e.g., two, three, four, five, six, seven, eight, nine, ten or more tags). In some embodiments, the encoded information is represented by more than one tag, wherein all encoding tags are included in the coding sequence (e.g., by using a specific tag combination to encode the information). In some embodiments, the encoded information is represented by more than one tag, wherein less than all encoding tags are included in the coding sequence (e.g., by using a tag from a set of more than one individual tags to encode in a separate coding sequence).

[0154] In any of the above embodiments, the header and / or oligonucleotide tag comprises about 5 to about 300 nucleotides. In some embodiments, the header 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, 10 to 100 nucleotides, 10 to 50 nucleotides, 10 to 25 nucleotides, 20 to 300 nucleotides, 20 to 250 nucleotides, 20 to 200 nucleotides, 20 to 150 nucleotides, 20 to 100 nucleotides, 20 to 50 nucleotides, 30 to 300 nucleotides, 30 to 250 nucleotides, 30 to 200 nucleotides, 30 to 150 nucleotides, 30 to 100 nucleotides, 30 to 50 nucleotides, 40 to 300 nucleotides, 40 to 250 nucleotides, 40 to 200 nucleotides, 40 to 150 nucleotides, 40 to 100 nucleotides, 40 to 75 nucleotides, 40 to 50 nucleotides, 50 to 30 0 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, 70 to 200 nucleotides, 70 to 150 nucleotides, 70 to 100 nucleotides, 80 to 300 nucleotides, 80 to 250 nucleotides, 80 to 200 nucleotides 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, 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.

[0155] 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.

[0156] In some embodiments, the method comprises coupling the structural unit to a functional site located on or in the particle. In some embodiments, the functional site is located on the magnetic core. In some embodiments, the functional site is located in the polymer gel. In some embodiments, the functional site can react with one or more reactive groups of the structural unit. In some embodiments, the functional site comprises a borate ester, an amine, an isocyanate, a carboxylic acid, or an aryl halide reactive group.

[0157] In some embodiments, the method of generating a DNA-encoded library comprises multiple rounds of incubation, wherein each round of incubation comprises incubating particles with building blocks attached to functional groups in the particles and oligonucleotide tags attached to the header DNA or the previous oligonucleotide tag. In some embodiments, one or more cleanup or purification steps are performed during or between each round. In some embodiments, the method results in particles comprising a plurality of building blocks and a polynucleotide sequence comprising a relevant tag for each building block. Thus, in some embodiments, the method comprises incubating the particles with a second oligonucleotide tag and a second building block, a third oligonucleotide and a third building block, and so on.

[0158] In some embodiments, the oligonucleotide sequence is associated with additional information, such as the concentration of functional sites or different batches of particles.

[0159] In certain embodiments, the method further comprises amplifying oligonucleotides. In certain embodiments, the oligonucleotides are amplified to produce a library. In certain embodiments, the oligonucleotides are sequenced to determine the structural units present on the particle. In certain embodiments, sequencing determines additional information, such as the concentration of functional sites or different batches of particles.

[0160] In yet another aspect, the invention features a method of screening a plurality of chemical entities, the method comprising: (a) contacting a target with any of the particles described herein and / or the libraries described herein; and (b) selecting one or more particles having a predetermined characteristic for the target compared to a control, thereby screening the chemical entities.

[0161] In order to create a large number of chemical entities within the library, the solution containing the head segment can be divided into multiple aliquots and then placed into multiple physically separated compartments (such as the wells of a multi-well plate). Generally speaking, this is a "splitting" step. Within each compartment or well, a series of chemical reactions and ligation steps are performed using the single-stranded tags within each aliquot. The relationship between the chemical reaction conditions and the single-stranded tag sequence is recorded. The reaction and ligation steps can be performed in any order. The reacted and ligated aliquots are then combined or "pooled" and optionally purified at this time. These splitting and pooling steps can be optionally repeated.

[0162] Next, the library can be tested and / or selected for a particular characteristic or function, as described herein. For example, a mixture of labeled chemical entities can be separated into at least two populations, wherein a first population binds to a specific biological target and a second population does not bind (e.g., by negative selection or positive selection). The first population can then be selectively captured (e.g., by elution on a column providing the target of interest or by incubating an aliquot with the target of interest) and optionally further analyzed or tested, such as using optional washing, purification, negative selection, positive selection, or separation steps.

[0163] In some embodiments, the library is contacted with a biological target under conditions suitable for binding of at least one member of the library to the target, followed by removal of library members that do not bind to the target, and analysis of one or more oligonucleotide tags associated with the target. This method may 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., ligands for receptors), receptor targets (e.g., GPCRs and RTKs), ion channels, bacteria, viruses, parasites, DNA, RNA, prions, and carbohydrates.

[0164] In another embodiment, the chemical entity bound to the target does not undergo amplification, but is directly analyzed. Exemplary analytical methods include: microarray analysis, including attenuated resonance photonic crystal analysis; bead-based methods for deconvolution of labels; label-free photonic crystal biosensor analysis; or hybridization-based methods.

[0165] In some embodiments, the present invention relates to a method for the identification and discovery of any number of chemical entities with a specific characteristic or function (e.g., in a selection step). The desired characteristic or function can be used as the basis for dividing a library into at least two parts, while enriching at least one of the member or related members in the library with the desired function. In a specific embodiment, the method includes identifying a small drug-like library member that binds or inactivates a protein with therapeutic significance. In any of these cases, the oligonucleotide tag encodes the chemical history of the library member, and in each case, the set of chemical possibilities can be represented by any specific tag combination. In certain embodiments, a series of chemical reactions are designed and a group of structural units are selected so that the reaction of the selected structural unit under the chemical conditions defined will produce a variety of molecules (or molecular libraries) of combination, wherein one or more molecules can be used as therapeutic agents for specific diseases.

[0166] V. In vitro translation methods

[0167] Also provided herein are in vitro translation methods utilizing the particles provided herein. In some embodiments, the method comprises incubating particles comprising a magnetic core coated with a polyacrylamide gel comprising an RNA template, ribosomes, and amino acids under conditions whereby the RNA is translated into a polypeptide.

[0168] In some embodiments, the RNA template is contained in or on the 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 at the periphery of the magnetic bead gel. In some embodiments, the RNA template is conjugated before polyacrylamide polymerization. In some embodiments, the RNA template is conjugated to the particle after polyacrylamide polymerization.

[0169] 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, such as a single-chain antibody. In some embodiments, the RNA template contains a sequence complementary to a gel-bound oligonucleotide.

[0170] In some embodiments, the methods include creating a translation system by isolating, refining, and mixing factors involved in the translational synthesis of proteins or peptides, such as ribosomes, translation factors, tRNAs, amino acids, and energy sources including ATP and GTP.

[0171] An example of a natural tRNA is a mixture of purified tRNA fractions obtained from collecting and crushing Escherichia coli, which is also commercially available. Some of the A, U, C, and G in the natural tRNA have been chemically modified by enzymes. Alternatively, tRNAs with naturally occurring sequences can also be used, although they are transcribed in a test tube. In contrast, artificial tRNAs, which are transcription products of tRNAs, are preferably used as orthogonal tRNAs to replace natural tRNAs. Artificial tRNAs can be prepared by in vitro transcription using template DNA and an appropriate RNA polymerase. Such artificial tRNAs do not contain any chemical modifications.

[0172] In some embodiments, the translation system is an E. coli S30 translation system (Promega, Madison, Wisconsin) for efficient in vitro translation. The E. coli S30 translation system provides favorable high-efficiency translation of various mRNA templates compared to other in vitro translation systems (e.g., wheat germ extract, rabbit reticulocyte lysate). In addition, the S30 system for in vitro translation is fully characterized and is very suitable for preparing very large reaction mixtures, thereby facilitating the construction of very large libraries by the method of the present invention. The S30 system can also be suitable for using tRNA molecules with non-natural amino acids to incorporate non-natural amino acids. Referring to PCT Patent Publication No. 90 / 05785, which is incorporated herein by reference. In some embodiments, the translation system is a synthetic mixture (e.g., PURExpress) of recombinantly expressed and purified proteins involved in translation. These systems are advantageous because different components can be eliminated to drive the construction of certain types of libraries (e.g., eliminating release factors).

[0173] 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 a HiBit, 6His, FLAG, HA, V5, a streptavidin tag, a GST tag, or an MBP tag. In some embodiments, the combination of the detection tag and the detection agent produces a detectable signal (e.g., fluorescence).

[0174] In some embodiments, the translated peptide or polypeptide is retained in the particle. In some embodiments, the translated peptide or polypeptide does not diffuse out of 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 displays a puromycin tag in various ways, which results in covalent or non-covalent attachment of the peptide to the mRNA.

[0175] In some embodiments, the RNA template encodes a peptide or polypeptide comprising multiple domains. For example, in some embodiments, the RNA template encodes a peptide or polypeptide of interest and a detection tag. In some embodiments, the RNA template encodes multiple detection tags.

[0176] 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 comprises the amino acid sequence (G n S) X, wherein 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).

[0177] 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 nucleotide modified with 3' puromycin.

[0178] 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.

[0179] 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, measuring density, detecting affinity tags, and / or detecting DNA sequences. In some embodiments, fluorescence microscopy and flow cytometry are used to visualize translation of specific epitopes within the particles.

[0180] In some embodiments, the translated gel particles exhibit uniform antibody-derived fluorescence throughout the periphery of the gel, whereas only magnetic bead autofluorescence is detected in the same particles prior to translation. In some embodiments, for selected epitopes (such as FLAG, HA, or V5), baseline separation is achieved by flow cytometry of the translated gel particle fluorescence compared to the untranslated particles.

[0181] In some embodiments, the translation of gel-immobilized RNA and the subsequent immobilization of the translated products in gel particles are verified by orthogonal detection of multiple epitope tags. In some embodiments, the translated products immobilized in the gel particles are peptides. In some embodiments, the translated products immobilized in the gel particles are therapeutic drugs. In some embodiments, the presence of each epitope of the translation products immobilized or captured in the gel particles is confirmed by specific epitope tagging and in-gel protein binding assays using immunofluorescence measurements. In some embodiments, the in-gel peptide capture yield is quantified via an in vitro translation efficiency assay based on HiBiT luminescence.

[0182] In some embodiments, the gel particle library prepared by the method provided herein is screened via FACS. For example, in some embodiments, the method provided herein obtains a gel particle library of peptide templates in which degenerate codons are fused to HiBiT tags for use in limited dilution emulsion PCR (emPCR) to template magnetic beads. In some embodiments, single bead qPCR analysis of templated library beads shows that approximately 30% of the beads in more than 4000 DNA templates / beads are templated. In some embodiments, the library beads (5 x 10 6 ) is combined with control epitope beads (e.g., 1% HA or V5), translated in the presence of puromycin oligonucleotide P4, detected with dye-labeled antibodies (e.g., anti-HA-647AF; anti-V5-CF488A), and sorted into two populations, high V5 and high HA, by FACS. The sorted hit particles are sorted to determine the enrichment rate for various control tags. The read pattern matches the epitope tag sequence or the degenerate sequence of the library. In some embodiments, the HA positive hit pool is 99% HA coding sequence, and the V5 hit pool is 50% V5 coding sequence, which are enriched 100 times and 50 times respectively compared to the library starting material.

[0183] In some embodiments, the library scale synthesis and sorting provided herein are compatible with engineered genetic codes. For example, in some embodiments, beads are functionalized with DNA templates encoding a single codon (e.g., a native Gln codon) fused to a HiBiT tag and encapsulated in a P1 functionalized gel. Azide functionality is displayed via IVTT using an engineered in vitro transcription and translation (IVTT) mixture lacking glutamine and glutamine-tRNA synthetase (GlnRS) and containing a CUGtRNA loaded using dFx flexizyme. Asn GGC In some embodiments, the HiBiT quantification yields similar levels of captured peptides (105 ± 7 nM) as the pure native peptide epitopes captured in some other embodiments and examples provided herein. In some embodiments, a gel particle library of peptide templates having engineered codons fused to the HiBiT tag and exhibiting AzK functionality is prepared using the methods described herein to obtain an engineered IVTT mixture.

[0184] In some embodiments, a peptide library template with 5NNT degenerate codons (excluding GAC codons) was used to template magnetic beads via emPCR to obtain peptides with 2 x 10 4 After gel encapsulation, the NNT library beads (3 x 106 ) was combined with AzK-templated beads prepared herein (approximately 0.1% of beads) and in-gel transcription / translation was performed using the engineered IVTT mixture with AzK functionality provided herein (without glutamine) and in the presence of P4. After translation, the entire library mixture was subjected to an in-gel CuAAC click reaction, where the AzK-presenting particles were labeled with 647AF-alkyne, and then the high 647AF population (approximately the top 2%) was sorted via FACS. After sequencing and decoding, approximately 6-fold enrichment of GAC-templated sequences was observed in the sorted population compared to the starting library pool.

[0185] In some embodiments, provided herein are methods for translating and detecting gel particle library beads containing non-canonical amino acids. For example, in some embodiments, NNU library particles (3 x 10 6 ) undergo an engineered in vitro translation (IVT) reaction that provides for the installation of azidolysine (AzK) at CUG codons. The translated particles are washed and treated with AF647-alkyne in a CuAAC reaction and then analyzed by flow cytometry. The resulting particles are sorted to isolate the top 2% of the AF647 population and sequences compared to the starting library. In some embodiments, a 6-fold enrichment is observed after one round of screening, with the percentage of AzK sequences increasing from approximately 0.09% in the original NNU library particles to approximately 0.58% after sorting and isolating the top 2% AF647 population using FACS.

[0186] In some embodiments, a standard PCR protocol for library generation is provided herein, and magnetic beads templated with several control epitope encoding sequences and spiked into a conventional library as background were successfully sorted as hits and showed significant enrichment compared to the library input.

[0187] In some embodiments, the coupled transcription / translation of mixed library / epitope particles is provided herein, without the need for additional compartmentalization, resulting in sufficiently pure in-gel epitope display for immunofluorescence-based screening. Translation products (such as RNA transcripts) are concentrated in a gel matrix of DNA-templated magnetic beads, indicating that all enzymatic steps are proximity-driven.

[0188] VI. Kits and Libraries

[0189] In some embodiments, provided herein are kits for preparing particles comprising a polymer coating and a magnetic core. In some embodiments, the kit comprises monomers. In some embodiments, the kit comprises acrylamide and bisacrylamide. In some embodiments, the kit comprises one or more polymerization initiators. In some embodiments, the kit comprises an oil solution and an aqueous solution. In some embodiments, the kit comprises: an oil solution comprising a polymerization initiator; and an aqueous solution comprising monomers. 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 oil solution is in one container and the aqueous solution is in another container. In some embodiments, emulsifying the oil solution with the aqueous solution results in polymerization of the monomers.

[0190] In some embodiments, the kit further comprises instructions for producing particles according to the methods provided herein. In some embodiments, the kit comprises instructions for producing a library of particles provided herein.

[0191] Also provided herein are particle libraries. In some embodiments, the library comprises particles with different oligonucleotide tags. In some embodiments, the library comprises particles with different building blocks. In some embodiments, the library comprises particles with different small molecules. In some embodiments, the library comprises particles with different mRNA sequences. In some embodiments, the library comprises particles with different peptides or polypeptides. In specific embodiments, the library comprises particles in about 10 2 to 10 10 Particles between (e.g., about 10 2 to 10 3 10 2 to 10 4 10 2 to 10 5 10 2 to 10 6 10 2 to 10 7 10 2 to 10 8 10 2 to 10 9 10 3 to 10 4 10 3 to 10 5 10 3 to 10 6 10 3 to 10 7 10 3 to 10 8 103 to 10 9 10 3 to 10 10 10 4 to 10 5 10 4 to 10 6 10 4 to 10 7 10 4 to 10 8 10 4 to 10 9 10 4 to 10 10 10 5 to 10 6 10 5 to 10 7 10 5 to 10 8 10 5 to 10 9 10 5 to 10 10 10 6 to 10 7 10 6 to 10 8 10 6 to 10 9 10 6 to 10 10 10 7 to 10 8 10 7 to 10 9 10 7 to 10 10 10 8 to 10 9 10 8 to 10 10 10 9 to 10 10 In some embodiments, each particle comprises a different oligonucleotide, peptide, small molecule, polypeptide, or building block group. In some embodiments, multiple particles within a library comprise the same oligonucleotide, peptide, small molecule, polypeptide, or building block group. In some embodiments, each particle in the library comprises an identification sequence that can be used to identify the building block group, peptide, small molecule, or polypeptide.

[0192] Examples

[0193] Example 1 Diversified functionalization of polyacrylamide hydrogel magnetic particles via copolymerization

[0194] This example demonstrates that diverse functionalization of polyacrylamide hydrogel-coated magnetic particles can be achieved via copolymerization. Free radical-mediated polymerization forms polyacrylamide hydrogels that introduce various functionalities into the polymer substrate by incorporating molecules with both the desired functionality and vinyl groups for copolymerization. Various copolymerization functionalities are employed, including but not limited to crosslinkers, cell adhesion promoters (e.g., alkylamines), affinity capture tags (e.g., chloroalkyl HaloTags), enzyme capture, probe capture, and oligonucleotides for hybridization (e.g., oligonucleotides for hybridization). Figure 18 ). The latter two can be used for labeling and characterization.

[0195] Using this methodology, polyacrylamide hydrogels with both chemical (e.g., amine, azide) and biochemical (e.g., oligonucleotide) groups were synthesized. These groups were also shown to be able to participate in reactions within the hydrogel matrix of the particle system.

[0196] method

[0197] Azido oligonucleotide preparation: Azide modification of 5'-NH2 P1 DNA oligonucleotide primer with EDC, NHS and 5-azidopentanoic acid to obtain 5'-N3P1, followed by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) of 5'-N3P1 to propargyl-modified magnetic microbeads (e.g., Dynabeads, 1.0 μm, 2.8 μm or 10.0 μm diameter) was performed as previously reported (Malone et al. ACS Comb. Sci. 2017, 19, 1, 9–14).

[0198] Methacrylamide DNA head segment (ac-HDNA) functionalization: Methacrylic acid (100 μmol) was added to DMF containing NHS (100 μmol) and EDC (100 μmol), and the esterification reaction was incubated at room temperature for 5 minutes. Methacrylic acid NHS ester solution (1 M, 15 μL) was added to phosphate buffer (0.4 M, pH 7.9) containing HDNA (100 nmol), and the reaction was incubated at room temperature for 1.5 hours, and then Tris buffer (20 μL, 1 M) was added for quenching. Methacrylamide-HDNA (ac-HDNA) was purified by reverse phase HPLC (Waters XTerra C18, 2.5 μm, 10 mm × 50 mm) using a gradient elution (mobile phase A: 97.5% 50 mM TEAA, pH 8; mobile phase B: ACN; 2.5% B 1 min, 2.5% to 12.5% ​​B 20 min, 4 mL / min) and absorbance detection (260 nm). Fractions containing the product of interest were pooled, dried, and re-dissolved in HPLC grade water for A260 quantification. Fractions of interest (1 μL) were spotted onto a MALDI-TOF MS target plate, dried, overlaid with THAP substrate solution (1 μL, 18 mg / mL THAP, 7 mg / mL diammonium citrate in 1:1 ACN:HO), dried, and analyzed via MALDI-TOF MS (Microflex, Bruker Daltonics, Inc., Billerica, MA).

[0199] Preparation of hydrogel particles: Carboxylic acid functionalized magnetic beads (M-270 carboxylic acid Dynabeads, 5×10 7 , ThermoFisher Scientific) were added to 1.5 mL microcentrifuge tubes and subjected to magnetic separation ( Figure 11). The supernatant was removed and the beads were suspended in an acrylamide monomer solution (300 μL, 0.5M 19:1, monoacrylamide: bisacrylamide, 0.6% APS). Oil (900 μL, 4 / 20 / 76 w / w / w, KF-6038 / mineral oil / DMF-A-6cs) was layered on the bead suspension and the separated oil / water sample was purged with Ar (10 min). TEMED (1 μL) was added to the oil layer, the head space 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 separated, the supernatant was removed, and the particles were washed with lysis buffer (breaking buffer) (4 × 1 mL) until no oil trace remained. The gel particles were suspended in bead buffer (1 mL) for storage. Various functionalizations were performed on the gel particles by adding the following reagents to the acrylamide monomer solution: propargyl methacrylate (20 μM, "alkyne" functional group), N-(3-aminopropyl) methacrylamide (0.002 mM to 20 mM, "amine" functional group), ac-P1 (20 μM, "reverse primer" functional group), or ac-HDNA (20 μM, HDNA functional group).

[0200] In-gel oligonucleotide hybridization: gel particles were mixed with FAM-P1' (1 μM, 20 pmol / 1×10 6 The gel particles were magnetically separated, washed (2×200 μL 2×SSC, 0.5% SDS), and suspended in bead buffer (250 μL) for storage and analysis.

[0201] In-gel hybridization of activity-based probes: Gel particles are copolymerized with oligonucleotides and then hybridized with trypsin activity-based probes conjugated to complementary oligonucleotides. The probe-hybridized gel beads are digested with trypsin to dequench the activity-based probes and obtain fluorescent gel particles ( Figure 19 ).

[0202] In-gel amine acylation: gel particles (1×10 7 ) were 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 separated, washed with bead buffer (2×200 μL), and suspended in bead buffer (200 μL) for storage and analysis.

[0203] In-gel CuAAC (Cu(I)-catalyzed azide-alkyne cycloaddition): Gel particles (1×10 7 ) were suspended in reaction buffer (1 M TEAA pH 7, 0.5% Tween-20, 100 μL) and combined with N3-647AF (5 nmol). A 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. The gel particles were magnetically separated, washed with bead buffer (2 x 200 μL), and suspended in bead buffer (200 μL) for storage and analysis.

[0204] In-gel DNA ligation: Gel particles (2×10 6 ) was suspended in T4 ligase buffer (20 μL, NEB), and enzymatic ligation substrate DNA oligonucleotides (L(+) and L(-), 50 μM each) were added, heated (2 min, 95°C), and cooled to room temperature. The heat-treated reaction mixture was split in half, T4 ligase (20 U, NEB) was added to one aliquot, and the samples were incubated at room temperature for 1 hour. The gel particles were magnetically separated, washed with bead buffer (2 × 200 μL), and suspended in bead buffer (200 μL) for storage and analysis.

[0205] Confocal fluorescence imaging (particle analysis): The gel particles hybridized with FAM-P10' were imaged via a confocal fluorescence microscope (Stellaris 8, Leica). The particles (approximately 10 6 ) were loaded into the imaging well and allowed to stand for 15 min. Particle fluorescence was collected (l ex / l em =488 / 520 nm) and used to calculate the object diameter (LAS).

[0206] Flow cytometry (particle analysis): Gel particles were analyzed by flow cytometry (NovoCyte, Agilent). The gel particles were suspended in bead buffer (1 to 5 × 10 4 Beads / μL) were collected for analysis. Particles were gated based on forward scatter (FSC) and side scatter (SSC) to isolate single particle populations ( Figure 7 ). Based on dye labels for gel particle detection reporting population fluorescence of various channels (FAMλ ex / λ em =488 / 530nm; TMRλ ex / λem =561 / 580nm;APCλ ex / λ em =640 / 660nm).

[0207] result

[0208] 2.8 μm magnetic beads were encapsulated in hydrogels showing diverse functionalities ( Figures 1A to 1E Specifically, oligonucleotides modified with commercially available 5'-acrylate or amino oligonucleotides modified with methacrylic acid ( Figure 2 ), using propargyl methacrylate (PMA) modified alkynes ( Figure 1D ), or using a primary amine of 3-(aminopropyl)methacrylamide (APMA) ( Figure 1E ). The gel particles were mixed with 5'-methacrylamide oligonucleotides ( Figure 1B ), methacrylamide-modified hairpin DNA (HDNA) ( Figure 1C ), propargyl methacrylate (PMA) ( Figure 1D ), or 3-(aminopropyl)methacrylamide (APMA) (20 μM additive) ( Figure 1E ) copolymerization. Gel functionalization is detected by chemical reactions involving complementary oligonucleotide hybridization ( Figure 1B ), Enzymatic ligation of dsDNA modules for DEL synthesis ( Figure 1C ), CuAAC with Alexa Fluor 488 Azide (488AF-N3) ( Figure 1D ), or acylation with fluorescein succinimidyl ester (FAM-OSu) ( Figure 1E Gel markers were detected by flow cytometry and compared with blank gel beads (Figure 1, right panel).

[0209] P1-functionalized gel particles were obtained by copolymerization with 5'-methacrylamide-modified DNA oligonucleotide P1 (acrydite), which was hybridized with 5'-fluorescein (FAM)-labeled DNA oligonucleotide complementary to P1 (FAM-P1') and analyzed by confocal fluorescence imaging ( Figure 10A A median diameter of 7 ± 2 μm was observed in nine preparations (32,000 sampled particles). Figure 10B Flow cytometric analysis of the FAM-P1′-probed gel particles revealed a unique set of signals with a 100-fold increase in fluorescence intensity compared to the unprobed gel particles, indicating that >95% of the magnetic beads were encapsulated in the hydrogel ( Figure 10C ).

[0210] By imaging microscopy ( Figure 9 ), increasing the magnetic bead size resulted in a larger and more uniform distribution of gel particles ( Figure 13 ), and all bead sizes (e.g., magnetic beads with diameters of 1.0, 2.8, and 10.0 μm, respectively, encapsulated in hydrogels) yielded populations that were easily gated by forward and side scatter in flow cytometry to allow comparison of hydrogel fluorescence upon probing ( Figure 7 ).

[0211] Particle functionalization was detected and characterized by flow cytometry, where appropriate fluorescent dye-labeled substrates (including complementary oligonucleotides, double-stranded oligonucleotide linker modules, azide or succinimidyl ester, respectively) were added. Depending on the reactivity, the relative fluorescence increased between 5-fold and 25-fold compared to native gel particles (Figure 1, right panel). Hybridization ( Figure 1B , right) and amine acylation ( Figure 1E , right panel) resulted in the largest shift; all functionalization reactions resulted in baseline separation of the products from the starting material by flow cytometry (Figure 1, right panel).

[0212] The hydrogel particles were also tested for enzyme activity-based assays. For example, the hydrogel particles were first copolymerized with oligonucleotides and then hybridized with trypsin activity-based probes conjugated to complementary oligonucleotides. The probe-hybridized gel beads were then digested with trypsin to dequench the activity-based probes and yield fluorescent gel particles. Figure 17A Time-dependent quantification of the fluorescence of hydrogel particles loaded with a fluorescent (on) green fluorescent probe with trypsin activity. Figure 17B Time-dependent quantification of hydrogel particles loaded with N-terminally labeled tryptic peptides with the red fluorescent dye Cy5. Following trypsin digestion, Cy5 is released from the gel, resulting in a time-dependent decrease (offset) in the fluorescence signal.

[0213] Hydrogel particles with increasing amine loading capacity were prepared by copolymerizing increasing concentrations of APMA (0.02 mM to 20 mM); acylation with FAM-OSu resulted in a log-linear increase in gel particle fluorescence over a four-order titration ( Figure 3A 、 3B ).

[0214] The hydrogels were diversely functionalized by incorporating substoichiometric amounts of methacrylamide-modified additives into the acrylamide:bisacrylamide monomer solution used for copolymerization. For example, the synthesis of hairpin DNA (HDNA) and subsequent proof-of-concept enzymatic oligonucleotide ligation reactions demonstrated the feasibility of standard DEL synthesis workflows using these particles.

[0215] The loading capacity of the particles for 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) carried approximately 4 fmol of amine sites.

[0216] in conclusion

[0217] In this example, magnetic core hydrogel particles successfully supported diverse functionalization via copolymerization and subsequent chemical and biochemical solid-phase synthesis procedures. The free radical-mediated polymerization reaction is mild and readily incorporates a variety of acryloyl and methacryloyl moieties. This example demonstrates some of the most common functionalities deployed in chemical library synthesis, but the technique is applicable to numerous other functionalities. This example should not be considered limiting the scope of the present invention.

[0218] In addition, time-dependent fluorescence data ( Figure 17A 、 17B ) demonstrated the utility of hydrogel scaffolds for facilitating biochemical enzyme activity assays. In-gel assay formats can be used to detect the presence of inhibitory elements (e.g., DNA-encoded library members, aptamers, peptides) that are also synthesized in the gel. In-gel analysis can also be used to probe catalytic activity in enzyme engineering experiments, where each particle contains a nucleic acid template for in vitro translation.

[0219] Example 2 In-gel DNA transcription followed by capture of the resulting mRNA via hybridization of magnetic beads to the hydrogel periphery

[0220] Enzymatic ligation plays an important role in the preparation of DNA-encoded chemical libraries, while 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 peripheral hydrogel by a complementary RNA sequence in the hydrogel.

[0221] method

[0222] Bead-templated PCR (single template): P1-functionalized magnetic microbeads (1×10 8 ) were suspended in a PCR mixture (0.4 mM dNTP, 4 μM forward primer P2, 0.1 U / μL Taq DNA polymerase in 1× standard Taq buffer) containing a DNA template (1 pmol / μL) and subjected to thermal cycling ([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] Prepare qPCR mixture (0.2mM each of dNTP, 0.5μM each of P1 and P2 primers, 0.25μM of qPCR probe, wherein the qPCR probe sequence is shown in SEQ ID NO:7, 0.05U / μL Taq polymerase, 1× standard Taq buffer) and aliquot (20μL each) into a 96-well PCR plate. Dilutions (1 / 100 and 1 / 1000) of the templated bead sample were prepared in bead buffer. qPCR wells were assembled by adding the diluted suspension (1μL). The reaction was thermally cycled ([95°C, 20s; 60°C, 20s; 68°C, 20s]×40 cycles) and fluorescence was monitored (530nm, QuantStudio3, Thermo Scientific). Serial dilutions of the template were used to prepare a standard curve, and a constant volume (1μL) was added to each standard reaction (100pg / μL to 0.1fg / μL in logarithmic terms). Beads were counted by hemocytometer to obtain the average amount of template loading per bead.

[0224] In-gel transcription: Untemplated beads labeled with 647AF and beads templated with DNA encoding the FLAG epitope (FLAG-templated beads) were encapsulated in a hydrogel copolymerized with P1 (50 μM) in separate emulsion polymerizations. The solidified gel particles (1×10 each of untemplated and FLAG-templated gel particles) were 7 ) were suspended in T7 RNAP reaction mixture (0.5 mM NTP, 5 mM DTT, 5 U / μL T7 RNAP, 0.4 U / μL TIPP in 1× NEB T7 buffer, 1 μM P3) and incubated (1 h, 37° C.). The particles were washed with lysis buffer (2×500 μL) and suspended in bead buffer (400 μL) for storage and analysis.

[0225] Flow cytometry (particle analysis): Gel particles were analyzed by flow cytometry (NovoCyte, Agilent). The gel particles were suspended in bead buffer (1 to 5 × 10 4 Beads / μL) were collected for analysis. Particles were gated based on forward scatter (FSC) and side scatter (SSC) to isolate single particle populations ( Figure 7 ). Based on dye labels for gel particle detection reporting population fluorescence of various channels (FAMλ ex / λ em =488 / 530nm; TMRλ ex / λ em =561 / 580nm;APCλ ex / λ em =640 / 660nm).

[0226] result

[0227] DNA-templated 2.8 μm magnetic beads were mixed with non-templated dye-labeled (Alexa Fluor 647, 647AF) beads as negative controls ( Figure 4A RNA transcripts are detected by a FAM-labeled DNA oligonucleotide probe incorporated into the 5' region (P3) of the RNA transcript. Figure 4B After in vitro transcription of the bead mixture, flow cytometric analysis indicated the presence of three bead populations: 38% high 647AF fluorescence (i.e., untemplated negative control beads), 59% high FAM fluorescence (i.e., DNA-templated beads), and 2% fluorescence in both channels ( Figure 4C ).

[0228] DNA oligonucleotide primer P1 ( Figure 4A ) functionalized magnetic beads ( Figure 4A ) and DNA oligonucleotide primer P2( Figure 4A ), DNA template beads were prepared by PCR. The DNA template contained T7 RNA polymerase promoter elements (T7 prom, Figure 4A The untemplated negative control beads were stained with Alexa Fluor 647 (647AF, Figure 4A Each bead set was encapsulated in a P2 copolymer hydrogel. In the presence of a FAM-labeled DNA oligonucleotide probe of the 5' region of the RNA transcript, in vitro transcription of the gel-encapsulated DNA-templated beads and the non-templated beads was performed. Figure 4B , P3) detected the presence of RNA transcripts hybridized via P2 in the gel. Two-dimensional flow cytometry analysis showed that most particles exclusively exhibited red fluorescence (38%) ( Figure 4C , 660 nm, negative control without template in Q1) or green fluorescence (59%) ( Figure 4C , 520 nm, templated and RNA-loaded beads in Q3).

[0229] in conclusion

[0230] Flow cytometry results demonstrated that RNA was trapped on the periphery of the magnetic bead hydrogel by hybridization. The results also showed that transcript migration onto the untemplated beads was not only infrequent but also formed a distinct population. Therefore, due to the low level of transcript migration, this method facilitates the generation of libraries with a large number of diverse templates.

[0231] Example 3 In-gel translation of peptides and capture of translated peptides on the hydrogel periphery of magnetic beads

[0232] This example demonstrates in-gel translation of a peptide and subsequent capture or immobilization of the peptide product on the hydrogel periphery of magnetic beads.

[0233] method

[0234] In-gel translation (bioamino acids): DNA templated beads were encapsulated in a hydrogel copolymerized with ac-P1 (50 μM) and acrylamide-modified BSA (50 μM). BSA (2 μmol) and N-acryloyloxysuccinimide (20 μmol) were combined in a 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 to 70 μL) were suspended in NEB containing puromycin capture oligonucleotide P4 (10 μM). The reaction mixture was incubated (3 h, 37°C), washed with lysis buffer (0.5 mL), washed with PBST (0.5 mL), and suspended in PBST (10 4 particles / μL).

[0235] 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).

[0236] In-gel immunofluorescence detection: The translated particles were suspended in detection antibody solution (4×10 4 particles / μL, 10 ng / μL APC anti-HA, 50 ng / μL CF488 anti-V5, 10% skim milk in PBST), incubated (16 h, 4°C), washed with PBST (0.5 mL) and suspended in PBST (1 mL) for flow cytometry.

[0237] Flow cytometry (particle analysis): Gel particles were analyzed by flow cytometry (NovoCyte, Agilent). Gel particles were suspended in bead buffer (1 to 5 × 104 beads / μL) for analysis. Particles were gated based on forward scatter (FSC) and side scatter (SSC) to isolate single particle populations (Figure S2). Population fluorescence in various channels was reported based on the dye label used for gel particle detection (FAMλ ex / λ em =488 / 530nm; TMRλ ex / λ em=561 / 580nm;APCλ ex / λ em =640 / 660nm).

[0238] HiBiT assay: Samples (5 μL solution or 2.5 × 10 4 Hydrogel (5 μL PBS) was combined with LgBiT / substrate mixture (5 μL, 2× LgBiT / substrate, in PBS) in a microtiter plate (black, 384 wells) and incubated (15 min, 37° C.). HiBiT peptide standard solution (0.1 to 1000 nM HiBiT peptide) was assembled and incubated in a similar manner. Luminescence was analyzed via a microplate reader (CLARIOstar Plus, BMG LABTECH), and the concentration of unknowns was determined based on the HiBiT standard analysis ( Figure 8 ).

[0239] result

[0240] Magnetic beads were 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 underwent an mRNA display in vitro transcription / translation reaction, incorporating a puromycin-modified peptide capture oligonucleotide P4 ( complementary to the RNA directly 3' of the stop codon). Figure 5A The translated beads were analyzed by imaging microscopy and flow cytometry to visualize translation of specific epitopes. The translated gel particles exhibited uniform antibody-derived fluorescence throughout the periphery of the gel ( Figure 5B ); before translation, only magnetic bead autofluorescence was detected in the same particles ( Figure 5C Fluorescence of the translated gel particles by flow cytometry achieved baseline separation compared to untranslated particles for all three exemplary epitopes ( Figure 5D In-gel peptide capture yields were quantified via HiBiT luminescence. Particles translated in the presence of the puromycin capture oligonucleotide P4 retained approximately 100 nM HiBiT peptide, whereas translations in the absence of P4 retained <1 nM peptide ( Figure 5E ).

[0241] in conclusion

[0242] In this example, orthogonal detection of multiple epitope tags successfully demonstrated translation of gel-immobilized RNA and subsequent capture or immobilization of translation products in gel particles. Immunofluorescence measurements confirmed the presence of each epitope by specific epitope tagging and demonstrated proof of concept for performing in-gel protein binding assays. In addition, HiBiT detection confirmed the presence of captured peptides and achieved high-sensitivity quantification of captured peptides. The in-gel peptide capture yield (approximately 100 nM) was sufficient for robust detection in immunofluorescence and HiBiT assays.

[0243] Example 4 Compatibility of polyacrylamide hydrogel magnetic particles with FACS analysis as a high-throughput screening strategy

[0244] This example demonstrates the compatibility of the novel gel particle format with FACS analysis as a high-throughput screening strategy, thereby demonstrating its utility. Furthermore, the data also demonstrate that library-scale synthesis and sorting are compatible with the engineered genetic code.

[0245] method

[0246] Bead-templated PCR (single template): P1-functionalized magnetic microbeads (1×10 8 ) were suspended in a PCR mixture (0.4 mM dNTP, 4 μM forward primer P2, 0.1 U / μL Taq DNA polymerase in 1× standard Taq buffer) containing a DNA template (1 pmol / μL) and subjected to thermal cycling ([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.

[0247] A qPCR mixture (0.2 mM each dNTP, 0.5 μM each of P1 and P2 primers, 0.25 μM qPCR probe, 0.05 U / μL Taq polymerase, 1× standard Taq buffer) was prepared and aliquoted (20 μL each) into a 96-well PCR plate. Dilutions (1 / 100 and 1 / 1000) of the templated bead sample were prepared in bead buffer. qPCR wells were assembled by adding the diluted suspension (1 μL). The reaction was thermally cycled ([95°C, 20 s; 60°C, 20 s; 68°C, 20 s] × 40 cycles) and fluorescence was monitored (530 nm, QuantStudio3, Thermo Scientific). Serial dilutions of the template were used to prepare a standard curve, and a constant volume (1 μL) was added to each standard reaction (100 pg / μL to 0.1 fg / μL in logarithmic terms). Beads were counted by hemocytometer to obtain the average amount of template loading per bead.

[0248] Bead templated by emPCR (NNK library template): P1 functionalized magnetic microbeads (1×10 8 ) is suspended in the PCR mixture (0.2mM dNTP, 8μM forward primer P2, 0.02% w / v KF-6102, 0.3U / μL Taq DNA polymerase, in 1× standard Taq buffer) containing NNK5 DNA template (1.2fg / μL). Oil (900μL, 4 / 20 / 76, KF-6038 / mineral oil / DMF-A-6CS, w / w / w) is added to the top of each aqueous reaction mixture. The reaction is emulsified (65s, 2500rpm) using a bead mill homogenizer (BeadBug, Benchmark Scientific). Using a wide-mouth pipette tip, aliquots (50μL) are transferred to 96-well PCR plates, and the sample is subjected to thermal cycling ([95°C, 20s; 60°C, 20s; 68°C, 30s] × 35 cycles, 68°C, 5min). The plate was placed on a magnet stand and incubated (30 min), the supernatant was removed, and the separated beads were transferred to a clean 1.5 mL tube in lysis buffer and washed with lysis buffer (4 x 1 mL). The washed beads were separated and suspended in bead buffer (1 mL) for analysis.

[0249] Prepare qPCR mixture (0.2mM each of dNTP, 0.5μM each of P1 and P2 primers, 0.25μM of qPCR probe, 0.05U / μLTaq polymerase, 1× standard Taq buffer) and aliquot (20μL each) into a 96-well PCR plate. Beads were counted by a hemocytometer to prepare a suspension (100 beads / μL and 1 bead / μL) in a bead buffer with a known density. Three 100-bead and 77 single-bead qPCR wells were assembled by adding the appropriate suspension (1μL). The reaction was thermally cycled ([95°C, 20s; 60°C, 20s; 68°C, 20s]×40 cycles) and fluorescence was monitored (530nm, QuantStudio3). Serial dilutions of template were used to prepare a standard curve, adding a constant volume (1 μL) to each standard reaction (100 pg / μL to 0.1 fg / μL on a log scale).

[0250] Flow cytometry (library screening): Gel particles were screened by FACS (FACSAria III, BD Biosciences). Based on the dye label used for gel particle detection, the gel particles were detected in various channels (FAMλ ex / λ em =488 / 530nm; APCλ ex / λ em =640 / 660 nm) were subjected to multispectral fluorescence analysis of single particle populations (gated by FSC / SSC correlation as described above).

[0251] In-gel immunofluorescence detection: The translated particles were suspended in detection antibody solution (4×10 4 particles / μL, 10 ng / μL APC anti-HA, 50 ng / μL CF488 anti-V5, 10% skim milk in PBST), incubated (16 h, 4°C), washed with PBST (0.5 mL) and suspended in PBST (1 mL) for flow cytometry.

[0252] NGS library preparation and analysis: A bead aliquot (approximately 1,000 beads) from each of the flow cytometry sorted populations was amplified using primers P1 and P2 and gel purified to isolate amplicons within the template range (180 to 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 sample were taken for end repair, adenylation, and adapter ligation reactions. The adapter-ligated products were cleaned up without library size selection using Agencourt AMPure XP beads (Beckman Coulter, Inc. Brea, CA). The cleaned ligated DNA was amplified by 10 PCR cycles to enrich for adapter-ligated products. Amplified PCR products were cleaned up by 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 Micro SR 300 cycle chemistry and dual indexing. Analysis was performed by aligning each sequence with a reference epitope or library sequence and counting the ratio of matches (up to 3 mismatches) to each aligned sequence.

[0253] aminoacylazidolysine-CUG-tRNA Asn Synthesis of: The aminoacylation reaction (20 μL, 20 μM tRNA and 20 μM dFx flexizyme in 0.1 M Bicine, pH 9.0) was heated (95°C, 3 min) and cooled to room temperature within 5 min. 20 mM MgCl2 was added and the mixture was cooled on ice (5 min). The reaction was initiated by adding azidolysine 3,5-dinitrobenzyl ester (25 mM in DMSO) and incubated on ice for 2 hours. After the acylation reaction, aminoacyl-Ne-azidolysine-CUG-tRNA Asn The pellet was precipitated (0.3 M NaOAc, pH 5.2, 100% EtOH, 10,000 g, 15 m). The pellet was washed (0.1 M NaOAc, pH 5.2, 70% EtOH) and dried.

[0254] In vitro translation of AzK-containing peptides: Peptides containing Ne-azidolysine were translated in a genetically reprogrammed in vitro translation system from recombinant E. coli that omitted glutamine and GlnRS to reprogram 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 of each amino acid), and 1.5 mg / mL E. coli total tRNA, with final protein concentrations 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 EF-G, and 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 ribosome. 5 The particles were suspended in a solution containing puromycin capture oligonucleotide P4 (10 μM) and Ne-azidolysine-CUG-tRNA. Asn The reaction was incubated (3 h, 37 ° C), washed with lysis buffer (0.5 mL), washed with PBST (0.5 mL), and suspended in PBST (10 4 particles / μL) for analysis.

[0255] Library-scale synthesis and sorting with an engineered genetic code: Beads were functionalized with a DNA template encoding a single GAC codon (which naturally encodes Gln) and a HiBiT tag and encapsulated in a P1-functionalized gel. An engineered IVT mixture was used in which glutamine was excluded and GlnRS loaded the non-canonical amino acid azidolysine (AzK) onto CUGtRNAAsn using dFx flexizyme, allowing for specific visualization of azide functionality using in vitro translation.

[0256] result

[0257] Using a peptide library template of 5 NNK degenerate codons fused to a HiBiT tag, magnetic beads were templated via limiting dilution emPCR. Single bead qPCR analysis of templated library beads indicated 4.2k DNA templates / bead, of which approximately 30% of the beads were templated. 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 ).

[0258] The sorted hit particles were sorted to determine the enrichment rate against various control tags. The read pattern matched the epitope tag sequence or the degenerate sequence of the NNK5 library. The HA positive hit pool was about 99% HA coding sequence, and the V5 hit pool was about 50% V5 coding sequence, which were enriched by about 100 times and about 50 times (compared to the library starting material) respectively. Figure 12 ).

[0259] The data also demonstrated that library-scale synthesis and sorting were compatible with the engineered genetic code. The levels of captured peptides quantified by HiBit (105 ± 7 nM) were similar to those of previously tested all-natural peptide epitopes ( Figure 5E ). Peptide library templates with 5 NNT degenerate codons (to exclude GAC codons) were designed, and encapsulated magnetic beads carrying these sequences were generated (bead QC). NNT library beads (3×10 6 ) was combined with a small amount of AzK templated beads of the invention (0.1% of the beads), wherein the azidolysine (AzK) peptide coding sequence is shown in SEQ ID NO: 18, and translated using an engineered IVT mixture in the presence of P4. After translation, the entire library mixture was subjected to in-gel CuAAC, wherein any AzK presenting particles were labeled with 647AF-alkyne, and the high 647AF population (top 2%) was sorted via FACS ( Figure 15 After high-throughput sequencing and decoding, approximately 6-fold enrichment of CUG templated sequences was observed in the sorted population compared to the starting library pool (Table 2 and Figure 14 ).

[0260] in conclusion

[0261] These experiments in this example have determined the feasibility of the high-throughput screening based on FACS of gel particle library.Use standard PCR scheme to carry out library generation, with some control epitope encoding sequence templates and be doped into the magnetic beads as background in conventional library through successful sorting as hit, and show significant enrichment compared with library input.Importantly, the transcription / translation of the coupling of mixed library / epitope particle, without the need for other compartmentalization, obtains enough pure epitope display in gel, for screening based on immunofluorescence.Translation product (such as RNA transcript) is through being concentrated in the gel periphery of the magnetic beads through DNA template.

[0262] Table 1. Oligonucleotide sequences

[0263]

[0264]

[0265]

[0266] Table 2. DNA Sequencing Analysis from Input Library and High APC Population Particles (AzK Sorting NGS Results)

[0267] group AZK library enter 0.09％ 99.91％ High APC 0.58％ 99.42％

[0268] Values ​​are % of total sequences matching the epitope or NNU5 library reference.

Claims

1. A composition comprising particles comprising a magnetic core encapsulated in 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 from 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 acrylamide and bisacrylamide in a ratio of between about 10:1 and about 40:

1.

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

1.

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

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. 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, wherein the total particle diameter is less than about 40 μm, optionally when the total particle diameter is 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 coefficient of variation in particle diameter of about 3% to about 50%.

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

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

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. A method of producing a composition comprising particles comprising a magnetic core encapsulated in a polymer gel, The method comprises: An aqueous solution containing monomers and magnetic beads is emulsified with a solution containing a polymerization initiator to cause polymerization of the monomers and thereby produce a composition containing magnetic cores encapsulated in the polymer gel.

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

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

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

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

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

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

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

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

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

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

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

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

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. The method of any one of claims 21 to 34, further comprising removing the supernatant that does not contain the particles.

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

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

41. A kit comprising: a composition comprising an aqueous solution comprising magnetic beads and acrylamide; and a composition comprising an oil solution comprising 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: a composition according to any one of claims 1 to 20, and instructions for use.

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