Methods and compositions for selecting functional aptamers

Abstract

The present disclosure describes compositions and methods for selecting functional aptamers. In certain embodiments, provided herein are methods of using aptamer cluster-containing particles to identify functional aptamers from an aptamer library. In certain embodiments, provided herein are populations of functionally enriched aptamers. In certain embodiments, provided herein are methods of selecting aptamers for use in personalized cancer therapy and methods of creating tumor delivery systems. In certain embodiments, provided herein are compositions comprising aptamer cluster-containing particles, target cells (e.g., cancer cells, immune cells, etc.), and / or detectable indicators of cellular function (e.g., apoptosis, cell proliferation, fluorescent indicators of gene or protein expression, etc.).

Description

[Technical field]

[0001] The present invention relates to methods and compositions for selecting functional aptamers.

[0002] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 738,235, filed Sep. 28, 2018, which is incorporated by reference in its entirety. [Background technology]

[0003] background Aptamers are short, single-stranded nucleic acid oligomers or peptides that can bind to specific target molecules. Aptamers are usually selected in an iterative process from a large random candidate population. Recently, aptamers have been successfully selected in cells, in vivo, and in vitro.

[0004] Aptamer selection, their structure-function relationships, and mechanisms of action are all poorly understood. Although over 100 aptamer structures have been solved and reported to date, few recurring structural motifs have been identified.

[0005] A variety of different aptamer selection processes have been described to enrich aptamer libraries for aptamers that can bind to specific targets. Some of the binding aptamers identified from such binding enrichment pools were later found to have the ability to participate in functional effects on cells. However, the fact that an aptamer binds to a cell does not mean that it induces a desired cell function. For example, many aptamers that only bind to a specific target cell may not affect the function of that cell or may induce a cell function that is completely different from the desired cell function. Furthermore, functional aptamers that bind weakly to target cells and / or functional aptamers that bind to antigens that are expressed at low levels on the surface of target cells are not enriched by conventional aptamer selection processes.

[0006] Thus, there is a great need in the art for compositions and methods that allow direct enrichment of aptamer libraries for aptamers involved in desired cellular functions. Importantly, such methods and compositions would allow direct identification of aptamers that can modulate desired functional effects on target cells of interest, which would have a profound impact on aptamer therapy. Summary of the Invention

[0007] overview Disclosed herein are compositions and methods that facilitate direct enrichment of aptamer libraries for aptamers that induce desired cellular functions.As disclosed herein, the methods described herein enrich aptamer libraries for aptamer sequences that are different from conventional binding-based aptamer enrichment processes, resulting in the generation of aptamer pools that are highly enriched for aptamers that induce desired cellular functions.This facilitates the identification of functional aptamers that are useful, for example, as aptamer therapeutics.Also provided herein are functionally enriched aptamer libraries produced according to the methods described herein, as well as functional aptamers identified using the methods described herein.

[0008] In a particular aspect, the present invention provides a method for producing a functionally enriched aptamer population.In a particular embodiment, the method includes the steps of: (a) contacting target cells with a number of particles ("aptamer cluster particles") that have a library of aptamer clusters immobilized on the surface, and at least a portion of the immobilized aptamer clusters binds to at least a portion of the target cells to form a cell-aptamer cluster particle complex; (b) incubating the cell-aptamer cluster particle complex for a sufficient period of time for at least a portion of the target cells in the cell-aptamer cluster particle complex to be affected by cellular function; (c) detecting the cell-aptamer cluster particle complex that is affected by cellular function; (d) isolating the cell-aptamer cluster particle complex that is detected in step (c) and that comprises the target cells that are affected by cellular function from other cell-aptamer cluster particle complexes; and (e) amplifying the aptamers in the separated cell-aptamer cluster particle complex to produce a functionally enriched aptamer population. In some embodiments, steps (c) and (d) are performed using a flow cytometer.

[0009] In some embodiments, the cell function is cell survival, cell death (e.g., apoptosis, unprogrammed cell death), cell proliferation, gene expression (e.g., cytokine expression), cell morphology, cell activation, phosphorylation, calcium mobilization, degranulation, cell migration, or cell differentiation.

[0010] In certain embodiments, the target cells are further contacted with a reporter of a cellular function (e.g., a fluorescent reporter of a cellular function) before and / or during step (b). In some embodiments, the target cells are contacted with a reporter of a cellular function before, during, or after contacting the target cells with the aptamer cluster particles. In certain embodiments, the target cells are modified to express a reporter of a cellular function (e.g., a fluorescent protein) when acted upon by a cellular function. In certain embodiments, the cell-adaptor-cluster particle complexes acted upon by a cellular function are detected by detecting the reporter of a cellular function in step (c). In certain embodiments, the reporter of a cellular function is a fluorescent dye. In some embodiments, the reporter of a cellular function is a luminescent dye. In some embodiments, the fluorescent and / or luminescent dye is a calcium-sensitive dye, a cell tracking dye, a lipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolite-sensitive dye, a pH-sensitive dye, a membrane potential-sensitive dye, a mitochondrial membrane potential-sensitive dye, or a redox potential dye. In certain embodiments, the reporter of cell function is an activation-related marker, an oxidative stress reporter, an immunogenic cell death marker, a necrotic marker, a cell differentiation marker, an angiogenesis marker, an apoptosis marker, an autophagy marker, a cell survival marker, or a marker of ion concentration.In certain embodiments, the target cell is not contacted with the reporter of cell function.In some embodiments, the cell that is affected by the cell function is detected by the change in morphology and / or behavior.

[0011] In some embodiments, the method further comprises separating the aptamer cluster particles from the target cells in the cell-aptamer-cluster particle complexes separated in step (d). In certain embodiments, the aptamer cluster particles in the cell-aptamer-cluster particle complexes are separated in step (d) by cell lysis and centrifugation.

[0012] In certain embodiments, the method further comprises dissociating the aptamers from the particles in the separated aptamer cluster particles. In some embodiments, the aptamers in the separated cell aptamer cluster particles are isolated by HPLC purification before step (e). In some embodiments, the method further comprises identifying the enriched population of aptamers by sequencing after step (e).

[0013] In some embodiments, the method further comprises preparing aptamer cluster particles before step (a).In certain embodiments, the step of preparing aptamer cluster particles comprises:(1) immobilizing a large number of aptamers from an aptamer library on a particle surface; and (2) amplifying the large number of immobilized aptamers locally on the particle surface to form aptamer cluster particles.In some embodiments, in step (2), the large number of immobilized aptamers are amplified using emulsion PCR.

[0014] In some embodiments, the method further comprises step (f): (i) forming aptamer cluster particles from the population of functionally enriched aptamers of step (e); and (ii) repeating steps (a)-(e) with the newly formed aptamer cluster particles to generate a further population of functionally enriched aptamers. In some embodiments, step (f) is repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 8 times. In some embodiments, the further enriched population of aptamers of step (f) has, for example, at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times less sequence diversity compared to the library of aptamer clusters of step (a). In some embodiments, each round of step (f) enriches the aptamer population for aptamers that modulate a cellular function, e.g., by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 fold.

[0015] In some embodiments, step (f) further comprises applying restrictive conditions in successive enrichment iterations. In some embodiments, the restrictive conditions are selected from (i) reducing the total number of particles, (ii) reducing the copy number of the aptamer per particle, (iii) reducing the total number of target cells, and / or (iv) shortening the incubation period. In some embodiments, the method comprises introducing errors into the aptamer sequence by amplifying the population of aptamers with an error-prone polymerase.

[0016] In some embodiments, the aptamers in the aptamer clusters comprise unexposed single-stranded nucleic acid sequences with molecular caps. In some embodiments, the aptamer clusters comprise aptamers that contain regions of conserved sequences and regions of randomized sequences. In some embodiments, the regions of randomized sequences are exposed and the regions of conserved sequences are capped. In certain embodiments, the aptamer clusters comprise aptamers that contain chemical modifications or non-natural nucleotides. In some embodiments, the aptamer clusters comprise aptamers that are DNA, RNA, or chemical modifications thereof. In some embodiments, the aptamer clusters are labeled with fluorescent markers or elements that allow visualization under an optical microscope. In some embodiments, the elements that allow visualization under an optical microscope are nanoparticles.

[0017] In certain embodiments, at least 85% (e.g., at least 90%, or at least 95%) of the aptamer cluster particles used in the method individually contain multiple copies of no more than three unique aptamer sequences. In certain embodiments, at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) of the aptamer cluster particles used in the method individually contain multiple copies of no more than two unique aptamer sequences. In certain embodiments, at least 60% (e.g., at least 65%, at least 70%, or at least 75%) of the aptamer cluster particles used in the method individually contain multiple copies of only one unique aptamer sequence.

[0018] In some embodiments, the aptamer cluster comprises at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10 3 , 10 4 , or 10 5In certain embodiments, the library of aptamer clusters collectively comprises at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , or 10 15 In certain embodiments, the library of aptamer clusters comprises 100 to 10 14 The aptamer sequence comprises different aptamer sequences.

[0019] In certain embodiments, the particles of the aptamer cluster particles are selected from polymer beads, agarose beads, polystyrene beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, controlled pore beads, microbeads, and nanoparticles. In certain embodiments, the particles have an average diameter in at least one dimension of about 3 nm to about 30 μm (e.g., about 25 nm to about 30 μm). In certain embodiments, the particles have an average diameter in at least one dimension of about 3 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 50 nm, 100 nm, 250 nm, 0.5 μm, 2 μm, 5 μm, 10 μm, 20 μm, or 30 μm.

[0020] In certain embodiments, the library of aptamer clusters comprises aptamers preselected through one or more processes selected from the group consisting of combined Cell SELEX, negative SELEX, and in vitro evolution. In some embodiments, the method further comprises enriching the initial library of aptamers for aptamers that bind to target cells to generate a binding-enriched population of aptamers, and then using the aptamer binding-enriched population to create the aptamer cluster particles of step (a). In some embodiments, the step of creating the aptamer cluster particles comprises: (1) immobilizing a number of aptamers from the binding-enriched population of aptamers on a particle surface; and (2) locally amplifying the immobilized number of aptamers on the particle surface to form aptamer cluster particles. In certain embodiments, the initial library of aptamers is enriched by repeating combined Cell SELEX one or more times (e.g., 1, 2, 3, 4, 5 times). In some embodiments, the initial library is not enriched for binding aptamers prior to step (a).

[0021] In certain embodiments, the time period of step (b) is substantially instantaneous. In certain embodiments, the time period is from 1 microsecond to about 1 month. In certain embodiments, the time period is from about 10 minutes to about 5 days. In certain embodiments, the time period is at least 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In certain embodiments, the period is 15 minutes or less, 20 minutes or less, 25 minutes or less, 30 minutes or less, 35 minutes or less, 40 minutes or less, 45 minutes or less, 50 minutes or less, 55 minutes or less, 1 hour or less, 2 hours or less, 3 hours or less, 4 hours or less, 5 hours or less, 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less, 10 hours or less, 11 hours or less, or 12 hours or less. In some embodiments, the period is about 10 minutes to about 1 month. In certain embodiments, the period is about 1 hour to about 24 hours. In some embodiments, the period is about 1 hour to about 2 hours. In some embodiments, the period is about 1.5 hours to about 24 hours. In certain embodiments, the period is about 1.5 hours to about 2 hours.

[0022] In certain embodiments, the target cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterium. In certain embodiments, the bacterium is a pathogenic bacterium.

[0023] In some embodiments, the target cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a cancer cell or an immune cell. In certain embodiments, the mammalian cell is a cell from a patient. In some embodiments, the cell from a patient is a cancer cell from a patient or an immune cell from a patient.

[0024] In some embodiments, the target cell is a cancer cell (e.g., a cancer cell from a patient). In certain embodiments, when the target cell is a cancer cell, the cellular function is cell death and / or apoptosis. In some embodiments, when the target cell is a cancer cell, the cellular function is modulation (e.g., inhibition) of ligand expression of an immune checkpoint protein (e.g., PD-L1, PD-L2).

[0025] In certain embodiments, the target cell is an immune cell (e.g., a T cell (helper T cell, cytotoxic T cell, regulatory T cell, etc.), a B cell, a macrophage, a dendritic cell). In some embodiments, when the target cell is an immune cell, the cell function is modulation (e.g., enhancement or suppression) of expression of an immune protein (e.g., a cell surface immune protein such as an immune checkpoint protein (e.g., PD-1, CTLA-4, immune activation markers)). In some embodiments, the immune protein is a cytokine (e.g., an inflammatory cytokine). In some embodiments, when the target cell is an immune cell, the function is cell proliferation. In some embodiments, when the target cell is an immune cell, the function is cell death (e.g., apoptosis). In certain embodiments, the function is increased cytotoxicity by the immune cell.

[0026] In certain embodiments, the cell-aptamer-cluster particle complexes contain, on average, about 1 to about 6 particles per target cell. In certain embodiments, the cell-aptamer-cluster particle complexes contain, on average, about 2 to about 4 particles per target cell.

[0027] In certain embodiments, during step (b), multiple cell-aptamer-cluster particle complexes are incubated in a single reaction volume.

[0028] In some embodiments, the cell-aptamer-cluster particle complexes are separated in step (d) via flow cytometry, fluorescence microscopy, optical tweezers, micropipette, microfluidic separation, micromanipulation, or separation seeding.

[0029] In certain aspects, provided herein is a functionally enriched aptamer population produced by the method described herein.In certain embodiments, the aptamer population is characterized by a functional enrichment of more than 1.1 times (e.g., a functional enrichment of more than 1.5 times) compared with the aptamers in the aptamer cluster library before enrichment.In some embodiments, the cellular function is the promotion of cancer cell death or apoptosis.In certain embodiments, the cellular function is the promotion of immune response.

[0030] In certain aspects, provided herein is a method for selecting aptamers for use in personalized cancer therapy, comprising selecting at least one aptamer candidate that promotes cell death or apoptosis of cancer cells from a functionally enriched aptamer population provided herein. In certain embodiments, the method further comprises sequencing the selected aptamer. In some embodiments, the method further comprises a sequenced aptamer. In some embodiments, the aptamer has been sequenced prior to selection. In certain embodiments, the selection process comprises a high-throughput functional assay and additional sequencing of the assay results. In some embodiments, the data of all candidates is then analyzed (e.g., to detect the best functional candidate in the population).

[0031] In certain aspects, provided herein is a method of creating a tumor delivery system, the method comprising selecting at least one candidate aptamer that promotes cancer cell death or apoptosis from the functionally enriched aptamer population provided herein, and combining the at least one aptamer with a tumor therapeutic agent for localized tumor delivery.

[0032] In some aspects, provided herein is a method for selecting an aptamer for use in personalized cancer therapy, the method comprising preparing a population of functionally enriched aptamers using the methods provided herein, and selecting at least one aptamer candidate from the functionally enriched aptamer population that promotes cell death or apoptosis of cancer cells from a patient. In some embodiments, the aptamer is sequenced prior to selection. In some embodiments, the method further comprises sequencing the selected aptamer. In certain embodiments, the method further comprises synthesizing the sequenced aptamer.

[0033] In certain aspects, provided herein are methods of treating cancer in a subject, the methods comprising: (a) preparing a functionally enriched aptamer population using the methods described herein, using cancer cells of the subject as target cells; (b) selecting at least one aptamer from the functionally enriched aptamer population that promotes cell death or apoptosis of the cancer cells; and (c) administering the selected aptamer to the subject. In some embodiments, the methods further comprise harvesting the cancer cells from the subject.

[0034] In certain embodiments, provided herein are compositions comprising a multiplicity of particles having immobilized libraries of aptamer clusters ("aptamer cluster particles"), target cells, and, optionally, a reporter of cellular function.

[0035] In some embodiments, the cell function reporter is a fluorescent reporter. In some embodiments, the fluorescent reporter is a membrane integrity reporter. In some embodiments, the fluorescent reporter is a capsid integrity reporter. In some embodiments, the fluorescent reporter is a protein integrity reporter. In certain embodiments, the fluorescent reporter is a protein denaturation reporter. In some embodiments, the fluorescent reporter is a cell death reporter. In some embodiments, the fluorescent reporter is a redox potential reporter.

[0036] In some embodiments, the composition comprises at least about 10, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , or 10 8 In some embodiments, the composition comprises 10 aptamer clusters. 6 ~10 11 (For example, 10 6 ~10 9 In certain embodiments, each aptamer cluster comprises at least about 10, 100, 10 3 , or 10 4 In certain embodiments, each aptamer cluster comprises about 10 copies of the aptamer. 4 ~10 6 Contains a copy of the aptamer.

[0037] In some embodiments, the aptamer cluster is labeled with a fluorescent marker. In some embodiments, the aptamer cluster is labeled with an element that allows visualization under an optical microscope. In certain embodiments, the element is a nanoparticle. In some embodiments, the aptamer cluster is labeled with an antisense strand. In certain embodiments, the antisense strand is displaced or removed upon target binding.

[0038] In some embodiments, the composition further comprises an enzyme, hi certain embodiments, the enzyme is a ligase, a polymerase, a nuclease, an editing enzyme, and / or a restriction enzyme.

[0039] In certain embodiments of the compositions described herein, the target cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterium. In certain embodiments, the bacterium is a pathogenic bacterium.

[0040] In certain embodiments of the compositions described herein, the target cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a cancer cell or an immune cell. In certain embodiments, the mammalian cell is a cell derived from a patient. In some embodiments, the cell derived from a patient is a cancer cell derived from a patient or an immune cell derived from a patient.

[0041] In certain embodiments of the compositions described herein, the target cell is a cancer cell (e.g., a cancer cell from a patient). In certain embodiments, when the target cell is a cancer cell, the cellular function is cell death and / or apoptosis. In some embodiments, when the target cell is a cancer cell, the cellular function is modulation (e.g., inhibition) of expression of a ligand of an immune checkpoint protein (e.g., PD-L1, PD-L2).

[0042] In certain embodiments of the compositions described herein, the target cell is an immune cell (e.g., a T cell (helper T cell, cytotoxic T cell, regulatory T cell, etc.), a B cell, a macrophage, a dendritic cell). In some embodiments, when the target cell is an immune cell, the cell function is modulation (e.g., enhancement or suppression) of expression of an immune protein (e.g., a cell surface immune protein such as an immune checkpoint protein (e.g., PD-1, CTLA-4, immune activation markers)). In some embodiments, the immune protein is a cytokine (e.g., an inflammatory cytokine). In some embodiments, when the target cell is an immune cell, the function is cell proliferation. In some embodiments, when the target cell is an immune cell, the function is cell death (e.g., apoptosis).

[0043] In certain aspects, provided herein is a method for selecting a population of aptamers that modulate cell function.In some embodiments, the method includes: (i) incubating a library of labeled aptamer cluster particles (e.g., beads, such as microbeads or nanobeads) with target cells in a single reaction volume under certain conditions and for a certain period of time to form cell-aptamer cluster particle complexes; (ii) separating the cell-aptamer cluster particle complexes that have changed cell function from cell-aptamer cluster particle complexes that have no desired effect, unbound particles, and unbound cells; (iii) isolating the aptamer cluster particles from the cell-aptamer cluster particle complexes that have changed cell function; (iv) dissociating the aptamers from the particles; and (v) amplifying individual aptamer sequences to provide a functionally enriched aptamer population. In certain embodiments, the method further comprises additionally functionally enriching the population of aptamers by repeating steps (i)-(v) (e.g., repeating steps (i)-(v) at least 1, 2, 3, 4, 5, or 6 or more additional times). In certain embodiments, additionally functionally enriching the population of aptamers comprises applying restrictive conditions (e.g., reducing the total number of particles, reducing the copy number of aptamer per particle, reducing the total number of target cells, shortening the incubation period) in successive rounds. In some embodiments, the functionally enriched population of aptamers has at least 2-fold (e.g., at least 3, 4, 5, 6, 7, 8, or 9-fold) reduced sequence diversity compared to the majority of aptamers resulting from the incubation step. In certain embodiments, the enriched population of aptamers has at least 10, ... 2 , 10 3 , 10 4 , 10 5 , or 10 6fold, resulting in a reduction in sequence diversity. In some embodiments, the aptamer population of each additional round of screening is enriched by at least 1.1, 1.2, 1.3 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 3 times. In some embodiments, the number of rounds performed is at least 2 times (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times). In certain embodiments, the method further comprises, after step (v), identifying the enriched population of aptamers by sequencing. In certain embodiments, the method further comprises, before step (i), providing a library of labeled aptamer cluster particles. In some embodiments, the library of aptamer cluster particles is prepared by a method comprising: (a) immobilizing a number of aptamers from an aptamer library on a particle surface; and (b) locally amplifying the number of immobilized aptamers on the particle surface to form a number of immobilized aptamer cluster particles.

[0044] In certain embodiments, the aptamer cluster particles comprise clusters of exposed single-stranded nucleic acids immobilized on the surface of a particle (e.g., a bead, such as a microbead or nanobead). In some embodiments, the exposed single-stranded nucleic acid is not naturally occurring. In some embodiments, the aptamer cluster particles further comprise a non-exposed single-stranded nucleic acid sequence with a molecular cap. In some embodiments, the molecular cap, 3' cap and / or 5' cap is an oligonucleotide (e.g., 18 nucleotides in length) having a sequence complementary to a specific PCR primer sequence. In some embodiments, the aptamer clusters are prepared using emulsion PCR. In some embodiments, each aptamer cluster particle comprises a cluster of less than 10 (e.g., less than 7, less than 5, less than 3, or less than 2) different aptamer sequences. In some embodiments, each aptamer cluster particle comprises multiple copies of a unique aptamer sequence as a cluster on the surface of the particle. In some embodiments, the aptamer is a peptide aptamer.

[0045] In some embodiments, each aptamer cluster contains at least two (e.g., at least 50) identical aptamers. In certain embodiments, a library of aptamer cluster particles contains 100-10 14 In some embodiments, the library of aptamer cluster particles comprises at least 10 different aptamers immobilized on the surface of the particle. 8 In some embodiments, the aptamer sequence comprises a region of conserved sequence and a region of randomized sequence. In some embodiments, the randomized sequence is exposed and the conserved sequence is capped.

[0046] In certain embodiments, the particles are selected from the group consisting of polymer beads, agarose beads, polystyrene beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, controlled pore beads, microbeads and nanoparticles. In some embodiments, the particles have an average diameter of about 25 nm, 50 nm, 100 nm, 250 nm, 0.5 μm, 2 μm, 5 μm, 10 μm, 20 μm, or 30 μm. In some embodiments, the particles have at least one dimension of about 25 nm, 50 nm, 100 nm, 250 nm, 0.5 μm, 2 μm, 5 μm, 10 μm, 20 μm, or 30 μm. In some embodiments, the aptamers are preselected through one or more processes selected from the group consisting of Cell SELEX, negative SELEX, and in vitro evolution. In certain embodiments, the aptamer sequence comprises a chemical modification. In some embodiments, the aptamer is a single-stranded nucleic acid (e.g., DNA, RNA, or chemical modifications thereof). In some embodiments, the aptamer is a single-stranded nucleic acid hybridized to a partial length complementary strand (e.g., the aptamer primer sequence is capped and the randomized sequence is single stranded). In some embodiments, the aptamer sequence comprises non-natural nucleotides.

[0047] In some embodiments, the period of time is sufficient for the aptamer clusters to affect the target cells. In some embodiments, the period of time is from about 10 minutes to about 5 days (e.g., from about 1.5 hours to about 24 hours, or from about 1.5 hours to about 2 hours).

[0048] In certain embodiments, the target cell is a whole cell bearing the native epitope. In some embodiments, the target cell is a prokaryotic cell (e.g., a bacterium). In some embodiments, the target cell is a eukaryotic cell (e.g., a mammalian cell, a cancer cell, an immune cell, or a cell derived from a patient). In certain embodiments, the target cell is in a substantially natural (native) environment.

[0049] In some embodiments, the target cells are detectably labeled. In certain embodiments, the target cells are labeled with a detectable label before, during, and / or after incubating the library of labeled aptamer cluster particles with the target cells. In some embodiments, the detectable label is a fluorescent dye (e.g., a calcium-sensitive dye, a cell tracer dye, a lipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolite-sensitive dye, a pH-sensitive dye, a membrane potential-sensitive dye, a mitochondrial membrane potential-sensitive dye, or an oxidation-reduction potential dye). In some embodiments, the detectable label is an activation-related marker, an oxidative stress reporter, an angiogenesis marker, an apoptosis marker, an autophagy marker, a cell survival marker, or a marker of ion concentration. In some embodiments, the cellular function that is modulated is cell survival, apoptosis, cell proliferation, gene expression, cell morphology, cell activation, phosphorylation, calcium mobilization, degranulation, cell migration, or cell differentiation. In some embodiments, the cellular function that is modulated is apoptosis. In certain embodiments, the cellular function that is modulated is apoptosis, and the target cells are cancer cells. In some other embodiments, the cellular function that is modulated is an immune function (e.g., immune cell activation, immune cell suppression, immune cell proliferation, cytokine expression, immune cell differentiation) and the target cell is an immune cell.

[0050] In certain embodiments, the cell-aptamer cluster particle complex contains about 2-4 particles per target cell. In some embodiments, the aptamer cluster particles contain about 1-6 clusters per particle. In some embodiments, multiple cell-aptamer cluster particle complexes are included in a single reaction volume. In some embodiments, the separation step is performed by quantifying the signal from individual cells and then physically separating the cell-aptamer cluster particle complexes having altered cell function (e.g., via flow cytometry, fluorescent microscopy, optical tweezers, micropipette, microfluidic separation, micromanipulation, or separation seeding). In some embodiments, the aptamer cluster particles are separated by cell lysis and centrifugation. In certain embodiments, the aptamers are isolated by HPLC purification after dissociation from the particles.

[0051] In certain aspects, provided herein are aptamers that induce apoptosis of cancer cells (e.g., triple-negative breast cancer cells). In certain embodiments, the aptamer comprises a sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs: 1-10. In certain embodiments, the aptamer is 100, 90, 80, 70, 60, 59, 58, 57, 56, 55, or 54 nucleotides or less in length. In some embodiments, the aptamer comprises RNA, DNA, and / or unnatural nucleotides. In some embodiments, the aptamer is chemically modified. In some embodiments, the aptamer is PEGylated. In certain embodiments, the aptamer induces apoptosis of triple-negative breast cancer cells (e.g., TNBC9) cells. In some embodiments, the aptamer consists of a sequence selected from one of SEQ ID NOs: 1-10.

[0052] In certain aspects, provided herein is a pharmaceutical composition comprising the aptamer described herein. In some embodiments, provided herein is a method for treating cancer, comprising administering the pharmaceutical composition described herein. In certain embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple-negative breast cancer. [Brief description of the drawings]

[0053] [Figure 1] FIG. 1 is a schematic diagram of the workflow for aptamer library synthesis, sequencing, and target identification according to certain embodiments described herein. [Diagram 2] Figure 2 is a schematic diagram of the workflow of the combination SELEX assay described herein. Figure 2 reveals SEQ ID NO:11. [Diagram 3] FIG. 3 is a schematic diagram showing the workflow of the functional SELEX assay described herein. [Figure 4] FIG. 4 is a schematic diagram of a particular aptamer structure according to certain exemplary embodiments described herein. [Diagram 5] Figure 5 shows a representative flow cytometry gating and sorting strategy. Panel A is a histogram of the clustered microbead library before incubation with target cells. Panel B is a scatter plot of the clustered microbead library after incubation with target cells and then incubation with caspase-3 / 7 probe (Cas-3 / 7). [Figure 6] Figure 6 shows an example of a flow cytometry gating and sorting strategy using two functional probes. Panel A is a scatter plot showing cells bound to a clustered bead library in black boxes. Panel B is a scatter plot of events from the black box in panel A probed with caspase-3 / 7 and mitochondrial membrane potential (MitoProbe Red). [Figure 7]FIG. 7 shows the results of functional enrichment of aptamer libraries by an exemplary method described herein for aptamers that induce apoptosis of the indicated cells. The bar graphs on the left of each panel show the fold increase in caspase-3 / 7 (Cas3 / 7) or mitochondrial membrane potential (DilC1(5)) per different rounds of functional enrichment. The histograms on the right of each panel overlay libraries enriched in the first (black) and final (gray) rounds of functional enrichment. All results shown, except for Kasumi-1, were obtained from functional enrichment starting from the third round of combined SELEX. Kasumi-1 results were obtained from functional enrichment starting from a random library (not combined enriched). Panel A shows results for the HCT116 human colon cancer cell line. Panel B shows results for the 4T1 mouse breast cancer cell line. Panel C shows results for the CT26 mouse colon cancer cell line. Panel D shows results for the Kasumi-1 human acute myeloid leukemia (AML) cell line. Panel E shows results from AML1 primary AML PBMCs taken from a donor. Panel F shows results from AML9 primary AML PBMCs taken from a donor. Panel G shows results from CLL1 primary chronic lymphocytic leukemia (CLL) PBMCs taken from a donor. [Figure 8]FIG. 8 shows Illumina sequencing abundance profiles of aptamer enriched libraries obtained from multiple rounds of combined Cell-SELEX and functional enrichment. The x-axis indicates the different selection rounds, with "B" corresponding to combined Cell-SELEX round and "F" corresponding to functional enrichment round. Functional enrichment started from combined Cell-SELEX round #3. The y-axis shows the relative abundance of individual aptamers on a logarithmic scale. The 10,000 most abundant aptamers in each combined Cell-SELEX round are shown as black lines. The 10,000 most abundant aptamers in each functional Cell-SELEX round are shown as blue lines. The 10 most abundant aptamers in each combined Cell-SELEX round are shown as thick solid lines. The 10 most abundant aptamers in each functional enrichment round are shown as dashed lines. Panel A shows the profile of an assay performed on AML1 primary human myeloblasts. Panel B is a profile of the assay performed in the HCT116 colon cancer cell line. [Figure 9] Figure 9. Comparison of caspase-3 / 7 activation after incubation of binding-enriched, clustered microbead libraries or function-enriched clustered microbead libraries with target cells. The percentage of cells bound to microbeads and caspase-3 / 7 positive cells was measured by flow cytometry. Panel A shows the results after incubation of the enriched library with AML1 target cells. Panel B shows the results after incubation of the enriched library with HCT116 target cells. Each data point was measured in four technical replicates and significance was calculated by Welch's one-way t-test. [Figure 10]Figure 10 shows the results for the identification of lead aptamer candidate E8 from a functionally enriched tumoricidal aptamer library. Panel A shows sequence abundance plots from multiple functional SELEX rounds. The plots show a random selection of 1,000 aptamer sequences from the 10,000 most abundant aptamer sequences from each round. The 10 most abundant aptamer sequences are highlighted. Panel B shows a representative screen of the 10 most abundant aptamers from Panel A for significant caspase-3 / 7 activation in TNBC9 cells compared to vehicle (V) and random oligonucleotides (R). Aptamer IDs are labeled 1-10 (E1, E2, ... E10). Panel C shows the selectivity of aptamer candidate E8 to induce cell death in TNBC9 cells (blue) compared to negative MCF10A target cells (red). STA, staurosporine; PAC, paclitaxel; Random, random oligonucleotide. Panel D shows the effect of E8 on MDA-MB-231 cells. Panel E shows the dose-response curves of E8 and PEGylated E8. Panel F shows the effect of E8 on TNBC cells in mouse serum. [Figure 11]Figure 11 shows results demonstrating the biodistribution and efficacy of E8 aptamer candidates in animal models. Panel A shows E8 fluorescence measured in vivo 0.1 h, 24 h, and 48 h after injection into NOD / SCID mice bearing MDA-MB-231-derived tumors. White arrows indicate tumor location. Panel B shows E8 retention at the tumor site 3 h after intravenous injection (Ve, vehicle; K, kidney; T, tumor). The inset on the right shows a magnification and the white arrow points to the tumor site. Panel C shows quantitative measurements of E8 fluorescence signal in tumors over a 48-h period. Panel D shows the efficacy of E8 in reducing tumor volume in mice. Asterisks indicate statistical differences of p < 0.05 (n = 8 mice / group). Panel E shows representative photographs of tumors excised from mice sacrificed on day 11. Panels FG show histochemical analysis of caspase-3 activity in tissue sections from tumors (Panel F, vehicle treatment; Panel G, E8 treatment). Panels HI show TUNEL analysis of tissue sections from tumors (H, vehicle treatment; I, E8 treatment). [Figure 12] FIG. 12 shows the results showing the efficacy of E8 in human ex vivo organ cultures (EVOCs). Panel A shows tissue specimens from patient 1 (P1). Panel B shows tissue specimens from patient 2 (P2). Pathological staging was performed by two blinded pathologists on a scale of 0 to 4. White asterisks indicate samples in which efficacy reached at least a grade of 3. Rnd, random. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Detailed Description Overview Provided herein are methods and compositions for identifying aptamers that regulate functional effects on target cells.In certain embodiments, the method comprises contacting target cells with a number of aptamer clusters immobilized on a surface (e.g., the surface of a particle such as a bead, including microbeads, nanobeads, etc.).Thus, in some embodiments, the method comprises incubating a library of aptamer cluster particles with target cells in a single reaction volume under certain conditions and for a period of time to form cell-aptamer cluster particle complexes, and isolating and identifying a population of aptamers that regulate cell function.

[0055] In some embodiments, aptamers that functionally modulate a cellular function are identified by providing a target cell with a detectable label indicative of the modulated function (e.g., a fluorescent dye such as a calcium-sensitive dye, a cell tracer dye, a lipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolite-sensitive dye, a pH-sensitive dye, a membrane potential-sensitive dye, a mitochondrial membrane potential-sensitive dye, or an oxidation-reduction potential dye), then measuring the signal of the detectable label, followed by physical separation of the cell-aptamer cluster particle complex that has altered the cellular function. Physical separation can be performed, for example, by flow cytometry, fluorescence microscopy, optical tweezers, micropipette, microfluidic separation, micromanipulation, or separation seeding, etc. The aptamer cluster particles can then be separated from the cell-aptamer cluster particle complex, for example, via cell lysis and centrifugation. Individual aptamer sequences can be dissociated from the particles, amplified, and sequenced.

[0056] In certain aspects, methods and compositions related to producing aptamer clusters immobilized on a surface (e.g., a particle surface) are also provided herein. In some embodiments, aptamers (e.g., from an aptamer library described herein) are immobilized on a particle. The particle can be made of any material. For example, in some embodiments, the particle is made of plastic, glass, polymer, or metal. In certain embodiments, the particle is a polymer bead, an agarose bead, a polystyrene bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, a glass bead, a controlled pore bead, a microbead, or a nanoparticle. In some embodiments, the particles have an average diameter in at least one dimension of about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 μm. In some embodiments, the particles are coated with a blocker, such as a polymer, protein, oligo, lipid, and / or chemical group. In some embodiments, the particles contain anchors of any length for binding target cells in the vicinity of the clusters. The anchors may be polymers, proteins, oligos, lipids, and / or chemical groups. In some embodiments, a local amplification process, such as emulsion PCR, is then performed to generate aptamer clusters. The complementary strand can be removed to generate single-stranded aptamer clusters. The aptamer cluster particles are then ready for use in the aptamer identification methods provided herein.

[0057] definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0058] The articles "a" and "an" are used herein to refer to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0059] As used herein, the term "aptamer" refers to a short (e.g., less than 200 bases) single-stranded nucleic acid molecule (ssDNA and / or ssRNA) that can specifically bind to a protein or peptide target or a topographical feature on a target cell.

[0060] As used herein, the term "aptamer cluster" refers to a collection (eg, at least 10) of locally immobilized aptamers of identical sequence.

[0061] The terms "bind" or "interact" refer to an association, which may be a stable bond between two molecules, e.g., between an aptamer and a target, due to electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions, e.g., under physiological conditions.

[0062] As used herein, two nucleic acid sequences are "complementary" or "complementary" to one another if they form base pairs with each other at every position.

[0063] As used herein, "contacting" refers to bringing two or more molecular entities together such that they can interact with each other.

[0064] As used herein, two nucleic acid sequences "correspond" to each other if both are complementary to the same nucleic acid sequence.

[0065] The terms "modulation" or "modulating," when used in reference to a functional property or biological activity or process (e.g., enzymatic activity or receptor binding), refer to the ability to upregulate (e.g., activate or stimulate), downregulate (e.g., inhibit or suppress), or otherwise alter the nature of such property, activity, or process. In certain instances, such modulation may be contingent on the occurrence of a specific event, such as activation of a signaling pathway, and / or may be manifest only in particular cell types.

[0066] As used herein, "specific binding" refers to the ability of an aptamer to bind to a predetermined target. In certain embodiments, an aptamer is characterized by its K for binding to its non-specific, unrelated target (e.g., BSA, casein, or unrelated cells such as HEK293 cells or E. coli cells when cells are not the target of the process and are used as a negative control for the process). D significantly less (e.g., at least 2-fold less, at least 5-fold less, at least 10-fold less, at least 50-fold less, at least 100-fold less, at least 500-fold less, or at least 1000-fold less) than D In certain embodiments, the aptamer binds to the target at about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M, about 10 -13 Less than M or about 10 -14 K under M D The antibody specifically binds to its target with an affinity corresponding to

[0067] As used herein, the Tm or melting temperature of two oligonucleotides is the temperature at which 50% of oligonucleotides / targets are bound and 50% of oligonucleotide target molecules are not bound.The Tm value of two oligonucleotides depends on the oligonucleotide concentration and is influenced by the concentration of monovalent and divalent cations in reaction mixture.Tm can be empirically determined or calculated using nearest neighbor formula, as described in Santa Lucia, J. PNAS (USA) 95:1460-1465(1998), which is incorporated herein by reference.

[0068] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein. These terms refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (gene loci) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be made before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified, such as by conjugation with a labeling component.

[0069] Aptamer Library In certain embodiments, the methods and compositions provided herein relate to identifying aptamers with desired properties among aptamers present in an aptamer library. As used herein, an aptamer library is a set of aptamers that are comprised of distinct sequences (e.g., at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 -15 Aptamers are a collection of nucleic acid molecules (e.g., DNA and / or RNA) having distinct sequences of aptamers (e.g., nucleotides, nucleotides, and sequences) where at least a subset of the nucleic acid molecules are structured such that they can specifically bind to a target protein, peptide, or cellular topographic feature. In some embodiments, any library of potential aptamers may be used in the methods and compositions provided herein.

[0070] In some embodiments, the aptamer library used in the methods and compositions provided herein has formula (I): P1-R-P2(I) comprising, consisting of, and / or consisting essentially of a nucleic acid molecule (e.g., DNA and / or RNA) having a sequence according to wherein P1 is a 5' primer site sequence of about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; P2 is a 3' primer site sequence of about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; and R is at least 10, 15, 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases in length and / or a sequence that contains randomly arranged bases up to about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50 bases in length.

[0071] In one embodiment, R is a sequence comprising about 25% A. In another embodiment, R is a sequence comprising about 25% T. In another embodiment, R is a sequence comprising about 25% G. In another embodiment, R is a sequence comprising about 25% C. In yet another embodiment, R is a sequence comprising about 25% A, about 25% T, about 25% G, and about 25% C.

[0072] In some embodiments, the aptamer library used in the methods and compositions provided herein has formula (I): P1-R"-P2(I) comprising, consisting of and / or consisting essentially of a nucleic acid molecule (DNA and / or RNA) having a sequence according to where P1 is a 5' primer site sequence of about 10-100 bases long, about 10-50 bases long, about 10-30 bases long, about 15-50 bases long, or about 15-30 bases long; P2 is a 3' primer site sequence of about 10-100 bases long, about 10-50 bases long, about 10-30 bases long, about 15-50 bases long, or about 15-30 bases long; and R" is a random sequence from a biased mixture. or any combination of random and repetitive or biased strings of characters, and are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases in length and / or up to about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length.

[0073] In some embodiments, the aptamer library used in the methods and compositions provided herein has Formula II: P1-S1-L1-S1*-S2-L2-S2*-P2(II) (An exemplary schematic is shown in FIG. 4A ), Where: P1 is a 5' primer site sequence of about 10 to 100 bases, about 10 to 50 bases, about 10 to 30 bases, about 15 to 50 bases, or about 15 to 30 bases; P2 is a 3' primer site sequence of about 10 to 100 bases, about 10 to 50 bases, about 10 to 30 bases, about 15 to 50 bases, or about 15 to 30 bases; S1 and S2 are each independently a 5' primer site sequence of at least one base ( For example, a stem region sequence of about 4 to 40 bases in length, or 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, or 40 bases in length); S1* is the complementary sequence of S1; S2* is the complementary sequence of S2; L1 and L2 is independently at least one base (e.g., about 1 to 50 bases in length or 1, 2, 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 or 50 bases in length); and S1-L1-S1*-S2-L2-S2* are, in total, at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length, and / or no more than about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length.

[0074] In some embodiments, the aptamer library used in the methods and compositions provided herein has Formula III: P1-S1-L1-S2-L2-S2*-L1-S1*-P2(III), (An exemplary schematic is shown in FIG. 4B ), Where: P1 is a 5' primer site sequence about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; P2 is a 3' primer site sequence about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; S1 and S2 are each independently a stem region sequence of at least one base (e.g., about 4-40 bases in length or 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, or 40 bases in length); S1* is the complementary sequence of S1; S2* is the complementary sequence of S2; L1 and L2 are each independently at least one base (e.g., a loop region sequence of about 1-50 bases in length or 1, 2, 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, or 50 bases in length); and S1-L1-S2-L2-S2*-L1-S1* is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases in total in length, and / or no more than about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50 bases in length.

[0075] In some embodiments, the aptamer library used in the methods and compositions provided herein has Formula IV: P1-Lib-M1 / M2-D-M1 / M2*-Lib-P2(IV) (An exemplary schematic diagram is provided in FIG. 4C ), Where: P1 is a 5' primer site sequence about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; P2 is a 3' primer site sequence about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; Lib is a sequence having a formula selected from: (i) R; (ii) R″; (iii) S1-L1-S1*-S2-L2-S2*; and (iv) S1-L1-S2-L2-S2*-L1-S1*; R is a sequence comprising randomly arranged bases that is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length and / or no more than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length; R″ is a sequence at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length and / or no more than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length, containing randomly placed bases from a biased mixture, or any combination of random and repeating or biased strings; S1 and S2 are each independently a stem region sequence of at least one base (e.g., about 4 to 40 bases in length or 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, or 40 bases in length). S1* is the complement of S1; S2* is the complement of S2; L1 and L2 are each independently a loop region sequence of at least 1 base (e.g., about 1 to 50 bases in length, or 1, 2, 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, or 50 bases in length); wherein S1-L1-S1*-S2-L2-S2* in total are at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length and / or no more than about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length; D is a spacer sequence comprising at least one base (e.g., about 1-20 bases in length, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases in length); M1 is a multimerization domain sequence about 10-18 bases in length, or 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases in length, where a strand of the sequence can interact with another strand that contains a complementary domain; and M2 is the complementary domain of M1 that contains the strand that interacts with a strand of the M1 sequence.

[0076] In some embodiments, the aptamer library used in the methods and compositions provided herein has the formula V: P1-Lib-T*-Lib-P2(V) (An exemplary schematic diagram is shown in FIG. 4D ), where: P1 is a 5' primer site sequence about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; P2 is a 3' primer site sequence about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; Lib is a sequence having a formula selected from: (i) R; (ii) R″; (iii) S1-L1-S1*-S2-L2-S2*; and (iv) S1-L1-S2-L2-S2*-L1-S1*; R is a sequence comprising randomly arranged bases that is at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length and / or no more than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length; R″ is a sequence at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length and / or no more than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length, comprising randomly placed bases from a biased mixture, or any combination of random and repeating or biased strings; S1 and S2 are each independently a stem region sequence of at least one base (e.g., about 4-40 bases in length, or 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, or 40 bases in length); S1* is the complement of S1; S2* is the complement of S2; L1 and L2 are each independently a loop region sequence of at least 1 base (e.g., about 1 to 50 bases in length, or 1, 2, 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, or 50 bases in length); wherein S1-L1-S1*-S2-L2-S2* in total are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length and / or no more than about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length; T is a second strand that is bound by Watson / Crick or Hoogsteen base pairing to any portion of the Lib sequence or T*, where this strand optionally contains unpaired domains at its 5' and 3' ends (e.g., to facilitate attachment of a functional moiety to the aptamer); and T* is a dedicated domain sequence (e.g., about 4-40 bases in length, or 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 or 40 bases in length).

[0077] In some embodiments, the aptamer libraries used in the methods and compositions provided herein include P1-R-P1*, P1-S1-R-S1-P2, and P1-R-S1-R-S1-R-P2 comprising, consisting of, and / or consisting essentially of a nucleic acid molecule (DNA and / or RNA) having a sequence of a formula selected from Where: P1 is a 5' primer site sequence of about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; P2 is a 3' primer site sequence of about 10 to 100 bases long, about 10 to 50 bases long, about 10 to 30 bases long, about 15 to 50 bases long, or about 15 to 30 bases long; and P1* is a sequence complementary to P1.

[0078] R is a sequence comprising randomly arranged bases that is at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length and / or no more than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length; S1 is a stem region sequence of at least one base (e.g., about 4 to 40 bases in length or 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, or 40 bases in length).

[0079] In some embodiments of the above formula, R, P, or S comprises a CpG island and / or a G-quadruplex sequence.

[0080] In some embodiments of the above formula, R is a randomly placed base from any random mixture (e.g., 25% A, 25% T, 25% G, 25% C for the reference bases) that is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length, and / or up to about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length.

[0081] In one embodiment of the above formula, R is a sequence comprising about 25% A. In another embodiment, R is a sequence comprising about 25% T. In another embodiment, R is a sequence comprising about 25% G. In another embodiment, R is a sequence comprising about 25% C. In yet another embodiment, R is a sequence comprising about 25% A, about 25% T, about 25% G, and about 25% C.

[0082] In some embodiments of the above formula, R" is a sequence that includes randomly placed bases from a biased mixture (e.g., any mixture that deviates from 25% per base for the reference base). In some embodiments, R" is a sequence that includes about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% As. In some embodiments, R″ is a sequence that contains about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% T. In some embodiments, R″ is a sequence that contains about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% C. In some embodiments, R″ is a sequence that contains about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% Gs. In some embodiments, R″ is a sequence that contains any combination of random strings (where a string is any sequence containing a single base) and repeating or biased strings.

[0083] In some embodiments of the above formula, R″ is any combination of randomly placed bases from a biased mixture (e.g., any mixture that deviates from 25% per base for the canonical base); or random strings (where a string is any sequence containing a single base) with repeating or biased strings of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 bases in length, and / or up to about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 bases in length.

[0084] In some embodiments of the above formula, S1 is a stem region sequence of at least one base, in other embodiments, S1 is a stem region sequence of about 4-40 bases in length, or 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, or 40 bases in length.

[0085] In some embodiments of the above formula, S2 is a stem region sequence of at least one or more bases, in other embodiments, S2 is a stem region sequence between about 4-40 bases in length or 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, or 40 bases in length.

[0086] In some embodiments of the above formula, L1 is a loop region sequence of at least one base. In other embodiments, L1 is a loop region sequence of about 1-50 bases in length or 1, 2, 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, or 50 bases in length.

[0087] In some embodiments of the above formula, L2 is a loop region sequence of at least one base. In other embodiments, L2 is a loop region sequence of about 1-50 bases in length or 1, 2, 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, or 50 bases in length.

[0088] In some embodiments of the above formula, T may include unpaired domains at its 5' and 3' ends, or may be a padlock tail (e.g., a loop between two domains paired with the library). The aptamers of the present disclosure may include any number of stems and loops, as well as other structures consisting of stems and loops (e.g., hairpins, bulges, etc.). In some embodiments, the loops of the aptamer include buried bases to form a stable loop-loop WC pairing that forms a stem orthogonal to the main library axis. In other embodiments, two loops in an aptamer together form an orthogonal stem. In still other embodiments, the loops of the aptamer include buried bases to form a stable Hoogsteen pairing with an existing stem along the main library axis. In other embodiments, the loops of the aptamer may form a Hoogsteen pairing with any stem of the aptamer.

[0089] In some embodiments of the above formula, the aptamer sequence further comprises one or more multimerization domains.

[0090] In some embodiments of the above formula, the aptamer sequence further comprises one or more spacers (e.g., about 1-20 bases in length or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases in length).

[0091] The aptamers of the present disclosure can be prepared in a variety of ways. In one embodiment, the aptamers are prepared by chemical synthesis. In another embodiment, the aptamers are prepared by enzymatic synthesis. In one embodiment, the enzymatic synthesis can be carried out using any enzyme that can add nucleotides to extend a primer, with or without the use of a template. In some embodiments, Aptamers are prepared by assembling together k-mers (eg, k≧2 bases).

[0092] In some embodiments, the aptamers of the present disclosure may comprise any combination of DNA, RNA, and natural and / or synthetic analogs thereof. In one embodiment, the aptamer comprises DNA. In one embodiment, the aptamer comprises RNA.

[0093] In other embodiments, the aptamers of the present disclosure may include any modification at the 5' end, 3' end, or internally. Aptamer modifications include, but are not limited to, spacers, phosphorylations, linkers, conjugation chemistries, fluorophores, quenchers, photoreactivity, and modified bases (e.g., LNA, PNA, UNA, PS, methylation, 2-O-methyl, halogenation, superbases, iso-dN, reverse bases, L-ribose, other sugars as backbones, etc.).

[0094] In some embodiments, the aptamers of the present disclosure can be attached at the 5' end, 3' end, or internally to an external non-nucleic acid molecule. Non-limiting examples of non-nucleic acid molecules include, but are not limited to, amino acids, peptides, proteins, small molecule drugs, mono- and polysaccharides, lipids, antibodies and antibody fragments, or combinations thereof.

[0095] The aptamers of the present disclosure may include any domain that has a biological function. Non-limiting examples of the biological functions of aptamers described herein include, but are not limited to, functioning as a template for RNA transcription, binding, recognizing, and / or regulating the activity of proteins, binding to transcription factors, specialized nucleic acid structures (e.g., Z-DNA, H-DNA, G-quad, etc.), acting as enzyme substrates for restriction enzymes, specific exonucleases and endonucleases, recombination sites, editing sites, or siRNA. In one embodiment, the aptamer regulates the activity of at least one protein. In another embodiment, the aptamer inhibits the activity of at least one protein.

[0096] In other embodiments, the aptamers of the present disclosure may include any domain for incorporation into nucleic acid nanostructures constructed by any one of several known methods (Shih et al, Nature 427:618-621 (2004); Rothemund, Nature 440:297-302 (2006); Zheng et al, Nature 461:74-77 (2009); Dietz et al, Science 325:725-730 (2009); Wei et al, Nature 485:623-626 (2012); Ke et al, Science 338:1177-1183 (2012); Douglas et al, Science 335:831-834 (2012), each of which is incorporated herein by reference). In still other embodiments, the aptamers of the present disclosure may include any domain that functions in molecular logic and computation (Seelig et al, Science 314:1585-1588 (2006); Macdonald et al, Nano Lett 6:2598-2603 (2006); Qian et al, Nature 475:368-372 (2011); Douglas et al, Science 335:831-834 (2012); Amir et al, Nat Nanotechnol 9:353-357 (2014), each of which is incorporated herein by reference).

[0097] In some embodiments, the aptamers of the disclosure are subjected to one or more cycles of negative selection against a negative control target (e.g., including but not limited to, a eukaryotic or prokaryotic cell, a virus or viral particle, a protein, a molecule, a tissue, or a whole organism, in vivo or ex vivo). In other embodiments, the aptamers of the disclosure are subjected to one or more cycles of positive selection against a positive target (e.g., a eukaryotic or prokaryotic cell, a virus or viral particle, a molecule, a tissue, or a whole organism, in vivo or ex vivo).

[0098] The aptamers of the present disclosure may be in solution or attached to a solid phase (e.g., a surface, a particle, a resin, a substrate, etc.). In some embodiments, the aptamer is attached to a surface. In one embodiment, the surface is a particle surface.

[0099] In some embodiments, the aptamers of the present disclosure are synthesized in an aptamer library. The aptamer library of the present disclosure can be prepared in a variety of ways. In one embodiment, the aptamer library is prepared by chemical synthesis. In another embodiment, the aptamer library is prepared by enzymatic synthesis. In one embodiment, the enzymatic synthesis can be performed using any enzyme that can add nucleotides to extend a primer, with or without a template.

[0100] In some embodiments, the aptamers synthesized in the aptamer library may comprise any combination of DNA, RNA, and natural and / or synthetic analogs thereof. In one embodiment, the aptamers synthesized in the aptamer library comprise DNA. In one embodiment, the aptamers synthesized in the aptamer library comprise RNA.

[0101] In some embodiments, the aptamers synthesized in the aptamer library are a collection of 10K species (K≧2) of nucleic acids (e.g., DNA, RNA, natural or synthetic bases, base analogs, or combinations thereof) with Z copies per species (1≦Z≦K-1).

[0102] In other embodiments, the aptamers synthesized in the aptamer library of the present disclosure may include any modification at the 5' end, 3' end, or internally. Aptamer modifications include, but are not limited to, spacers, phosphorylations, linkers, conjugation chemistries, fluorophores, quenchers, photoreactive modifications, and modified bases (e.g., LNA, PNA, UNA, PS, methylation, 2-O-methyl, halogenation, superbases, iso-dN, reverse bases, L-ribose, other sugars as backbones).

[0103] In some embodiments, aptamers synthesized in an aptamer library may be linked to an external non-nucleic acid molecule at the 5' end, 3' end, or internally. Non-limiting examples of non-nucleic acid molecules include, but are not limited to, amino acids, peptides, proteins, small molecule drugs, mono- and polysaccharides, lipids, antibodies and antibody fragments, or combinations thereof.

[0104] The aptamers synthesized in the aptamer library may include any domain that has a biological function. Non-limiting examples of the biological functions of the aptamers described herein include, but are not limited to, functioning as a template for RNA transcription, binding to, recognizing, and / or regulating the activity of a protein, binding to a transcription factor, specialized nucleic acid structures (e.g., Z-DNA, H-DNA, G-quad, etc.), acting as an enzyme substrate for a restriction enzyme, specific exonucleases and endonucleases, recombination sites, editing sites, or siRNA. In one embodiment, the aptamers synthesized in the aptamer library regulate the activity of at least one protein. In another embodiment, the aptamers synthesized in the aptamer library inhibit the activity of at least one protein.

[0105] In other embodiments, the aptamers synthesized in the aptamer library may include any domain for incorporation into a nucleic acid nanostructure constructed by one of several known methods (Shih et al, Nature 427:618-621 (2004); Rothemund, Nature 440:297-302 (2006); Zheng et al, Nature 461:74-77 (2009); Dietz et al, Science 325:725-730 (2009); Wei et al, Nature 485:623-626 (2012); Ke et al, Science 338:1177-1183 (2012); Douglas et al, Science 335:831-834 (2012), each of which is incorporated herein by reference). In still other embodiments, the aptamers of the present disclosure may include any domain that functions in molecular logic and computation (Seelig et al, Science 314:1585-1588 (2006); Macdonald et al, Nano Lett 6:2598-2603 (2006); Qian et al, Nature 475:368-372 (2011); Douglas et al, Science 335:831-834 (2012); Amir et al, Nat Nanotechnol 9:353-357 (2014), each of which is hereby incorporated by reference).

[0106] In some embodiments, the aptamers synthesized in the aptamer library are subjected to one or more negative selection cycles against a target (e.g., a eukaryotic or prokaryotic cell). In other embodiments, the aptamers of the disclosure are subjected to one or more positive selection cycles against a target (e.g., a eukaryotic or prokaryotic cell).

[0107] The aptamers synthesized in an aptamer library can be in solution or attached to a solid phase (e.g., a surface, a particle, a resin, a substrate, etc.). In some embodiments, the aptamers synthesized in an aptamer library are attached to a surface. In one embodiment, the surface is a particle surface.

[0108] Immobilized aptamer clusters In certain aspects, provided herein is a method for identifying aptamers that modulate target cells by incubating a sample containing the target cells with a plurality of aptamer clusters immobilized on a particle surface (e.g., clusters of aptamers from an aptamer library provided herein). In some embodiments, the surface is a bead (e.g., a microbead, a nanobead).

[0109] Any known method known in the art may be used to generate aptamer clusters immobilized on the surface of a particle. In certain embodiments, the surface-immobilized aptamer clusters are generated by first immobilizing aptamers (e.g., from an aptamer library disclosed herein) to a surface (e.g., the surface of a particle). In some embodiments, the aptamer clusters are generated by immobilizing aptamers (e.g., from an aptamer library disclosed herein) to a surface (e.g., the surface of a particle) at least about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10Each individual aptamer is immobilized on a surface (e.g., on the surface of a particle). Following immobilization of the aptamers, a local amplification process (e.g., emulsion PCR, bridge amplification, or rolling circle amplification) is performed to generate a cluster of copies of each immobilized aptamer located near the immobilization site of the original immobilized aptamer. In certain embodiments (e.g., those in which rolling circle amplification is performed), the aptamer clusters are not directly immobilized on the surface, but are accommodated in nanopits or pores on the surface. In some embodiments, the aptamer clusters can be prepared using emulsion PCR. In some embodiments, the amplification is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10,000, 20,000, 30,000, 4 This results in each aptamer cluster containing 0,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1000,000 identical aptamer molecules. In certain embodiments, the aptamer clusters are then sequenced (e.g., by Illumina sequencing or Polonator sequencing). If present, complementary strands may be removed from the aptamer clusters by washing the surface under conditions that are not conducive to strand hybridization (e.g., by salt concentration and / or temperature) to generate clusters of single-stranded aptamers. The surface (e.g., particle surface) is then prepared for use in the aptamer identification methods provided herein. In some embodiments, immobilized aptamer clusters are prepared and / or sequenced on one instrument and then transferred to a separate instrument for aptamer identification. In other embodiments, aptamer clusters are prepared and / or sequenced on the same instrument used for aptamer identification.

[0110] In some embodiments, each aptamer cluster particle comprises a cluster of less than 1000 different aptamer sequences, for example, less than 900, less than 800, less than 700, less than 600, less than 500, less than 400, less than 300, or less than 200, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, or less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2 different aptamer sequences. In some embodiments, each aptamer cluster particle comprises a unique aptamer sequence in multiple copies as a cluster on the surface of the particle. In some embodiments, each aptamer cluster comprises at least two identical aptamers (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9, 000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 identical aptamers).

[0111] In some embodiments of the above method, the aptamer or aptamer cluster (e.g., from an aptamer library) includes an adapter that allows the aptamer to be brought to the surface level (e.g., when the surface is not flat, such as a particle containing pores). In some embodiments, the aptamer or aptamer cluster is immobilized inside the pores of the particle surface, and the adapter is used to bind the aptamer to the surface to bring the aptamer to the surface level. In some embodiments, the adapter is a nucleic acid adapter (e.g., a sequence at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 bases in length). In some embodiments, a sequence complementary to the adapter sequence is hybridized to the adapter prior to aptamer screening. In some embodiments, the adapter is a chemical adapter (e.g., a polymer that connects the aptamer to the surface).

[0112] Aptamer library screening In certain embodiments, provided herein are methods for identifying one or more aptamers that specifically regulate a function of a target cell, the method generally comprising: (i) incubating the library with target cells in a single reaction volume under conditions and for a period of time to form a cell-aptamer cluster particle complex; (ii) separating the cell-aptamer cluster particle complex that has altered cell function from cell-aptamer cluster particle complexes that do not have the desired effect, unbound particles, and unbound cells; (iii) separating the aptamer cluster particles from the cell-aptamer cluster particle complex that has altered cell function; (iv) dissociating the aptamers from the particles; and (v) amplifying individual aptamer sequences to provide a functionally enriched population of aptamers.

[0113] In certain embodiments, the method further comprises enriching the population of functional aptamers by repeating steps (i)-(v) in additional rounds to obtain a specific, enriched, functional aptamer population. In certain embodiments, enriching the population of functional aptamers comprises applying restrictive conditions (e.g., reducing the total number of particles) in successive rounds. In some embodiments, the enriched aptamer population has at least 1.5-fold (e.g., about 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0-fold) reduced sequence diversity compared to the majority of aptamers from the incubation step. In some embodiments, the aptamer population for each additional round of screening is enriched by at least 1.1-fold (e.g., about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7. 1.8, 1.9, or 2.0-fold). The number of rounds of enrichment can be as many as desired. For example, in some embodiments, the number of rounds is at least 2 (e.g., at least 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 110, 111, 112, 113, 114, 115, 7, 48, 49, 50, 51. 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 times). In certain embodiments, the method further comprises, after step (v), identifying the enriched population of functional aptamers by sequencing.

[0114] The library of aptamer cluster particles can be incubated with target cells under any conditions that are conducive to forming cell-aptamer cluster particle complexes and allowing the aptamer cluster particles to affect the target cells. Conditions include, but are not limited to, for example, a controlled period of time, optimal temperature (e.g., 37°C), and / or incubation medium (e.g., target cell medium). The incubation period can be from about 10 minutes to about 5 days, from about 30 minutes to about 4 days, from about 1 hour to about 3 days, from about 1.5 hours to about 24 hours, or from about 1.5 hours to about 2 hours. In some embodiments, the incubation period can be, for example, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0115] The target cells and the library of aptamer cluster particles can be mixed in a ratio such that the cell-aptamer cluster particle complex formed contains about 1-10 particles per target cell (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 particles per target cell). In certain embodiments, the cell-aptamer cluster particle complex formed contains about 2-4 particles per target cell. In some embodiments, the aptamer cluster particles in the cell-aptamer cluster particle complex formed contain about 1-10 clusters per particle (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 clusters per particle). In certain embodiments, the aptamer cluster particles in the cell-aptamer cluster particle complex formed contain about 1-6 clusters per particle.

[0116] In some embodiments, the target cells are labeled with and / or include a detectable label. The target (e.g., target cells) are labeled with and / or include a detectable label. The target may be detectably labeled directly (e.g., via a direct chemical linker) or indirectly (e.g., using a detectably labeled target-specific antibody). In embodiments where the target is a cell, the target cells may be labeled by incubating the target cells with a detectable label under conditions such that the detectable label is internalized by the cell. In some embodiments, the target is detectably labeled prior to performing the aptamer screening methods described herein. In some embodiments, the target is labeled during the performance of the aptamer screening methods provided herein. In some embodiments, the target is labeled after binding to the aptamer cluster (e.g., by contacting the bound target with a detectably labeled antibody). In some embodiments, any detectable label may be used. Examples of detectable labels include, but are not limited to, fluorescent moieties, radioactive moieties, paramagnetic moieties, luminescent moieties, and / or colorimetric moieties. In some embodiments, a target described herein is linked to, contains, and / or is bound by a fluorescent moiety.Examples of fluorescent moieties include, but are not limited to, allophycocyanin, fluorescein, phycoerythrin, peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, and CyPet.

[0117] The target may be a non-molecular target or a supramolecular target. Non-limiting examples of targets that the aptamers of the present disclosure may bind and / or regulate include, but are not limited to, cells, bacteria, fungi, archaea, protozoa, viruses, virion particles, synthetic and naturally occurring microscopic particles, and liposomes. In some embodiments, the target that is contacted with the aptamer cluster particles is live / natural. In other embodiments, the target that is contacted with the aptamer cluster particles is fixed or in solution.

[0118] In some embodiments, the target cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell. Non-limiting examples of bacteria include Aspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Gonococcus, Histoplasma, Klebsiella, Legionella, Leishmania, Meningococci, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma, and Vibriocholerae.It contains antibiotics (Neisseria gonorrhea), Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis.B canis、Fetal Campylobacter、Fetal Campylobacter intestinalis、Leptospira pomona、Listeria monocytogenes、Brucella ovis、Chlamydia psittaci、Trichomonas fetus、Toxoplasma gondii、Actinobacteria (Escherichia coli) Actinobacillus equuli、Salmonella abortus ovis、Salmonella abortus equi、Elective (Pseudomonas aeruginosa)、Corynebacterium equi、Corynebacterium pyogenes、Actinobaccilus seminis、Mycoplasma bovigenitalium、Aspergillus fumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, and Clostridium botulinum.

[0119] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is an animal cell (e.g., a mammalian cell). In some embodiments, the cell is a human cell. In some embodiments, the cell is from a non-human animal, such as a mouse, rat, rabbit, pig, cow (e.g., cow, bull, buffalo), deer, sheep, goat, llama, chicken, cat, dog, ferret, or primate (e.g., marmoset, rhesus monkey). In some embodiments, the cell is a parasitic cell (e.g., a malaria cell, a leishmania cell, a cryptosporidium cell, or an amoeba cell). In some embodiments, the cell is a fungal cell, such as, for example, a Paracoccidioides brasiliensis cell.

[0120] In some embodiments, the cells are cancer cells (e.g., human cancer cells or patient-derived cancer cells). In some embodiments, the cells are from any cancerous or precancerous tumor. Non-limiting examples of cancer cells include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lymph node, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. Furthermore, the cancer may specifically be of the following histological types, but is not limited to: neoplasm, malignant, carcinoma, carcinoma, undifferentiated, giant cell and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, gastrinoma, malignant, cholangiocarcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma of adenomatous polyps, adenocarcinoma, familial adenomatous polyposis, solid tumors, carcinoid tumors, Malignant, branching alveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, eosinophilic carcinoma, eosinophilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, non-encapsulated sclerosing carcinoma, adrenocortical carcinoma, endometrial carcinoma, cutaneous adnexal carcinoma, apocrine adenocarcinoma, Sebaceous carcinoma, ceruminous carcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cyst cell carcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease, mammary gland, acinar cell carcinoma, adenosquamous carcinoma, squamous carcinoma. Adenocarcinoma with squamous metaplasia, Thymoma, Malignant, Ovarian stromal tumor, Malignant, Theca tumor, Malignant, Granulosa cell tumor, Malignant and blastoma, Malignant, Sertoli cell carcinoma, Leydig cell tumor, Malignant, Lipid cell tumor, Malignant, Paraganglioma, Malignant, Extramammary paraganglioma, Malignant, Pheochromocytoma, Gloman's sarcoma, Malignant melanoma, Amelanotic melanoma, Superficial spreading melanoma, Malignant melanoma Giant pigmented nevus, Epithelioid cell melanoma, Blue nevus, Malignant, Sarcoma, Fibrosarcoma, Fibrous histiocytoma, Malignant, Myxosarcoma, Liposarcoma , leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, mixed tumor, malignant, mixed mullerian tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, mesenchymoma, malignant, brenner tumor, malignant, phyllodes tumor, malignant, synovial sarcoma, mesothelioma, malignant, blastoma, embryonal carcinoma, teratoma, malignant, ovarian goiter, malignant, choriocarcinoma, mesonephroma, malignant, angiosarcoma, hemangioendothelioma, malignant, Kaposi's sarcoma, hemangiopericytoma, malignant, lymphangiosarcoma, osteosarcoma, paracortical osteosarcoma, chondrosarcoma,Chondroblastoma, malignant, Mesenchymal chondrosarcoma, Giant cell tumor of bone, Ewing's sarcoma, Odontogenic tumor, malignant, Ameloblastic odontosarcoma, Ameloblastoma, malignant, Ameloblastic fibrosarcoma, Pinealoma, malignant, Chordoma, Glioma, malignant, Ependymoma, Astrocytoma, Protoplasmic astrocytoma, Fibrous astrocytoma, Astroblastoma, Glioblastoma, Oligodendroglioma, Primitive neuroectodermal, Cerebellar sarcoma, Soft tissue sarcoma, Ganglioblastoma, Neuroblastoma, Retinoblastoma, Olfactory neurogenic tumor, Meningioma, malignant, Neurofibrosarcoma, Schwannoma, malignant, Granular cell Neoplasm, malignant, malignant lymphoma, Hodgkin's disease, Hodgkin's lymphoma, paragranulomatous, malignant lymphoma, small lymphocytic, malignant lymphoma, large cell, diffuse, malignant lymphoma, follicular, mycosis fungoides, other specified non-Hodgkin's lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryocytic leukemia, myeloid sarcoma, and hairy cell leukemia.

[0121] In some embodiments, the target cell is an immune cell (e.g., human immune cell or patient-derived immune cell).The term "immune cell" as used herein refers to the cell that plays a role in immune response.Immune cells are hematopoietic cells, including lymphocytes such as B cells and T cells; natural killer cells; myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0122] In some embodiments, the target cell is infected with a virus. For example, in some embodiments, the virus is HIV, hepatitis A, hepatitis B, hepatitis C, herpes virus (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, dengue virus, papilloma virus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, or Ebola virus.

[0123] In some embodiments, the cellular function that is modulated is cell survival, cell proliferation, gene expression, cell morphology, cell activation, phosphorylation, calcium mobilization, degranulation, cell migration, and / or cell differentiation. In certain embodiments, the target is linked to, bound by, or includes a detectable label that allows detection of a biological or chemical effect on the target. In some embodiments, the detectable label is a fluorescent dye. Non-limiting examples of fluorescent dyes include, but are not limited to, calcium-sensitive dyes, cell tracer dyes, lipophilic dyes, cell proliferation dyes, cell cycle dyes, metabolite-sensitive dyes, pH-sensitive dyes, membrane potential-sensitive dyes, mitochondrial membrane potential-sensitive dyes, and redox potential dyes. In certain embodiments, the target is labeled with a calcium-sensitive dye, a cell tracer dye, a lipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolite-sensitive dye, pH-sensitive dyes, membrane potential-sensitive dyes, mitochondrial membrane potential-sensitive dyes, or redox potential dyes.

[0124] In certain embodiments, the target cells are labeled with an activation-associated marker, an oxidative stress reporter, an angiogenesis marker, an apoptosis marker, an autophagy marker, a cell survival marker, or a marker of ion concentration. In yet another embodiment, the target cells are labeled with an activation-associated marker, an oxidative stress reporter, an angiogenesis marker, an apoptosis marker, an autophagy marker, a cell survival marker, or a marker of ion concentration prior to exposing the aptamer to the target.

[0125] In some embodiments, the target cells are labeled after exposing the aptamer to the target.In some embodiments, the target is labeled with fluorescently labeled antibodies, annexin V, antibody fragments and artificial antibody-based constructs, fusion proteins, sugars, or lectins.In another embodiment, the target cells are labeled after exposing the aptamer to the target with fluorescently labeled antibodies, annexin V, antibody fragments and artificial antibody-based constructs, fusion proteins, sugars, or lectins.

[0126] In some embodiments, the target cells are labeled with a fluorescent dye, non-limiting examples of which include calcium-sensitive dyes, cell tracer dyes, lipophilic dyes, cell proliferation dyes, cell cycle dyes, metabolite-sensitive dyes, pH-sensitive dyes, membrane potential-sensitive dyes, mitochondrial membrane potential-sensitive dyes, and redox potential dyes.

[0127] In some embodiments, the target cells are labeled with a calcium-sensitive dye, a cell tracer dye, a lipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolite-sensitive dye, a pH-sensitive dye, a membrane potential-sensitive dye, a mitochondrial membrane potential-sensitive dye, or an oxidation-reduction potential dye. In certain embodiments, the target cells are labeled with an activation-associated marker, an oxidative stress reporter, an angiogenesis marker, an apoptosis marker, an autophagy marker, a cell survival marker, or an ion concentration marker. In yet another embodiment, the target cells are labeled with an activation-associated marker, an oxidative stress reporter, an angiogenesis marker, an apoptosis marker, an autophagy marker, a cell survival marker, or a marker of ion concentration before exposing the aptamer to the cells. In some embodiments, the target cells are labeled after exposing the aptamer to the target. In certain embodiments, the target cells are labeled with a fluorescently labeled antibody or antigen-binding fragment thereof, annexin V, a fluorescently labeled fusion protein, a fluorescently labeled sugar, or a fluorescently labeled lectin. In certain embodiments, the target cells are labeled with a fluorescently labeled antibody or antigen-binding fragment thereof, Annexin V, a fluorescently labeled fusion protein, a fluorescently labeled sugar, or a fluorescently labeled lectin after exposing the aptamer to the cells.

[0128] In some embodiments, any reporter of cellular function can be used in the methods described herein.

[0129] In some embodiments, the cellular function is bacterial membrane integrity. An example of a bacterial membrane integrity reporter includes, but is not limited to, the LIVE / DEAD BacLight Bacterial Viability Kit. In certain embodiments, the cellular function is bacterial oxidase and reductase expression. An example of a bacterial oxidase and reductase expression reporter includes, but is not limited to, the BacLight RedoxSensor Bacterial Vitality Assay. In certain embodiments, the cellular function is bacterial membrane potential change. An example of a bacterial membrane potential change reporter includes, but is not limited to, the BacLight Bacterial Membrane Potential Kit.

[0130] In one embodiment, the cellular function is apoptosis. Examples of apoptosis reporters are listed in Table 14.

[0131] In some embodiments, the reporter of cell function is an antibody, hi certain embodiments, the antibody is labeled with a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, allophycocyanin, fluorescein, phycoerythrin, peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet, Emerald, Cerulean, and CyPet.

[0132] In certain embodiments, the cellular function is autophagy and the antibody binds to a marker of autophagy (e.g., LC3, p62).

[0133] In some embodiments, the cellular function is cell proliferation and the antibody binds to a proliferation marker (eg, Ki67, MCM2, PCNA).

[0134] In some embodiments, the cellular function is expression of a tumor antigen and the antibody binds to the tumor antigen (e.g., prostate specific antigen (PSA), prostate membrane antigen (PSMA), cancer antigen 15-3 (CA-15-3), carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), alpha fetoprotein (AFP), NY-ESO-1, MAGEA-A3, WT1, hTERT, tyrosinase, gp100, MART-1, MelanA, beta catenin, CDC27, HSP70-2-m, HLA-A2-R17OJ, AFP, EBV-EBNA, HPV16-E7, MUC-1, HER-2 / neu, mammaglobin A).

[0135] In some embodiments, the cellular function is expression of an immune checkpoint protein and the antibody is directed to an immune checkpoint receptor and / or an immune checkpoint receptor ligand (e.g., B7-H3 (CD276), B7-H4 (VTCN1), B7-H5 (VISTA), BTLA (CD272), CD96 (Tactile), CD112 (Nectin-2), CD134 (OX40), CD137 (4-1BB), CD137L (4-1BBL), CD1 52(CTLA-4), CD155(PVR), CD223(LAG3), CD226(DNAM1), CD252(OX40L), CD258(LIGHT), CD273(PD-L2), CD274(PD-L1), CD278(I Binds to COS), CD279 (PD-1), CD357 (GITR), DR3 (TNFRSF25), galectin 9, GITRL, HVEM, ICOSL (B7-H2), IDO, TIGIT, TIM3, TL1A, VSIG4).

[0136] In some embodiments, the cellular function is cytokine expression and the antibody binds to a cytokine (e.g., IL-6, TNF-α, IFN-γ, IL-1β, IL-3, IL-4, IL-5, IL-13, GM-CSF, IL-2, IL-8, GRO-α, IL-10, MCP-1, MIP-1, MCP-3, MIG, IL-12, CCL5).

[0137] In some embodiments, the cellular function is a macrophage function and the antibody binds to a marker of macrophage function (e.g., IDO, CD163, CD206, Arginase 1, CD204 (MSR-1), CD369, GPNMB, VSIG4, Marco, MerTK, osteopontin, Axl, VISTA).

[0138] In some embodiments, the cell function is a dendritic cell function and the antibody binds to a marker of dendritic cell function (eg, CD103, CD11b, XCR1, CD80, CD86).

[0139] Figure 1 provides a typical workflow illustrating a particular embodiment of the method described herein. The workflow begins with an initial aptamer library (e.g., an aptamer library described herein), which is as selected and prepared for Illumina sequencing. This library may, for example, be newly synthesized, but may also be the product of a previous selection process. This process may include one or more positive selection cycles, one or more negative selection cycles, or both, together and in sequence.

[0140] Figure 3 is a diagram illustrating some steps of the process. Figure 3I shows a series of combined SELEX steps to select binding specific aptamers for target cells, and functional SELEX performed to enrich the population with binding aptamers that provide a functional effect on cells. Many options are possible, since combined and functional SELEX can be repeated any number of times. Figure 3II illustrates the steps of combining aptamer cluster particles (see B) and cells (see A) described herein to form detectably labeled aptamer cluster particle cell complexes (see C). Figure 3III shows the results of flow cytometry to detect the intensity of fluorescent markers of cell function (y-axis) as well as particle complexes with cells (x-axis), which allows the separation of cell aptamer cluster particle complexes with altered cell function (labeled ii) from cell aptamer cluster particle complexes without the desired effect (labeled iv), unbound particles (v), and unbound cells with both the desired effect (labeled i) and without the desired function (labeled iii).

[0141] The prepared library is immobilized on particles such as beads. Emulsion PCR (ePCR) amplification converts each single sequence from the initial library into a cluster of at least, for example, 10,000 copies of the same sequence. The library of aptamer cluster particles is then incubated with target cells. The target cells can be labeled with a fluorescent dye to report a biological or chemical effect on the target cells before being introduced into the aptamer cluster particles. The target cells and the library of aptamer cluster particles can be incubated for a period of time to allow the effect to occur. A fluorescent dye or marker to report a biological or chemical effect (e.g., cell activation, apoptosis, etc.) can then be delivered to the target cells (see FIG. 1). In some embodiments, a reporter is added to the cells before incubation. In some embodiments, a reporter is added during incubation. In certain embodiments, a reporter is added after incubation. In some embodiments, a second reporter is used (e.g., before incubation) to mark cells that express the desired phenotype (e.g., apoptosis) independent of the incubation process with the aptamer. In certain embodiments, the second reporter helps to distinguish false positives. In some embodiments, a second (or third) reporter (e.g., a reporter acting by a different mechanism) is used to confirm that the detected phenotype is not a false positive. Then, the clusters with positive effects are sorted from the clusters with negative effects, and the corresponding functional aptamer sequences are analyzed. The sorted positive clusters can be amplified and immobilized on the surface of particles as an initial library for additional rounds of screening. After each round of screening, a portion of the enriched functional aptamers undergoes output sampling and comparative functional analysis, followed by aptamer identification by sequencing.

[0142] Other Compositions and Methods In certain aspects, also provided herein are compositions comprising a population of functionally enriched aptamers, such as a population of aptamers selected using the aptamer library screening methods described herein. In some embodiments, the population of functionally enriched aptamers is characterized by greater than a 1.1-fold increase in function compared to a control aptamer, e.g., greater than 1.2-fold, greater than 1.3-fold, greater than 1.4-fold, greater than 1.5-fold, greater than 1.6-fold, greater than 1.7-fold, greater than 1.8-fold, greater than 1.9-fold, greater than 2.0-fold, greater than 2.1-fold, greater than 2.2-fold, greater than 2.3-fold, greater than 2.4-fold, greater than 2.5-fold, greater than 2.4-fold, greater than 2.5-fold, greater than 2.6-fold, greater than 2.7-fold, greater than 2.8-fold, greater than 2.9-fold, greater than 3-fold, greater than 3.5-fold, greater than 4-fold, greater than 4.5-fold, greater than 5-fold, greater than 5.5-fold, greater than 6-fold, greater than 6.5-fold, greater than 7-fold, greater than 7.5-fold, greater than 8-fold, greater than 8.5-fold, greater than 9-fold, greater than 9.5-fold, greater than 10-fold improvement in function compared to a control library. The control library can be, for example, one or more non-functional aptamers, a random pool of aptamers, an aptamer cluster library before functional screening or enrichment, or a population of aptamers that do not regulate a specific cellular function of interest.In some embodiments, the function is measured as the quantitative fluorescence of a detectable label of a cellular function, as the quantitative luminescence of a detectable label of a cellular function, or as a change in cellular morphology.In some embodiments, the function measured is cancer cell death or apoptosis, for example, cell death or apoptosis of tumor-derived cells.Therefore, the aptamer population that modifies tumor-derived individualized cells is also encompassed by the present invention.In some other embodiments, the function measured is the activation / deactivation or other phenotype conversion / skewing / polarization of immune cells.

[0143] In certain aspects, provided herein are compositions comprising aptamer clusters (e.g., clustered aptamer libraries generated during the practice of the methods described herein). In certain embodiments, the aptamer clusters are immobilized on a solid support (e.g., a particle surface). In certain embodiments, the composition further comprises a target cell (e.g., a cancer cell, an immune cell, a bacterial cell). In certain embodiments, the composition further comprises a reporter of a cell function. In certain embodiments, the reporter is a fluorescent reporter (e.g., a membrane integrity reporter, a capsid integrity reporter, a protein integrity reporter, a protein denaturation reporter, a cell death reporter, or a redox potential reporter).

[0144] In some embodiments, the composition has a pH of at least about 10. 2 aptamer clusters (e.g., at least about 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , or 10 9 In certain embodiments, the composition comprises at least about 10 aptamer clusters. 6 In some embodiments, the composition comprises 10 aptamer clusters. 5 ~10 10 aptamer clusters (e.g., 10 5 ~5×10 9 , 10 5 ~10 9 , 10 5 ~5×10 8 , 10 5 ~10 8 , 10 5 ~5×10 7 , 10 5 ~10 7 , 105 ~5×10 6 , 10 5 ~10 6 , 10 5 ~5×10 5 , 5×10 5 ~10 10 , 10 6 ~10 10 , 5×10 6 ~10 10 , 10 7 ~10 10 , 5×10 7 ~10 10 , 10 8 ~10 10 , 5×10 8 ~10 10 , 10 9 ~10 10 , 5×10 9 ~10 10 , 5×10 5 ~5×10 9 , 10 6 ~10 9 , 5×10 6 ~5×10 8 , 10 7 ~10 8 In certain embodiments, the composition comprises 10 6 ~10 9 In some embodiments, each aptamer cluster comprises at least about 2 copies of an aptamer (e.g., at least about 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , or 10 6 In certain embodiments, each aptamer cluster comprises at least about 10 copies of the aptamer. 4 In some embodiments, each aptamer cluster comprises 10 copies of the aptamer. 3 ~10 7 aptamers (e.g., 10 per cluster) 3 ~5×10 6 , 10 3 ~10 6 , 103 ~5×10 5 , 10 3 ~10 5 , 10 3 ~5×10 4 , 10 3 ~10 4 , 10 3 ~5×10 3 , 5×10 3 ~10 7 , 10 4 ~10 7 , 5×10 4 ~10 7 , 10 5 ~10 7 , 5×10 5 ~10 7 , 10 6 ~10 7 , 5×10 6 ~10 7 , 5×10 3 ~5×10 6 , 10 4 ~10 6 , or 5 × 10 4 ~5×10 5 In certain embodiments, each aptamer cluster comprises 10 4 ~10 6 The antibody comprises aptamers.

[0145] In some embodiments, the target can be any type of cell (e.g., a prokaryotic cell, such as a bacterium or archaea, or a eukaryotic cell, such as an animal cell, a plant cell, a protozoan cell, a mammalian cell, etc.), a virus, or the like.

[0146] In some embodiments, the reporter of cell function is a fluorescent reporter. In some embodiments, the reporter of function is a cell death reporter, a redox potential reporter, a membrane integrity reporter. Examples of cell death reporters are 7-AAD and annexin V fluorophores. In some embodiments, the reporter of function is a viral reporter, such as a capsid integrity reporter (e.g., a reporter for measuring the capsid integrity and / or function of a virus). In some embodiments, the reporter of function is a protein reporter, such as a protein integrity reporter (i.e., a reporter for measuring the structural integrity and stability of a protein) or a protein denaturation reporter (i.e., a reporter for detecting protein denaturation).

[0147] In some embodiments, a method for selecting aptamers for cancer treatment is also provided.For example, in some embodiments, the method for selecting aptamers for use in personalized cancer treatment comprises: a) providing a population of aptamers characterized by increasing the promotion of cell death or apoptosis of patient-derived cancer cells by more than 1.5 times compared with control aptamers; b) selecting at least one aptamer candidate from the aptamer population; and c) preparing at least one aptamer for use in personalized cancer treatment.In some embodiments, the population of functionally enriched aptamers is prepared using the aptamer library screening method described herein.

[0148] In some embodiments, the method for preparing a tumor-targeting delivery system includes: a) providing a population of aptamers characterized by a greater than 1.1-fold (e.g., 1.5-fold) increase in promoting cell death or apoptosis of patient-derived cancer cells compared to a control aptamer; b) selecting at least one candidate aptamer from the population of aptamers; and c) combining at least one aptamer with a tumor therapeutic agent for tumor-localized delivery. In some embodiments, the population of functionally enriched aptamers is prepared using the aptamer library screening method described herein.

[0149] Aptamer that induces apoptosis in cancer cells In certain embodiments, provided herein are aptamers that selectively bind to and / or kill cancer cells (e.g., breast cancer cells, such as triple-negative breast cancer cells), including by inducing apoptosis. In some embodiments, provided herein are pharmaceutical compositions comprising such aptamers, methods of using such aptamers to treat cancer and / or kill cancer cells, and methods of making such aptamers.

[0150] In certain aspects, provided herein are aptamers comprising a nucleic acid sequence that is at least 60% identical (e.g., at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical) to any one of SEQ ID NOs: 1-10 (Table 1). In some embodiments, the aptamer comprises a nucleic acid sequence of any one of SEQ ID NOs: 1-10. In certain embodiments, the aptamer comprises at least 20 (e.g., at least 25, at least 30, at least 35, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53) consecutive nucleotides of any one of SEQ ID NOs: 1-10. In certain embodiments, the aptamer provided herein comprises any one of the nucleic acid sequences of SEQ ID NOs: 1 to 10. In some embodiments, the aptamer provided herein has a sequence essentially consisting of SEQ ID NOs: 1 to 10. In certain embodiments, the aptamer provided herein has a sequence consisting of SEQ ID NOs: 1 to 10.

[0151] [Table 1]

[0152] The term "identical" or "percent identity" with respect to two or more nucleic acids refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity when compared over a comparison window or designated region and aligned for maximum correspondence), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, as described below, or by manual alignment and visual inspection (see, e.g., the NCBI website at http: / / www.ncbi.nlm.nih.gov / BLAST / , etc.).

[0153] In certain embodiments, aptamers are 100 nucleotides or less in length (e.g., 90 nucleotides or less in length, 85 nucleotides or less in length, 80 nucleotides or less in length, 75 nucleotides or less in length, 70 nucleotides or less in length, 65 nucleotides or less in length, 60 nucleotides or less in length, 59 nucleotides or less in length, 58 nucleotides or less in length, 57 nucleotides or less in length, 56 nucleotides or less in length, 55 nucleotides or less in length, or 54 nucleotides or less in length.

[0154] In some embodiments, the aptamers provided herein can bind to cancer cells (e.g., human cancer cells). In some embodiments, the aptamers provided herein can induce cell death (e.g., apoptosis) of cancer cells (e.g., human cancer cells) upon contact with the cancer cells. In some embodiments, the cancer cells are cancer cells from a patient. In some embodiments, the cancer cells are solid tumor cells. In certain embodiments, the cancer cells are (epithelial) cancer cells. In some embodiments, the cancer cells are breast cancer cells. In some embodiments, the cancer cells are triple-negative breast cancer cells (i.e., breast cancer cells that do not express the estrogen receptor (ER), progesterone receptor (PR) and HER / 2neu genes). In some embodiments, the aptamers induce cell death upon contact with cancer cells in vitro. In certain embodiments, the aptamers induce cell death upon contact with cancer cells in vivo (e.g., in humans and / or animal models).

[0155] In some embodiments, the aptamers provided herein comprise one or more chemical modifications. Examples of modifications are shown in Table 2.

[0156] [Table 2]

[0157] In certain embodiments, the aptamer comprises a terminal modification. In some embodiments, the aptamer is chemically modified (e.g., attached to the 5' end of the aptamer) with polyethylene glycol (PEG) (e.g., 0.5-40 kDa). In some embodiments, the aptamer comprises a 5' end cap (e.g., reverse thymidine, biotin, albumin, chitin, chitosan, cellulose, terminal amine, alkyne, azide, thiol, maleimide, NHS). In certain embodiments, the aptamer comprises a 3' end cap (e.g., reverse thymidine, biotin, albumin, chitin, chitosan, cellulose, terminal amine, alkyne, azide, thiol, maleimide, NHS).

[0158] In certain embodiments, the aptamers provided herein comprise one or more (e.g., at least 1, 2, 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, 51, 52, 53, or 54) modified sugars. In some embodiments, the aptamers comprise one or more 2' sugar substitutions (e.g., 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions). In certain embodiments, the aptamer comprises locked nucleic acid (LNA), unlocked nucleic acid (UNA) and / or 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'-F-ANA) sugars in its backbone.

[0159] In certain embodiments, the aptamer contains one or more (e.g., at least 1, 2, 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, 51, 52, 53, or 54) methylphosphonate internucleotide linkages and / or phosphorothioate internucleotide linkages. In certain embodiments, the aptamer comprises one or more (e.g., at least 1, 2, 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, 51, 52, 53, or 54) triazole internucleotide linkages. In certain embodiments, the aptamer is modified (e.g., at its 5' end) with cholesterol or a dialkyl lipid.

[0160] In some embodiments, the aptamer comprises one or more modified bases (eg, BzdU, naphthyl, tryptamino, isobutyl, 5-methylcytosine, alkyne (dibenzocyclooctyne), azide, maleimide).

[0161] In certain embodiments, the aptamers provided herein are DNA aptamers (e.g., D-DNA aptamers or R-DNA aptamers). In some embodiments, the aptamers provided herein are RNA aptamers (e.g., D-RNA aptamers or R-RNA aptamers). In some embodiments, the aptamers comprise a mixture of DNA and RNA.

[0162] Aptamers can be synthesized by methods well known to those skilled in the art. For example, aptamers can be chemically synthesized, for example, on a solid support. Solid-phase synthesis can use phosphoramidite chemistry. Briefly, nucleotides on a solid support are detritylated and then coupled with appropriately activated nucleoside phosphoramidites to form phosphite triester bonds. Capping is then performed, followed by oxidation of the phosphite triester with an oxidizing agent, usually iodine. The cycle can then be repeated to build the aptamer.

[0163] Treatment method In certain aspects, a pharmaceutical composition is provided that includes an aptamer provided herein (e.g., a therapeutically effective amount of the aptamer). In some embodiments, the pharmaceutical composition further includes a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0164] In certain embodiments, the pharmaceutical composition is for the treatment of cancer. In some embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is an (epithelial) cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer.

[0165] "Pharmaceutically acceptable carrier" refers to a substance that aids in the administration of an active agent to a subject and the absorption of the active agent by the subject, and that can be included in the compositions described herein without causing significant toxicological adverse effects to the patient. Non-limiting examples of pharma-ceutically acceptable excipients include water, NaCl, saline, lactated Ringer's, normal sucrose, normal glucose, binders, bulking agents, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, saline solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose, starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such formulations can be sterilized and, if necessary, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic agents that do not adversely react with the compositions described herein. Those skilled in the art will recognize that other pharmaceutical excipients are useful.

[0166] In some embodiments, provided herein is a method for treating cancer, comprising administering a pharmaceutical composition comprising one or more aptamers as described herein.In certain embodiments, the cancer is breast cancer.In some embodiments, the cancer is triple-negative breast cancer.Thus, in certain aspects, provided herein is a method for delivering the aptamer and / or pharmaceutical composition as described herein to a subject.

[0167] In certain embodiments, the pharmaceutical compositions and aptamers described herein can be administered in conjunction with any other conventional anti-cancer treatments, such as, for example, radiation therapy and surgical removal of tumors. These treatments may be administered as needed and / or as indicated, and can occur before, simultaneously with, or after administration of the pharmaceutical compositions, dosage forms, and kits described herein.

[0168] In certain embodiments, the method includes multiple administrations of the aptamer. Separate administrations can include any number of administrations (e.g., multiple administrations) of 2 or more, including 2, 3, 4, 5, or 6 administrations. For the therapeutic methods and other monitoring methods provided herein, those skilled in the art can easily determine the number of administrations to be performed, or the desirability of performing one or more additional administrations, according to methods known in the art. In some embodiments, the dosages can be separated by at least 1, 2, 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, or 30 days, or 1, 2, 3, or 4 weeks. Thus, the methods provided herein include methods of providing one or more doses to a subject, the number of doses being determined by monitoring the subject, and based on the results of the monitoring, it can be determined whether or not to provide one or more additional doses. The decision of whether or not to administer one or more additional doses can be based on various monitoring results, including, but not limited to, signs of tumor growth or inhibition of tumor growth, the appearance of new metastases or inhibition of metastases, the subject's anti-bacterial antibody titer, the subject's anti-tumor antibody titer, the subject's overall health, and / or the subject's body weight.

[0169] The period between administrations can be any of a variety of periods. The period between administrations can be any of a variety of factors, including the number of administrations, the period in which the subject initiates an immune response, and / or the period in which the subject clears bacteria from normal tissue, as described above, including the monitoring step. In one example, the period can be a function of the period in which the subject initiates an immune response; for example, the period can be longer than the period in which the subject initiates an immune response, such as more than about 1 week, more than about 10 days, more than about 2 weeks, or more than about 1 month; in another example, the period can be shorter than the period in which the subject initiates an immune response, such as less than about 1 week, less than about 10 days, less than about 2 weeks, or less than about 1 month. In yet another example, the period can be a function of the period in which the subject clears bacteria from normal tissue; for example, the period can be longer than the period in which the subject clears bacteria from normal tissue, such as more than about 1 day, more than about 2 days, more than about 3 days, more than about 5 days, or more than about 1 week.

[0170] The effective dose of the aptamer described herein is the amount of the aptamer that is effective for a particular patient, composition, and administration method to achieve a desired therapeutic response while minimizing toxicity to the patient. Effective dosage levels can be determined using the methods described herein, and are dependent on a variety of pharmacokinetic factors, including the activity of the particular composition administered, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, disease, general health, and medical history of the patient receiving treatment, etc. In general, the effective dose of a cancer therapeutic agent is the amount of the therapeutic agent that is the lowest effective dose to produce a therapeutic effect. Such an effective dose generally depends on the factors described above.

[0171] Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Administration by injection includes intravenous (IV), intralesional, peritumor, intramuscular (IM) and subcutaneous (SC) administration. The compositions described herein can be administered in any form and by any effective route, including oral, parenteral, enteral, intravenous, intratumoral, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ocular, nasal (intranasal), topical, parenteral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intraarterial and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g., transvaginal and perivaginal)), implantable, intravesical, intrapulmonary, intraduodenal, intragastric and intrabronchial. In preferred embodiments, the bacterial compositions described herein are given orally, rectally, topically, intravesically, by injection into or near a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.

[0172] Dosage regimens may take any of a variety of methods and amounts and can be determined by those skilled in the art according to known clinical factors. As is known in the medical arts, the dosage for any one patient may depend on numerous factors, including the subject's species, size, body surface area, age, sex, immune competence, tumor size and general health, the specific microorganism administered, the duration and route of administration, the type and stage of disease, e.g., tumor size, and other compounds, such as drugs, administered concomitantly.

[0173] The therapeutic methods described herein are considered to be suitable for treating primary tumors, secondary tumors or metastases, as well as recurrence of tumors or cancer. The dosage of the pharmaceutical compositions described herein can be appropriately set or adjusted depending on the dosage form, administration route, the degree or stage of the target disease, etc.

[0174] In some embodiments, the dose administered to a subject is sufficient to prevent cancer, delay the onset of cancer, delay or stop the progression of cancer, prevent the recurrence of cancer, or contribute to the overall survival of the subject.Those skilled in the art will recognize that the dosage depends on various factors, such as the strength of the individual compound used, as well as the age, species, disease, and weight of the subject.The magnitude of the dosage is also determined by the presence, nature, and extent of adverse side effects associated with the administration of the individual compound, as well as the route, timing, and frequency of administration, and the physiological effect required.

[0175] The appropriate dosage and administration schedule can be determined by conventional dose-response detection techniques known to those skilled in the art. Generally, treatment is started with a small dose that is less than the optimal dose of the compound. The dosage is then increased in small increments until the optimal effect is achieved under the circumstances. Effective dosages and treatment protocols can be determined by routine methods, such as starting with a low dose in laboratory animals, increasing the dosage while confirming the effect, and systematically changing the administration schedule. Animal experiments are often used to determine the maximum tolerated dose (MTD) of a biologically active substance per kg of body weight. Those skilled in the art routinely estimate the dosage for efficacy while avoiding toxicity in other species, including humans.

[0176] In accordance with the above, dosages of the aptamers described herein for therapeutic use may vary depending on the particular aptamer, the age, weight and clinical condition of the recipient patient, and the experience and judgment of the treating clinician or practitioner, among other factors that influence the selected dosage. In general, the dosage should be sufficient to result in slowing, and preferably causing regression of tumor growth, and most preferably causing complete regression of the cancer.

[0177] Examples of cancers that can be treated by the methods described herein include hematological malignancies, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocytic leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myelogenous leukemia, leukemia cutis, fetal cell leukemia, eosinophilic leukemia, Gross leukemia, Reeder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, Anaplastic cell leukemia, Hairy cell leukemia, Hemocytoblastic leukemia, Hemoblastic leukemia, Histiocytic leukemia, Stem cell leukemia, Acute monocytic leukemia, Leukopenic leukemia, Lymphatic leukemia, Lymphoblastic leukemia, Lymphocytic leukemia, Lymphogenous leukemia, Lymphoid leukemia, Lymphosarcoma cell leukemia, Mast cell leukemia, Megakaryocytic leukemia, Micromyeloblastic leukemia, Monocytic leukemia, Myeloblastic leukemia, Myelogenous leukemia, Myelomonocytic leukemia, Naegeli leukemia, Plasma cell cell) leukemia, plasmacytic leukemia, promyelocytic leukemia, acinic cell carcinoma, lobular carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar epithelial carcinoma, basal cell carcinoma, carcinoma basocellulare), basal cell carcinoma, basal squamous cell carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, choriocarcinoma, colloid carcinoma comedonal carcinoma, endometrial carcinoma, cribriform carcinoma, armor-like carcinoma, skin cancer, columnar cell carcinoma, ductal carcinoma, hard carcinoma, embryonal carcinoma, encephaloid carcinoma carcinoma), epidermoid carcinoma, adenoid carcinoma, exophytic carcinoma, ulcerative carcinoma, fibrocarcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, globular cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, beaded carcinoma, telangiectatic carcinomataticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, choriocarcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematopoietic carcinoma, hepatocellular carcinoma, Hürthle cell tumor, hyaline cell carcinoma, adrenal-like carcinoma, infantile embryonal carcinoma, carcinoma in situ (cancer in normal position, carcinoma in situ), intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kurticytsky cell (neuroendocrine cell) carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma carcinoma, melanoma, soft carcinoma, mucinous carcinoma, carcinoma muciparum, mucous cell carcinoma, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxoma-like carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, medullary carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, Schneider's carcinoma, scirrhous carcinoma, scrotal carcinoma, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, liposarcoma sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromatous sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multifocal pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cell, lymphoma, immunoblastic sarcoma of T cell, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcomaReticulum cell sarcoma, rhabdomyosarcoma, serous cystic sarcoma, synovial sarcoma, telangiectatic sarcoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant islet cell tumor, malignant carcinoid, precancerous skin lesion, testicular cancer, lymphoma, thyroid cancer, These include, but are not limited to, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical carcinoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral lentigo melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, nodular melanoma, subungual melanoma, superficial spreading melanoma, plasmacytoma, colon cancer, and rectal cancer.

[0178] In some embodiments, the methods and compositions described herein relate to the treatment of sarcomas. The term "sarcoma" generally refers to tumors composed of embryonic connective tissue-like entities and generally consisting of closely packed cells organized in a fibrillar, heterogeneous or homogeneous entity. Sarcomas include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, liposarcoma (adipose tissue sarcoma), and sarcoma (cell sarcoma). sarcoma), liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromatous sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multifocal pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cell, lymphoma, immunoblastic sarcoma of T cell, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulum cell sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0179] Additional examples of neoplasms that can be treated using the methods and compositions described herein include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant insulinoma, malignant carcinoid, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenocortical carcinoma.

[0180] In some embodiments, the cancer to be treated is melanoma.The term "melanoma" is taken to mean the tumor arising from the melanocyte system of skin and other organs.Non-limiting examples of melanoma are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral lentigo melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0181] Particular categories of tumors that can be treated using the methods and compositions described herein include lymphoproliferative diseases, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, thyroid cancer, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, and metastatic cancer of all of the above. Individual tumor types include hepatocellular carcinoma, liver cancer, hepatoblastoma, rhabdomyosarcoma, esophageal cancer, thyroid cancer, ganglioneuroblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, invasive ductal carcinoma of the breast, papillary adenocarcinoma, melanoma, lung squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well-differentiated, moderately differentiated, poorly differentiated, or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, adrenal gland tumor (Gravitz tumor, hypernephroma), adrenal gland-like adenocarcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, and embryonal cancer, Wilms' tumor, testicular cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, and large cell lung cancer), bladder cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon cancer, rectal cancer, hematopoietic malignancies (including all types of leukemia and lymphoma, such as acute myeloid leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.).

[0182] Cancers addressed in certain embodiments also include precancerous lesions, such as actinic keratosis (solar keratosis), moles (dysplastic nevi), actinic cheilitis (farmer's lip), skin horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, iron deficiency dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis, and cervical dysplasia.

[0183] Cancers addressed in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal, or mesenchymal origin, including, but not limited to, cholangiomas, colon polyps, adenomas, papillomas, cystadenomas, hepatocellular adenomas, hydatidiform moles, renal tubular adenomas, squamous cell papillomas, gastric polyps, hemangiomas, osteomas, chondromas, lipomas, fibromas, lymphangiomas, leiomyomas, rhabdomyomas, astrocytomas, nevi, meningiomas, and ganglioneuromas. (Example) EXAMPLES

[0184] Reagents used in Examples 2 and 3 To prepare the binding buffer (x10), 50 mg of tRNA was weighed and distributed into a 50 ml tube, and 5 ml of azide solution (10% azide in PBSX1), 0.5 ml MgCl2 (1M), and 44.5 ml PBSX1 were added. The medium containing 10% human serum was prepared by adding 10% human serum to the standard growth medium of the target cancer cells (DMEM, IMDM, RPMI, etc.). The random DNA library was prepared by dissolving the DNA library in DNase / RNase-free ultrapure water (UPW) to a final concentration of 1 mM and dispensing it into an Eppendorf tube. An oligonucleotide sequence complementary to the primer sequence was prepared, and each of the cap 3' and cap 5' was dissolved in DNase / RNase-free UPW to a final concentration of 100 mM and dispensed into an Eppendorf tube to prepare the aptamer capping (cap 3' and cap 5') solution. Tubes containing both Cap 3' and Cap 5' at a concentration of 50 mM were prepared. The above solutions were freshly prepared and stored at 4°C, and then warmed to room temperature before each experiment. EXAMPLES

[0185] Combined CELL-SELEX: Selection of aptamers that bind to target cells (see Figure 2) a. Cancer cell binding SELEX, round 1 Cells were prepared one day prior. Cells were split at a ratio of 1:2 or 1:3 depending on the proliferation rate of the cells being tested. Cells were counted with PI and run on CytoFLEX.

[0186] Aptamer pools were prepared in DNase / RNase-free Eppendorf tubes as described in Table 3.

[0187] [Table 3]

[0188] The aptamer pool was denatured by heating at 95°C for 5 min, spun down, and cooled on ice for 10 min. 438.21 μl of medium supplemented with 10% human serum was added to the aptamer pool and the tube was incubated at room temperature for at least 10 min. 5.0E6 cells were transferred to an Eppendorf tube and centrifuged at 300xg for 5 min. The supernatant was discarded and the cells were resuspended in 1 ml of medium. Centrifuged again at 300xg for 5 min and the supernatant was discarded. The aptamer pool was then added to the cell pellet, mixed gently, and incubated at 37°C, 50 rpm for 1 h. The cells were then centrifuged at 300xg for 5 min and most of the supernatant was removed (leaving only about 10 μl of supernatant in the tube) to remove unbound sequences. The cells were then washed twice with 1 ml of medium by suspending the cells in 1 ml of medium, centrifuging at 300xg for 5 min, removing most of the supernatant (leaving only about 10 μl of the supernatant in the tube), and transferring the cells to a new tube. To elute the bound DNA, 590 μL of UPW was added to the pellet, which was then heated at 95°C for 10 min. The reaction was then centrifuged at 13.1xg for 5 min at room temperature, and the supernatant was transferred to a new tube.

[0189] PCR calibration was performed with the following conditions: 30% and 60% template, 30 and 36 cycles, primer ratio 1:10, and Tm of the lower of the two primers. Reactions were assembled according to Table 4, vortexed, and spun down. The experiment was performed on ice and immediately after the addition of Herculase.

[0190] [Table 4]

[0191] Eight PCR tubes were prepared, four for 60% template and four for 30% template. 12 μl UPW was added to the negative 60% template and 6 μl UPW to the negative 30% template. The same amount of elution was added to the tubes. 8 μl of PCR calibration mix was added to each 60% template tube and 14 μl of mix was added to each 30% template tube. Lib markers were prepared by diluting to a concentration of 10 μM in UPW and mixing with loading dye. The markers can be stored at 4°C and used for library calibration. Perform PCR reaction using the following program (Tm can be changed depending on the primer Tm):

[0192] [Table 5]

[0193] 20 μl of each sample was loaded onto a 3% agarose gel with 4 μl of loading dye. The most intense single-stranded band at the size of the marker was isolated. A large-scale PCR reaction was performed using the PCR reaction conditions that produced the most intense band. For example, for large-scale PCR, the following conditions were used: 30% template, 30 cycles, 1:10, Tm=56°C.

[0194] [Table 6]

[0195] Samples were concentrated with a 10k Amicon® Ultra Centrifugal Filter. Columns were inserted into the designated tubes and 500 μl of sample was added to each tube. Tubes were centrifuged at 14,000 x g for 20 minutes. Each column was inverted and transferred to a new Eppendorf tube, and the new tube was centrifuged at 1000 x g for 2 minutes. Samples from all tubes were mixed, resulting in a total of approximately 80-100 μl of sample.

[0196] An HPLC vial with an insert was prepared. The volume of the sample was measured and up to 102 μl of sample was transferred to the insert. The sample was run through the HPLC vial, which took approximately 20 minutes to complete. The HPLC results were analyzed. Fractions were run on a 3% agarose gel and the fractions of interest were mixed. The DNA was washed and the concentration was measured using a NanoDrop.

[0197] b. Cancer cell binding SELEX, round 2 Round 2 was performed by repeating the procedure of round 1 with the following modifications: Aptamer pools were prepared in DNase / RNase-free Eppendorf tubes as described in Table 7.

[0198] [Table 7]

[0199] 5.0E6 cells were transferred to an Eppendorf tube (this number may vary depending on cell type, but should be more limiting than the previous conditions). The Eppendorf tube was incubated at 37°C, 50 rpm for 50 min (instead of 1 h as in round 1). Washing and elution steps were the same as in round 1. Negative selection was performed, for example, on PBMC. 80 μl Binding Buffer X1 was added to the pellet without azide. Bound DNA was eluted by heating to 95°C for 10 min and centrifuging at 13.1xg for 5 min at room temperature. The supernatant was transferred to a new tube. Eluted DNA was denatured by heating to 95°C for 5 min (centrifugation) and then cooling on ice for 10 min. 220 μl medium supplemented with 10% human serum was added. A vial of PBMC was thawed and counted, and the total amount of 5.0E6 cells was aliquoted into an Eppendorf tube. The cells were centrifuged at 300xg for 5 min. The supernatant was discarded and 1 ml of fresh medium (without additives) was added to suspend the cells. The cells were centrifuged again at 300xg for 5 minutes. The supernatant was discarded and fresh medium was added to the pellet. The Eppendorf tube was incubated at 37°C and 50 rpm for 1 hour. The cells were centrifuged at 300xg for 10 minutes. The supernatant was transferred to a new tube. The round 1 procedure was repeated using 150 μl of the eluate for amplification and purification (500 μl sample in total).

[0200] c. Cancer cell binding SELEX, round 3 Round 3 was performed by repeating the steps of round 2 but with more restrictive conditions, e.g., incubation with 3.0E6 cells was used and the incubation time was reduced to 40 min. EXAMPLES

[0201] Functional SELEX on Suspension Cells: Selection of Functional Aptamers Targeting Cells (See Figure 3) Log PCR calibration was performed on the supernatant from round 3 of Example 3. Samples were prepared using Table 8 with 8, 10, 12 and 14 cycles of PCR (first 3 cycles at Tm 56°C, remaining at primer Tm). 20 μl of each sample was loaded onto a 3% agarose gel with 4 μl of loading dye.

[0202] [Table 8] Large scale PCR reactions were carried out under the following conditions, for example: 15% template, 12 cycles, and Tm=56°C.

[0203] [Table 9]

[0204] The samples from all PCR tubes were mixed and 102 μl of the sample was purified by HPLC. The concentration of the purified DNA was measured on the Qubit.

[0205] The beads were prepared according to Table 10 using 2 nM Template beads for ion Torrent (trademark) manufactured by Invitrogen.

[0206] [Table 10]

[0207] The purified DNA samples were denatured by heating to 95°C for 5 min, spun down and chilled on ice for 10 min. The tubes were incubated at room temperature for 30 min at 90 / 60 rpm. 800 μl of medium supplemented with 10% human serum was added to the pool.

[0208] 5.0E6 cells were transferred to an Eppendorf tube. Two controls were prepared: negative (cells only) and positive (cells and toxin). Cells were centrifuged at 300xg for 5 min, the supernatant was discarded and 1 ml of fresh medium (no additives) was added. Cells were centrifuged at 300xg for 5 min. The supernatant was discarded and the bead pool was added to the pellet and mixed gently. The Eppendorf tube was incubated at 37°C for 1 h at 90 / 60 rpm, then centrifuged at 300xg for 5 min and the supernatant was removed. 800 μl of medium supplemented with 10% human serum and 0.8 μl of caspase 3 / 7 green were added. The tube was incubated at 37°C for 15 min. Two FACS tubes were prepared, labeled with dead and live cell complexes, coated with BSA. Samples were run on a BD FACSMelody™ cell sorter (controls were run first to adjust gain). Samples were sorted based on yield in round 1 and purity from round 2 onwards. Two types of sorted cells were obtained: dead cancer cells complexed with bead aptamers in tube #1 and live cancer cells complexed with bead aptamers in tube #2. Bound DNA was eluted by heating at 95°C for 10 min and centrifuged at 300xg for 5 min at room temperature. The supernatant was transferred to a new tube.

[0209] A PCR calibration was prepared using the following conditions: 15% template (upscaled to 30% or more if necessary), 14 to 22 cycles (Table 11) with the first three cycles unchanged, and the lower Tm of the two primers used as the Tm.

[0210] [Table 11] The PCR reaction was carried out according to the following program.

[0211] [Table 12] 20 μl of each sample was loaded onto a 3% agarose gel with 4 μl of loading dye. Large-scale PCR was performed as follows.

[0212] [Table 13]

[0213] An HPLC vial with an insert was prepared. The volume of the sample was measured and the volume (102 μl) was transferred to the insert. The vial was closed with a cap and placed in position P1A1. The sample was run through the HPLC, which took about 20 min to complete. The HPLC results were analyzed and, if necessary, fractions were run on a 3% agarose gel. The concentration was measured on the Qubit and 2 nM beads were prepared. The round 2 procedure was repeated as many times as necessary. The most abundant functional aptamers from the resulting pool were sequenced according to standard methods. The identified aptamers were validated in an apoptosis assay using cancer cells. EXAMPLES

[0214] Example of functional Cell-SELEX Three rounds of binding SELEX against cancer cells were performed (e.g., as described in Example 2). Specifically, an ssDNA library composed of a random core flanked by constant regions was folded in the presence of oligonucleotides (called caps) complementary to the constant regions. Folding was performed by incubating at 95°C for 5 min, followed by cooling on ice for 10 min and incubating at 37°C for an additional 10 min. The folded library and cells were incubated together for 1 h in target cell medium supplemented with 10% human serum. The concentration of the library during the incubation step was 500 nM. After each round, the samples were washed and unbound candidates were removed by centrifugation using a 10% ELISA kit (100 μg / ml ELISA kit) containing 10% human serum. 4 x10 from the second selection round onwards 6The bound fraction was eluted by incubation at 95°C for 10 min to prepare the input library for the next round. From the second round onwards, negative selection was added. The eluted library was refolded and incubated with non-target cells as above, but in this case the unbound fraction served as input for the asymmetric PCR (aPCR) process. Samples of the output libraries from all rounds were saved for evaluation.

[0215] After three rounds of binding SELEX, the binding enrichment library is amplified onto microbeads in a water-in-oil emulsion, so that each picoliter droplet contains, on average, one sequence of the binding enrichment library. Emulsion PCR is then performed using an IonTorrent OneTouch to amplify each aptamer onto the bead surface in the droplet picoreactor. The emulsion is then broken. The aptamer bead library is amplified on a 10 8 The library consists of 100 microbeads, each covered with a cluster of multiple copies of a single oligo. This library is used in the first round of functional SELEX. The bead library is prepared so that it can be incubated with cells in a rapid temperature gradient, including oligo caps that complement the flanking regions of the library, which contain fluorescent functional labels (Figure 5A).

[0216] Beads can hold multiple candidates. The more candidates held on each bead, the more diverse candidates can be screened with a given number of beads. However, increasing the number of candidates on one bead reduces the effective concentration of each candidate. To create clustered beads, we used the Ion Proton Sample Preparation Ion PI™ Hi-Q™ OT2 200 Kit and the Ion OneTouch™ Automated Sample Preparation System. The protocol provided with this kit is optimized for Proton sequencing technology, where creating a high percentage of monoclonal beads is a priority. Instead of using 6-8 μl of the 100 pM stock as recommended by the manufacturer to create approximately 10% templated beads, we used 1 μl of the 2 nM stock to create approximately 40% templated beads. A Poisson distribution fit to this percentage of templated beads indicates that greater than 0.75 of the population of templated beads are monoclonal, approximately 0.195 of the templated beads are biclonal, and only approximately 0.05 of this population carries three or more oligo candidates. With the number of beads per reaction as the limiting factor, this is approximately 500 x 10 6 Pieces of beads shown above: Approx. 642 x 10 6 The Ion PI® Hi-Q® OT2 200 Kit user manual instructions were followed, except that the recommended amount of template was exceeded. Enrichment QC was performed using the Ion Sphere® Quality Control Kit according to the manufacturer's instructions. Ion Spheres were labeled with a Cy5-conjugated cap to aid detection on the FACSMelody.

[0217] Target cancer cells were prepared 1 day prior by splitting cells at a ratio of 1:2 / 1:3 to reach 90% confluence in culture dishes. Two types of controls were prepared: cells only (negative control) and cells with toxin added (positive control). One hour before adding the bead library, the cell culture medium was changed to conditioned medium. The medium was aspirated, and the aptamer bead library diluted in medium containing 10% human serum was added to the cells and incubated at 37°C for 1.5-2 hours under gentle shaking conditions. Unbound aptamers were collected from the medium supernatant and transferred to a new tube. Target cells containing bound aptamers were gently removed from the plate and pooled in one collection tube. The cells were incubated with 1.5 μL of functional probe CellEvent Caspase-3 / 7 at a stock concentration of 500 nM for 15 minutes at 37°C in the dark. In some cases, two functional probes, such as CellEvent Caspase-3 / 7 and MitoProbe Dilc1, were incubated with the target cells and bead library mixture simultaneously (Figure 6). Functional labeling was also performed in which cells were first stained with one functional probe and then incubated with a second functional probe to better distinguish between signal and noise in FACS analysis. Examples of alternative probes that can be used in the functional Cell Selex method are listed in Table 14.

[0218] [Table 14]

[0219] Two FACS tubes precoated with BSA were labeled for dead cell complexes (tube #1) and live cells (tube #2). Samples were run on a BD FACSMelody® cell sorter (a control was run first to adjust the gain). Samples were sorted based on yield in round 1 and purity from round 2 onwards. Only morphologically intact cells were gated on during sorting. Cells bound to beads and staining positive for caspase-3 / 7 (clust+ / cas+) were designated as “positive events”. Cells bound to beads but staining negative for caspase-3 / 7 (clust+) were also collected for further analysis and classified as “negative events”. A representative gating strategy is shown in Figure 5B. Two types of sorted cells were obtained: dead cancer cells complexed with the bead aptamer in tube #1 and live cancer cells complexed with the bead aptamer in tube #2. Bound DNA was eluted by heating at 95°C for 10 min, followed by centrifugation at 300xg for 5 min at room temperature. The supernatant was transferred to a new tube. Events that were positive for the caspase-3 / 7 dye and bead label were amplified by emulsion PCR to obtain a bead library for the next round of functional SELEX. Functional SELEX and emulsion PCR were repeated until functional enrichment was observed in the target cell population incubated with the beads. EXAMPLES

[0220] Validation of cytotoxicity of functionally enriched aptamer libraries against different tumor cells Multiple rounds of the functional Cell-SELEX process, as described in Example 7, were performed on the following cancer cell lines: A. HCT116 human colon cancer cell line B. 4T1 mouse breast cancer cell line C. CT26 mouse colon cancer cell line D. Kasumi-1 human acute myeloid leukemia (AML) cell line E. AML1 primary AML myeloblasts from donor F. AML9 primary myeloblasts from donors G. CLL1 Primary Chronic Lymphocytic Leukemia (CLL) Lymphocytes from Donors

[0221] Except for Kasumi-1 cells, where functional Cell-SELEX was initiated after three rounds of binding SELEX and caspase-3 / 7 apoptosis was measured, functional SELEX was initiated from a random (non-binding enriched) library and mitochondrial membrane potential (MitoProbe DilC1(5)) was measured.

[0222] Functional enrichment is verified by functional assays, in which bead libraries from all / most rounds of functional SELEX are incubated with target cell populations and apoptosis enrichment is compared across different bead populations (Figure 7). All of the displayed functional processes, except Kasumi-1, were performed with Caspase 3 / 7 as the apoptosis probe, starting from binding-enriched libraries after three rounds of binding SELEX. Kasumi-1 was performed with the MitoProbe DilC1(5) mitochondrial membrane potential probe, and the initial functional library was a random library (not binding-enriched).

[0223] In the final round of functional SELEX, the final clustered microbead library was incubated with positive target cells followed by incubation with negative counterselected cells. For human suspension cells (primary or cell lines), PBMCs from healthy donors were used as negative counterselected cells. For human adherent cell lines, MCF10a cell line or PBMCs from healthy donors were used as negative counterselected cells. For mouse adherent cell lines, freshly isolated splenocytes were used as negative counterselected cells. Positive events that emerged during counterselection were used to remove fortuitous functional reads during Illumina sequencing analysis. EXAMPLES

[0224] Comparison of binding-enriched and function-enriched aptamer libraries The libraries generated above from all functional SELEX rounds, including the negative events from each round and the positive events from the counterselected cells, were prepared for Illumina sequencing. Sequencing was performed on an Illumina NextSeq 500 sequencer using the NextSeq 500 / 550 High Output Kit. Sequencing abundance profiles were contrasted between aptamers from the binding Cell-SELEX round and those from the functional Cell-SELEX round for selections performed on AML1 primary human myeloblasts and HCT116 colon cancer cell lines. Each SELEX process was run for 10 -6 Starting from log abundance, we used 10 for the final enriched aptamer library. -3 or 10 -2 The final SELEX rounds were performed using the same sequence of the 10,000 most abundant aptamers as the 10 most abundant aptamers, ending with log abundance. As can be seen in Figure 8, little crossover was observed between the binding-enriched and function-enriched libraries for the 10,000 most abundant aptamers in either tissue origin in the final SELEX round. Also, no crossover was observed between the binding-enriched and function-enriched libraries for the 10 most abundant aptamers in the final SELEX round.

[0225] We also compared the final enriched libraries from combined SELEX and functional SELEX after selection on AML1 and HCT116 tissue / cell origins for caspase-3 / 7 activation ability. Combined or functional enriched libraries were clustered on microbeads and incubated with target cells for 2 hours at 37°C, followed by incubation with caspase-3 / 7 probe. The percentage of cells bound to microbeads and caspase-3 / 7 positive cells was gated and measured by flow cytometry. The functional enriched library showed increased caspase-3 / 7 activity. Comparing round 7 of combined enriched library to round 7 of functional enriched library for AML1 target cells, incubation with functional enriched library showed a 1.5-fold increase in caspase-3 / 7 (Figure 9, Panel A). For HCT116 target cells, comparing round 7 of the binding enriched library with round 8 of the functional enriched library, incubation with the functional enriched library showed a two-fold increase in caspase-3 / 7 (Figure 9, panel B). EXAMPLES

[0226] Selection and functional validation of lead aptamer oligonucleotide candidates Following functional enrichment of aptamers involved in apoptosis of triple-negative breast cancer (TNBC) cells derived from patient-derived xenografts (PDXs) called TNBC9 (Example 8), lead molecule selection was performed. Based on sequence analysis (Figure 10, Panel A), the 10 most abundant sequences in the functionally enriched aptamer population were selected, synthesized, and folded. The sequences of the selected candidate aptamers are shown in Table 15.

[0227] [Table 15]

[0228] The target killing efficacy of the selected candidate aptamers was measured in TNBC9 cells. Specifically, the candidate aptamers were synthesized, folded, and directly verified to induce caspase-3 / 7 activation in TNBC9 cells. As shown in Figure 10, panel B, all of the selected candidate aptamers induced significant levels of apoptosis in these cells compared to vehicle alone or random oligonucleotides.

[0229] Among the selected aptamers, aptamer E8 was identified as the most effective in inducing apoptosis (Figure 10, panel B) and was selected for further analysis. Notably, the levels of direct target killing by E8 observed in vitro and ex vivo ranged from 20-40% in independent biological replicates, which is comparable to the levels observed with approved anti-cancer biologics. (See, e.g., Yamashita, M. et al. A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flow cytometry using cryopreserved human peripheral blood mononuclear cells. Sci. Rep. 6 , 19772 (2016); Romano, E. et al. Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients. Proc. Natl. Acad. Sci. USA 112, 6140-6145 (2015); Kumar, R. et al. PD-1 blockade restores impaired function of ex vivo expanded CD8 T cells and enhances apoptosis in mismatch repair deficient EpCAMPD-L1 cancer cells. Onco. Targets. Ther. 10 , 3453-3465 (2017), all of which are incorporated herein by reference.) E8 showed remarkable selectivity at the target cell level, killing TNBC9 cells but not MCF10A cells, which were used as negative targets in the in vitro evolution process (Figure 10, Panel C). E8 was not limited to TNBC9, and showed a significant effect on MDA-MB-231 cells as well (Figure 10, Panel D).In preparation for in vivo studies, these effects were re-examined using E8 modified with polyethylene glycol (PEG), a modification that increases the in vivo stability and half-life of oligonucleotides, and demonstrated that the effects were maintained with PEG (Figure 10, panel E). Furthermore, E8 retained functionality in mouse serum (Figure 10, panel F). EXAMPLES

[0230] Biodistribution and efficacy of lead aptamer candidates in animal models The in vivo biodistribution of aptamer E8 (described in Example 10) was measured using fluorescently labeled E8. E8 was labeled as previously described for aptamer in vivo imaging probes (Bouvier-Mueller, A. & Duconge, F. Application of aptamers for in vivo molecular imaging and theranostics. Adv. Drug Deliv. Rev. 134, 94-106 (2018); Kryza, D. et al. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas. PLoS One 11, e0149387 (2016); Theodorou, I. et al. In Vitro and In Vivo Imaging of Fluorescent Aptamers. Methods Mol. Biol. 1380, 135-150 (2016), all of which are incorporated herein by reference in their entirety). The aptamer molecules were modified at the 5' with Cy5.5 and at the 3' with polyethylene glycol (PEG), modifications that enhance the in vivo stability and extend the half-life of the oligonucleotides. Fluorescently labeled E8 was intravenously injected twice (6 and 60 mg / kg) into NOD / SCID mice with MDA-MB-231 tumors induced in the right hind limb. Fluorescence was measured in vivo at 0.1, 24, and 48 hours after injection. The E8 lead aptamer candidate was localized and significantly retained in the tumor at 24 and 48 hours after injection (Figure 11, panels A-C). Specifically, the retention level of E8 peaked at 1-3 hours after injection and then declined, but was still maintained up to 48 hours after injection.

[0231] Various concentrations of E8 aptamer candidates were incubated with red blood cells and with PBMCs from healthy donors separately to confirm that E8 administration is applicable in a clinical setting. No effects of blood aggregation or red blood cell hemolysis were measured. Only a slight release of specific cytokines was detected after cytokine antibody array assay on PBMCs.

[0232] To evaluate the efficacy of E8 on tumor burden, the PEGylated aptamer was injected every 2 days for 11 days at a dose of 100 mg / kg (equivalent to standard chemotherapy agents on a molar basis). During this 11-day period, tumor volume was significantly reduced in E8-treated animals compared to vehicle-treated animals, and tumors excised from E8-treated animals showed visible signs of tissue death (final volumes on day 11: 168 ± 39 and 301 ± 51 mm in E8-treated and vehicle-treated animals, respectively). 3 ) (Figure 11, Panel D). Tumors excised from E8-treated animals showed visible signs of tissue death (Figure 11, Panel E). Histochemical analysis of caspase-3 activity in tissue sections from tumors of vehicle- and E8-treated animals showed prominent staining in tumors from E8-treated animals (Figure 11, Panels FI). TUNEL analysis, measuring apoptotic DNA fragmentation, further demonstrated increased cell death in tissue sections from tumors of E8-treated animals. No significant changes in physical appearance or body weight were observed following injection of E8 compared to PBS controls. EXAMPLES

[0233] Validation of lead aptamer candidates in human ex vivo organ cultures (EVOCs) The efficacy of aptamer E8 (described in Example 10) was evaluated in human ex vivo organ cultures (EVOCs). EVOCs were freshly obtained from two representative TNBC patients and prepared by Curesponse. Tissue slices 250 μm wide were plated in 24-well plates in culture medium under high oxygen conditions. Primary tumor cancer cells were allowed to survive for up to 14 days.

[0234] E8 aptamer and other chemotherapeutic agents (palbociclib, everolimus, fulvestrant) were administered to EVOCs at concentrations ranging from 20 to 50 μM. After the first day, the samples were replaced with a new medium and given a second dose of the therapeutic agents at the same concentrations. After 5 days, samples were fixed in 4% w / v paraformaldehyde, and tissue sections were prepared and stained with hematoxylin and eosin. Efficacy was graded on a 0-4 scale by two blinded pathologists. Pathological evaluation showed that E8 candidate had a significant effect on tumor cells (grade 3-4 on a 0-4 scale) in EVOC samples obtained from two patients who had developed resistance to at least one chemotherapeutic agent (Figure 12).

[0235] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the case of conflict, the present application, including definitions herein, will control.

[0236] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. The present disclosure includes the following embodiments. [1] A method for generating a functionally enriched population of aptamers, the method comprising: (a) contacting a target cell with a plurality of particles having a library of aptamer clusters immobilized on their surface ("aptamer cluster particles") and a reporter of cell function, where at least a portion of the immobilized aptamer clusters bind to at least a portion of the target cell to form a cell-aptamer cluster particle complex; (b) incubating the cell-aptamer-cluster particle complex for a period of time sufficient for at least a portion of the target cells in the cell-aptamer-cluster particle complex to be affected by a cellular function; (c) detecting the cell-aptamer-cluster particle complexes affected by the cellular function using a reporter of the cellular function; (d) separating the cell aptamer-cluster particle complexes that contain target cells affected by the cellular function detected in step (c) from other cell aptamer-cluster particle complexes; and (e) amplifying the aptamers in the separated cell-aptamer-cluster particle complexes to generate a functionally enriched population of aptamers; The method comprising: [2] The method of embodiment 1, wherein steps (c) and (d) are carried out using a flow cytometer. [3] The method of embodiment 1 or 2, further comprising separating the aptamer cluster particles from the target cells in the cell-aptamer cluster particle complexes separated in step (d). [4] The method of embodiment 3, further comprising a step of dissociating aptamers from the particles in the separated aptamer cluster particles. [5] The method of any one of embodiments 1 to 5, further comprising step (f): (i) forming aptamer cluster particles from the population of functionally enriched aptamers of step (e); and (ii) repeating steps (a) to (e) using the newly formed aptamer cluster particles to generate a further population of functionally enriched aptamers. [6] The method of embodiment 5, wherein step (f) is repeated at least twice. [7] The method of embodiment 5, wherein step (f) is repeated at least three times. [8] The method of embodiment 5, wherein step (f) is repeated at least four times. [9] The method of any one of embodiments 5 to 8, wherein step (f) further comprises applying limiting conditions in successive enrichment iterations.

[10] The method of embodiment 9, wherein the restrictive conditions are selected from (i) reducing the total number of particles, (ii) reducing the copy number of the aptamer per particle, (iii) reducing the total number of target cells, (iv) shortening the incubation period, and / or (v) introducing errors into the aptamer sequence by amplifying the population of aptamers using an error-prone polymerase.

[11] A method according to any one of embodiments 5 to 10, wherein the further enriched population of aptamers in step (f) exhibits a two-fold reduction in sequence diversity compared to the library of aptamer clusters in step (a).

[12] The method of any one of embodiments 5 to 10, wherein each round of step (f) enriches the aptamer population by at least 1.1-fold for aptamers that regulate cellular functions.

[13] The method of any one of embodiments 1 to 12, further comprising, after step (e), identifying the enriched population of aptamers by sequencing.

[14] The method of any one of embodiments 1 to 13, further comprising a step of preparing aptamer cluster particles prior to step (a).

[15] The steps of producing an aptamer cluster particle include: (1) immobilizing a large number of aptamers from an aptamer library on a particle surface; and (2) locally amplifying a large number of immobilized aptamers on the particle surface to form aptamer cluster particles; 15. The method of embodiment 14, comprising:

[16] The method of embodiment 15, wherein in step (2), the multiple immobilized aptamers are amplified using emulsion PCR.

[17] The method of any one of embodiments 1 to 16, wherein the aptamers in the aptamer cluster comprise an unexposed single-stranded nucleic acid sequence having a molecular cap.

[18] The method according to any one of embodiments 1 to 17, wherein the aptamer clusters immobilized on the aptamer cluster particles are prepared using emulsion PCR.

[19] The method of any one of embodiments 1 to 18, wherein at least 95% of the aptamer cluster particles individually contain multiple copies of 10 or fewer unique aptamer sequences.

[20] The method of any one of embodiments 1 to 18, wherein at least 95% of the aptamer cluster particles individually contain multiple copies of seven or fewer different aptamer sequences.

[21] The method of any one of embodiments 1 to 18, wherein at least 95% of the aptamer cluster particles individually contain multiple copies of no more than five different aptamer sequences.

[22] The method of any one of embodiments 1 to 18, wherein at least 95% of the aptamer cluster particles individually contain multiple copies of no more than three different aptamer sequences.

[23] The method of any one of embodiments 1 to 18, wherein at least 95% of the aptamer cluster particles individually contain multiple copies of no more than two different aptamer sequences.

[24] The method of any one of embodiments 1 to 18, wherein at least 95% of the aptamer cluster particles individually contain multiple copies of no more than one distinct aptamer sequence.

[25] The method of any one of embodiments 1 to 18, wherein the aptamer cluster particles individually contain multiple copies of no more than one distinct aptamer sequence.

[26] The method of any one of embodiments 1 to 25, wherein the aptamer cluster comprises at least 50 identical aptamers.

[27] The method of any one of embodiments 1 to 26, wherein the library of aptamer clusters comprises 100 to 10 14 different aptamer sequences.

[28] The method of any one of embodiments 1 to 27, wherein the library of aptamer clusters comprises at least 108 different aptamer sequences.

[29] The method of any one of embodiments 1 to 28, wherein the aptamer cluster comprises aptamers containing regions of conserved sequence and regions of randomized sequence.

[30] The method of embodiment 29, wherein the regions of randomized sequence are exposed and the regions of conserved sequence are capped.

[31] The method of any one of the preceding claims, wherein the particles are selected from polymer beads, agarose beads, polystyrene beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, controlled pore beads, microbeads, and nanoparticles.

[32] The method of any one of embodiments 1 to 31, wherein the particles have an average diameter in at least one dimension of from about 25 nm to about 30 μm.

[33] The method of any one of embodiments 1 to 31, wherein the particles have an average diameter in at least one dimension of about 25 nm, 50 nm, 100 nm, 250 nm, 0.5 μm, 2 μm, 5 μm, 10 μm, 20 μm, or 30 μm.

[34] The method of any one of embodiments 1 to 33, wherein the library of aptamer clusters comprises aptamers preselected via one or more processes selected from the group consisting of combined Cell SELEX, negative SELEX, and in vitro evolution.

[35] The method of any one of embodiments 1 to 33, further comprising enriching the initial library of aptamers for aptamers that bind to the target cells to generate a binding-enriched population of aptamers, and then using the aptamer-binding-enriched population to generate the aptamer cluster particles of step (a).

[36] The method of embodiment 35, wherein the initial library of aptamers is enriched by performing one or more rounds of combinatorial Cell SELEX.

[37] The steps of producing an aptamer cluster particle include: (1) immobilizing a number of aptamers from a binding-enriched population of aptamers onto a particle surface; and (2) A large number of immobilized aptamers are locally amplified on the particle surface to form aptamer cluster particles. 37. The method of embodiment 35 or 36, comprising:

[38] The method of any one of embodiments 1 to 37, wherein the aptamer cluster comprises aptamers containing chemically modified or non-natural nucleotides.

[39] The method of any one of embodiments 1 to 38, wherein the aptamer cluster comprises an aptamer that is DNA, RNA, or a chemically modified version thereof.

[40] The method of any one of embodiments 1 to 39, wherein the aptamer clusters are labeled with a fluorescent marker or an element to enable visualization under an optical microscope.

[41] The method of any one of embodiments 1 to 40, wherein the period is from about 10 minutes to about 5 days.

[42] The method of any one of embodiments 1 to 40, wherein the period is from about 1.5 hours to about 24 hours.

[43] The method of any one of embodiments 1 to 40, wherein the period is from about 1.5 hours to about 2 hours.

[44] The method of any one of embodiments 1 to 43, wherein the target cell is a prokaryotic cell.

[45] The method of embodiment 44, wherein the target cell is a bacterium.

[46] The method of any one of embodiments 1 to 43, wherein the target cell is a eukaryotic cell.

[47] The method of embodiment 46, wherein the eukaryotic cell is a mammalian cell.

[48] ​​The method of embodiment 47, wherein the mammalian cell is a cancer cell or an immune cell.

[49] The method of any one of embodiments 46 to 48, wherein the mammalian cells are cells derived from a patient.

[50] The method of embodiment 49, wherein the patient-derived cells are patient-derived cancer cells or patient-derived immune cells.

[51] The method of any one of embodiments 1 to 50, wherein the target cells are contacted with a reporter of cell function before, during, or after contacting the target cells with the aptamer cluster particles.

[52] The method of any one of embodiments 1 to 50, wherein the target cells are contacted with a reporter of cell function before, during, or after step (b).

[53] The method of any one of embodiments 1 to 52, wherein the reporter of cell function is a fluorescent dye.

[54] The method of embodiment 53, wherein the fluorescent dye is a calcium-sensitive dye, a cell tracking dye, a lipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolite-sensitive dye, a pH-sensitive dye, a membrane potential-sensitive dye, a mitochondrial membrane potential-sensitive dye, or an oxidation-reduction potential dye.

[55] The method of any one of embodiments 1 to 54, wherein the reporter of cell function is an activation-related marker, an oxidative stress reporter, an angiogenesis marker, an apoptosis marker, an autophagy marker, a cell survival marker, or a marker of ion concentration.

[56] The method of any one of embodiments 1 to 55, wherein the cell function is cell survival, apoptosis, cell proliferation, gene expression, cell morphology, cell activation, phosphorylation, calcium mobilization, degranulation, cell migration, or cell differentiation.

[57] The method of embodiment 56, wherein the target cell is a cancer cell.

[58] The method of embodiment 57, wherein the cellular function is apoptosis.

[59] The method of embodiment 57, wherein the cellular function is modulation of ligand expression of an immune checkpoint protein.

[60] The method of any one of embodiments 1 to 55, wherein the target cell is an immune cell.

[61] The method of embodiment 59, wherein the cellular function is modulation of expression of an immune protein.

[62] The method of embodiment 60, wherein the immunity protein is a cell surface immunity protein.

[63] The method of embodiment 62, wherein the cell surface protein is an immune checkpoint protein.

[64] The method of embodiment 61, wherein the immune protein is a cytokine.

[65] The method of embodiment 60, wherein the cell function is proliferation.

[66] The method of embodiment 60, wherein the cellular function is apoptosis.

[67] The method of any one of embodiments 1 to 66, wherein the cell-aptamer-cluster particle complex comprises about 2 to about 4 particles per target cell.

[68] The method of any one of embodiments 1 to 58, wherein the aptamer cluster particles contain an average of 1 to 6 aptamer clusters per particle.

[69] The method of any one of embodiments 1 to 68, wherein a plurality of cell-aptamer-cluster particle complexes are contained within a single reaction volume in step (b).

[70] The method of any one of embodiments 1 to 69, wherein in step (d), the cell-aptamer-cluster particle complex is separated via flow cytometry, fluorescence microscopy, optical tweezers, micropipette, microfluidic separation, micromanipulation, or separation seeding.

[71] The method of any one of embodiments 1 to 70, further comprising separating the aptamer cluster particles in the cell-aptamer cluster particle complex separated in step (d) by cell lysis and centrifugation.

[72] The method of any one of embodiments 1 to 71, wherein the aptamers in the separated cell aptamer cluster particles are isolated by HPLC purification prior to step (e).

[73] A functionally enriched population of aptamers produced by the method of any one of embodiments 1 to 72.

[74] A functionally enriched aptamer population as described in embodiment 73, wherein the aptamer population is characterized by greater than two-fold functional enrichment compared to the aptamers in the aptamer cluster library prior to enrichment.

[75] The functionally enriched aptamer population of embodiment 73 or 74, wherein the cellular function is promotion of cancer cell death or apoptosis.

[76] A functionally enriched aptamer population described in any one of embodiments 73 to 75, wherein the cellular function is promotion of an immune response.

[77] A method for selecting an aptamer for use in personalized cancer treatment, comprising selecting at least one aptamer candidate that promotes cell death or apoptosis of cancer cells derived from a patient from the functionally enriched aptamer population described in embodiment 75.

[78] The method of embodiment 77, further comprising sequencing the selected aptamers.

[79] The method of embodiment 78, further comprising synthesizing the sequenced aptamer.

[80] A method for creating a tumor delivery system, comprising selecting at least one candidate aptamer that promotes cancer cell death or apoptosis from the functionally enriched aptamer population described in embodiment 75, and combining the at least one aptamer with a tumor therapeutic agent for localized tumor delivery.

[81] A method for selecting an aptamer for use in personalized cancer therapy, comprising preparing a population of functionally enriched aptamers according to any one of embodiments 1 to 72, and selecting from the functionally enriched aptamer population at least one candidate aptamer that promotes cell death or apoptosis of cancer cells derived from a patient.

[82] The method of embodiment 81, further comprising sequencing the selected aptamers.

[83] The method of embodiment 82, further comprising synthesizing the sequenced aptamer.

[84] A method of treating cancer in a subject, comprising: (a) obtaining cancer cells from a subject; (b) preparing a functionally enriched aptamer population according to any one of embodiments 1 to 72 using the cancer cells as target cells; (c) selecting at least one aptamer from the functionally enriched population of aptamers that promotes cell death or apoptosis in cancer cells; and (d) administering the aptamer to a subject; The method comprising:

[85] A composition comprising a number of particles having a library of aptamer clusters immobilized thereon (“aptamer cluster particles”), target cells, and a reporter of cellular function.

[86] The composition of embodiment 85, wherein the reporter of cell function is a fluorescent reporter.

[87] The composition of embodiment 86, wherein the fluorescent reporter is a membrane integrity reporter.

[88] The composition of embodiment 86, wherein the fluorescent reporter is a capsid integrity reporter.

[89] The composition of embodiment 86, wherein the fluorescent reporter is a protein integrity reporter.

[90] The composition of embodiment 86, wherein the fluorescent reporter is a protein denaturation reporter.

[91] The composition of embodiment 86, wherein the fluorescent reporter is a cell death reporter.

[92] The composition of embodiment 86, wherein the fluorescent reporter is a redox potential reporter.

[93] The composition of any one of embodiments 85 to 92, wherein the composition comprises at least about 106 aptamer clusters.

[94] A composition described in any one of embodiments 85 to 92, wherein the composition comprises 106 to 109 aptamer clusters.

[95] A composition described in any one of embodiments 85 to 94, wherein each aptamer cluster contains at least about 104 copies of the aptamer.

[96] A composition described in any one of embodiments 85 to 94, wherein each aptamer cluster contains approximately 104 to 106 copies of the aptamer.

[97] A composition described in any one of embodiments 85 to 96, wherein the aptamer cluster is labeled with a fluorescent marker.

[98] The composition of any one of embodiments 85 to 96, wherein the aptamer cluster is labeled with an element to enable visualization under an optical microscope.

[99] The composition of embodiment 98, wherein the element is a nanoparticle.

[0100] A composition described in any one of embodiments 85 to 99, wherein the aptamer cluster is labeled with the antisense strand.

[0101] The composition described in embodiment 100, wherein the antisense strand is displaced or removed upon target binding.

[0102] A composition described in any one of embodiments 85 to 101, further comprising an enzyme.

[0103] The composition described in embodiment 102, wherein the enzyme is a ligase, polymerase, nuclease, editing enzyme, and / or restriction enzyme.

[0104] A composition described in any one of embodiments 85 to 103, wherein the target cell is a prokaryotic cell.

[0105] A composition described in embodiment 104, wherein the target cell is a bacterium.

[0106] A composition described in any one of embodiments 85 to 103, wherein the target cell is a eukaryotic cell.

[0107] The composition described in embodiment 106, wherein the eukaryotic cell is a mammalian cell.

[0108] The composition described in embodiment 107, wherein the mammalian cell is a cancer cell or an immune cell.

[0109] The composition described in embodiment 107 or 108, wherein the mammalian cells are cells derived from a patient.

[0110] The composition described in embodiment 109, wherein the patient-derived cells are cancer cells or immune cells derived from the patient.

Claims

1. 1. A method for generating a functionally enriched population of aptamers, the method comprising: (a) contacting a target cell with a plurality of particles having a library of aptamer clusters immobilized on their surface ("aptamer cluster particles") and a reporter of cell function, where at least a portion of the immobilized aptamer clusters bind to at least a portion of the target cell to form a cell-aptamer cluster particle complex; (b) incubating the cell-aptamer-cluster particle complex for a period of time sufficient for at least a portion of the target cells in the cell-aptamer-cluster particle complex to be affected by a cellular function; (c) detecting a cellular aptamer-cluster particle complex affected by a cellular function by detecting a reporter of the cellular function present in the cellular aptamer-cluster particle complex; (d) separating the cell aptamer-cluster particle complexes that contain target cells affected by the cellular function detected in step (c) from other cell aptamer-cluster particle complexes; and (e) amplifying the aptamers in the separated cell-aptamer-cluster particle complexes to generate a functionally enriched population of aptamers; The method comprising:

2. 2. The method of claim 1, wherein steps (c) and (d) are carried out using a flow cytometer.

3. The method of claim 1 or 2, further comprising step (f): (i) forming aptamer cluster particles from the population of functionally enriched aptamers of step (e); and (ii) repeating steps (a) to (e) using the newly formed aptamer cluster particles to generate a further population of functionally enriched aptamers.

4. 4. The method of claim 3, wherein step (f) further comprises applying limiting conditions in successive enrichment iterations.

5. The method of claim 4, wherein the restrictive conditions are selected from (i) reducing the total number of particles, (ii) reducing the copy number of the aptamer per particle, (iii) reducing the total number of target cells, (iv) shortening the incubation period, and / or (v) introducing errors into the aptamer sequence by amplifying the population of aptamers using an error-prone polymerase.

6. The method of any one of claims 1 to 5, further comprising, after step (e), identifying the enriched population of aptamers by sequencing.

7. The method of any one of claims 1 to 6, further comprising the step of producing aptamer cluster particles prior to step (a).

8. The steps of producing aptamer cluster particles include: (1) immobilizing a large number of aptamers from an aptamer library on a particle surface; and (2) locally amplifying a large number of immobilized aptamers on the particle surface to form aptamer cluster particles; The method of claim 7, comprising:

9. The method of claim 8, wherein in step (2), the multiple immobilized aptamers are amplified using emulsion PCR.

10. The method of any one of claims 1 to 9, wherein the aptamer cluster comprises aptamers that contain regions of conserved sequence and regions of randomized sequence.

11. The method of claim 10, wherein the regions of randomized sequence are exposed and the regions of conserved sequence are capped.

12. 12. The method of any one of claims 1 to 11, wherein the particles are selected from polymer beads, agarose beads, polystyrene beads, acrylamide beads, solid core beads, porous beads, paramagnetic beads, glass beads, controlled pore beads, microbeads, and nanoparticles.

13. The method of any one of claims 1 to 12, wherein the library of aptamer clusters comprises aptamers preselected via one or more processes selected from the group consisting of combined Cell SELEX, negative SELEX, and in vitro evolution.

14. The method according to any one of claims 1 to 13, wherein the target cell is a prokaryotic cell.

15. The method according to any one of claims 1 to 13, wherein the target cell is a eukaryotic cell.

16. The method of claim 15, wherein the eukaryotic cell is a mammalian cell.

17. A method according to any one of claims 1 to 16, wherein the target cells are contacted with a reporter of cell function before, during or after contacting the target cells with the aptamer cluster particles.

18. The method of any one of claims 1 to 17, wherein the reporter of cell function is a fluorescent dye.

19. 20. The method of claim 18, wherein the fluorescent dye is a calcium-sensitive dye, a cell tracking dye, a lipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolite-sensitive dye, a pH-sensitive dye, a membrane potential-sensitive dye, a mitochondrial membrane potential-sensitive dye, or an oxidation-reduction potential dye.

20. The method of any one of claims 1 to 19, wherein the reporter of cell function is an activation-associated marker, an oxidative stress reporter, an angiogenesis marker, an apoptosis marker, an autophagy marker, a cell survival marker, or a marker of ion concentration.

21. The method of any one of claims 1 to 20, wherein the cell function is cell survival, apoptosis, cell proliferation, gene expression, cell morphology, cell activation, phosphorylation, calcium mobilization, degranulation, cell migration, or cell differentiation.

22. 1. A method for selecting an aptamer for use in personalized cancer therapy, comprising: The following steps (a) to (e): (a) contacting a target cell with a plurality of particles having a library of aptamer clusters immobilized on their surface ("aptamer cluster particles") and a reporter of cell function, where at least a portion of the immobilized aptamer clusters bind to at least a portion of the target cell to form a cell-aptamer cluster particle complex; (b) incubating the cell-aptamer-cluster particle complex for a period of time sufficient for at least a portion of the target cells in the cell-aptamer-cluster particle complex to be affected by a cellular function; (c) detecting a cellular aptamer-cluster particle complex affected by a cellular function by detecting a reporter of the cellular function present in the cellular aptamer-cluster particle complex; (d) separating the cell aptamer-cluster particle complexes that contain target cells affected by the cellular function detected in step (c) from other cell aptamer-cluster particle complexes; and (e) amplifying the aptamers in the separated cell-aptamer-cluster particle complexes to generate a functionally enriched population of aptamers; wherein the cellular function is promoting cancer cell death or apoptosis; A method for selecting an aptamer for use in the personalized cancer treatment, comprising selecting at least one candidate aptamer that promotes cell death or apoptosis of cancer cells derived from a patient from the functionally enriched aptamer population generated.

23. A composition comprising a multiplicity of particles having immobilized a library of aptamer clusters ("aptamer cluster particles"), target cells, and a reporter of cell function, The composition, wherein the reporter of cellular function is a reporter of cellular function present in a cellular aptamer cluster particle complex formed when a target cell is contacted with the aptamer cluster particle and the reporter of cellular function, and at least a portion of the immobilized aptamer cluster binds to at least a portion of the target cell.

24. 24. The composition of claim 23, wherein the reporter of cell function is a fluorescent reporter.

25. The composition has at least about 10 6 25. The composition of claim 23 or 24, comprising aptamer clusters.

26. Each aptamer cluster has at least about 10 4 A composition according to any one of claims 23 to 25, comprising an aptamer copy.

27. The composition according to any one of claims 23 to 26, wherein the target cell is a prokaryotic cell.

28. The composition according to any one of claims 23 to 26, wherein the target cell is a eukaryotic cell.

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