WO2025059212A1

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Info

Publication number: WO2025059212A1
Application number: PCT/US2024/046251
Authority: WO
Inventors: James L. BANAL; Paul BLAINEY
Original assignee: Cache Dna Inc
Current assignee: Cache Dna Inc
Priority date: 2023-09-12
Filing date: 2024-09-11
Publication date: 2025-03-20
Anticipated expiration: 2026-03-12

Abstract

The present disclosure relates in some aspects to compositions and methods for signal generation. A composition for signal generation may comprise (a) a particle comprising a barcode that identifies said particle; and (b) a signal-generation scaffold associated with said barcode; wherein said barcode is present on said particle at a density of less than or equal to 1,000,000, and wherein said signal-generation scaffold comprises a circular nucleic acid construct.

Description

MULTIPLEXED SIGNAL AMPLIFICATION FOR SELECTION OF BARCODED MICROPARTICLES

CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Patent Application 63/582,167, filed September 12, 2023, which is incorporated by reference herein in its entirety for all purposes.

BACKGROUND

[0002] Barcoded particles may be used for a variety of applications. A barcoded particle may comprise a biological material (e.g., a protein or a nucleic acid). Barcodes associated with particles may be detected to identify the particle or to identify contents within the particle. A barcode may be a nucleic acid barcode, and the nucleic acid barcode may be detected.

SUMMARY

[0005] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0006] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0008] FIG. 1 provides a flowchart for an example method for identifying a barcoded particle.

[0009] FIG. 2 shows an example workflow for using a composition to generate a signal.

[0010] FIG. 3 shows an example workflow for using a composition to retrieve a barcoded particle and generate a signal.

[0011] FIGs. 4A-4B provide schematics of exemplary padlock probes used in making circular nucleic acid constructs in signal-generation scaffolds.

[0012] FIGs. 5A-5C provide schematics of compositions comprising exemplary signalgeneration scaffolds. [0013] FIGs. 6A-6B provide schematics of compositions comprising exemplary signalgeneration scaffold precursors.

[0014] FIG. 7 shows an example workflow for generating a branched signal-generation scaffold.

[0015] FIG. 8 shows an example workflow for generating a branched signal-generation scaffold.

[0016] FIG. 9 shows an example workflow for utilizing a signal-generation scaffold to identify contents inside a microparticle.

[0017] FIG. 10A shows example flow cytometry results from sorting microparticles based on signal generated using a method described herein. FIG. 10B shows example short-read sequencing data of internal barcodes extracted from sorted microparticles from FIG. 10A.

[0018] FIG. 11 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

DETAILED DESCRIPTION

[0019] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0020] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "at least," "greater than" or "greater than or equal to" applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0021] Whenever the term "no more than," "less than," or "less than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "no more than," "less than," or "less than or equal to" applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0022] Recognized herein is a need for improved methods for identifying and retrieving barcoded particles. Provided herein are compositions and methods that address such and other needs. Barcoded particles can be used in a host of applications in areas such as room-temperature storage of sequence-controlled polymers used for multi-omics and information storage. In these applications, unique single-stranded DNA barcodes can be used to identify the contents within these barcoded particles and, optionally, to select and retrieve these barcoded for downstream storage or analysis. In such cases, it is important to have a distinguishable signal that can differentiate the barcoded particles from non-selected particles. This can be challenging to achieve when multiple barcodes are present in the solution and target probes exhibit low copy numbers relative to the probes. Non-specific binding exacerbates the challenge of discerning selected and unselected particles, particularly for selecting low-abundance barcodes.

[0023] In some applications, barcoded particles comprise highly dense DNA barcodes that represent metadata information. A limitation associated with the use of highly dense DNA barcodes in these particles is the potential hindrance of hybridization with additional selection probes. This issue can be attributed to the steric hindrance and electrostatic repulsion that may occur when numerous DNA strands are in close proximity, thereby limiting the accessibility of selection probes to their target sequences. One way to address this limitation is reducing the barcode density per particle. Reducing the density of DNA barcodes on each particle can mitigate steric hindrance and electrostatic repulsion, thereby improving the hybridization efficiency of selection probes. However, this approach can come at the cost of signal: lowering the number of barcodes that can be detected can reduce the overall signal intensity, which can make the identification or selection process more challenging.

[0024] The present disclosure provides a way to improve the accuracy and efficiency of barcoded particle identification by lowering barcode density per particle while still generating high levels of signal. The present disclosure provides compositions and methods that can produce a robust signal from a low-abundance DNA barcode on a barcoded particle by utilizing a signal-generation scaffold associated with the barcode. In some cases, the compositions and methods described herein are further compatible with high levels of multiplexing, enabling multiple different types of signal to be generated or enabling different ways to identify or retrieve selected particles.

I. COMPOSITIONS

[0025] In one aspect, the present disclosure provides a composition comprising (a) a particle comprising a barcode that identifies the particle. The particle can comprise a biological particle, for example, a nucleic acid, a protein, a peptide, or a cell. In some cases, the particle is synthetic. In other cases, the particle is biologically derived. The particle can comprise genomic DNA. In some cases, the particle comprises viral nucleic acid. In other cases, the particle comprises mammalian nucleic acid. In further cases, the particle comprises a synthetic sequence-defined polymer. The particle can comprise a genome, such as, for example, genomes of up to 1, 2, 3, 10, or 200 gigabases. In some cases, the particle comprises up to 1 pg, up to 3 pg, up to 5 pg, up to 10 pg, or up to 15 pg of nucleic acid. The particle can comprise a hydrodynamic diameter of from 1 to 20 micrometers, from 1 to 15 micrometers, or from 1 to 10 micrometers.

[0026] In some cases, the particle comprises a bead that encapsulates the nucleic acid, protein, or peptide. The bead can comprise a polymer network, for example, a crosslinked polymer network. In some cases, the bead comprises a gel or a colloid. In some cases, the particle comprises a silica particle. The silica particle can be non-porous. In some cases, the silica particle can comprise surface functional groups (e.g., hydroxy groups or amino groups).

[0028] In some cases, the barcode is present on the particle at a density of no more than 200,000, no more than 500,000, no more than 750,000, no more than 1,000,000, no more than 1,250,000, no more than 1,500,000, no more than 1,750,000, no more than 2,000,000, or no more than 2,500,000 barcodes per particle.

[0029] The composition can further comprise (b) a signal -generation scaffold associated with the barcode. The signal-generation scaffold can comprise a nucleic acid molecule. In some cases, the signal-generation scaffold comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten nucleic acid molecules.

[0031] In other cases, the signal-generation scaffold or a precursor thereof comprises a padlock probe, wherein the two ends are hybridized to a splint molecule. The padlock probe can further comprise a nucleic acid primer. In some cases, the padlock probe comprises chemical affinity pairs (e.g., alkyne-azide, dibenzocyclooctyne-azide, alkyne-thiol, alkene-thiol, or tetrazine-/ra//.s-cyclooctene) at the two ends such that it is configured to generate a circular nucleic acid upon ligation. In some cases, the 5’ and 3’ ends comprise crosslinking molecules that form covalent linkages using heat or light.

[0032] In some cases, a nucleic acid molecule in the signal-generation scaffold comprises a modified sugar, backbone, or nucleobase. For example, the nucleic acid can be a xenonucleic acid. In some cases, a nucleic acid primer that is hybridized to the circular nucleic acid construct comprises a modified nucleotide. Non-limiting examples of modified nucleotides include a locked nucleic acid (LNA), a hexitol nucleic acid (HNA), a cyclohexene nucleic acid (CeNA), morpholino phosphoramidates, a threose nucleic acid (TNA), a glycol nucleic acid (GNA), a 2’- fluoroarabinonucleic acid (FANA), an arabinonucleic acid (ANA), a (l'-3')-P-L-ribulo nucleic acid (riboNA), 3 '-2' phosphonomethyl-threosyl nucleic acid (tPhoNA), altritol nucleic acids (AltNA), or a 2'-deoxyxylonucleic acid (dXNA). In some cases, the nucleic acid primer comprises a modified backbone, for example, a phosphorothiate, a phosphonate, triazole, or a boranophosphate. In some cases, the nucleic acid primer is a chimera comprising a combination of a modified nucleotide and a modified backbone.

[0035] In some cases, a nucleic acid adapter molecule is situated between the circular nucleic acid construct and the barcode. An exemplary composition is shown in in FIG. 5B. The nucleic acid adapter molecule can comprise a linear nucleic acid adapter or a circular nucleic acid adapter. In some cases, the adapter comprises an L-probe or a padlock probe. The nucleic acid adapter can comprise a first adapter region and a second adapter region, wherein the first adapter region is hybridized to an adapter-binding region of the circular nucleic acid construct, while the second adapter region is coupled to the barcode. The second adapter region can be directly coupled to the barcode. For example, the second adapter region can be directly coupled to the barcode via hybridization. Alternatively, the second adapter region can be indirectly coupled to the barcode, wherein one or more other adapters are situated between the second adapter region and the barcode. For example, the second adapter region can be hybridized to an intermediate adapter nucleic acid molecule, wherein the intermediate adapter nucleic acid molecule is coupled to the barcode.

[0038] In some embodiments, the signal-generation scaffold is a branched signal-generation scaffold comprising multiple circular nucleic acid constructs. A branched signal-generation scaffold can comprise multiple branching levels of adapter nucleic acid molecules. An exemplary composition is illustrated in FIG. 7, wherein the branched signal-generation scaffold 704 comprises a first level of branching adapters 771 and 772 that branch from nucleic acid adapter 781. The branched signal-generation scaffold 704 further comprises a second level of branching adapters (e.g., 761, 762, 763, or 764) that are each hybridized to an adapter in the first level of branching adapters. Another exemplary composition is illustrated in FIG. 8, wherein the branched signal-generation scaffold 804 comprises a first level of branching adapters 841 and 842, each of which is hybridized to a second level branching adapter. A branched signalgeneration scaffold can comprise at least 2, 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 branching levels of adapter nucleic acid molecules. In some cases, a branching level of adapter nucleic acid molecules comprises at least 2, 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 adapter nucleic acid molecules. In some cases, an adapter nucleic acid molecule in a branching level comprises at least 2, 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 adapter regions that are each hybridized to a separate nucleic acid molecule.

[0040] Circular nucleic constructs in the signal-generation scaffold can be hybridized to separate adapter regions of a plurality of adapter regions in the signal-generation scaffold. In some cases, the plurality of adapter regions comprise the same adapter sequence. In other cases, the plurality of adapter regions comprise different adapter sequences.

[0041] Multiple circular nucleic acid constructs in the signal-generation scaffold can be hybridized to a same adapter nucleic acid molecule. Alternatively, multiple circular nucleic acid constructs in the signal-generation scaffold can be hybridized to separate adapter nucleic acid molecules. For example, in FIG. 7, circular nucleic acid constructs 761 and 762 are hybridized to a same adapter nucleic acid molecule 771, while circular nucleic acid construct 763 is hybridized to a different adapter nucleic acid molecule 772.

[0042] In some embodiments, the composition further comprises a concatemer comprising a signal sequence of a circular nucleic acid construct in the signal-generation scaffold. In some cases, the concatemer comprises a complement of the signal sequence of a circular nucleic acid construct in the signal-generation scaffold. The concatemer can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 copies of the signal sequence or complement thereof.

[0043] The composition can further comprise a probe hybridized to a sequence of the concatemer. In some cases, the probe is hybridized to the signal sequence or complement thereof in the concatemer. The hybridized probe can comprise a detectable moiety, such as an optically- active molecule (e.g., a fluorescent molecule) or a radiolabel. In some cases, the hybridized probe comprises a magnetically-active molecule (e.g., an iron oxide nanoparticle) or other affinity molecule (e.g., biotin, streptavidin, avidin, neutravidin, hexahistidine, or nickel chelate). In other cases, the hybridized probe comprises an orthogonal ssDNA barcode. In some cases, the hybridized probe comprises a peptide nucleic acid. In some cases, the hybridized probe comprises an alkyne, azide, dibenzocyclooctyne, an alkyne, a thiol, or an alkene.

[0044] In some embodiments, the composition further comprises a plurality of probes hybridized to sequences of the concatemer. The plurality of probes can comprise optically active molecules. The plurality of probes can comprise fluorescent molecules that emit at the same wavelength. Alternatively, the plurality of probes comprises fluorescent molecules that emit at the different wavelengths. In some cases, the plurality of probes comprises magnetically-active molecules, affinity molecules (e.g., biotin, streptavidin, avidin, neutravidin, hexahistidine, or nickel chelate), orthogonal ssDNA barcodes, or a combination thereof. In some cases, the plurality of probes comprises an optically active molecule and a magnetically-active molecule. In some cases, the plurality of probes comprises an optically active molecule and an affinity molecules (e.g., biotin, streptavidin, avidin, neutravidin, hexahistidine, or nickel chelate).

[0045] In some embodiments, the composition further comprises a probe hybridized to a sequence of a circular nucleic acid construct in the signal -generation scaffold, wherein the probe comprises a detectable moiety, a magnetically-active molecule or an affinity molecule. For example, a detectable moiety can comprise an optically-active molecule or a radiolabel. In some cases, a plurality of probes, each comprising a detectable moiety, a magnetically-active molecule or an affinity molecule is hybridized to circular nucleic acid constructs in the signal-generation scaffold.

II. METHODS

[0046] In some cases, the present disclosure provides methods of using a composition described elsewhere herein to generate signal. The methods described herein can be compatible with the identification and retrieval of barcoded particles and can improve the accuracy and efficiency of barcoded particle identification by lowering barcode density per particle while still generating high levels of signal.

[0047] In one aspect, the present disclosure provides a method, comprising (a) providing a composition comprising (i) a particle comprising a barcode (e.g., a single-stranded nucleic acid barcode) that identifies the particle. In some cases, the barcode is present on the particle at a density of no more than 200,000, no more than 500,000, no more than 750,000, no more than 1,000,000, no more than 1,250,000, no more than 1,500,000, no more than 1,750,000, no more than 2,000,000, no more than 2,500,000 barcodes per particle, no more than 5,000,000 barcodes per particle, or no more than 10,000,000 barcodes per particle. As described elsewhere herein, the particle can comprise a biological particle, for example, a nucleic acid, a protein, or a peptide. In some cases, the method comprises encapsulating the biological particle in a bead, a gel, a polymer matrix, or a silica particle. The composition provided can further comprise (ii) a signal-generation scaffold associated with the barcode, as described elsewhere herein. For example, the signal-generation scaffold can comprise a circular nucleic acid construct comprising a signal sequence and optionally, one or more adapter nucleic molecules.

[0048] The method can further comprise coupling the barcode with the particle. The method can comprise covalently linking the barcode with the particle, for example by crosslinking to another component (e.g., a polymer) in the particle. In some cases, one end of the barcode is ligated to another nucleic acid in the particle). Alternatively, the method can comprise noncovalently associating the barcode with the particle, for example, via hybridization of the barcode to another nucleic acid in the particle or by electrostatic interaction with a charged moiety. As another example, the method can comprise associating the barcode with a polypeptide domain on the surface of the particle via sequence or secondary structure recognition (e.g., coupling an aptamer with an aptamer-binding domain).

[0049] In some cases, the method further comprises, prior to (a), generating the signalgeneration scaffold. This can comprise hybridizing a plurality of nucleic acid molecules to generate a signal-generation scaffold precursor. In some cases, the method comprises associating the circular nucleic acid construct with the barcode. This can comprise directly associating the circular nucleic acid construct with the barcode. For example, a region of the circular nucleic acid construct can be hybridized to a region of the barcode. As another example, the adapter nucleic acid molecule can be covalently linked to the barcode. Alternatively, associating the circular nucleic acid construct with the barcode can comprise indirectly associating the adapter nucleic acid molecule with the barcode. For example, a region of the circular nucleic acid construct can be hybridized to a first region of an adapter nucleic acid molecule, and a second region of the adapter nucleic acid molecule can be further associated to the barcode.

[0051] In some cases, generating the signal-generation scaffold comprises associating a plurality of circular nucleic acid constructs with the barcode. In some cases, the method comprises generating a branched signal-generation scaffold, as described elsewhere herein. Generating a branched signal -generation scaffold can comprise hybridizing multiple different adapter nucleic acid molecules together to generate at least 2, 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 branching levels of adapter nucleic acid molecules. For example, the method can comprise hybridizing at least 2 first-level branching adapters to a base level adapter. The method can further comprise hybridizing one or more second-level branching adapters to each of the first-level branching adapters. In some cases, the method can further comprise hybridizing a third-level branching adapter to one of the second- level branching adapters. In some cases, a branching adapter comprises a padlock probe. Hybridizing a branching adapter to an additional adapter can comprise hybridizing the two ends of the padlock probe to the additional adapter. In some cases, generating the branched signalgeneration scaffold comprises circularizing one or more adapter padlock probes by linking the two ends of a padlock probe together. This can result in a branched signal-generation scaffold comprising one or more circular adapter nucleic acid molecules that associate the circular nucleic acid construct with the barcode.

[0052] In some embodiments, the method further comprises, prior to (a), generating the circular nucleic acid construct. For example, the method can comprise hybridizing a splint nucleic acid molecule to the padlock probe, wherein, upon hybridizing, the padlock probe comprises (i) a first end hybridized to a first region of the splint nucleic acid molecule, and (ii) a second end hybridized to a second region of the splint nucleic acid molecule. The first end and the second end of the padlock probe can be linked together to generate the circular nucleic acid construct. The two ends of the padlock probe can be linked together enzymatically using a ligase. Alternatively, the two ends of the padlock probe can be linked together using chemical affinity pairs (e.g., alkyne-azide, dibenzocyclooctyne-azide, alkyne-thiol, alkene-thiol, or tetrazine-/ra//.s cyclooctene) or a bifunctional crosslinking molecule attached to the two ends of the padlock probe. In some cases, a copper catalyst is used to link the two ends of the padlock probe. In other cases, heat or light is used to mediate crosslinking of the two ends of the padlock probe that are modified with crosslinking molecules that form covalent linkages using heat or light.

[0053] In some cases, prior to circularization, the method comprises associating the padlock probe with the barcode. For example, this can comprise hybridizing a region of the padlock probe to a region of the barcode. As another example, the method can comprise indirectly associating the padlock probe with the barcode via one or more nucleic acid adapters. For example, the padlock probe can be hybridized to a first region of an adapter nucleic acid molecule and a second region of the adapter nucleic acid molecule can be further associated to the barcode.

[0054] In some cases, generating the signal-generation scaffold can comprise hybridizing at least 1, at least 2, 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 padlock probes to different adapter nucleic acid molecules and associating the adapter nucleic acid molecules to the barcode. Alternatively, generating the signal-generation scaffold can comprise hybridizing at least 1, at least 2, 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 padlock probes to the same adapter nucleic acid molecule and associating the adapter nucleic acid molecule to the barcode.

[0055] In some cases, associating the one or more padlock probes with the barcode generates a signal-generation scaffold precursor, which upon, circularization of the one or more padlock probes, generates the signal -generation scaffold.

[0056] In some embodiments, the method further comprises (b) generating a signal using the signal-generation scaffold. This can comprise using the circular nucleic acid construct and a primer to generate a concatemer. The concatemer can be generated, for example, in a rolling circle amplification reaction. A polymerase can be used to generate the concatemer. In some cases, the polymerase is a strand-displacing polymerase. A polymerase can be a reverse transcriptase. A polymerase can be a QB replicase.

[0057] In some embodiments, the method comprises using a 3’ end of a barcode molecule comprising the barcode that is provided in (a) as a primer to generate the concatemer in (b). For example, in (a), the particle can comprise a barcode molecule comprising the barcode, and a 3’ end of the barcode molecule can be hybridized to a primer-binding region of the circular nucleic acid construct, as illustrated in FIG. 5A. In this example, generating the concatemer in (b) can comprise using the 3’ end of the barcode molecule as a nucleic acid primer to amplify the circular nucleic acid construct, thereby generating a concatemer comprising a sequence of the circular nucleic acid construct or complement thereof. [0058] In other embodiments, the method comprises using a primer in an adapter nucleic acid molecule to generate the concatemer in (b). In some cases, prior to (a), the method comprises hybridizing the adapter nucleic acid molecule comprising the primer to the barcode, providing a composition such as in FIG. 5B. In other cases, prior to (a), the method comprises hybridizing the adapter nucleic acid molecule comprising the primer to an intermediate adapter that is hybridized to the barcode, providing a composition such as in FIG. 5C. In either case, the primer in the adapter nucleic acid molecule can be hybridized to the circular nucleic acid construct. The method can further comprise using the primer to amplify the circular nucleic acid construct, thereby generating the concatemer, as exemplified in FIG. 2. In some cases, the primer in the adapter nucleic acid molecule is first hybridized to a precursor of the circular nucleic acid construct (e.g., a padlock probe) prior to generating the circular nucleic acid construct.

[0059] In some embodiments, the circular nucleic acid construct comprises a signal sequence; and (b) comprises generating the signal by amplifying the circular nucleic acid construct in an amplification reaction to generate a concatemer comprising the signal sequence or complement thereof. In some cases, the concatemer generated in (b) comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 copies of the signal sequence or complement thereof. Generating the signal in (b) can further comprise hybridizing a probe to the signal sequence or complement thereof in the concatemer. In some cases, the concatemer is dissociated from the particle via dehybridization from the barcode or an adapter nucleic acid prior to probe hybridization. In other cases, the concatemer is dissociated from the particle via dehybridization from the barcode or an adapter nucleic acid following probe hybridization. In some cases, the concatemer remains associated with the particle during signal generation.

[0061] In some embodiments, the signal-generation scaffold provided in (a) comprises a plurality of circular nucleic acid constructs. The method can comprise, in (b), generating the signal from circular nucleic acid constructs of the plurality of circular nucleic acid constructs. Generating the signal can comprise generating a plurality of concatemers. In some cases, the method comprises generating at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten concatemers. The plurality of concatemers can be generated from different circular nucleic acid constructs. Alternatively, the plurality of concatemers can be generated from the same circular nucleic acid construct.

[0062] In some cases, the method comprises generating concatemers of the plurality of concatemers simultaneously. For example, a plurality of primers can each be hybridized to a circular nucleic acid construct of the circular nucleic acid constructs and subsequently contacted with a polymerase and dNTPs, enabling the generation of concatemers from the primers simultaneously.

[0063] In other cases, the method comprises generating concatemers of the plurality of concatemers in sequentially. For example, the method can comprise hybridizing a first set of primers to circular nucleic acid constructs or precursors thereof and generating a first set of concatemers from the first set of primers. Following the generation of the first set of concatemers, the method can further comprise hybridizing a second set of primers to circular nucleic acid constructs or precursors thereof and subsequently, generating a second set of concatemers from the second set of primers.

[0064] In some embodiments, the particle provided in (a) is among a plurality of particles. In some cases, the method further comprises (d) identifying the particle from other particles of the plurality of particles using the barcode identified in (c). This can be done, for example, by detecting a fluorescent signal from the particle and sorting the particle based on the signal using flow cytometry. In some cases, identifying the particle comprises detecting two or more fluorescent signals of different emission wavelengths and sorting the particle based on the two or more fluorescent signals. This can be done when probes comprising two or more fluorescent labels capable of producing different emission wavelengths are hybridized to the concatemer associated with the particle. As another example, identifying the particle can comprise retrieving the particle from other particles of the plurality of particles using a probe comprising a magnetically-active molecule that is hybridized to the concatemer associated with the particle. As another example, identifying the particle can comprise retrieving the particle from other particles of the plurality of particles using a probe comprising a hexahistidine using a nickel column. In some cases, a magnetically-active molecule or an affinity molecule may be dissociated with the concatemer following particle retrieval. This can be done, for example, by dehybridzing the probe comprising magnetically-active molecule or an affinity molecule from the concatemer. As another example, the magnetically-active molecule or an affinity molecule may be attached to the probe via a cleavable linker (e.g., a peptide linker), and can be released from the probe following particle retrieval. In other cases, the barcode is disassociated from the particle after particle retrieval. For example, the barcoded particle can be placed in conditions that allow the barcode to be dehybridized from a nucleic acid in the particle or de-coupled from a protein on the surface of the particle. In some cases, the barcode remains associated with particle during retrieval and signal detection.

[0065] The method can further comprise (e) extracting the contents (e.g., a nucleic acid, a protein, or a peptide) from the particle. In some cases, extracting the contents comprises subjecting the particle to conditions that modifies the particle and releases the contents. This can comprise contacting the particle with a chemical agent that modifies the particle to release the contents. For example, this can be done by contacting a gel bead with an agent that permeabilizes the gel bead. As another example, a buffered oxide etch can be used to dissolve silica particle and release the encapsulated contents. In some cases, contacting the particle with the chemical agent does not degrade or affect the integrity of the contents. In some cases, the particle comprises a cell and (e) extracting the contents from the particle further comprises lysing the cell and extracting a biological particle from the cell. In some cases, the particle comprises a biological particle that is attached to the particle via a cleavable linker (e.g., a disulfide linker) and extracting the biological particle from the particle comprises cleaving the linker to release the biological particle.

[0066] The method can further comprise (f) analyzing the contents. The contents can be analyzed by sequencing a nucleic acid in the contents or analyzing a nucleic acid or protein by gel electrophoresis. In some cases, a nucleic acid or protein in the contents can be analyzed by associating a probe to the nucleic acid or protein and detecting the probe.

[0067] The method can further comprise storing the particle prior to extracting the contents. In some cases, the particle is stored at room temperature. In some cases, the particle is stored at from 15 to 37 degrees Celsius, from 15 to 30 degrees Celsius, or from 20 to 30 degrees Celsius. In some cases, storing encapsulated contents in the particle helps protect the integrity of the contents or helps prevent the contents from degradation compared to unencapsulated controls.

III. COMPUTER SYSTEMS

[0068] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 11 shows a computer system 1101 that is programmed or otherwise configured to prepare a composition comprising a signal-generation scaffold described elsewhere herein or to implement a method for generating a signal using a signal-generation scaffold described elsewhere herein. The computer system 1101 can regulate various aspects of preparing a composition or implementing a method of the present disclosure, such as, for example, designing nucleic acid sequences (e.g., adapter nucleic acid sequences or padlock probes) for preparing a signal -generation scaffold or a signal-generation scaffold precursor, as described elsewhere herein. The computer system 1101 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0069] The computer system 1101 includes a central processing unit (CPU, also "processor" and "computer processor" herein) 1105, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1101 also includes memory or memory location 1110 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1115 (e.g., hard disk), communication interface 1120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1125, such as cache, other memory, data storage and/or electronic display adapters. The memory 1110, storage unit 1115, interface 1120 and peripheral devices 1125 are in communication with the CPU 1105 through a communication bus (solid lines), such as a motherboard. The storage unit 1115 can be a data storage unit (or data repository) for storing data. The computer system 1101 can be operatively coupled to a computer network ("network") 1130 with the aid of the communication interface 1120. The network 1130 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1130 in some cases is a telecommunication and/or data network. The network 1130 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1130, in some cases with the aid of the computer system 1101, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1101 to behave as a client or a server. [0070] The CPU 1105 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1110. The instructions can be directed to the CPU 1105, which can subsequently program or otherwise configure the CPU 1105 to implement methods of the present disclosure. Examples of operations performed by the CPU 1105 can include fetch, decode, execute, and writeback.

[0071] The CPU 1105 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1101 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0072] The storage unit 1115 can store files, such as drivers, libraries and saved programs. The storage unit 1115 can store user data, e.g., user preferences and user programs. The computer system 1101 in some cases can include one or more additional data storage units that are external to the computer system 1101, such as located on a remote server that is in communication with the computer system 1101 through an intranet or the Internet.

[0073] The computer system 1101 can communicate with one or more remote computer systems through the network 1130. For instance, the computer system 1101 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1101 via the network 1130.

[0074] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1101, such as, for example, on the memory 1110 or electronic storage unit 1115. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1105. In some cases, the code can be retrieved from the storage unit 1115 and stored on the memory 1110 for ready access by the processor 1105. In some situations, the electronic storage unit 1115 can be precluded, and machine-executable instructions are stored on memory 1110.

[0075] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion. [0076] Aspects of the systems and methods provided herein, such as the computer system 1101, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0077] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0078] The computer system 1101 can include or be in communication with an electronic display 1135 that comprises a user interface (UI) 1140 for providing, for example, a design of a signal-generation scaffold or a signal-generation scaffold precursor described elsewhere herein. Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0079] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1105. The algorithm can, for example, perform sequencing or identify a barcode or particle based on a detected signal.

[0080] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

IV. EXAMPLES

[0081] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1: Signal generation using a signal-generating scaffold

[0082] FIG. 1 and FIG. 2 show examples of methods for signal generation to identify a barcoded particle, according to some embodiments. FIG. 1 shows a flowchart representing a method for generating a signal using a signal-generation scaffold associated with a barcode molecule on a particle to generate a signal to identify the barcode molecule and the particle. The method comprises providing composition comprising (i) a particle comprising a barcode molecule that identifies the particle; and (ii) a signal-generation scaffold associated with the barcode molecule, wherein the barcode molecule is present on the particle at a density of less than or equal to 1,000,000, and wherein the signal-generation scaffold comprises a circular nucleic acid construct. The method further comprises generating a signal using the signalgeneration scaffold. Finally, the method comprises detecting the signal, thereby identifying the barcode molecule and the particle.

[0084] The composition 261 is then contacted with a ligase to generate the composition 152. The ligase ligates the first end and the second end of the padlock probe together to generate a circular nucleic acid construct 212 comprising the signal sequence 221. This operation generates the composition 262, which comprises (i) the particle 201 comprising the barcode molecule 202, and (ii) a signal-generation scaffold 203 associated with the barcode molecule 202, wherein the barcode molecule is present on the particle at a density of less than or equal to 1,000,000. The signal-generation scaffold 203 comprises the circular nucleic acid construct 212 and the adapter nucleic acid molecule 231. The first region of the adapter nucleic acid molecule 231 is still hybridized to the barcode molecule 202 and the primer 232 is hybridized to the circular nucleic acid construct 212.

[0085] Next, the composition 262 is contacted with a strand-displacing polymerase and dNTPs (at a concentration of 1-500 pM) and used in a rolling circle amplification reaction to generate the composition 263. The primer 232 is amplified using the circular nucleic acid construct 212 as a template produce a single-stranded DNA concatemer 241, comprising multiple copies of the complementary sequence 222 of the signal sequence 221 as a rolling circle amplification product. This operation generates the composition 263, which comprises (i) the particle 201 comprising the barcode molecule 202, (the) a signal-generation scaffold 203 associated with the barcode molecule 202, and (iii) the concatemer.

[0086] Following the rolling circle amplification reaction, the composition 263 is contacted with a plurality of detectable probes 251 at a concentration of 1 nM to 1 mM, wherein multiple probes of the plurality of probes hybridize to the complementary sequences 222 in the concatemer 241. The detectable probes 251 are then detected by imaging, thereby detecting the barcode molecule 202 and the particle 201.

Example 2: Signal generation and selection of barcoded microparticle using a signal-generation scaffold

[0087] FIG. 3 shows an example workflow illustrating the method represented in FIG. 1, further comprising magnetic retrieval the barcoded microparticle. First, a composition comprising (i) a particle 301 comprising a barcode molecule 311 is provided, wherein the barcode molecule is present on the particle at a density of less than or equal to 1,000,000. The composition further comprises a (ii) signal-generation scaffold 312 associated with the barcode molecule 311. The signal -generation scaffold 212 comprises a circular nucleic acid construct 321 comprising a first signal sequence 331 and a second signal sequence 332. The composition further comprises an adapter nucleic acid molecule 341, wherein the adapter nucleic acid comprises a first region that is hybridized to the barcode molecule 311 and a primer 342 that is hybridized to the circular nucleic acid construct 321. The particle 301 is among a plurality of particles, including particle 302 and particle 303.

[0088] Next, the composition is contacted with a DNA polymerase and dNTPs and used in a rolling circle amplification reaction. The primer 342 is amplified using the circular nucleic acid construct 321 as a template produce a single-stranded DNA concatemer 351, comprising multiple copies of a first complementary sequence 333 of the first signal sequence 331 and multiple copies of a second complementary sequence 234 of the second signal sequence 332 as a rolling circle amplification product.

[0089] Following the rolling circle amplification reaction, the single-stranded DNA concatemer 351 is contacted with a plurality of fluorescently labeled probes 361, wherein a fluorescently labeled probe 361 of the plurality of fluorescently labeled probes hybridizes to the first complementary sequences 333 in the concatemer 351. The single-stranded DNA concatemer 351 is also contacted with a plurality of magnetically-active probes 362, wherein a magnetically active probe 362 of the plurality of magnetically-active probes hybridizes to the second complementary sequences 334 in the concatemer 351.

[0090] Following probe hybridization, the concatemer 351 that is hybridized to the magnetically active probes is magnetically retrieved along with the associated barcoded particle 301, thereby identifying the particle 301 from the other particles 302 and 303. The fluorescently labeled probes 361 are detected by imaging, thereby detecting the barcode molecule 301 and the particle 311.

Example 3: Exemplary padlock probes used in making signal-generation scaffolds

[0091] FIGs. 4A and 4B show examples of padlock probes used in making different signalgeneration scaffolds. FIG. 4A depicts a padlock probe comprising a primer-binding region 401, a first region 411 configured to hybridize to a first portion of a splint molecule, and a second region 412 configured to hybridize to a second portion of a splint molecule. The padlock probe further comprises five signal sequences 421, wherein each signal sequence 421 has the same sequence. The signal sequence 421 can be configured such that the signal sequence 421 or a complement thereof binds to a detectable probe. The padlock probe illustrated in FIG. 4A can be used in making a circular nucleic acid construct in a signal -generation scaffold described elsewhere herein. For example, the padlock probe can be hybridized to a splint molecule and the two ends can be ligated to produce a circular nucleic acid construct comprising the primer binding region 401 and five signal sequences 421. The resulting circular nucleic acid construct can be used to generate a concatemer comprising multiple copies of the five signal sequences 421 or complement thereof.

[0092] FIG. 4B depicts a padlock probe comprising a primer-binding region 401, a first region 411 configured to hybridize to a first portion of a splint molecule, and a second region 412 configured to hybridize to a second portion of a splint molecule. The padlock probe further comprises five signal sequence: 421, 422, 423, 423, and 425, wherein each of the five signal sequences comprises a different sequence from the others. Each of the five signal sequences can be configured such that the signal sequence or a complement thereof binds to a different probe (e.g., a fluorescent probe, a magnetically active probe). The padlock probe illustrated in FIG. 4B can be used in making a signal -generation scaffold described elsewhere herein. For example, the padlock probe can be hybridized to a splint molecule and the two ends can be ligated to produce a circular nucleic acid construct comprising the primer binding region 401 and the five signal sequences 421, 422, 423, 423, and 425. The resulting circular nucleic acid construct can be part of a signal-generation scaffold that is used to generate a concatemer comprising a multiple copies of the five signal sequences 421, 422, 423, 423, and 425 or complement thereof.

Example 4: Exemplary signal-generation scaffolds

[0093] FIGs. 5A-5C depict exemplary signal-generation scaffolds, according to some embodiments. FIG. 5A depicts an exemplary composition, according to some embodiments, comprising a (i) particle 501 comprising a barcode molecule 511 and (ii) a signal-generation scaffold 521 associated with the barcode molecule 511. The signal -generation scaffold 521 comprises a circular nucleic acid construct 531 comprising a signal sequence 541. In this exemplary composition, the circular nucleic acid construct 531 is directly coupled to the barcode molecule 511. The barcode molecule 511 comprises a primer 551 that is hybridized to a portion of the circular nucleic acid construct 531.

[0094] FIG. 5B depicts an exemplary composition, according to some embodiments, comprising a (i) particle 502 comprising a barcode molecule 512 and (ii) a signal-generation scaffold 522 associated with the barcode molecule 512. The signal-generation scaffold 522 comprises a circular nucleic acid construct 532 comprising a signal sequence 542. In this exemplary composition, the circular nucleic acid construct 532 is indirectly coupled to the barcode molecule 512. The signal-generation scaffold 522 comprises an adapter nucleic acid molecule 562 that couples the circular nucleic acid construct and the barcode molecule 512. The adapter nucleic acid molecule 562 comprises a primer 552 and a barcode molecule-binding region 562a. The primer 552 in the adapter nucleic acid molecule 561 is hybridized to a portion of the circular nucleic acid construct 532. The barcode molecule-binding region 562a is hybridized to a portion 512b of the barcode molecule 512.

Example 5: Exemplary signal-generation scaffold precursors

[0096] In FIG. 6A, the secondary adapter nucleic acid molecule 612 couples a plurality primary adapter nucleic acid molecules (including 621, 622, and 623) to the barcode molecule 602. The secondary adapter nucleic acid molecule 631 comprises a barcode binding region 631b that is hybridized to a portion of the barcode molecule 602. The secondary adapter nucleic acid molecule 631 further comprises a plurality of secondary adapter regions, each comprising a same sequence 631a. Each secondary adapter region is hybridized to a primary adapter nucleic acid molecule. [0097] Each primary adapter nucleic acid molecule is further coupled to its associated padlock probe via hybridization of a primer in the primary adapter nucleic acid molecule to a primer binding domain in a padlock probe.

[0098] The ends of each padlock probe in the signal-generation precursor 603 are hybridized to a splint molecule, such that the padlock probe is configured to generate a circular nucleic acid construct upon ligation of the two ends. As such, the signal-generation scaffold precursor 603 depicted in FIG. 6A is configured to generate a signal-generation scaffold comprising a plurality of circular nucleic acid constructs, each of which is indirectly coupled to the barcode molecule 602.

[0100] In FIG. 6B, the secondary adapter nucleic acid molecule 681 couples a plurality of different primary adapter nucleic acid molecules to the barcode molecule 652. The secondary adapter nucleic acid molecule 681 comprises a barcode binding region 681b that is hybridized to a portion of the barcode molecule 652. The secondary adapter nucleic acid molecule 681 further comprises a plurality of secondary adapter regions (including 681al, 681a2, and 681a3). In FIG. 6B, the secondary adapter regions of the plurality of secondary adapter regions comprise different sequences. Each secondary adapter region is hybridized to a different primary adapter nucleic acid molecule. As shown in FIG. 6B, the secondary adapter sequence 681al is hybridized to primary adapter molecule 671, the secondary adapter sequence 681a2 is hybridized to primary adapter molecule 672, and the secondary adapter sequence 681a3 is hybridized to primary adapter molecule 673.

[0101] Each primary adapter nucleic acid molecule is further coupled to its associated padlock probe via hybridization of a primer in the primary adapter nucleic acid molecule to a primer binding domain in a padlock probe. [0102] The ends of each padlock probe in the signal-generation precursor 653 are hybridized to a splint molecule, such that the padlock probe is configured to generate a circular nucleic acid construct upon ligation of the two ends. As such, the signal-generation scaffold precursor 653 depicted in FIG. 6B is configured to generate a signal-generation scaffold comprising a plurality of circular nucleic acid constructs, each of which is indirectly coupled to the barcode molecule 652

Example 6: Method of generating a branched signal-generation scaffold

[0103] FIG. 7 depicts an exemplary method of generating a branched signal-generation scaffold comprising a plurality of circular nucleic acid adapters, according to some embodiments. In FIG. 7, a composition comprising (a) a particle 701 comprising a barcode molecule 702 and (b) a branched signal-generation scaffold precursor 703 associated with the barcode molecule 702 is provided. The branched signal-generation scaffold precursor 703 comprises (i) a plurality of primer molecules (e.g., 711), (ii) a plurality of primer-binding padlock probes (e.g., 721), (iii) a plurality of adapter padlock probes (e.g., 731 or 741), and (iv) a linear adapter nucleic acid molecule 751.

[0104] Each primer molecule is hybridized to a primer-binding domain in a primer-binding padlock probe. Each primer-binding padlock probe is indirectly coupled to the barcode molecule 702 via two adapter padlock probes and the linear adapter nucleic acid molecule 751.

[0106] The second adapter padlock probe 741 couples the first adapter padlock probe 731 to the linear adapter nucleic acid molecule 751. The second adapter padlock probe 741 is directly coupled to the linear adapter nucleic acid molecule 751 via hybridization of the two ends of the second adapter padlock probe 741 to two linear adapter regions 751al and 751a2 in the linear adapter nucleic acid molecule 751. The linear adapter molecule 751 couples the second adapter padlock probe 741 and the barcode molecule 702. The linear adapter molecule 751 is directly coupled to the barcode molecule via hybridization of a barcode binding region 751b to a portion of the barcode molecule. [0107] The branched signal-generation scaffold precursor 703 is used to generate a branched signal-generation scaffold 704, comprising a plurality of circular nucleic acid constructs. For each padlock probe in the branched signal -gen eration scaffold precursor 703, a ligase is used to ligate the two ends of the padlock probe together. For example, the two ends of the primerbinding padlock probe 721 is ligated together to generate a primer-binding circular nucleic acid construct 761. The two ends of the adapter padlock probe 731 are ligated together to generate the circular nucleic acid adapter 771, and the two ends of the adapter padlock probe 741 are ligated together to generate the circular nucleic acid adapter 781. The ligation reactions produce a composition comprising (a) the particle 701 comprising the barcode molecule 702 and (b) the branched signal-generation scaffold 704 associated with the barcode molecule 702. In the branched signal-generation scaffold 704, a plurality of primer molecules are hybridized to primer-binding domains in a plurality of primer-binding circular nucleic acids. Each primerbinding circular nucleic acid is indirectly coupled to the barcode molecule 702 via two circular nucleic acid adapters and the linear adapter nucleic acid molecule 751.

[0108] As shown in FIG. 7, primer molecule 711 is hybridized to a primer-binding domain in primer-binding circular nucleic acid 761. Primer-binding circular nucleic acid 761 is directly coupled to a first circular nucleic acid adapter 771, which is further directly coupled to a second circular nucleic acid adapter 781. The second circular nucleic acid adapter 781 is further directly coupled to a linear adapter region 751a3 of the linear adapter nucleic acid molecule 751. The linear adapter molecule 751 is directly coupled to the barcode molecule via hybridization of a barcode binding region 751b to a portion of the barcode molecule.

[0109] The branched signal-generation scaffold 704 is then used to generate a signal. The branched signal-generation scaffold is contacted with a DNA polymerase and dNTPs and used in rolling circle amplification reactions to produce a plurality of single-stranded DNA concatemers from the plurality of primers in the branched signal -generation scaffold. For each primer in the branched signal-generation scaffold, its associated primer-binding circular nucleic acid is used as the template in a rolling circle amplification reaction to produce a single-stranded DNA concatemer of the plurality of single-stranded DNA concatemers. The plurality of singlestranded DNA concatemers are then used to generate the signal, for example, through hybridization of detectable probes to the plurality of single-stranded DNA concatemers.

[0110] In FIG. 7, the branched signal-generation scaffold 704 can be used to generate a plurality of different signals. Each primer-binding circular nucleic acid in the branched signalgeneration scaffold 704 can comprise a different signal sequence, such that the rolling circle amplification reactions produce a plurality of single-stranded DNA concatemers, each comprising multiple copies of a different signal sequence or complement thereof. For example, primer-binding circular nucleic acid 761 and primer-binding circular nucleic acid 762 can comprise different signal sequences. The associated concatemer generated from primer-binding circular nucleic acid 761 can be hybridized to magnetically active probes, while the associated concatemer generated from primer-binding circular nucleic acid 762 can be hybridized to fluorescently labeled probes for the purpose of magnetic retrieval of particle 701 and identification by fluorescent detection. A third primer-binding circular nucleic acid 763 can optionally be used to generate an associated concatemer comprising a different signal sequence or complement thereof that hybridizes to fluorescently labeled probes with a different emission wavelength for the purpose of multi-channel detection.

Example 7: Method of generating a branched signal-generation scaffold

[0111] FIG. 8 depicts an exemplary method of generating a branched signal-generation scaffold comprising a plurality of linear adapter nucleic acid molecules, according to some embodiments. In FIG. 8, a composition comprising (a) a particle 801 comprising a barcode molecule 802 and (b) a branched signal-generation scaffold precursor 803 associated with the barcode molecule 802 is provided. The branched signal-generation scaffold precursor 803 comprises (i) a plurality of padlock probes (e.g., 811), (iii) a plurality of primary adapter nucleic acid molecules (e.g., 831), (iv) a plurality of secondary adapter nucleic acid molecules (e.g., 841 or 851), and (v) a barcode binding adapter nucleic acid molecule 861.

[0112] Each padlock probe (e.g., 811) in the branched signal -generation scaffold precursor 803 is hybridized to a splint molecule 821, such that the padlock probe is configured to generate a circular nucleic acid construct upon ligation of the two ends. Each padlock probe (e.g., 811) is indirectly coupled to the barcode molecule 802 via a primary adapter nucleic acid molecule (e.g., 831), (iv) two intermediate adapter nucleic acid molecules (e.g., 841 and 851), and (v) a barcode binding adapter nucleic acid molecule 861.

[0114] The secondary adapter nucleic acid molecule 841 couples the plurality of primary adapter nucleic acid molecule to a tertiary adapter nucleic acid molecule 851. As shown in FIG. 8, a region of the secondary adapter nucleic acid molecule 841 is directly hybridized to a region 851a2 of the tertiary adapter nucleic acid molecule 851. In this example, the tertiary adapter nucleic acid molecule 851 has multiple adapter binding regions that (851al and 851a2) that can comprise different sequences and are each hybridized to a different secondary adapter nucleic acid molecule.

[0115] The tertiary adapter nucleic acid molecule 851 couples the plurality of secondary adapter nucleic acid molecules to the barcode binding adapter nucleic acid molecule 861. As shown in FIG. 8, a region 851a3 of the secondary intermediate adapter nucleic acid molecule 851 is directly hybridized to a region 861a of the barcode binding adapter nucleic acid molecule 861

[0116] The barcode binding adapter nucleic acid molecule 861 couples the tertiary intermediate adapter nucleic acid molecule 851 and the barcode molecule 802. The barcode binding adapter nucleic acid molecule 861 is directly coupled to the barcode molecule via hybridization of a barcode binding region 861b to a portion of the barcode molecule.

[0117] The branched signal-generation scaffold precursor 803 is used to generate a branched signal-generation scaffold 804, comprising a plurality of circular nucleic acid constructs. For each padlock probe in the branched signal-generation scaffold precursor 803, a ligase is used to ligate the two ends of the padlock probe together. For example, the two ends of the padlock probe 821 is ligated together to generate a primer-binding circular nucleic acid construct 871. The ligation reactions produce a composition comprising (a) the particle 801 comprising the barcode molecule 802 and (b) the branched signal -generation scaffold 804 associated with the barcode molecule 802. In the branched signal-generation scaffold 804, each circular nucleic acid constructs is indirectly coupled to the barcode molecule 802 via a primary adapter nucleic acid molecule (e.g., 831), (iv) two intermediate adapter nucleic acid molecules (e.g., 841 and 851), and (v) a barcode binding adapter nucleic acid molecule 861. Each circular nucleic acid construct comprises a primer-binding region that is hybridized to a primer in a primary adapter nucleic acid molecule (e.g., 831).

[0118] The branched signal-generation scaffold 804 is then used to generate a signal. The branched signal-generation scaffold is contacted with a DNA polymerase and dNTPs and used in rolling circle amplification reactions to produce a plurality of single-stranded DNA concatemers from the plurality of primers in the branched signal -generation scaffold. For each primer in the branched signal-generation scaffold, its associated circular nucleic acid construct is used as the template in a rolling circle amplification reaction to produce a single-stranded DNA concatemer of the plurality of single-stranded DNA concatemers. The plurality of single-stranded DNA concatemers are then used to generate the signal, for example, through hybridization of detectable probes to the plurality of single-stranded DNA concatemers.

[0119] In FIG. 8, the branched signal-generation scaffold 804 can be used to generate a plurality of different signals. Each circular nucleic acid construct in the branched signalgeneration scaffold 804 can comprise a different signal sequence, such that the rolling circle amplification reactions produce a plurality of single-stranded DNA concatemers, each comprising multiple copies of a different signal sequence or complement thereof. For example, circular nucleic acid construct 871 and circular nucleic acid construct 872 can comprise different signal sequences. The associated concatemer generated from circular nucleic acid construct 871 can be hybridized to magnetically active probes, while the associated concatemer generated from circular nucleic acid construct 872 can be hybridized to fluorescently labeled probes for the purpose of magnetic retrieval of particle 801 and identification by fluorescent detection. A third primer-binding circular nucleic acid 873 can optionally be used to generate an associated concatemer comprising a different signal sequence or complement thereof that hybridizes to fluorescently labeled probes with a different emission wavelength for the purpose of multichannel detection.

Example 8: Utilization of signal-generation scaffold for identification of particle contents

[0120] FIG. 9 and FIGs. 10A-B depict an experiment utilizing a signal-generation scaffold according to some embodiments to identify contents inside a microparticle.

[0121] In this example, a population of 96 barcoded microparticles was provided, featuring 30 external barcodes and 96 unique internal barcodes. FIG. 9 shows a schematic of the experimental workflow using an external barcode Bl to select and identify a target population of microparticles comprising external barcode Bl. As shown in FIG. 9, an external barcode Bl on microparticle 901 was associated with a signal-generation scaffold 904 comprising a circular nucleic acid construct comprising a signal sequence. The signal-generation scaffold 904 was used to generate a concatemer 905 comprising multiple copies of the signal sequence complement through rolling circle amplification. Following amplification, the population of barcoded microparticles were contacted with detectable probes, which were hybridized to the signal sequence complement in the amplified concatemer 905 associated with barcode Bl. The signal detected from the probes was then used to sort the target particles comprising external barcode Bl from the non-target particles using flow cytometry. Following flow cytometry, the internal barcodes were extracted from the sorted particles and sequenced, generating short-read sequencing data.

[0122] Separately, a control population of 96 barcoded microparticles was also sorted using barcode Bl selection without amplification of the signal sequence.

[0123] FIG. 10A shows the flow cytometry results from sorting the microparticles. As shown by the data, utilizing signal sequence amplification allowed for a distinct separation between the target microparticles comprising barcode Bl and the non-target microparticles that do not comprise barcode Bl. The control group that was sorted without signal sequence amplification resulted in an indistinguishable population.

[0124] FIG. 10B shows the short-read sequencing data of the internal barcodes obtained from the sorted populations. The data show that utilizing signal sequence amplification correctly selected for the internal barcodes 48, 56, and 92 that were encapsulated in the target microparticles comprising the external barcode Bl. On the other hand, the control group that was sorted based on external barcode Bl without signal sequence amplification led to misidentification of the target microparticles and did not select for the correct internal barcodes associated with external barcode Bl.

[0125] These results showed that a utilizing a signal-generation scaffold described herein enables accurate and robust selection of barcoded microparticles, even when barcodes are present at a low density.

[0126] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A composition comprising: a) a particle comprising a barcode that identifies said particle; and b) a signal-generation scaffold associated with said barcode; wherein said barcode is present on said particle at a density of less than or equal to 1,000,000, and wherein said signal-generation scaffold comprises a circular nucleic acid construct.

2. The composition of claim 1, wherein said circular nucleic acid construct comprises a primerbinding region that is hybridized to a nucleic acid primer.

3. The composition of claim 2, wherein a portion of said circular nucleic acid construct is hybridized to a splint nucleic acid molecule.

4. The composition of claim 2, wherein said nucleic acid primer comprises a modified nucleotide.

5. The composition of claim 4, wherein said modified nucleotide is a locked nucleic acid (LNA), a hexitol nucleic acid (HNA), a cyclohexene nucleic acid (CNA), morpholino phosphoramidates, a threose nucleic acid (TNA), a glycol nucleic acid (GNA), a 2’- fluoroarabinonucleic acid (FANA), an arabinonucleic acid (ANA), a (l'-3')-P-L-ribulo nucleic acid (riboNA), 3 '-2' phosphonom ethyl -threosyl nucleic acid (tPhoNA), altritol nucleic acids (AltNA), or a 2'-deoxyxylonucleic acid (dXNA).

6. The composition of claim 2, wherein said nucleic acid primer comprises a modified backbone.

7. The composition of claim 6, wherein said modified backbone is a phosphorothiate, a phosphonate, triazole, or a boranophosphate.

8. The composition of claim 2, wherein said nucleic acid primer comprises a combination of modified nucleotide and modified backbone.

9. The composition of claim 1, wherein said circular nucleic acid construct is directly coupled to said barcode.

10. The composition of claim 9, wherein said circular nucleic acid construct comprises a barcode-binding region that is hybridized to said barcode.

11. The composition of claim 1, wherein said circular nucleic acid construct is indirectly coupled to said barcode.

12. The composition of claim 11, wherein said signal-generation scaffold comprises an adapter nucleic acid molecule, wherein said adapter nucleic acid molecule comprises a first adapter region and a second adapter region, wherein: a) said first adapter region is hybridized to an adapter-binding region of said circular nucleic acid construct; and b) said second adapter region is coupled to said barcode.

13. The composition of claim 12, wherein said second adapter region is hybridized to said barcode.

14. The composition of claim 12, wherein said second adapter region is hybridized to an intermediate adapter nucleic acid molecule, wherein said intermediate adapter nucleic acid molecule is coupled to said barcode.

15. The composition of claim 12, wherein said adapter nucleic acid molecule comprises an additional circular nucleic acid construct.

16. The composition of claim 1, wherein said signal-generation scaffold comprises at least two circular nucleic acid constructs associated with said barcode.

17. The composition of claim 16, further comprising a concatemer comprising a signal sequence of said circular nucleic acid construct or complement thereof.

18. The composition of claim 14, wherein said concatemer comprises at least 2 copies of said signal sequence or complement thereof.

19. The composition of claim 16, further comprising a probe hybridized to a sequence of said concatemer.

20. The composition of claim 19, wherein said probe comprises an optically-active molecule.

21. The composition of claim 19, wherein said probe comprises a magnetically-active molecule or an affinity molecule.

22. The composition of claim 19, further comprising a plurality of probes hybridized to sequences of said concatemer.

23. The composition of claim 22, wherein probes of said plurality of probes comprise optically- active molecules.

24. The composition of claim 22, wherein probes of said plurality of probes comprise magnetically-active molecules or affinity molecules.

25. The composition of claim 1, further comprising a probe hybridized to a sequence of said circular nucleic acid construct, wherein said probe comprises a detectable moiety, a magnetically-active molecule or an affinity molecule.

26. The composition of claim 25, wherein said detectable moiety comprises an optically-active molecule.

27. The composition of claim 1, wherein said particle comprises a nucleic acid, a protein, or a peptide.

28. The composition of claim 27, wherein said particle comprises a bead that encapsulates said nucleic acid, said protein, or said peptide.

29. A method, the method comprising:

(a) providing a composition, the composition comprising:

(i) a particle comprising a barcode that identifies said particle; and

(ii) a signal-generation scaffold associated with said barcode; wherein said barcode is present on said particle at a density of less than or equal to 1,000,000, and wherein said signal-generation scaffold comprises a circular nucleic acid construct; and

(b) generating a signal using said signal-generation scaffold; and

(c) detecting said signal, thereby identifying said barcode and said particle.

30. The method of claim 29, further comprising, prior to (a), generating said circular nucleic acid construct.

31. The method of claim 30, wherein generating said circular nucleic acid construct comprises hybridizing a splint nucleic acid molecule to a padlock probe, wherein, upon said hybridizing, said padlock probe comprises (i) a first end hybridized to a first region of said splint nucleic acid molecule, and (ii) a second end hybridized to a second region of said splint nucleic acid molecule.

32. The method of claim 31, further comprising, prior to (a), linking together said first end and said second end of said padlock probe.

33. The method of claim 29, wherein, in (a), said particle comprises a barcode molecule comprising said barcode, and wherein, in (a), a 3’ end of said barcode molecule comprising said barcode is hybridized to a primer-binding region of said circular nucleic acid construct; and (b) comprises using said 3’ end of said barcode molecule as a nucleic acid primer to amplify said circular nucleic acid construct, thereby generating a concatemer comprising a sequence of said circular nucleic acid construct or complement thereof.

34. The method of claim 29, further comprising, prior to (a), hybridizing an adapter nucleic acid molecule to said barcode, wherein said adapter nucleic acid molecule comprises a primer.

35. The method of claim 34, further comprising, prior to (a), hybridizing said circular nucleic acid construct or precursor thereof to said primer.

36. The method of claim 34, wherein said primer is hybridized to said circular nucleic acid construct or precursor thereof.

37. The method of claim 36, wherein (b) comprises using said primer to amplify said circular nucleic acid construct, thereby generating a concatemer.

38. The method of claim 29, wherein, in (a), said circular nucleic acid construct comprises a signal sequence; and (b) comprises generating said signal by amplifying said circular nucleic acid construct in an amplification reaction to generate a concatemer comprising said signal sequence or complement thereof.

39. The method of claim 38, wherein said concatemer generated in (b) comprises at least two copies of said signal sequence or complement thereof.

40. The method of claim 38, wherein (b) comprises hybridizing a probe to said signal sequence or complement thereof in said concatemer; and wherein (c) comprises detecting said probe, thereby detecting said concatemer, thereby identifying said circular nucleic acid construct, thereby identifying said barcode in said particle.

41. The method of claim 40, wherein said probe comprises an optically-active molecule and (c) comprises detecting said optically-active molecule.

42. The method of claim 40, wherein said probe comprises a magnetically-active molecule or an affinity molecule and (c) comprises detecting said probe using said magnetically-active molecule or said affinity molecule.

43. The method of claim 29, wherein, in (a), said signal -generation scaffold comprises a plurality of circular nucleic acid constructs.

44. The method of claim 43, further comprising, in (b), generating said signal from circular nucleic acid constructs of said plurality of circular nucleic acid constructs.

45. The method of claim 29, wherein, in (a), said particle is among a plurality of particles, and wherein said method further comprises (d) identifying said particle from other particles of said plurality of particles using said barcode identified in (c).

46. The method of claim 45, wherein said particle comprises contents, and said method further comprises (e) extracting said contents from said particle.

47. The method of claim 46, wherein said contents comprise a nucleic acid, a protein, or a peptide.