WO2015200384A1 - Multiplex dna nanoparticle protein assay and quantitation - Google Patents

Abstract

Disclosed are DNA nanoparticles (DeNAno) comprising rolling circle replicated circular oligonucleotides. Also disclosed are methods of multiplexed protein assay and quantitation, the method comprising the steps of creating a DeNAno particle by rolling circle replication of one or more circular oligonucleotide templates; creating one or more libraries of DeNAno particles by using templates with random sequences; selecting the DeNAno particles that bind specifically to magnetic beads coated with antibodies against a target of interest; synthesizing bead specific DeNAno particles and using the same to mask the beads; pooling DeNAno masked beads against multiple target antigens, and adding the pooled DeNAno masked beads to an analyte containing sample, wherein the analytes recognized by their cognate antibodies displace the masking DeNAno particles in a concentration dependent fashion; removing the beads from the sample; and amplifying and sequencing the displaced DeNAno particles. Further disclosed are kits comprising: a plurality of DeNAno particles; instructions for their use; and analysis algorithms.

Description

MULTIPLEX DNA NANOPARTICLE PROTEIN ASSAY AND QUANTITATION

RELATED APPLICATIONS

[001] The present application claims priority to the U.S. Provisional Serial Application Serial No. 62/015,592, filed on June 23, 2014 by Mesmer et al., and entitled "MUDPAQ ASSAY," the entire disclosure of which, including all the drawings, are incorporated herein by reference.

FIELD OF THE INVENTION

[002] The present invention is in the field of proteomics and identification and quantitation assay for proteins in a sample.

BACKGROUND OF THE DISCLOSURE

[003] Proteomics studies have the power to deliver pivotal new insights into cancer cells, biomarkers, and host responses. Unlike the field of genomics, the field of proteomics has been constrained by the lack of simple and affordable analytical tools. Multiplex proteomic analysis methods remain massive, expensive, cumbersome and specialized.

[004] Proteomics is a subfield of systems biology consisting of techniques for the large-scale study of the proteome, i.e. the set of proteins in an organism, a cell, or any biological system, at a given time and under certain conditions. Cancer proteomics can be used to study the protein content of tumor and support cells, biomarkers and drug response indicators in bodily fluids, and proteins in experimental systems. While the tools and technologies for proteomic studies have improved considerably, there have not been equivalent revolutionary leaps forward in proteomic methods as has been seen in genomics with the advent of high throughput DNA sequencing.

[005] Modern discovery proteomics research relies a great deal on mass spectrometry (MS) based techniques, which permit the identification of peptide sequences and proteins based on known genomic information. MS based approaches are geared towards unbiased discovery because the user does not need to know in advance what he/she is looking for. With complex biological samples, MS-based proteomics requires that the proteins or peptides or both be fractionated prior to MS analysis either by two- dimensional gel electrophoresis (2DGE), liquid chromatography (LC), or a combination of the two. Recently, MudPIT (or shotgun proteomics) was developed for rapid, multi- dimensional protein identification in a complex mixture. Notably, although MS-based proteomics approaches may be multiplex (many analytes are detected), they are inherently unsuitable for high-throughput screening (multiple samples or specimens).

[006] Protein microarrays enable simultaneous analysis of different proteins in a single experiment and yield information on protein identity, quantity, interaction, and function. Protein microarrays are useful for measuring protein-protein interactions or small molecule binding for drug discovery, biomarker identification, and molecular profiling of cellular material. Unlike the MS based techniques, studies using protein microarrays are biased because they often require the user to have some knowledge of what proteins to look for and also have affinity reagents to probe for these proteins of interest. Antibody microarrays for capturing proteins or peptides are a specific example of a protein microarray. Advantages of this technique include its low-volume requirements, multiplexed detection capability, and the fact that it is rapid and highly amenable to automation. However wider application of antibody microarrays has been limited, partly because of the cost of producing antibodies and the limited availability of antibodies with high specificity and high affinity for the target. In addition, preserving proteins in a biologically active conformation before analysis with protein arrays is challenging. This technique requires specialized equipment to read the array and each array can be costly.

[007] ImmunoPCR uses PCR based detection of oligonucleotide-antibody conjugates to quantify proteins. It is a very sensitive method with multiplexing capability, but requires expensive custom reagents, significant optimization for multiplexing, and is moderate throughput. As such, the technology has been most useful for highly sensitive detection of pathogens in very dilute samples. The bio-barcode assay is similar in leveraging PCR amplification to achieve high sensitivity. There are many nanotechnology device based platforms that can achieve similar high sensitivity with some multiplexing. These include nano-cantilevers, nano-plasmonics, and nano waveguides among others but device based approaches are intrinsically low to moderate throughput and the sensitivity is often far greater than relevant for biological samples.

[008] Luminex® xMAP® technology is currently the state of the art in many research laboratories. xMAP® is a multiplexing platform that uses beads that are internally dyed with fluorescent dyes to produce a specific spectral address. Biomolecules such as an oligopeptide or antibody can be conjugated to the surface of beads to capture analytes of interest. This technology requires a dedicated instrument, similar to a flow cytometer, to identify the analyte being measured and then assess the amount of analyte bound to the bead. Luminex® bead assays require the user to have insight into what proteins to look for and have affinity reagents for these proteins of interest. Advantages of Luminex® are its multiplexing (it can detect up to 500 proteins per well in microtiter plate) and the need for very small sample volumes. However, key disadvantages of this technology are its requirement for specialized and expensive detection equipment, a limit on the degree of multiplexing due to its dependence on dyes, and the throughput limitations of a flow based assay.

[009] Therefore, there is a growing need for proteomic assays that are easy to use and cost effective. Moreover, there is a clear need for improved methodologies for performing massively parallel multiplex assays that are cost-effective, do not require complex detection equipment, and have an unlimited multiplexing capability.

SUMMARY OF THE INVENTION

[0010] Disclosed are DNA nanoparticles (DeNAno) comprising a rolling circle replicated circular oligonucleotides. Also disclosed are methods of multiplexed protein assay and quantitation, the method comprising the steps of creating a DeNAno particle by rolling circle replication of one or more circular oligonucleotide templates; creating one or more libraries of DeNAno particles by using templates with random sequences; selecting the DeNAno particles that bind specifically to magnetic beads coated with antibodies against a target of interest; synthesizing bead specific DeNAno particles and using the same to mask the beads; pooling DeNAno masked beads against multiple target antigens, and adding the pooled DeNAno masked beads to an analyte containing sample, wherein the analytes recognized by their cognate antibodies displace the masking DeNAno particles in a concentration dependent fashion; removing the beads from the sample; and amplifying and sequencing the displaced DeNAno particles. Further disclosed are kits comprising: a plurality of DeNAno particles; instructions for their use; and analysis algorithms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 illustrates an overview of the Multiplexed DeNAno Protein Assay and Quantitation (MuDPAQ) technique. FIG. 1A shows that DeNAno particles are created by rolling circle replication of circular oligonucleotide templates. The resulting single strand DNA forms a nanoparticle whose size is tunable as a function of the polymerization reaction kinetics. FIG. IB shows that libraries of DeNAno particles are created by using templates with random sequences. FIG. 1C shows that DeNAno particles binding specifically to magnetic beads coated with antibodies against a target of interest are selected by biopanning. FIG. ID shows that bead specific particles are synthesized and used to mask the cognate bead. FIG. IE shows that DeNAno masked beads against multiple target antigens are pooled and added to analyte containing sample. FIG. IF shows the analytes recognized with high affinity by their cognate antibodies displace the masking DeNAno particles in a concentration dependent fashion. FIG. 1G shows that the beads are removed by magnet, and the displaced DeNAno particles amplified and sequenced. The population frequency of any given particle is reflective of its competitive analyte' s concentration.

[0012] FIG. 2 illustrates the selection and characterization of streptavidin binding DeNAno particles. FIG. 2A shows the selection of streptavidin-binding clones. FIG. 2B shows the streptavidin binding results of four clones. FIG. 2C shows the competitive displacement with biotin after the particles were bound to the streptavidin coated beads. FIG. 2D shows the competitive displacement of clone D8 with biotin or modified biotins. FIG. 2E shows the biotin competition titration. FIG. 2F shows the Streptavidin competition titration.

[0013] FIG. 3 illustrates the selection of DeNAno particles specific for rituximab and trastuzumab (Herceptin). FIG. 3A shows that the parent library and each round of selected particles were labeled and incubated with antibody coated polystyrene beads, washed, and measured. FIG. 3B shows that seven unique sequences were recovered from the final rounds of two separate selections against rituximab coated beads. FIG. 3C shows that nine unique sequences were recovered from the trastuzumab selection, six of which shared the motif shown. FIG. 3D shows that a rituximab binding clone was tested for competitive displacement by a rituximab binding mimetope peptide conjugated to BSA. No competitive displacement was seen with control peptide. The mimetope has weak affinity (μΜ) for rituximab and is a poor competitor.

[0014] FIG. 4 illustrates the stability of DeNAno coated streptavidin beads. Streptavidin coated beads were coated with the indicated, fluorescently labeled streptavidin binding DeNAno particles (D7 and D8) or a biotinylated control, washed extensively, and incubated at 4C for one week. The fluorescence was measured before and after another round of washes. [0015] FIG. 5 illustrates the selection method for DNA nanoparticles that bind to target coated beads. A 100 base library with a 60 base random region flanked by 2 20 base primer sites was ligated and amplified with Rolling Circle Amplification to produce nanoparticles. The nanoparticles were incubated with the target cells and washed. Remaining nanoparticles are amplified by PCR with a polymerase that lacks 5' to 3' exonuclease activity, such as the Stoffel fragment of Taq polymerase, and then asymmetrically by PCR (using only one primer) to generate an excess of the template strand. The template strand was re-ligated and the cycle repeated.

[0016] FIG. 6 illustrates the high-throughput sequencing of DeNAno libraries during a cell based selection. FIG. 6A shows the global changes in particle frequency distribution as the selection against leukemia cells proceeds. Most sequences in the initial library were unique. As the selection proceeded, an increase in the fraction of sequences with greater population frequency was observed. FIG. 6B shows the technical control replicate. The same sample was independently amplified in duplicate and the population frequencies were compared. FIG. 6C shows the biological control replicate. The second round of selection was performed in duplicate and independently amplified for sequencing.

[0017] FIG. 7 shows the process for the selection of streptavidin-binding DeNAno. FIG. 7A is a schematic of selection process. FIG. 7B shows the results of staining of streptavidin selection rounds 1-5. Probe-only, library, and positive control (biotinylated library) are also shown. FIG. 7C shows the results of staining of 4 selected streptavidin clones on streptavidin beads and BSA beads. Negative control clone (GlOneg) and biotinylated positive control clone (GlObio) also shown.

[0018] FIG. 8 shows the results of the imaging of DeNAno and staining different size DeNAno. FIG. 8A is a photograph of SA-D8 DeNAno roughly 75 nm in diameter as observed by transmission electron microscopy (TEM) using negative staining. FIG. 8B is the atomic force micrograph (AFM) of dried DeNAno SA-D8 on poly-L-lysine coated mica. Fig 8C shows the results of the binding of streptavidin DeNAno made by alteration of dNTP concentration. DeNAno particles were made with 3 nmol dNTPs for 30 min at 30 °C (the standard conditions), or 93.8 pmol dNTPs for 30 min at 30 °C. A control DeNAno from a different library was also made for both of these conditions and used as an internal control in the staining and subsequent PCR. The ratio of the bound particles (streptavidin DeNAno :control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed. [0019] FIG. 9 shows the results of the competitive titration and competitive release of DeNAno with biotin and biotin derivatives. FIG. 9A shows the results of free biotin (top), desthiobiotin (middle), and 2-iminobiotin (bottom) competition titrations were done by pre-incubating streptavidin beads with one of the biotin/biotin derivatives (or buffer for the baseline), then adding DeNAno particles. FIG. 9B shows the results of biotin (top), desthiobiotin (middle), and 2-iminobiotin (bottom) competitive release assays were done by staining streptavidin beads with DeNAno particles, then adding biotin/biotin derivative (or buffer for baseline).

[0020] FIG. 10 shows the results of the dissociation of streptavidin-binding DeNAno over time. Streptavidin magnetic beads were stained with DeNAno particles. The stained beads were then incubated in 10ml buffer for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and % release is graphed (DeNANo in supernatant/DeNANo in total * 100%). At day 21, a biotin knockoff was also done (filled symbols), in which excess biotin was added to an aliquot of total sample, incubated, then beads were removed via magnet to obtain supernatant only.

[0021] FIG. 11 shows the results of the streptavidin competitive titration. Free streptavidin competition titration of SA-D7 and SA-D8 clones and GlObio positive control. DeNAno particles were pre-incubated with varying concentrations of free streptavidin, then streptavidin beads were added.

[0022] FIG. 12 shows the results of experiments with antibody- specific DeNAno. FIG. 12A shows the results of the staining of dominant clones from rituximab (3Ritl) and bevacizumab (Aval) on specific monoclonal-, irrelevant monoclonal-, and human polyclonal IgG antibody-coated beads. FIG. 12B shows the results of the competitive titration with rituximab-specific (Rit pept) or irrelevant peptide (irr pept) was done by pre-incubating peptide with rituximab-coated polystyrene beads, followed by incubation with 3Ritl DeNAno. FIG. 12C shows the results of the competitive release with peptide was done by pre-incubating rituximab-coated polystyrene beads with 3Ritl, followed by incubation with Rit pept, irr pept, or buffer. Total sample and sample released into the supernatant were measured by qPCR and % released is graphed. FIG. 12D shows that protein G was pre-incubated for lhr with different concentrations of rituximab or bevacizumab. Alexa Fluor647-labeled 3Ritl DeNAno, Aval DeNAno, or Lib-neg DeNAno (left y axis), or Alexa Fluor488-labeled anti-kappa human light chain antibody (right y axis) were then added and incubated for an additional 2hr, then washed and measured for fluorescence. For anti-kappa light chain samples only: rituximab and bevacizumab samples <l-fold free antibody were diluted with mouse IgG2b κ to equal 1- fold total antibody. This was done to bind all free protein G sites before addition of anti- kappa antibody so it would not bind non-specifically.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0024] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more than one standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, of a given value, for example value+20%, or value+15%, or value+10%, or value+5%.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] Disclosed herein are novel assays termed MuDPAQ, which occupy the niche for high throughput, highly multiplexed protein or biomarker assays, which niche is currently poorly served with existing technologies. There is a growing need for assays of this type as tissue banks become more extensive, patterns of markers rather than single markers become validated, and pharmacologic responses to therapy become more utilized. By reverse translating a protein detection event into a DNA signal, the MuDPAQ assay leverages the power and commoditization of sequencing to enable massively parallel analyses that can be cheaply outsourced to any academic or commercial facility. [0027] Thus, in one aspect disclosed herein, is a DNA nanoparticle (DeNAno) comprising a rolling circle replication of circular oligonucleotides. The term DeNAno, as used herein refers to a DNA particle produced by a rolling circle replication of circular oligonucleotide templates. Random templates of oligonucleotides are used to produce highly diverse libraries of DeNAno particles. These oligonucleotides are composed of concatemeric repeats of single stranded DNA strands, whose size is tunable as a function of the polymerization reaction kinetics. The concatemeric size of the DeNAno particle is used to tune the avidity of the DeNAno in detecting different levels of competing antigen.

[0028] In some embodiments, the DeNAno technology is a novel biomolecular affinity reagent that replaces single or bivalent affinity with hyper-avidity. Rolling circle replication of circular oligonucleotide templates produces a concatemeric single strand of DNA that is composed of many copies of the template sequence.

[0029] In some embodiments, under physiological conditions, this ssDNA forms a nanoparticle whose size and number of template copies is tuned by the reaction conditions. When random sequences are inserted into a template circle, massively diverse libraries are made with each particle containing many copies of a unique sequence. As with aptamer libraries, some of these sequences adopt secondary and tertiary structures that give rise to specific binding interactions. Unlike aptamers, those interactions need not be high affinity since the presence of many copies allows avidity to compensate if the target is also multimeric or a polymer.

[0030] In some embodiments, DeNAno particle binds specifically to antibody coated beads. In some embodiments, biopanning technique is used to recover the DeNAno particles that bind specifically to magnetic beads coated with antibody. In some embodiments disclosed herein, the biopanning technique is iterative. The term "biopanning" as used herein, refers to an affinity selection technique for selecting binding partners. In some embodiments, the binding partners are selected from phage display libraries.

[0031] In some embodiments, bead specific DeNAno particles are synthesized and used to mask the cognate bead. In certain embodiments, the DeNAno particles, composed of concatemeric repeats, bind to the cognate antibody coated bead through highly avid but individually low affinity interactions. In some embodiments, the avidity of the DeNAno particle can be tuned through its concatemeric size such that a given particle can be made to detect different relative levels of the competing antigen. [0032] In some embodiments, the DeNAno particles are competitively displaced from the DeNAno masked beads by the antibody's cognate antigen. In some embodiments, the beads are removed by magnet and the displaced DeNAno particles are amplified and sequenced. In some embodiments, the population frequency of any given DeNAno particle is reflective of its competitive analyte's concentration.

[0033] Referring to FIG. 1, in one aspect, disclosed herein is a multiplexed DeNAno Protein Assay and Quantitation (MuDPAQ) technique comprising: creating a DeNAno particle by rolling circle replication of circular oligonucleotide templates as shown in (A); creating libraries of DeNAno particles by using templates with random sequences as shown in (B); selecting DeNAno particles that bind specifically to magnetic beads coated with antibodies against a target of interest as shown in (C); synthesizing bead specific DeNAno particles and using the same to mask the cognate bead as shown in (D); pooling DeNAno masked beads against multiple target antigens, and adding the same to analyte containing sample wherein the analytes recognized with high affinity by their cognate antibodies displace the masking DeNAno particles in a concentration dependent fashion as shown in (E, F); removing the beads by magnet, amplifying, and sequencing the displaced DeNAno particles as shown in (G).

[0034] In some embodiments, the MuDPAQ assay is used for high throughput analysis of multiple multiplexed samples. In some embodiments, the multiplexing potential is essentially unlimited, or limited only by the physical amount of sample and beads because the readout for a particular analyte occurs at the level of population frequency of a unique sequence.

[0035] Most protein analysis techniques require specialized instrumentation that raise cost and limit throughput. The MuDPAQ assay leverages the power, availability, and low cost of high throughput sequencing to enable massively parallel analyses. Thus, in one aspect disclosed herein, is a kit comprising: DeNAno particles; instructions for their use; and analysis algorithms. In some embodiments, the end user sequences the results of the MuDPAQ assay at a sequencing facility of their choice, and foregoes the need to purchase any additional instrumentation to perform the assay. In some embodiments, the MuDPAQ assay does not require washing once the beads are incubated with sample. In some embodiments, if magnetic beads are used, the entire assay is automated. Selection, production and biopanning of DeNAno libraries:

[0036] In some embodiments, the selection, production and biopanning of DeNAno libraries are conducted essentially as described in Steiner JM et al. J Biotechnol. 2010;145:330-3, incorporated by reference herein in its entirety, and as shown in FIG. 5. A 100 base library with a 60 base random region flanked by two 20-base primer sites is ligated and amplified with Rolling Circle Amplification to produce nanoparticles. The nanoparticles are incubated with the target cells and washed. Remaining nanoparticles are amplified by PCR with a polymerase that lacks 5' to 3' exonuclease activity, such as the Stoffel fragment of Taq polymerase, and then asymmetrically by PCR (using only one primer) to generate an excess of the template strand. The template strand is re-ligated and the cycle is repeated.

[0037] In some embodiments, this selection method is used to select DeNAno particles against commercially available antibody coated beads. The beads are chosen from the Bio-Plex Pro Human Cancer Biomarker panel available from Bio-Rad and can be read with any Luminex compatible reader. In some embodiments, 3 to 5 rounds of selection are required and the success of selection is indicated by monitoring the amplification step using real-time PCR. A large shift towards an earlier C_T (threshold cycle) value indicates that DeNAno particles that associate with the bead have been enriched. In some embodiments, the selected pool of particles is further confirmed by labeling the particles with a fluorescent probe and comparing the bead bound fluorescence with either the original library or a random negative control particle (as in FIG. 2A).

Particle analysis:

[0038] In some embodiments, following a successful selection, the pool of particles is cloned into bacteria and sequenced. Individual clones are regenerated as DeNAno particles from synthetic oligo templates or by asymmetrical amplification of cloned plasmids. In some embodiments, selected particles are checked for binding specificity to their cognate bead and competitive displacement with a high concentration of the intended analyte.

[0039] In some embodiments, if DeNAno selections performed against protein coated beads yield a few candidate particle sequences, several clones are tested for their competitive response to analyte. In some embodiments, it is preferable to use several distinct DeNAno particles against a particular bead, just as polyclonal antibodies are often superior for certain immunoassays. In some embodiments, different particles with different relative ease of competitive displacement expands the dynamic range of the assay for a particular analyte. Competitive displacement is measured by real time PCR of the particles in the supernatant of pre-coated beads incubated with analyte.

[0040] In some embodiments, the DeNAno particles are made in a range of sizes by controlling the synthesis reaction kinetics. Since the avidity of a DeNAno particle is directly proportional to size, the sensitivity to competitive displacement also relate to size. Therefore, selected particles are tested for stability once coated, sensitivity to displacement at our standard size (the product of a 30 minute reaction, about 30 kb and about 250 nm by dynamic light scattering) as well as anywhere between about 1% to about 99% of that size. In some embodiments, the size refers to the length of the DNA and thus template copies, not necessarily physical diameter or hydrodynamic radius. In some embodiments, if the DeNAno particles are made from unique templates that contained a small sequence tag (one or two bases), different sized versions of the same particle are distinguished in the final sequencing output.

[0041] In some embodiments, the ratio of the number of a given bead to the expected analyte concentration is optimized for different concentration ranges because competitive displacement of highly multivalent DeNAno particles requires that a high density of analyte binding sites become occupied. In some embodiments, the relationship of competitive displacement to bead number at various analyte concentrations is analyzed. At low analyte concentration, fewer beads ensure a sufficient density of binding sites are occupied whereas at higher analyte concentrations, saturation of binding sites require that more beads be used to obtain a greater dynamic range.

[0042] Multiplexed protein assays are usually conducted on processed biospecimens or biofluids. Therefore, in some embodiments, the assays are performed in buffers containing detergent and a high concentration of urea as well as serum, plasma, urine, and tissue culture media containing fetal bovine serum. In some embodiments, dilution of the recovered DeNAno containing samples and DNA extraction are separately evaluated for loss of sensitivity.

Multiplex Validation

[0043] In some embodiments, the DeNAno particle populations are sequenced on the MiSeq platform from Illumina. A given pool of DeNAno particles are amplified by tagged primers that add the sequencing primer sites as well as a tag for multiplexing on the sequencer. Referring to FIG. 6, in some embodiments, this technique is used to evaluate a selection on primary leukemia cells as well as several controls and technical replicates.

[0044] In some embodiments, the multiplex performance of the MuDPAQ assay is evaluated by using a matrix of eight samples containing various concentrations of each of 10 analytes. In an exemplary embodiment, the concentrations span, by half log increments, four logs increasing from the limit of detection. In another exemplary embodiment, each sample is assayed independently multiple times, such as two, three, four, five, six, seven, eight, nine, ten, or more times.

[0045] In some embodiments, recombinant extracellular Her2 protein, the target of trastuzumab (Herceptin), is spiked at a defined concentration as a positive control for the trastuzumab beads, and rituximab beads are used as a negative control. In some embodiments, each analyte is tested alone over the same concentration range (with the positive and negative controls) to establish a calibration curve for that analyte.

[0046] In some embodiments, multiple detection beads and antibody coated controls are incubated with each sample and the displaced DeNAno particles collected, amplified, and sequenced. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the number of detection beads may be one to ten or more. In some embodiments, the population frequency of each DeNAno particle in each sample is determined and the ratio between the positive control and each analyte' s relative frequency is used as a measure of concentration.

[0047] In some embodiments, Luminex® beads are used to select DeNAno particles. In some embodiments, polystyrene coated beads are used, as in FIG. 3.

[0048] In some embodiments, the product of the initial selections are several candidate particle sequences, each of whom have a different level of sensitivity to competitive displacement and is used to expand the dynamic range.

[0049] In some embodiments, the product of the initial selection is a single dominant clonal sequence. In some embodiments, the selection is repeated. Since every template molecule in the initial library is unique, repeating the selection should produce a different result every time. In some embodiments, different libraries are used. In some embodiments, a panel of particles from different libraries against a given bead is an effective counter for amplification or sequencing bias.

[0050] In some embodiments, the size of the particles is tuned via the polymerization reaction kinetics. In some embodiments, ligation based or Click chemistry based schemes are used to assemble DeNAno particles through sequential concatenation with precise control over the copy number. In some embodiments, samples from biological fluids or tissue culture media are diluted or DNA purified prior to amplification of the DeNAno particles. In some embodiments, standard DNA purification techniques are used. In some embodiments, DNA purification is performed by using biotinylated capture probes with streptavidin coated beads.

EXAMPLES

[0051] While the methods and protocols described herein use specifically identified reagents, such as specific DeNAno particles, proteins, peptides, antibodies, or small molecules, these methods and protocols are exemplary only, and similar methods and analogous reagents may be utilized to arrive at analogous results.

EXAMPLE 1: PROOF OF CONCEPT

[0052] Initial proof-of-concept experiments were performed with human dendritic cells (DC). Particles that bound specifically to human DC, but not to other mouse or human cells, were obtained. Thereafter, particles that selectively bound to mouse pancreatic cell line PANC02, human breast cancer cell line MDA-MB-231, and primary chronic lymphocytic leukemia cells, have been developed. In all cases the selection and binding function were tested in serum.

EXAMPLE 2: SELECTION AND CHARACTERIZATION OF STREPTAVIDIN BINDING DENANO PARTICLES.

[0053] DeNAno particles that bound a defined protein target were selected by screening a DeNAno library against streptavidin coated magnetic beads. Referring to FIG. 2, (A.) shows the selection of streptavidin-binding clones. DeNAno particles were made from each round of selection, hybridized with a fluorescently-labeled complimentary oligo, and compared for binding to the streptavidin coated beads. After the final round of selection, four unique sequences were recovered. (B.) shows the four clones that were recovered from the final selection round by sequencing 16 random colonies from the cloned final round. D7 was found in 3/16, D8 in 11/16, and E6 and F4 were found once each. G10 and Gi l were random particles from the same parent library. The G10 particle hybridized to a biotinylated probe (bio-GlO) was used as a positive control. Clones were incubated with streptavidin coated magnetic beads alone or with biotin. (C.) shows the competitive displacement with biotin after the particles were bound to the streptavidin coated beads. (D.) shows the competitive displacement of clone D8 with biotin or modified biotins. The titration in (E.) shows that less biotin (~10-fold) was required to inhibit the binding of D7 and D8, compared to the GlO+bio probe positive control. (F.) shows that Streptavidin-binding clones were only weakly inhibited by very high concentrations of free streptavidin.

[0054] When regenerated as DeNAno particles, the clones bound to the streptavidin coated beads and were completely inhibited by free biotin pre-incubated with the beads, confirming that they bind streptavidin at or near the biotin binding site. A biotinylated particle used as control was similarly inhibited but needed an order of magnitude higher biotin concentration for equivalent degree of competitive inhibition. When the particles bound the streptavidin coated bead first, free biotin competitively displaced the selected particles but not the biotinylated particle control. Accordingly, the selected particles were binding through a collection of relatively weak interactions, unlike the biotinylated control. When the reciprocal experiment was done using free streptavidin, the inverse relationship was seen. The biotinylated control particles were completely inhibited from binding to the beads by as little as ΙΟΟηΜ free streptavidin, whereas the selected particles were only partially inhibited by 10μΜ. Collectively, these data demonstrate that the DeNAno particles bind to the streptavidin coated beads through a highly multivalent collection of weak interactions that are readily displaced by a high affinity ligand to the same site.

EXAMPLE 3: SELECTION OF DENANO PARTICLES SPECIFIC FOR RITUXIMAB AND TRASTUZUMAB (HERCEPTIN

[0055] DeNAno particles that bind to several other protein targets including thrombin, fibrinogen, the extracellular domain of CD3, and the therapeutic monoclonal antibodies trastuzumab (Herceptin) and rituximab (Rituxan) were selected. (FIG. 3). Peptide mimetopes were identified and generated as described in the disclosure of the International Publication WO 2009/121024 and U.S. provisional application number 61/979,123, both of which are incorporated herein by reference in their entirety. The selected DeNAno particles were competitive blocked from binding to the monoclonal antibody (mAb) by the cognate peptide, indicating that the DeNAno particles interact at or near the antigen combining site of the antibody. [0056] Referring to FIG. 3, the selection of DeNAno particles specific for rituximab and trastuzumab (Herceptin) are shown. (A.) shows that the parent library and each round of selected particles were labeled and incubated with antibody coated polystyrene beads, washed, and measured. (B.) shows that seven unique sequences were recovered from the final rounds of two separate selections against rituximab coated beads. By using the evolutionary weighting and conservation masking software, MEMEfinder®, a highly significant (e value = 6.5e-4) motif was identified in all seven sequences. (C.) shows that nine unique sequences were recovered from the trastuzumab selection, six of which shared the motif shown (e value = 1.7e-7). (D.) shows a rituximab binding clone that was tested for competitive displacement by a rituximab binding mimetope peptide conjugated to BSA. No competitive displacement was seen with control peptide. The mimetope was found to have weak affinity (μΜ) for rituximab and was a poor competitor.

EXAMPLE 4: STABILITY OF DENANO COATED STREPTAVIDIN BEADS

[0057] Stability study was performed using the streptavidin binding particle, D8, to test whether the DeNAno particles remain bound to the detecting bead unless analyte is present. Streptavidin coated beads were incubated with an excess of fluorescently labeled D8 particles and washed extensively. After one week at 4 °C, the particles were measured before and after another series of washes. There was only a minimal decrease in fluorescent signal following the washes. This loss in fluorescent signal reflects the loss of beads during the wash.

[0058] Referring to FIG. 4, the stability of DeNAno coated streptavidin beads are shown. Streptavidin coated beads were coated with the indicated, fluorescently labeled streptavidin binding DeNAno particles (D7 & D8) or a biotinylated control, washed extensively, and incubated at 4C for one week. The fluorescence was measured before and after another round of washes.

EXAMPLE 5: SELECTION METHOD FOR DNA NANOP ARTICLES THAT BIND TO TARGET COATED BEADS

[0059] The selection, production and biopanning of DeNAno libraries was conducted essentially as described in Steiner JM et al. J Biotechnol. 2010;145:330-3 and as shown in FIG. 5. A 100 base library with a 60 base random region flanked by 2 20 base primer sites was ligated and amplified with Rolling Circle Amplification to produce nanoparticles. The nanoparticles were incubated with the target cells and washed. Remaining nanoparticles were amplified by PCR with a polymerase that lacks 5' to 3' exonuclease activity, such as the Stoffel fragment of Taq polymerase, and then asymmetrically by PCR (using only one primer) to generate an excess of the template strand. The template strand is re-ligated and the cycle repeated.

EXAMPLE 6: HIGH-THROUGHPUT SEQUENCING OF DENANO LIBRARIES DURING A CELL BASED SELECTION

[0060] High throughput sequencing of DeNAno libraries was used to evaluate a selection on primary leukemia cells and several controls and technical replicates. A total of 32 different DeNAno pools were prepared and sequenced simultaneously, yielding -100,000 complete sequences per pool with excellent concordance between both technical and biological replicates. (A.) shows global changes in particle frequency distribution as the selection against leukemia cells proceeds. Most sequences in the initial library were unique. As the selection proceeds, an increase in the fraction of sequences with greater population frequency was observed. (B.) shows the technical control replicate. The same sample was independently amplified in duplicate and the population frequencies compared. (C.) shows the biological control replicate. The second round of selection was performed in duplicate and independently amplified for sequencing.

EXAMPLE 7: SELECTION OF DNA NANOPARTICLES WITH PREFERENCE FOR AGGREGATED PROTEIN TARGET

INTRODUCTION

[0061] DeNAno DNA particles are a novel multivalent reagent that rely on high overall avidity instead of high affinity to bind to targets. DeNAno particles that specifically bind to primary human dendritic cells and the mouse pancreatic cancer cell line Panc-02 have been selected previously. (Steiner, J. M. et al. DeNAno: Selectable deoxyribonucleic acid nanoparticle libraries. /. Biotechnol. 145, 330-3 (2010); Ruff, L. E., Marciniak, J. Y., Sanchez, A. B., Esener, S. C. & Messmer, B. T. Targeted and reversible cancer cell-binding DNA nanoparticles. Nanotechnol. Rev. 3, 569-578 (2014).) The selection process is a biopanning strategy akin to that used in aptamer selection (systemic evolution of ligands by exponential enrichment— SELEX), in which a highly diverse library of DNA particles is incubated with the target to capture binders followed by amplification and iteration of the process. While aptamers are generally small pieces of DNA or RNA (<100 bp) that bind in a monovalent fashion with high affinity, DeNAno are concatemers of up to several hundred copies in length made by rolling circle amplification (RCA), and sizes that can be several hundred nanometers. As with aptamers, DeNAno selection does not require prior knowledge of the target, thus selection on complex targets such as cells is possible. Previous work has shown it is possible to multimerize aptamers via RCA, standard nucleic acid chemistry, or attachment to nanoparticles. (Zhao, W. et al. Bioinspired multivalent DNA network for capture and release of cells. Proc. Natl. Acad. Sci. U. S. A. 109, 19626-31 (2012); Musumeci, D. & Montesarchio, D. Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer. Pharmacol. Ther. 136, 202-15 (2012); Levy- Nissenbaum, E., Radovic-Moreno, A. F., Wang, A. Z., Langer, R. & Farokhzad, O. C. Nanotechnology and aptamers: applications in drug delivery. Trends Biotechnol. 26, 442- 449 (2008); Zhu, J., Huang, H., Dong, S., Ge, L. & Zhang, Y. Progress in aptamer- mediated drug delivery vehicles for cancer targeting and its implications in addressing chemotherapeutic challenges. Theranostics 4, 931-44 (2014).) However, aptamers are, by definition, high affinity, and particles selected in the multivalent format of DeNAno may bind in a different fashion than these multimerized aptamers. Thus, selection with DeNAno libraries may identify different types of binding molecules than the monovalent, high-affinity aptamers. Specifically, a DeNAno particle may have many low, monovalent affinity interactions that equal a high overall avidity, or the DeNAno may require a minimum copy number to produce the 3D structure required for binding. Aptamers that are made multivalent (even by RCA) would not possess these same qualities.

[0062] The selection process for aptamers and DeNAno is similar. Briefly, in SELEX, a library of 10¹²-10¹⁵ oligonucleotides (DNA or RNA) is incubated with a target, washed or otherwise purified, and re-amplified via defined primer sites at the 5' and 3' ends of the aptamer. The random region of the aptamer is generally 60-80 bp in length. This process is repeated until binding clones dominant the pool. (Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-10 (1990); Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-22 (1990).) The selected aptamers are cloned, sequenced and analyzed, and a binding motif is often identified. These aptamers can have nM-pM affinity, similar to an antibody. Aptamers have been shown to bind via the 3D structure of their primary sequence. Binding is achieved through a combination of van der Waals forces, hydrogen bonding, salt bridges, hydrophobic, and electrostatic interactions. (Banerjee, J. & Nilsen- Hamilton, M. Aptamers: multifunctional molecules for biomedical research. /. Mol. Med. (Bed). 91, 1333-42 (2013); Hermann, T. Adaptive Recognition by Nucleic Acid Aptamers. Science (80-. ). 287, 820-825 (2000).) Selection of DeNAno particles occurs in a similar fashion. DeNAno are made via RCA of circularized oligonucleotide template, using the DNA polymerase, phi29. The resulting DeNAno is a concatemer of single-stranded DNA with sequence complementary to the circularized oligonucleotide template. 10¹⁰-10ⁿ particles are incubated with a target, washed, and re-amplified via defined primer sites at the 5' and 3' ends of the oligonucleotide template. The template strand is enriched via PCR, this template is then circularized, and the selection process is repeated until binding particles dominate the pool. Cell binding DeNAno with primary sequence motifs have been identifie. These particles could also be removed from their target by incubation of an oligo complementary for this motif.

[0063] This example discusses the evaluation of DeNAno particles that bind to specific proteins. Previous DeNAno were selected against unknown targets on the surface of cells, and thus a thorough analysis of the particles' binding properties was not possible. Streptavidin/biotin were used because they are a well-characterized system, and monoclonal antibodies were chosen to confirm these results because of their potential use in immunoassays.

MATERIAL AND METHODS

DENANO PARTICLES AND CHARACTERIZATION

[0064] DeNAno particles were made as previously described. (Steiner, J. M. et al. J. Biotechnol. 145, 330-3 (2010).) Briefly, a lOObp template oligo (Integrated DNA Technologies; IDT, Coralville, IA, USA, all oligos from IDT unless otherwise specified) was circularized via a 40 bp complementary oligo and ligated with T4 ligase (New England Biolabs; NEB, Ipswich, MA, USA). RCA was then performed on this template, using the complementary oligo as the initiating oligo and phi29 DNA polymerase (NEB). RCA was performed at 30 °C for 30 min, with a dNTP concentration of 3 nmol or 93.75 pmol. Enzyme was heat inactivated at 65 °C for 10 min or 95 °C for 5 min. The resulting DeNAno particles are concatemers complementary to the circularized template. Their size is influenced by the amount of time the reaction is run and the concentration of dNTPs (NEB). For fluorescent readouts, particles were labeled with 1/10 molar ratio Alexa Fluor 647-labeled complementary oligo (see Supplemental Table 1 for oligo sequences).

BEADS

[0065] Streptavidin-coated magnetic beads (NEB) were used for selections/staining for strep tavidin- specific DeNAno with no modifications. For rituximab and bevacizumab selections/staining, coated beads were made as follows: 6 μιη polystyrene beads (Polysciences, Warrington, PA, USA) were washed with 20 mM sodium phosphate buffer pH 7.5 (Boston BioProducts, Ashland, MA, USA), then coated with 100 μg/mL rituximab (Genentech, South San Francisco, USA), bevacizumab (Genentech), or polyclonal human IgG (Thermo Fisher Scientific, Waltham, MA, USA) diluted in 20 mM sodium phosphate pH 7.5. Beads and antibody were incubated for 2 hr at room temperature (RT) or overnight at 4 °C. Non-adsorbed antibody was removed with 3x1 mL washes with 20 mM sodium phosphate pH 7.5. Finally, beads were resuspended in PBS supplemented with 0.02% NaN₃ (Ricca Chemical Company, Arlington, TX, USA).

[0066] In one control experiment, streptavidin PMMA (Sapidyne Instruments, Boise, ID, USA) and streptavidin sepharose (GE Healthcare Life Sciences, Piscataway, NJ, USA) were used in place of streptavidin magnetic beads.

DENANO SELECTIONS

[0067] DeNAno selections were performed as previously described, with minor modifications. The nanoparticles were incubated with target beads for 20 min at RT (streptavidin and rituximab selections) or overnight at 4 °C (bevacizumab selection). For streptavidin, the target was streptavidin-coated magnetic beads. For rituximab and bevacizumab selections, the target was rituximab- or bevacizumab-coated beads (described above). Non-binding particles were removed by repeated washes. Bound DeNAno particles were amplified by Hemo KlenTaq (NEB) back to the 100 bp oligo, and the template strand was amplified by asymmetric PCR. The template strand was then re- circularized as above and the entire process repeated through 4-5 rounds of selection. 100 bp oligos were then cloned into pGEM T-easy vector (Promega, Madison, WI, USA), transformed into NEB 5-alpha high efficiency competent cells (NEB) and sequenced via colony PCR (Eton Bioscience Inc, San Diego, CA, USA).

DENANO STAINING

[0068] For initial experiments, clones of selected particles were generated via PCR of the pGEM T-easy insert, followed by asymmetric PCR to amplify the template strand. Circularization and RCA were then performed, as above. RCA conditions were 30 °C for 30 min with 3 nmol dNTP, followed by heat inactivation for 10 min at 65 °C— these are the standard RCA conditions used, unless otherwise noted. Templates for clones of interest were synthesized (IDT).

[0069] All stainings were performed in a pre-blocked 96-well v-bottom plate. Pre-block was PBS (without calcium and magnesium, Mediatech, Manassas, VA, USA) 1% BSA (Sigma, St. Louis, MO, USA) supplemented with 10 mM MgCl₂ (Teknova, Hollister, CA, USA; PBS 1% BSA 10 mM MgCl₂). Unless otherwise noted, approximately 3x10¹⁰ labeled particles were incubated with 2 μL· coated beads for 20 min at RT in PBS 10 mM MgCl₂. Beads were then washed once with PBS 10 mM MgCl₂, twice by Tris-buffered saline (Mediatech) 0.05% Tween-20 (Thermo Fisher Scientific) 10 mM MgCl₂ (TBST 10 mM MgCl₂), once with PBS 10 mM MgCl₂, and resuspended in PBS 10 mM MgCl_2. Washes were performed by magnetic pulldown (streptavidin beads) or centrifugation at lOOOxg for 3 min (rituximab and bevacizumab beads). Fluorescence was measured with multimode microplate reader (TEC AN, Mannedorf, Switzerland).

TRANSMISSION ELECTRON MICROSCOPY.

[0070] All transmission electron microscopy (TEM) Images were taken on a FEI Technai G Sphera transmission electron microscope (FEI, Hillsboro, OR, USA) operating at 200 kV. Copper grids (formvar/carbon-coated, 400 mesh copper, Ted Pella, Inc., Redding, CA, USA) were prepared by glow discharging the surface at 20 mA for 1.5 minutes followed by treatment with 20 μΐ_^ 100 mM MgCl₂ for lmin in order to prepare the surface for DNA nanoparticle adhesion. The solution was wicked away and 4 μL· of DeNAno sample was deposited on the grid and allowed to sit for 30 s. All grids were treated with three drops of 1% w/w uranyl acetate (Mallinckrodt, Dublin, Ireland) to provide negative staining. DeNAno particles were dialyzed into 10 mM Tris 10 mM MgCl₂ pH 8.5 prior to imaging and loading on the grid, using a slide-a-lyzer 10K MWCO dialysis cassette (Thermo Fisher Scientific).

ATOMIC FORCE MICROSCOPY

[0071] Samples were prepared on freshly cleaved muscovite mica (Ted Pella, Inc.). Mica disks were nicked with a scalpel and vacuum cleaved then coated with a 0.005% w/v aqueous solution of poly-L-lysine (PLL, MW 30-70 kDa, Sigma), rinsed with deionized water, and dried overnight in a dessicator. Dialyzed SA-D8 particles (prepared as in TEM) were adsorbed to the PLL-mica for 30 min, rinsed with deionized water, and dried in a dessicator until imaged.

[0072] Images were acquired using ScanAsyst Si3N4 probes (Al-coated, 0.4 N/m spring constant, Bruker, Billerica, MA, USA) with PeakForce Tapping Mode on a MultiMode 8 AFM (NanoScope V Controller, Bruker).

DNTP DILUTION

[0073] For dNTP dilution experiment, standard particles were made - 30 min/30 °C/3 nmol dNTP, as well as 30 min/30 °C/93.8 pmol dNTP. These particles were all heat inactivated at 95 °C for 5 min. Control particles from another library (V10 control) with the same conditions were also made and used as an internal control in the staining. Unlabeled DeNAno particles were mixed with the internal control particles of the same size/condition and incubated with 2 μL· streptavidin beads for 2.5 hr at RT in PBS 1% BSA 10 mM MgCl₂. Samples were resuspended and an aliquot was taken before proceeding to the wash step. Beads were then washed via magnetic pulldown, once with PBS 1% BSA 10 mM MgCl₂, three times with TBST 1% BSA 10 mM MgCl₂, once with PBS 1% BSA 10 mM MgCl₂, and resuspended in PBS 1% BSA 10 mM MgCl₂. Bound and total samples were analyzed by qPCR. A standard was run for each library (a plasmid containing the 100 bp template used to make DeNAno). The ratio of the bound particles (streptavidin DeNAno:control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed.

COMPETITIVE TITRATION

[0074] For competitive titration experiments, biotin, -desthiobiotin, or 2- iminobiotin (all from Sigma) were pre-incubated with streptavidin beads for 20min in DPBS (with calcium and magnesium; Mediatech) 1% fetal bovine serum (FBS; Omega Scientific, Tarzana, CA, USA) or rituximab-specific peptide or irrelevant peptide were pre-incubated with rituximab beads for 20 min in PBS 10 mM MgCl₂. Fluorescently- labeled particles were then added and allowed to incubate for a further 20 min. Washes were performed via magnetic pulldown for streptavidin beads or centrifugation at lOOOx g for 3 min for rituximab beads. A biotinylated particle was used as a positive control for streptavidin experiment.

[0075] For streptavidin competitive titration experiment, free streptavidin was pre-incubated with fluorescently-labeled DeNAno for 20 min in DPBS 1% FBS, followed by addition of streptavidin magnetic beads for a further 20 min. Washes were performed via magnetic pulldown. A biotinylated particle was used as a positive control.

COMPETITIVE RELEASE

[0076] For biotin and streptavidin competitive release experiments, fluorescently-labeled particles were pre-incubated with streptavidin beads in DPBS 1 FBS for 20min at RT. Biotin, -desthiobiotin, 2-iminobiotin, or streptavidin (ProSpec, East Brunswich, NJ, USA) were then added and incubated for an additional 20min. Washes were performed as above. A biotinylated particle was used as a positive control.

[0077] For rituximab/peptide competitive release experiments, particles were pre-incubated with rituximab beads in PBS 10 mM MgCl₂ for 20 min at RT. 50μg/ml rituximab peptide, irrelevant peptide, or buffer were then added and incubated for an additional 1 hr. An aliquot of this total sample was taken, then the beads were spun down at lOOOx g for 3 min and the supernatant was collected. The supernatant was spun down an addition two times to ensure that all beads were removed from the sample. DeNAno content of total and supernatant samples were then measured by qPCR, using a plasmid containing lOObp oligo from the same library as a standard. % release was measured as supernatant DeNAno/total DeNAno * 100%.

DENANO DISSOCIATION

[0078] For dissociation experiment, standard conditions were used to make DeNAno particles. Unlabeled DeNAno particles were incubated with 2 μL· streptavidin beads for 2.5 hr at RT in PBS 1% BSA lOmM MgCl₂ and washed as in the dNTP dilution experiment. Samples were then resuspended in 10 mL PBS 1% BSA 10 mM MgCl₂ and stored at 4 °C. At days 1, 8, 14, 21, 28, and 35 aliquots of total sample (beads+supernatant) and supernatant-only were taken. To acquire supernatant- only, 100 μL· of total sample was incubated on a magnet for 5 min to remove beads from sample. Supernatant was removed to a new well and the procedure was repeated twice more. At day 21, an additional aliquot of total sample was taken and incubated with an excess of free biotin for 30 min. Supernatant from these samples was then acquired, as above. Total and dissociated samples were analyzed by qPCR and quantitated with a standard for that library. % released DeNAno is graphed (dissociated/total* 100%).

PROTEIN G SANDWICH ASSAY

[0079] For protein G sandwich assay, fluorescently-labeled rituximab- specific, bevacizumab-specific, or library negative DeNAno particles or anti-human kappa light chain antibody were used. Protein G magnetic beads (Thermo Fisher Scientific) were incubated with rituximab or bevacizumab for 1 hr at RT in PBS 1% BSA 10 mM MgCl₂. For anti-kappa samples only: mouse IgG2b (eBioscience, San Diego, CA, USA) was added to rituximab and bevacizumab samples that would be less than saturating (<l-fold on graph) to bind all the protein G so anti-kappa antibody would not bind to these non-specifically. DeNAno particles or anti-human kappa light chain antibody Alexa Fluor 488 (Thermo Fisher Scientific Inc) were then added and incubated for 2 hr at RT (no washing step was performed prior to their addition). After incubation, all samples were washed via centrifugation (900x g, 3 min), once with PBS 1% BSA 10 mM MgCl₂, three times with TBST 1% BSA 10 mM MgCl₂, once with PBS 1% BSA 10 mM MgCl₂, and resuspended in PBS 1% BSA 10 mM MgCl₂. Sample fluorescence was read on multimode microplate reader for both Alexa Fluor 488 (anti-kappa) and Alexa Fluor 647 (DeNAno).

DNA MODELING

[0080] mFold (The RNA Institute, College of Arts and Sciences, SUNY Albany, NY, USA) was used for DNA modeling. DNA conditions used were: 4°C conditions with 0.15 m Na⁺ and 0.01 m Mg²⁺ ionic conditions. Representative structures are shown when the output provided more than one structure. Motif analysis was performed with the MEME suite program. (Bailey, T. L. et al. MEME SUETE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202-8 (2009).) RESULTS AND DISCUSSION

STREPTAVIDIN-BINDING DENANO

[0081] A library of DeNAno particles was made as described in Materials and Methods and used in a selection on streptavidin-coated magnetic beads, which is outlined in FIG.. 7 A. All oligonucleotides and primers used are listed in Supplemental Table 1 (MJ library and primers). Following 5 rounds of selection, a population of streptavidin- binding particles emerged, as indicated by a > 10-fold increase in staining by fluorescently-labeled DeNAno from the initial library to the round 5 pool (FIG.. 7B). As a positive control, library particles were incubated with a biotinylated complementary oligo and incubated with the streptavidin beads (Library-bio). qPCR was performed on the round 5-selected particles to reduce the concatemers to their lOObp core. This lOObp PCR product was cloned into pGEM T-easy vector and sequenced. Four sequences were identified, including 1 dominant clone, SA-D8, which represented 11 of the 16 sequences (Table 1).

Table 1. Streptavidin clones

[0082] When tested individually, all four clones bound to streptavidin beads, but not BSA-coated polystyrene beads (FIG. 7C). A random clone from the same library was used as a negative control (GlOneg). This same clone was incubated with a biotinylated complementary oligo and used as a positive control (GlObio). The dominant clone (SA-D8) was also tested on streptavidin-coated sepharose and streptavidin-coated PMMA to confirm binding to streptavidin and not the bead (FIG. 7A, B). Sequence analysis was performed on these clones using MEME suite program and a motif emerged, ACGACGCA (FIG. 8B, C). DNA modeling put this motif in part of a stem-loop structure for each of the 4 clones (FIG. 8A). Interestingly, similar binding motifs or low- homology motifs with conserved nucleotides in the binding region have been reported for streptavidin-binding aptamers from four other laboratories. (Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202-8 (2009); Bing, T., Yang, X., Mei, H., Cao, Z. & Shangguan, D. Conservative secondary structure motif of streptavidin-binding aptamers generated by different laboratories. Bioorg. Med. Chem. 18, 1798-805 (2010); Wang, C, Yang, G., Luo, Z. & Ding, H. In vitro selection of high-affinity DNA aptamers for streptavidin. Acta Biochim. Biophys. Sin. (Shanghai). 41, 335-40 (2009); Bittker, J. A., Le, B. V & Liu, D. R. Nucleic acid evolution and minimization by nonhomologous random recombination. Nat. Biotechnol. 20, 1024-9 (2002); Stoltenburg, R., Reinemann, C. & Strehlitz, B. FluMag-SELEX as an advantageous method for DNA aptamer selection. Anal. Bioanal. Chem. 383, 83-91 (2005).) In these cases, the motif was identified to be important for aptamer binding to streptavidin, and this binding was inhibited by biotin.

[0083] The dominant DeNAno clone, SA-D8, was imaged using TEM (FIG. 8A) and AFM (FIG. 8B). In both cases, discreet 'balls' of DNA were observed, with a diameter of roughly 75 nm (TEM) or 100-250 nm (AFM), similar in size to previous reports using nanoparticle tracking system (Nanosight). The compactness of the imaged DeNAno is partly determined by the amount of salt present in the sample and the preparation method.

[0084] Next, DeNAno particles of different sizes were made to assess their binding capability. dNTP concentration was varied to create two different sized particles. SA-D7 and SA-D8 particles were made, as well as GlOneg, GlObio, and VIOcontrol. VIOcontrol is a clone from a different library which was made in the two different sizes and mixed with the experimental particles for use as an internal control in the subsequent PCR. Bound and total samples were analyzed by qPCR. A standard was run for each library (a plasmid containing the lOObp template used to make DeNAno). The ratio of the bound particles (streptavidin DeNAno:control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed (FIG. 8C). At the standard particle size (labeled 30 m/3 nmol), all particles except GlOneg bound to the streptavidin beads. GlOneg does not bind the beads, thus its ratio with VIOcontrol should be about 1, which is what was observed. At the small particle size (30 m/93.8 pmol), SA-D7 binding dramatically drops off, and is only slightly above background (GlOneg); SA-D8 and GlObio binding are not affected. This result may indicate that SA-D7 DeNAno requires a higher copy number to bind target than SA-D8, or that SA-D7 begins with a lower copy number. However, TEM imaging of the particles shows that SA-D7 does still make a particle at this small size, as does SA-D8 and G10. In summary, at least some DeNAno particles require a minimum copy number to bind their target. This suggests DeNAno are not simply aptamers made into concatemers by RCA, which has been previously demonstrated.

[0085] SA-D8 and SA-D7 were selected for thorough testing of buffer conditions amenable to DeNAno binding. GlOneg and GlObio were used as negative and positive controls. Binding was tested in a variety of buffer conditions: MgCl₂ concentration, NaCl concentration, a panel of standard buffers (mostly Good's buffers), and biologic buffers of different concentrations. For MgCl₂ concentration, binding was observed in >5 mM MgCl₂, with no adverse effects up to 40 mM MgCl₂ (FIG. 9A). For NaCl concentrations from 0-300 mM, NaCl had no effect on particles' ability to bind (FIG. 9B). Next, binding was tested in a panel of standard buffers with pH range from 4- 9.2 (Table 2).

Table 2. Rituximab clones

[0086] High levels of fluorescence were observed in most buffers (MES, HEPES, bicine, CAPSO, carbonate, sodium phosphate, PBS, and TBS), and lower fluorescence signal was observed in PIPES buffer, citrate buffer, and water (FIG. 9C). Citrate may be used as an anti-coagulant to reversibly bind calcium, however, in this assay, it would also compete for Mg²⁺, probably leading to the decrease in binding observed. Surprisingly, all particles bound in DMEM (including GlOneg). Finally, particle binding was tested in biologic buffers— urine, FBS, and human serum, at 1.1, 3.3, 10, and 30% concentration (FIG. 9D). SA-D8 bound streptavidin beads in all conditions except 30% urine, while SA-D7 was more sensitive, failing to bind in 30% urine, 30% FBS, 30% human serum, or 10% human serum. Overall, these results indicate the DeNAno particles bind in a variety of buffers and conditions, and perhaps unsurprising, are suited for the conditions they were selected in, namely 10 mM MgCl_2, 150 mM NaCl, and tris or sodium phosphate buffer. Additionally, the particles' ability to bind in high concentrations of biologic buffers demonstrates potential for diagnostic or in vivo use.

[0087] Next, the dominant DeNAno clone, SA-D8, was tested for binding in the presence of competitor oligo (FIG. 10). An excess of 100 bp oligo the same sequence as the SA-D8 DeNAno was pre-incubated with streptavidin beads, followed by addition of fluorescently-labeled DeNAno. There was 18.75-fold excess oligo over biotin-binding capability of the beads (150 pmol oligo vs 8pmol biotin-binding capacity). Additionally, this was at least 50-fold excess oligo versus total oligos that make up the DeNAno (3xl0⁹ particles added to reaction x 600 copies/particle— based on optimal reaction rate/temperature of the phi29 polymerase). No inhibition of SA-D8 was observed in the presence of SA-D8 competitor oligo or irrelevant oligo, suggesting the DeNAno requires multiple copies of the oligo to bind or that the avidity of the DeNAno is sufficiently greater than the affinity of the individual oligos for streptavidin.

[0088] As streptavidin- specific aptamers have been shown to be removed by biotin, SA-D8 and SA-D7 DeNAno were tested for binding to streptavidin in the presence of biotin and biotin derivatives (competitive titration and competitive release FIG. 9A and 9B). Particles affected by biotin likely bind at/near the biotin binding site, or their binding site is otherwise affected by conformational changes that occur when streptavidin binds biotin. In the competitive titration assay, different concentrations of biotin, desthiobiotin, or 2-iminobiotin were pre-incubated with streptavidin beads, then fluorescently-labeled DeNAno particles were added, further incubated, washed, and fluorescence measured on a multimode microplate reader (GlObio was used as a positive control, FIG. 9A). Biotin has a K_d of about 10^"15 M, desthiobiotin about 10^"11 M, and 2- iminobiotin about 10 -^"8 M (at pH 7.5). All particles were inhibited by high doses of biotin or biotin derivative. In the case of biotin and desthiobiotin, this occurred slightly below the estimated stoichiometric dose of 1: 1 biotin: streptavidin sites (53.3 nM biotin). For 2- iminobiotin, whose K_d decreases with pH due to the ratio of protonated to nonprotonated form, approximately 10000-fold excess was required to inhibit DeNAno binding. Also of note, the SA-D8 and SA-D7 clones' inhibition assumed a different pattern, with SA-D8 exhibiting a sharp decrease in fluorescence, while SA-D7 was a gradual decline. These same clones were also tested in a competitive release experiment, in which streptavidin beads were pre-incubated with fluorescently-labeled DeNAno particles, followed by addition of biotin, desthiobiotin, or 2-iminobiotin, further incubated, washed, and fluorescence measured on a multimode microplate reader (FIG. 9B). GlObio particle, the positive control, showed no decrease in fluorescence, due to the exceptionally slow dissociation kinetics of biotin- strep tavidin. SA-D7 and SA-D8 showed decreased fluorescence for all biotin derivatives at the same concentrations observed for the competitive titration experiment. The same pattern was also observed, in which SA-D8 showed a sharp decrease, with SA-D7 still a gradual decline. Thus, these streptavidin- binding DeNAno particles are inhibited by ligand (biotin or derivatives), but they may also be removed with the ligand. Their removal by ligand essentially transforms a protein binding event into a DNA-based signal that has potential use in high throughput, highly multiplexed protein or biomarker detection assays. With DeNAno particles, each target/ligand binding could become a sequence-able event, amenable to massively parallel analysis approaches. Proteomics data is increasingly used to detect panels of markers on tissues or in disease, rather than individual markers, however, current methods, such as MS, protein microarrays, and bead-based detection assays have certain limitations. These limitations include: expense, specialized equipment, and/or limitations in high-throughput screening of multiple samples/analytes. .

[0089] Surface plasmon resonance (SPR) and kinetic exclusion assays (KCA) were unsuccessfully attempted to obtain a K_d value for the DeNAno particles. In the case of SPR, no reading was obtained, perhaps due to the spacing of the target (strep tavidin) on the chip, as it is likely that one DeNAno binds multiple streptavidin. For KCA, binding to the streptavidin PMMA beads was observed, however, the binding could not be inhibited by free streptavidin, and thus no value could be obtained (data not shown). Instead, a dissociation timecourse was performed, using SA-D7, SA-D8, and positive control GlObio (FIG. 10). Standard size DeNAno were incubated with streptavidin beads, washed extensively, then the DNA-coated beads were resuspended in 10 mL buffer. At days 1, 8, 14, 21, 28, and 35, total and supernatant samples were taken. At day 21, biotin knockoff was also performed, to show the particles could still be removed from the beads, as in FIG. 9. DeNAno binding to the streptavidin beads was remarkably durable, with about 12% unbound at day 35. More impressively, at day 35, this was only 9.9-fold (SA-D7) and 11-fold (SA-D8) above the dissociation of GlObio (FIG. 11). In FIG. 11, the % release was normalized to GlObio. An initial spike in dissociation was observed at day 1 for SA-D7 and SA-D8, but this dropped by day 8, and continued to decrease over time.

[0090] As free biotin/biotin derivatives could inhibit DeNAno binding and binding was not inhibited by free streptavidin in the KCA test, we next tested whether free streptavidin could inhibit DeNAno binding, using the competitive titration assay (FIG. 11). In this experiment, free streptavidin was pre-incubated with fluorescently- labeled DeNAno prior to addition of streptavidin beads. Again, GlObio performed as expected, exhibiting a sharp decrease in fluorescence at the estimated 1 : 1 stoichiometric dose. SA-D7 and SA-D8 exhibited no such decrease; only 100-1000-fold excess streptavidin induced a drop in signal. At this high dose, large aggregates appeared to form, and it was sometimes difficult to pull down the magnetic beads. Thus, the DeNAno particles' behavior appears contrary— they are removed or inhibited by ligand (biotin and derivatives), yet they are not inhibited by 100-1000-fold excess of target (streptavidin). In fact, they seem to bind preferentially to aggregated streptavidin (bead) versus free streptavidin. Particles that bind in the presence of high concentrations of free target may be useful in assays limited by the 'high-dose hook effect'. This effect is observed most often in lateral flow assays (LFA) or other assays that do not employ an intermediate wash step. When there is an overabundance of target in the assay, the detection and/or capture antibodies are limiting, leading to target that is only bound to one antibody and not both (leading to a false negative). (Namburi, R., Ponnala, A. & Kancherla, V. High- dose hook effect. J. Dr. NTR Univ. Heal. Sci. 35 (2014); Butch, A. W. Dilution Protocols for Detection of Hook Effects/Prozone Phenomenon. Clin. Chem. 46, 1719-1720 (2000); Landsteiner, K. The Specificity of Serological Reactions. (Harvard University Press, 1946).)These particles could also be used to identify clusters of protein in the presence of large amounts of free protein, such as amyloid plaques in Alzheimer's, fibrin in clots in the presence of free fibrinogen, and cell- surface antibody in the presence of serum.

RrrUXIMAB- AND BEVACIZUMAB-BINDING DENANO

[0091] Phage display has previously identified peptides that bind specifically to monoclonal antibodies. The DeNAno selection technique was applied to monoclonal antibody-coated polystyrene beads to determine if DNA specific for monoclonal antibodies could also be identified with similar properties to the streptavidin-binding DeNAno. Selection on rituximab-coated beads was done the same as the selection on streptavidin. Bevacizumab selection differed in these ways: round 1 selection was performed with 10-times more particles (3xlOⁿ unique particles) and selection was performed overnight at 4 °C. Subsequent selections rounds were performed for 2-4hr at RT, but with standard number of DeNAno particles (3xl0¹⁰). These conditions were employed because multiple selection attempts with the standard conditions failed. After four rounds, a positive-staining population emerged for both selections and one dominant clone was identified for each selection (FIG. 12 and Tables 2 and 3).

Table 3. Bevacizumab clones

[0092] These clones were tested on specific and irrelevant monoclonal antibody-coated polystyrene beads, as well as polyclonal human IgG-coated polystyrene beads (FIG. 12A). 3Ritl particle (rituximab-specific) bound with >10-fold signal above control beads, and Bevl particle (bevacizumab -specific) bound with >30-fold signal above control beads.

[0093] Next, competitive titration and competitive release experiments were performed with 3Ritl particle and a previously-identified rituximab-specific mimetope peptide, to determine if these DeNAno could also be used as a ligand-receptor sensor, as the streptavidin-binding DeNAno could. This peptide has been shown to bind at the antigen-binding site of rituximab and compete with target cell surface receptor for mAb binding. The K_d of surface immobilized peptide- whole antibody was reported to be 131 nM and 3.99 μΜ for surface immobilized peptide-Fab fragment. Peptide was used in the place of recombinant CD20 due to the amount required for titration and release experiments. For the competitive titration experiment, rituximab peptide or irrelevant peptide was pre-incubated with the rituximab beads, followed by addition of fluorescently-labeled 3Ritl particle. The final concentration of peptide was 0, 0.5, 5, or 50 μg/mL. 3Ritl DeNAno exhibited a decrease in fluorescence in the presence of 50 μg/mL rituximab peptide, and no decrease with irrelevant peptide (FIG. 12B). In the competitive release experiment, fhiorescently-labeled 3Ritl DeNAno was pre-incubated with rituximab beads, followed by addition of 50 μg/mL rituximab peptide, irrelevant peptide, or buffer. Total samples and supernantant samples were compared via qPCR to determine the percent of DeNAno removed from the bead (% release, FIG. 12C). Like the streptavidin- specific DeNAno, rituximab- specific DeNAno was also removed from target by the ligand. This supports the possibility that ligand binding on drug, antibody, or receptor could be detected by removal of DeNAno specific for that same drug, antibody, or receptor, giving a protein binding event an amplifiable DNA signature.

[0094] Finally, 3Ritl and Bevl DeNAno and anti-kappa light chain antibody were tested in a protein G sandwich assay to assess DeNAno's ability to overcome the high-dose hook effect. Wash steps were not used until the end of the protocol, similar to conditions used in an LFA. In this assay, varying concentrations of monoclonal antibody (rituximab or bevacizumab) were pre-incubated with protein G magnetic beads. Mouse IgG was mixed with rituximab and bevacizumab samples that were less than saturating for anti-kappa samples. This was done to fill the open protein G binding sites prior to addition of anti-kappa, so the antibody would only bind the rituximab or bevacizumab, and not the protein G. DeNAno particles or anti-kappa antibody were then added (with no washing) and further incubated. After this incubation, beads were washed via magnetic pulldown and analyzed by multimode microplate reader. The results are graphed as fold-excess free antibody versus fluorescence (FIG. 12D). Even in conditions of 100-fold excess free antibody, both DeNAno particles were able to bind their target with no decrease in signal (left y axis). Also, no increase in background staining was observed on irrelevant beads at these high concentrations and no staining was observed with non-selected particles (lib-neg). Anti-kappa staining, however, peaked at 0.1-fold (bevacizumab) or 1-fold (rituximab) and steadily decreased in fluorescence intensity with increased free antibody (right y axis). These data parallel the results obtained in the free streptavidin competition experiment, and confirms that DeNAno preferentially bind aggregated target over free target.

[0095] DeNAno particles have previously been selected against cellular targets. However, characterization of these particles has been limited by the anonymity of the target. Selection of the particles against a well-characterized protein, streptavidin, has allowed for analysis of binding in a variety of conditions, binding competition, size required for binding, half-life, and to identify unique features of DeNAno. Two key unique features were observed: 1) DeNAno were displaced from target by the ligand and this event could be quantitated by fluorescence or qPCR, (or in the future, high- throughput sequencing) and 2) DeNAno displayed a preference for binding to aggregated versus free target, and they were able to utilize this preference to overcome the high-dose hook effect in the presence of 100-1000-fold excess free target. These particles could be used in LFA assays which do not contain an intermediate wash step, in an assay seeking to bind only aggregated target in the presence or absence of free target, or as a highly multiplexed, DNA readout for protein binding events.

[0096] While the present disclosure has been described and illustrated herein, it is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of multiplexed protein assay and quantitation, the method comprising the steps of:

creating a DNA nanoparticle (DeNAno) particle by rolling circle replication of one or more circular oligonucleotide templates;

creating one or more libraries of DeNAno particles by using templates with random sequences;

selecting the DeNAno particles that bind specifically to magnetic beads coated with antibodies against a target of interest;

synthesizing bead specific DeNAno particles and using the same to mask the beads;

pooling DeNAno masked beads against multiple target antigens, and adding the pooled DeNAno masked beads to an analyte containing sample, wherein the analytes recognized by their cognate antibodies displace the masking

DeNAno particles in a concentration dependent fashion;

removing the beads from the sample; and

amplifying and sequencing the displaced DeNAno particles.

2. The method of claim 1, wherein the DeNAno is produced by rolling circle replication of at least one circular oligonucleotide template.

3. The method of claim 1, wherein the DeNAno is composed of concatemeric repeats of single stranded DNA strands.

4. The method of claim 2, wherein the template comprises random sequences, resulting in a library wherein each nanoparticle comprises a plurality of copies of a unique sequence.

5. The method of claim 1, wherein the DeNAno binds specifically to antibody coated beads.

6. The method of claim 1, wherein the recognition of the analyte by the cognate antibody is with high affinity.

7. The method of claim 1, wherein the DeNAno is produced by ligating a 100 base library with a 60 base random region flanked by two 20-base primer sites, and amplifying the resulting sequence with rolling circle amplification.

8. The method of claim 1, further comprising cloning the DeNAno into bacteria and sequencing the same.

9. A kit comprising: a plurality of DeNAno particles; instructions for their use; and analysis algorithms.

10. A DeNAno particlecomprising a rolling circle replicated circular oligonucleotide.