Next-generation sequencing for protein measurement

Abstract

A method for detecting and quantifying a target molecule, such as a protein, in a biological sample is provided. The disclosed method includes capturing the target molecule with an aptamer, replacing the aptamer with an aptamer-identifying sequence, and sequencing the aptamer-identifying sequence using next-generation sequencing technology.

Description

[Technical Field]

[0001] Related applications

[0002] The following specification and materials are incorporated herein by reference in their entirety for all purposes: U.S. Provisional Patent Application No. 63 / 294,964, filed December 30, 2021.

[0003] Technical field This disclosure relates to a system and method for quantitatively measuring proteins in a biological sample. More specifically, the disclosed embodiments relate to capturing a target protein with a specially designed aptamer, preparing an elution of the aptamer capturing the target protein, and then replacing the aptamer in the elution with a “reporter” DNA molecule that can be sequenced more easily than the aptamer itself.

[0004] Overview Traditionally, various attempts to evaluate gene activity and / or decipher biological processes such as disease processes or the biological processes of pharmacological action have focused on genomics. However, proteomics can provide further information about the biological functions of cells and organisms. Proteomics involves qualitatively and quantitatively measuring gene activity by detecting and quantifying expression at the protein level rather than the gene level. Proteomics also includes the study of non-genetically encoded events, such as post-translational modifications of proteins and protein-protein interactions.

[0005] Currently, it is possible to obtain vast amounts of genomic information. DNA chips are being put into practical use as molecular arrays for this purpose, and the price of direct DNA sequencing continues to fall sharply. Similarly, the demand for high-throughput proteomics is also increasing. For health monitoring, proteomics is far superior to genomics because the genome is static and only indicates medical potential, while the proteome changes dynamically in response to a patient's medical condition and can even be said to define the patient's medical condition. However, while the detection and quantification of proteins is difficult, the detection and quantification of nucleic acids is relatively easy because, at least in part, proteins have more complex and diverse biological functions than DNA. For this reason, many efforts are underway to measure mRNA (messenger RNA) concentration as a surrogate for protein concentration. However, mRNA concentration has been shown not to correlate well with protein concentration. Proteomics seems to rely on its ability to directly detect proteins.

[0006] One method for detecting and quantifying the presence of specific proteins in biological samples is through the use of protein capture SOMAmer® (Slow Off-rate Modified Aptamer) reagents. SOMAmer reagents consist of chemically modified nucleotides, which greatly expand the physicochemical diversity of the large, randomized nucleic acid library from which the SOMAmer reagent is selected. Assays using SOMAmer reagents measure native proteins in a complex matrix by converting the individual protein concentrations to the corresponding SOMAmer reagent concentrations, which are then quantified by standard DNA techniques such as microarrays or qPCR.

[0007] SOMAmer reagents are single-stranded DNA-based protein affinity reagents containing chemically modified nucleotides that mimic amino acid side chains, expanding the chemical diversity of standard aptamers and enhancing the specificity and affinity of protein-nucleic acid interactions. These modified nucleotides are incorporated into a nucleic acid library used in a repeated selection and amplification process called SELEX (Systematic Evolution of Ligands by Exponential Enhancement), from which SOMAmer reagents are selected. Using a SELEX-type process, SOMAmer reagents can be generated to capture proteins that were resistant to selection by unmodified nucleic acids (ACTG conventional aptamers). SOMAmer reagents can also be adapted to select desired properties such as specificity and slow off-rate, as well as to mimic the assay conditions in which the reagent is used.

[0008] SOMAmer-based assays convert the presence of a protein in a sample into a specific SOMAmer-based DNA signal. Following the SOMAmer-protein binding step, a series of distribution and washing steps are performed, where the relative protein concentration is converted into a measurable nucleic acid signal. This nucleic acid signal is then quantified using DNA detection techniques, such as hybridization of fluorescently labeled SOMAmers with a custom DNA microarray. When the microarray is laser-scanned, the reading in relative fluorescence units (RFU) is directly proportional to the amount of the target protein in the initial sample.

[0009] Quantifying protein detection using microarray hybridization has several drawbacks. These include limited scalability, constant assay costs, and limited commercial supply of microarrays. Therefore, it is desirable to develop alternative methods for quantifying SOMAmer molecules in the eluate after capture. [Overview of the project]

[0010] This disclosure provides systems, apparatus, and methods for the detection and quantification of proteins using parallel sequencing techniques known as next-generation sequencing or NGS. More specifically, this disclosure relates to a hybridization capture (HC) technique in which a SOMAmer eluting molecule that signals protein capture is replaced with a “reporter” DNA molecule containing a SOMAmer-specific identification tag or “SOMA ID,” which can then be sequenced using NGS techniques.

[0011] In some embodiments, the present disclosure is a system and method for quantifying the abundance of a target protein in a biological sample, comprising: capturing the target protein by exposing the biological sample to a plurality of aptamers, each configured to bind to a specific protein; isolating an aptamer that captures one of the target proteins in an aptamer-containing eluate; and forming a plurality of tripolecular complexes by exposing the aptamers in the eluate to a plurality of capture probes, each configured to hybridize to a specific aptamer, wherein each tripolecular complex hybridizes one of the aptamers from the eluate with a first portion of the aptamer. The present invention relates to a system and method comprising: forming a tripolecular complex comprising a first capture probe containing a portion and a second capture probe containing a portion hybridized to a second portion of the aptamer, a DNA primer region, and an aptamer ID sequence corresponding to the aptamer; separating the tripolecular complex from the capture probe not bound to the aptamer; dissociating the capture probe in the tripolecular complex from the corresponding aptamer; amplifying the aptamer ID sequence in the dissociated capture probe; sequencing the aptamer ID sequence by next-generation sequencing; and determining the abundance of a target protein in a biological sample based on the data obtained from sequencing the aptamer ID sequence.

[0012] In some embodiments, the present disclosure relates to a system and method for quantifying the abundance of two or more target proteins in a biological sample, comprising: capturing the target proteins by exposing the biological sample to a plurality of aptamers, each configured to capture a specific protein; forming an aptamer-containing eluate by isolating an aptamer that has captured one of the target proteins in the biological sample; forming a plurality of tripolecular complexes, each comprising a specific aptamer present in the aptamer-containing eluate, a first probe hybridized to a corresponding first portion of the specific aptamer, a portion hybridized to a corresponding second portion of the specific aptamer, at least one DNA primer region, and a second probe comprising an aptamer ID sequence corresponding to the specific aptamer; amplifying the aptamer ID sequence; sequencing the aptamer ID sequence; and quantifying the abundance of the target protein based on the sequenced aptamer ID sequence.

[0013] In some embodiments, the present disclosure relates to a system and method for detecting a target protein in a biological sample, comprising: capturing a target protein with an aptamer by combining the biological sample with a plurality of aptamers, each configured to capture a specific protein; forming a tripolecular complex comprising: a first probe comprising the aptamer capturing the target protein, a portion hybridized to a corresponding first portion of the aptamer capturing the target protein, and a second probe comprising a portion hybridized to a corresponding second portion of the aptamer capturing the target protein, at least one DNA primer region, and an aptamer ID sequence corresponding to the aptamer capturing the target protein; amplifying the aptamer ID sequence; and sequencing the aptamer ID sequence to identify the aptamer ID sequence, thereby identifying the aptamer capturing the target protein and the target protein.

[0014] In some embodiments of the present disclosure, the aptamer used to capture the target protein may be directly sequenced, for example, using next-generation sequencing technology, without using tripolecular complexes, in order to convert the aptamer into a simpler sequence.

[0015] In some embodiments of this disclosure, the aptamer used to capture the target protein may be a SOMAmer.

[0016] In some embodiments of the present disclosure, the aptamer-containing eluate may be divided into groups before and / or after exposure to the hybridized probe region. In some examples, some or all of the eluate groups may be diluted to a desired extent.

[0017] In some embodiments of the present disclosure, a quantitative spike reporter may be added at a desired assay step to compensate for complementary changes in the count proportionality of the analyte.

[0018] The features, functions, and advantages may be achieved independently in various embodiments of this disclosure, or in combination in even more embodiments, and further details can be found by referring to the following description and drawings. [Brief explanation of the drawing]

[0019] [Figure 1] This is a schematic diagram of a tripolecular complex comprising an aptamer and two probes hybridized to the aptamer, according to an aspect of the present disclosure. [Figure 2A] This flowchart shows the steps of an exemplary method for preparing a hybridization probe necessary to prepare the tripolecular complex shown in Figure 1, according to an embodiment of the teachings of the present invention. [Figure 2B] This flowchart shows the steps of an exemplary method for producing the tripolecular complex shown in Figure 1, according to an embodiment of the teachings of the present invention. [Figure 3A]A flowchart showing steps of an exemplary method for performing a next-generation sequencing assay using a trimolecular complex such as the complex shown in FIG. 1, according to an aspect of the teachings of the present invention. [Figure 3B] A flowchart showing steps of an exemplary method for performing a next-generation sequencing assay that includes capturing a target protein and then forming a trimolecular complex such as the complex shown in FIG. 1, according to an aspect of the teachings of the present invention. [Figure 4] A flow diagram showing steps and by-products of an exemplary next-generation sequencing assay that includes four hybridization groups, according to an aspect of the teachings of the present invention. [Figure 5] A flow diagram showing steps and by-products of an exemplary next-generation sequencing assay that includes a single hybridization group and four PCR groups, according to an aspect of the teachings of the present invention. [Figure 6] A flow diagram showing steps and by-products of an exemplary next-generation sequencing assay that includes four hybridization groups and four PCR groups, according to an aspect of the teachings of the present invention. [Figure 7] A graph showing the results of a virtual simplified two-analyte assay of two individual samples in histogram form. [Figure 8] A graph showing the results of the assay of FIG. 7 in which a quantitative spike (qSpike) control reporter added at the same concentration was added to both samples, according to an aspect of the teachings of the present invention. [Figure 9] A graph showing in histogram form the raw results of three analyte SOMAmer assays of eight separate samples at different analyte concentrations, in which four different qSpike reporters were added to each sample, according to an aspect of the teachings of the present invention. [Figure 10] A graph showing in histogram form the normalized results of the assay of FIG. 9, according to an aspect of the teachings of the present invention. [Figure 11]This flowchart shows some steps and byproducts of an exemplary next-generation sequencing assay, including four PCR groups and a qSpike control reporter added to each group, according to an embodiment of the teachings of the present invention. [Figure 12] This is a graph of relative fluorescence units (RFU) versus temperature obtained by superimposing experimentally obtained single-phase thermal fusion data and a two-state theoretical model curve fitting for a SOMAmer probe double-chain according to the teaching aspects of the present invention. [Figure 13] This is a graph of relative fluorescence units (RFU) versus temperature, obtained by superimposing experimentally obtained biphasic thermal melting data for a SOMAmer probe double-strand according to an embodiment of the teachings of the present invention with a biphasic theoretical model curve fitting. [Modes for carrying out the invention]

[0020] Various embodiments and examples of hybridization-capture, next-generation sequencing assay systems for protein detection and quantification, as well as related methods, are described below and illustrated in the relevant drawings. Unless otherwise specified, the protein assays and / or various components thereof according to the teachings of the present invention may include at least one of the structures, components, functions, and / or variations described, illustrated, and / or incorporated herein. Furthermore, unless otherwise specifically excluded, the process steps, structures, components, functions, and / or variations described, illustrated, and / or incorporated in connection with the teachings of the present invention may be included in other similar devices and methods, including being interchangeable between the disclosed embodiments. The descriptions of the various examples below are purely descriptive and are not intended to limit the disclosure, application, or use thereof. Furthermore, the benefits provided by the examples and embodiments described below are purely illustrative, and not all examples and embodiments will have the same or to the same degree of benefits.

[0021] The section "Modes for Carrying Out the Invention" includes the following sections immediately following it: (1) Definitions; (2) Overview; (3) Examples, Components, and Alternatives; (4) Advantages, Functions, and Benefits; and (5) Conclusion. The section "Examples, Components, and Alternatives" is further divided into subsections, each of which is appropriately presented.

[0022] definition Unless otherwise specified, the following definitions apply in this book.

[0023] "Comprising," "including," and "having" (and their conjugations) are used interchangeably to mean that "comprising" is not necessarily limited to these, and are unrestricted terms not intended to exclude additional, unlisted elements or steps of method.

[0024] Terms such as "first," "second," and "third" are used to distinguish or identify different members of a group, etc., and are not intended to indicate a sequential or numerical limitation.

[0025] "AKA" means "also known as" and can be used to indicate an alternative or corresponding term for a given element or group of elements.

[0026] Terms indicating direction, such as "up," "down," "vertical," and "horizontal," should be understood within the context of the specific object being referred to. For example, an object can be oriented around defined X, Y, and Z axes. In these examples, the XY plane defines horizontal, up is defined as the positive Z direction, and down is defined as the negative Z direction.

[0027] In the context of a method, “providing” may include receiving, obtaining, purchasing, manufacturing, producing, processing, pre-processing, and / or similar actions so that the provided object or material is in a state and configuration for performing the other steps.

[0028] "NGS" stands for "Next Generation Sequencing."

[0029] "HC" stands for "hybridization - capture".

[0030] "SOMAmer" refers to the "Slow Off-rate Modified Aptamer" reagent developed and manufactured by SomaLogic Operating Co., Inc. ("SomaLogic") (Boulder, Colorado).

[0031] A "SOMAmer ID sequence," "SOMA ID," or "reporter" refers to a portion of a tripolecular complex containing SOMAmer-specific DNA strands that can be sequenced using NGS technology.

[0032] A "quantitative spike," "reporter spike," or "qSpike" refers to an amplified reporter used to normalize complementary read counts between samples, allowing for the recognition of true signal changes.

[0033] This disclosure may incorporate by reference one or more publications, patents, and / or patent applications. However, such materials are incorporated only to the extent that there is no inconsistency between the incorporated materials and the descriptions and drawings contained herein. In the event of any such inconsistency, including any inconsistency in terminology, this disclosure shall prevail.

[0034] overview Generally, this disclosure relates to methods for detecting and quantifying target molecules, such as proteins, in biological samples. The disclosed methods may include capturing a target molecule with an aptamer, replacing the aptamer with an aptamer identification sequence, and then sequencing the aptamer identification sequence using next-generation sequencing technology. Alternatively, the disclosed methods may include directly sequencing the aptamer after capturing the target molecule with it.

[0035] Examples, components, and alternatives The following sections describe selected embodiments, related systems, and / or methods for the detection and quantification of proteins using aptamers such as SOMAmer reagents, where the SOMAmer is replaced by hybridization capture with a reporter DNA molecule containing a SOMAmer-specific segment that can be sequenced using next-generation sequencing technology. The examples in these sections are for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Each section may include one or more distinct embodiments or examples, and / or contextual information or related information, functions, and / or structures.

[0036] A. Exemplary aptamers This section describes SOMAmers (Slow Off-rate Modified Aptamers), which are exemplary examples of aptamers that are suitably used in conjunction with the examples of systems and methods described herein.

[0037] The method known as "Systematic Evolution of Ligands by Exponential Enrichment," sometimes called the SELEX process, has revealed that nucleic acids possess three-dimensional structural diversity very similar to that of proteins. The SELEX process is a method for evolving nucleic acid molecules in vitro with respect to specific desired activity. Here, we describe SELEX for generating nucleic acid molecules that bind highly specifically to target molecules. The SELEX process provides any class of products called nucleic acid ligands or aptamers, each possessing a unique sequence and the property of specifically binding to a desired target compound or molecule. Each nucleic acid capture reagent identified by SELEX is a specific ligand for a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient ability to form a variety of two-dimensional and three-dimensional structures and possess sufficient chemical versatility to be available within their monomers, thereby acting as ligands (forming specific binding pairs) with virtually any chemical compound, whether monomer or polymer. Molecules of any size or composition can act as targets.

[0038] The SELEX method, applied to high-affinity binding applications, involves selecting from a mixture of candidate oligonucleotides and, using the same general selection scheme, sequentially repeating binding, distribution, and amplification to achieve substantially any desired criteria for binding affinity and selectivity. Preferably, the SELEX method includes the steps of: starting with a mixture of nucleic acids containing any segment of a randomized sequence; contacting the mixture with a target under binding-favorable conditions; distributing unbound nucleic acids from nucleic acids specifically bound to the target molecule; dissociating the nucleic acid-target complex; amplifying the nucleic acids dissociated from the nucleic acid-target complex to produce a ligand-rich mixture of nucleic acids; and then repeating the binding, distribution, dissociation, and amplification steps for a desired number of cycles to produce a highly specific, high-affinity nucleic acid ligand for the target molecule. In this way, aptamers suitable for binding to substantially any target protein can be discovered.

[0039] More specifically, SOMAmers are protein-binding aptamers discovered through improvements to the SELEX process, and generally exhibit a dissociation rate of 30-240 minutes (t 1 / 2 SOMAmers have a time interval (SOMA), which is the average time required for half of the protein-aptamer complex to dissociate. Furthermore, SOMAmers contain modified nucleosides that provide different integration functionalities. These functionalities may include tags for immobilization, labels for detection, means to facilitate or control separation, amino acid-like side chains to provide better affinity to proteins, and / or similar. Modifications to improve affinity to proteins are typically chemical groups that bind to the 5th position of the pyrimidine base. Functionalizing the 5th position with a protein-like moiety (e.g., benzyl, 2-naphthyl) expands the chemical diversity of SOMAmers, thereby enabling high-affinity binding to an even wider range of target molecules. In addition, some polymerases can still transcribe DNA with modifications at these positions, thus enabling the amplification required for the SELEX process.

[0040] While SOMAmer-containing binding aptamers are generally discovered by the SELEX process, it should be noted that other means of selection may exist. For example, as computer modeling of molecular interactions improves, it may become possible to directly calculate the ideal nucleic acid sequence for an aptamer and the relevant chemical modifications for a SOMAmer to generate capture reagents that are specific to a given target molecule. Other chemical techniques besides SELEX are also possible for screening aptamers and SOMAmers.

[0041] Assays aimed at detecting and quantifying physiologically important molecules in biological and other samples are essential tools in scientific research and healthcare. Each SOMAmer can bind to target molecules in a sample very specifically and with very high affinity. After appropriate washing and distribution steps, unbound proteins are first removed, followed by unbound SOMAmers, and then the SOMAmers are eluted from the resulting SOMAmer-protein complex. The SOMAmer eluate is then contacted with a microarray containing SOMAmer complements to enable determination of the presence, absence, quantity, and / or concentration of target molecules in the sample.

[0042] B. Exemplary Approaches to Hybridization Capture Assays This section describes a targeted hybridization-capture (HC) assay in which the SOMAmer signal from the assay eluate is replaced with a "reporter" DNA molecule containing a SOMAmer-specific identification sequence or "SOMA ID" for sequencing.

[0043] Prior to the HC assay described in this section, the SOMAmer binding step has already been performed, yielding an eluate containing a SOMAmer reagent that indicates the presence of the corresponding target protein in the sample. For example, but not limited to, the following steps may be performed to obtain a SOMAmer-containing eluate. (1) Immobilize a protein-specific SOMAmer reagent labeled with a 5' fluorescent dye, a photodegradable linker, and biotin onto streptavidin (SA) coated beads and incubate it with one or more samples containing a complex mixture of proteins; (2) SOMAmer-target protein complexes are formed on the beads; (3) Wash the beads to remove unbound proteins and tag the bound proteins with biotin. (4) The SOMAmer-protein complex is released from the beads by photodegradation of the linker by ultraviolet light; (5) By incubating in a buffer containing a polyanion competitive substance, the recombination of dissociated proteins is prevented, thereby kinetically enriching the target protein-specific complex that binds at a slow off-rate compared to the fast off-rate interaction that represents the binding of the target protein to the corresponding SOMAmer; (6) The SOMAmer-protein complex is recaptured on a second set of streptavidin-coated beads via a biotin-tagged protein, and then an additional washing step is performed to facilitate further removal of nonspecifically bound SOMAmer reagent; and (7) The SOMAmer reagent is released from the beads in the denaturation buffer, forming a SOMAmer-containing eluate suitable for quantitative analysis.

[0044] Now, turning to the hybridization-capture approach, the main focus of this section, Figure 1 schematically shows a tripolecular complex, typically denoted as 100, which can be used in next-generation sequencing assays to identify target proteins. Complex 100 contains SOMAmers after the assay, i.e., SOMAmer 102, which is one of the SOMAmers that remain in the SOMAmer-containing eluate after exposure to the biological sample and (e.g.) the other steps described above. In other words, the presence of SOMAmer 102 in the post-assay eluate indicates the presence of the corresponding target protein (or other target molecule) in the sample.

[0045] The complex 100 further comprises a first probe 104 and a second probe 106. The first probe 104 includes a hybridization region H1 complementary to the left portion of SOMAmer 102 in Figure 1. The second probe 106 includes a hybridization region H2 complementary to the right portion of SOMAmer 102 in Figure 1, common primer regions P1 and P2, and a unique SOMAmer identification sequence I corresponding to SOMAmer 102, as will be described in more detail below. s Alternatively, it may also include "SOMA ID". The positions of H1 and H2 may be reversed, and the common primer regions P1 and P2, as well as the unique SOMAmer identification sequence I in H2, may be reversed. S By appropriately inverting them, H1 hybridizes to the right side of SOMAmer102, and H2 hybridizes to the left side of SOMAmer102.

[0046] Hybridization regions H1 and H2 are configured to specifically bind to their respective complementary portions of the corresponding SOMAmers and may be designed to have similar melting temperatures (Tm) to achieve uniform hybridization under a given set of assay conditions. The hybridization regions of complex 100 may be designed and fabricated, for example, according to the following steps.

[0047] First, the target region of the probe is determined on the SOMAmer. For truncated SOMAmers, the target region may be the complete SOMAmer sequence containing the five bases of the fixed region used for amplification by SELEX from each end of the random region. For full-length SOMAmers, the SOMAmer may be truncated in silico (i.e., computationally) to any desired length, such as 50mer, and then the hybridization completion is determined.

[0048] Next, a boundary is determined to divide the target region into two parts. In some embodiments, the melting temperature of a 25-mer (e.g.) double chain between the SOMAmer and both H1 and H2 is determined by computer to achieve similar melting temperatures in the two hybridization regions, and then the boundary between the two regions is modified stepwise until the melting temperatures are maximally balanced between the H1-SOMAmer double chain and the H2-SOMAmer double chain. In other embodiments, different melting temperatures may be intentionally selected, for example, a first melting temperature (e.g., 45°C) for the H1 probe and a second melting temperature (e.g., 35°C) for the H2 probe.

[0049] Length constraints may also be imposed on the hybridization regions. For example, a minimum length of 18 mer may be set for both H1 and H2. Similarly, a maximum length of 30 mer may be set for H2, for example, to ensure that the sum of H2 and the remaining reporter portion of the second probe remains below the desired maximum length for subsequent synthesis, for example, 100 base pairs. Under these constraints, the hybridization regions H1 and H2 can be generated by computer.

[0050] Common primer regions P1 and P2, and SOMAmerID sequence I of the second probe 106 S When generating a sequence, various factors can be considered. For example, in the design of a sequencing amplicon for counting applications, it is necessary to find a good balance between the need for short and inexpensive reads and the need for sequences that are long enough and contain enough information to serve as identifier sequences such as SOMAmer IDs used for counting and barcode sequences used for multiplexing. For these reasons, when scaling the content, the length of the reporter region, which ultimately becomes the largest part of the sequencing template, may be limited. For example, the length of primer regions P1 and P2 may be limited to 24mers, and the SOMAmer ID sequence I SThe length may be restricted to 15mers, the edit distance is at least 5, and the length of any homopolymer does not exceed 2mers. Other length constraints and selections are possible for both the primer region and the SOMAmer ID sequence.

[0051] Figure 2A is a flowchart illustrating the steps of an exemplary method 200 for preparing hybridization probes H1(104) and H2(106) used to form the tripolecular complex in Figure 1. Step 202 provides a set of SOMAmer sequences to be used in the assay.

[0052] In step 204, hybridization probe regions H1 and H2 are fabricated. These hybridization probe regions can be determined by computer under various length and / or other constraints, for example, as described above. Also, as previously mentioned, in some examples, the hybridization regions may be allocated from a single SOMAmer complementary structure based on factors such as the balance of melting temperatures of each region.

[0053] Step 206 is dedicated to each SOMAmer and assigns a unique SOMAmer ID (I S A region is created. The SOMAmer ID region can be designed in various ways. For example, I S The regions can be designed "by eye" (for example, to have the maximum edit distance) or they can be computer-generated in combination with the computer generation of hybridization regions H1 and H2. After the library of SOMAmer ID regions is generated, SOMAmer IDs are assigned to SOMAmers randomly or in any other preferred method to create a unique reporter corresponding to each SOMAmer.

[0054] In step 208, universal primer regions P1 and P2 are prepared. Universal primers may be designed for stability and to reduce the risk of downstream bias. For example, in some cases, the primers may have an estimated melting temperature of about 70°C and a length of 24mer or 25mer. In some cases, the primers may be terminated with guanine (G) at the 3' end for stability. In some cases, the primers may be evaluated with an oligonucleotide (oligo) analyzer to reduce the potential risk of dimerization. In some cases, the primers may be further modified to avoid nonspecific interactions with functional oligos used in known sequencing techniques.

[0055] In step 210, first and second SOMAmer-specific probes (sometimes called "capture probes") are prepared. Each first probe includes one or more elements suitable for binding to assay beads, for example, a hybridization region H1 with biotin for binding to streptavidin-coated beads. The first probe may also include additional elements such as a photodegradable linker. Each second probe includes a hybridization region H2, universal primer regions P1 and P2, and SOMAmer ID sequence I S This includes the following. As part of preparing a second probe, the SOMAmer ID region may be added to a universal primer to prepare an amplified reporter.

[0056] Figure 2B is a flowchart illustrating the steps of an exemplary method 250 for preparing tripolecular complexes, such as the tripolecular complex in Figure 1. In step 252, a SOMAmer-containing eluate is provided, and the SOMAmer in the eluate indicates the presence of one or more target proteins or other target molecules in one or more biological samples exposed to the SOMAmer library, as described above.

[0057] In step 254, the set or library of SOMAmer-specific capture probes prepared by method 200 is combined with the post-assay SOMAmer-containing eluate. For example, 25 μl of SOMAmer-containing eluate is combined with 25 μl of probe-containing solution to obtain a hybridization volume of 50 μl. To facilitate hybridization, the capture probes may have a concentration equal to or higher than the concentration of SOMAmer in the eluate. For example, a suitable probe concentration may fall in the range of 0.05 nM to 5.0 nM, such as 0.5 nM (nM = nanomoles per liter).

[0058] In some examples, in optional step 253 of Method 250, the SOMAmer-containing eluate may be split and selectively diluted before hybridization and then recombined before sequencing. More specifically, the post-assay eluate may be split into two or more dilution groups (e.g., four dilution groups) based on the expected relative abundance of SOMAmers within each group. The least concentrated (most diluted) sample may contain the most abundant SOMAmer in the eluate. Conversely, the most concentrated (least diluted) sample may contain the least abundant SOMAmer in the eluate. In this way, the SOMAmer count may be "leveled" to improve accuracy and precision in detecting less abundant SOMAmers. Each dilution group may be hybridized individually by exposure to a corresponding subset of probes. Further details regarding the use of dilution groups are provided in later sections of this disclosure.

[0059] Furthermore, leveling may be achieved by introducing H1 probes with and without capture tags in fixed ratios to specific high abundances of SOMAmer in the eluate. SOMAmer that form tripolecular complexes with the H1 probe without capture tags are removed during the washing step, as detailed below in Method 300.

[0060] In step 256, the first probe and the second probe hybridize to the SOMAmer, forming a trimolecular complex each containing (i) the SOMAmer, (ii) the first probe bound to the SOMAmer via hybridization region H1, and (iii) the second probe bound to the SOMAmer via hybridization region H2. Each of the second probes contains the SOMAmer ID sequence I S which can be sequenced to show the presence of the corresponding SOMAmer in the eluate and thus the presence of the corresponding protein captured by that SOMAmer in the original biological sample. Hybridization of the capture probe and the SOMAmer can be performed using any suitable technique, such as appropriate thermal cycling, and may include additives to enhance the hybridization rate.

[0061] FIG. 3A is a flowchart illustrating the steps of an exemplary method 300 for performing a next-generation sequencing assay using a trimolecular complex produced by a method such as the complex depicted in FIG. 1 and a method such as the method of FIG. 2B. In step 302, hybridized trimolecular complexes corresponding to a desired library of SOMAmer after the assay (e.g., each having a structure such as the structure of complex 100 and produced according to a method such as method 250) are provided for the sequencing assay.

[0062] In step 304, the trimolecular complex is captured on magnetic beads. For example, the complex can be captured via the binding of biotin bound to hybridization region H1 of the first probe and streptavidin on the beads. Capture can be achieved using any suitable technique. For example, in one example, the hybridization volume can be combined with a 30 μl solution containing beads at a concentration of 20 mg / ml and then mixed in a thermomixer at 45° C. and 1200 rpm for 30 minutes.

[0063] In step 306, the solution containing the bead-capture probes is washed one or more times to remove unbound H2 probe reporters, i.e., probes that have not hybridized to the corresponding SOMAmer. For example, washing may be carried out with a suitable buffer such as 20 mM phosphate buffer containing 1 mM EDTA and 0.05% SDS (sodium dodecyl sulfate). The washing step may be carried out statically, dynamically using a thermomixer, or a combination of both types in sequence. In one example, there may be two static 5-minute washing phases and two 10-minute dynamic washing phases at 1200 rpm. In any case, after washing, the resulting solution should contain the tripolecular complex bound to the beads, with at most a small amount of unbound H2 probe / reporter remaining.

[0064] In certain embodiments of SOMAmer elution leveling, or dynamic range compression, tripolecular complexes without bead-capture tags on H1 are removed in step 306, similar to unbound H2 probe reporters. These complexes reduce the number of copies of these SOMAmers in the final NGS sequencing, thereby lowering the count on these more abundant SOMAmers.

[0065] In step 308, the bound trimolecular complex is eluted from the bound beads, for example, by exposure to a solvent, heat, or any other suitable elution method. As part of this step, components of the complex may dissociate, yielding separated SOMAmer and probe. In one example, the complex is eluted by adding 85 μl of 20 mM NaOH to the eluate containing the bound complex, then mixing in a thermomixer at 1200 rpm for 3 minutes, and then mixing for 5 minutes to distribute the complex. The solution containing the eluted complex may then be combined with 20 μl of HCl.

[0066] In some examples, the elution solution obtained from step 308 (i.e., the eluate) may be divided and / or diluted into two or more groups, such as four dilution groups or primer amplification groups, in the optional step 309 of Method 300. As previously stated in the context of Method 250, using multiple dilution groups or primer amplification groups (which may not be diluted in some examples) corresponding to subsets of SOMAmers with different expected abundances helps to equalize the relative abundances of the overall set of SOMAmers or compress broad distributions to improve accuracy and precision in detecting relatively rare target molecules. Dividing into such groups can be done before hybridization (as in step 253 of Method 20), after hybridization (as in step 309 described herein), or both. Further details of possible dilution and recombination techniques are described in later sections of this disclosure.

[0067] In step 310, the solution(s) obtained from step 308 and the optional step 309 are prepared for next-generation sequencing (NGS). This involves the relevant SOMAmer ID sequence I S The preparation of the NGS may also include PCR amplification of a reporter region containing common primers (P1 and P2 in Figure 1). If elutes from multiple samples are combined before sequencing, the preparation may also include conjugating adapter sequences and / or barcode sequences for demultiplexing. The preparation of the adapter sequences and barcode sequences and their conjugation to the reporter region can be achieved by any preferred method known in the art, as is common when preparing samples for next-generation sequencing. In some examples, the barcode sequence may be added as part of a first preparation step, and the NGS adapter may be added as part of a second preparation step.

[0068] In the optional step 311, any groups that remained separated after step 310 may be recombined for preparation for sequencing.

[0069] In step 312, the prepared sample(s) are sequenced using next-generation sequencing technology. In some examples, the prepared sample(s) may be sequenced using a next-generation sequencing platform developed by Illumina, Inc. (San Diego, California). However, the method disclosed herein is also suitable for use with other NGS sequencing platforms.

[0070] After NGS, the data obtained by sequencing may be analyzed or otherwise processed in optional step 314 to determine the concentration of analytes, such as target proteins, in the original biological samples. Generally, this analysis involves demultiplexing the sequencing data using barcodes corresponding to each original sample (if multiple samples are multiplexed), counting reporter sequences, and scaling and / or normalizing the data to extract accurate results. For analytical purposes, the sequencing data may be written to a data file in a standard format, such as the ADAT format developed by SomaLogic. Possible quantitative analysis methods are discussed in detail below.

[0071] Figure 3B is a flowchart illustrating the steps of exemplary Method 350 for performing a next-generation sequencing assay, which includes capturing a target protein with an aptamer, forming a tripolecular complex from the aptamer, and using the tripolecular complex as a basis for identifying the captured target protein. Note that some details may not be repeated, as each step of Method 350 may be similar to the corresponding steps of the previously described methods (i.e., Methods 200 and 300).

[0072] In step 352 of method 350, the target protein is captured by exposing the biological sample to multiple aptamers, e.g., SOMAmers, each configured to bind to a specific protein. By exposing the sample to a library of numerous such SOMAmers, a large number of target protein species can be detected in a single assay.

[0073] In step 354, an aptamer that has captured one of the target proteins is isolated in an aptamer-containing eluate. Forming an aptamer-containing eluate may be achieved, for example, in a so-called SomaScan assay process performed by SomaLogic, which includes binding the aptamer to assay beads, capturing the protein with the aptamer, washing away the unbound protein, tagging the bound protein with biotin, releasing the aptamer from the beads, capturing the tagged protein on new beads, removing the unbound aptamer, denaturing the aptamer from the captured protein, and then separating the aptamer into the eluate.

[0074] In the optional step 356, the aptamer-containing eluate may be divided into multiple groups, which may be dilution groups, with respect to Figures 4-6 and 11, as described in more detail below.

[0075] In step 358, multiple tripolecular complexes are formed by exposing aptamers in the eluate (or each eluate dilution group) to multiple capture probes, each configured to hybridize to a specific aptamer. For example, each complex may have a structure similar to that of complex 100 shown in Figure 1. Thus, each tripolecular complex includes (i) one specific aptamer from the eluate, (ii) a first capture probe containing a portion hybridized to a first portion of the aptamer, and (iii) a second capture probe containing a portion hybridized to a second portion of the aptamer, further containing one or more DNA primer regions and an aptamer ID sequence corresponding to a specific aptamer. If separate dilution groups are formed, each dilution group is exposed to a different set of capture probes corresponding to a different subset of aptamers. Optionally, some of the H1 probes may lack bead capture tags for further leveling of SOMAmer counts.

[0076] In step 360, the groups formed in step 356 (if any) may be combined again.

[0077] In step 362, the trimolecular complex is separated from the capture probe that is not bound to the aptamer. For example, the hybridized complex may be captured on magnetic beads and then washed to remove the unbound probe.

[0078] In step 364, the captured probe in the tripolecular complex is dissociated from the corresponding aptamer. This may involve eluting the complex from the beads, but in any case, the result of step 362 is that the captured probe is no longer bound to the aptamer.

[0079] In step 366, the eluate containing the unbound capture probe may be optionally split and / or diluted (perhaps a second time, as discussed below with respect to Figure 6) to spasticize multiple PCR groups.

[0080] In step 368, the aptamer ID sequences in the dissociated capture probe of the eluate are amplified, for example, by PCR amplification of the DNA primer region(s) and associated ID sequences. At this stage, further preparations for NGS may be performed, for example, by binding of adapter sequences and / or demultiplexing of barcode sequences.

[0081] In step 370, the separated PCR groups (if any) may be combined again.

[0082] In step 372, the aptamer ID sequence is sequenced using next-generation sequencing technology. In some examples, this can be achieved using a next-generation sequencing platform developed by Illumina, Inc. (San Diego, California).

[0083] In step 374, the data obtained from sequencing the aptamer ID sequence may be used to determine the abundance of the target protein in the original biological sample.

[0084] C. Exemplary Dilution Groups or Dynamic Range Compression for Next-Generation Sequencing This section describes possible methods for diluting assay eluents to obtain higher assay efficiency, reproducibility, performance, and / or production feasibility in next-generation sequencing systems, in accordance with aspects of the teachings of the present invention; see Figures 4-6.

[0085] First, it is important to understand that it is possible to perform NGS on SOMAmer-containing eluates in a single elution solution that has never been divided into dilution groups, i.e., never divided, diluted, or recombined. Such assays are within the scope of the teachings of the present invention and have the advantage of requiring less elution and only one hybridization plate for every 96 samples. However, such assays have challenges in sensitivity and accuracy, for example, because the abundance of SOMAmers in the original eluate can be in an extreme range, perhaps by several orders of magnitude. For example, the target protein in the sample may have concentrations in the range of fM to μM (i.e., a range of about 9 orders of magnitude), resulting in SOMAmer concentrations in the eluate of 5 orders of magnitude or more. Performing the assay on such an eluate can result in overcounting of more abundant SOMAmers and corresponding undercounting of less abundant SOMAmers. Therefore, it may be desirable to normalize or compress the range of SOMAmer abundance before sequencing and counting.

[0086] The systems and methods of this disclosure address this problem by subdividing the SOMAmers into subpopulations before counting, which, when combined with dilution, results in leveling of counts in the subpopulations, or compression of the dynamic range, when the subpopulations are recombined for sequencing and counting. In some examples, a set of SOMAmer probes is subdivided into subpopulations based on the abundance of SOMAmer eluate (e.g., a first group containing rare SOMAmers, a second group containing moderately rare SOMAmers, a third group containing abundant SOMAmers). The dynamic range of each subpopulation is smaller than, and in some examples considerably smaller than, the dynamic range of the original (undivided) eluate. As described below, the diluted groups may be formed before and / or after hybridization of SOMAmers and probes, i.e., before and / or after the formation of tripolectures suitable for NGS.

[0087] In addition to leveling by dilution, high-abundance SOMAmers may be "leveled" by introducing an H1 probe without a bead-capture tag together with a tagged probe. The ratio of bead-capture tagged H1 probe to untagged H1 probe reduces the trimolecular complexes captured in step 304 of Method 300 and step 358 of Method 350 by an amount corresponding to that ratio. For example, if the ratio of untagged probe to tagged probe is 10:1, only 10 percent of the trimolecular complexes will be captured, reducing the NGS output count by any order of magnitude compared to an assay without an untagged probe. The ratio of untagged probe to tagged probe may vary for each SOMAmer, depending on the expected count of each SOMAmer.

[0088] 1. Four Hybridization Groups Figure 4 shows the steps and by-products of an exemplary NGS assay, generally indicated as 400, with four hybridization groups. In step 402, a SOMAmer-containing eluate 404 is provided. Elution 404 contains SOMAmers resulting from prior exposure to a biological sample and separation from the target molecule, as already described.

[0089] In step 406, eluate 404 is divided into four equal parts or aliquots 408, 410, 412, and 414. In the assay shown in Figure 4, aliquot 408 is diluted in a 1:16 ratio, i.e., 16 parts buffer to 1 part eluate; aliquot 410 is diluted in a 1:4 ratio; and aliquots 412 and 414 are diluted in a 1:2 ratio, respectively. In some examples, the aliquots may not need to be diluted.

[0090] In step 416, four aliquots are combined with groups of hybridization capture probes 418, 420, 422, and 424, each labeled “Group 1,” “Group 2,” “Group 3,” and “Group 4.” In this example, capture probe group 418 contains a capture probe configured to combine with the most diluted eluate and thus with the most common SOMAmer in the eluate. Similarly, capture probe group 420 contains a probe configured to combine with the next most common SOMAmer, and capture probe groups 422 and 424 each contain probes configured to combine with different subsets of SOMAmers in relatively low abundance. Thus, the result of step 416 is four separate solutions, each configured to yield a set of trimolecular compounds containing a SOMAmer and the corresponding probe hybridized to the SOMAmer during hybridization. Each compound may be generally similar to compound 100 in Figure 1.

[0091] Each of the four groups of hybridization capture probes may contain a fixed ratio of bead-captured tagged H1 probes and untagged H1 probes for a subset of SOMAmers within each group, in order to further equalize the count.

[0092] In step 426, the four solutions generated in step 416 are hybridized, captured by beads, washed, and eluted, respectively. This can be achieved, for example, as previously described with respect to steps 304, 306, and 308 of method 300 shown in Figure 3A.

[0093] In step 428, the individual elution solutions obtained from step 426 are recombined into a single elution solution 430. In some examples, the individual solutions may be recombined in different volumes, resulting in further dilution of relatively abundant capture groups (i.e., corresponding to abundant SOMAmers and target molecular species in the original biological sample). The result is a normalized combined solution with less overall variation in the concentrations of the different trimolecular compounds, which can be analyzed with relatively fewer sequencing "reads". For example, the combination of dilution and normalization reduces the number of reads required per sample from approximately 200 million to less than 5 million, thereby enabling multiplexing of a large number of samples per sequencing run and reducing the cost per sample while achieving an acceptable accuracy measured by the coefficient of variation (CV) of the results.

[0094] Step 432, which can be considered as a combination of the aforementioned steps 310, 312, and 314 of Method 300, involves preparing the solution 430 obtained from step 428 for next-generation sequencing (NGS), sequencing it, writing the results to a data file, and analyzing them as desired. The preparation involves the relevant SOMAmer ID sequence I SThis may include PCR amplification of a reporter region containing common primers (P1 and P2 in Figure 1). As previously mentioned, the preparation may also include ligating adapter sequences and / or barcode sequences for demultiplexing into a trimolecular compound. The prepared solution is then sequenced using NGS technology, such as a next-generation sequencing platform developed by Illumina, Inc. (San Diego, California) or other NGS sequencing platforms. After NGS, the data obtained by sequencing may be analyzed or otherwise processed to determine the concentration of analytes, such as target proteins, in the original biological samples. This may include demultiplexing the sequencing data using barcodes corresponding to each original sample (if multiple samples are combined), counting reporter sequences, and scaling and / or normalizing the data to extract accurate results. The sequencing data may be written to a data file in a standard format, such as the ADAT format developed by SomaLogic.

[0095] 2. A single hybridization group with four PCR groups Figure 5 shows the steps and byproducts of an exemplary NGS assay, typically indicated as 500, with one hybridization group and four PCR groups. In step 502, a SOMAmer-containing eluate 504 is provided. Elution 504 contains SOMAmers resulting from prior exposure to the biological sample and separation from the target molecule, as described above.

[0096] In step 506, the eluate 504 is combined with a complete set of hybridization capture probes 508, i.e., a set of probes configured to combine with all SOMAmers in the eluate. The set of probes may also contain a fixed ratio of tagged H1 probes to untagged H1 probes, as described above.

[0097] In step 510, the solution obtained in step 506 is hybridized, captured by beads, washed, and eluted. This can be achieved as described with respect to steps 304, 306, and 308 of method 300 shown in Figure 3A. However, in this example, four sets of universal primers may be used instead of just one set, with each set of primers associating with a different set of SOMAmer IDs corresponding to a set of SOMAmers that fall within a specific expected concentration range. In other words, step 510 yields four distinct groups of trimolecular compounds corresponding to different abundance groups of SOMAmers and thus different abundance groups of target molecules in the SOMAmer eluate from the original biological sample, each containing a different PCR primer and therefore capable of being amplified individually.

[0098] In step 512, the eluate obtained from step 510 is divided into four equal portions or aliquots 514, 516, 518, and 520. Each aliquot may be diluted at this stage to any desired degree, at the optional choice, in order to normalize the expected concentration of the SOMAmer ID sequence to be amplified in the next step. However, none of the dilutions in step 512 are shown in Figure 5.

[0099] In step 522, the individual eluates obtained from step 512 are prepared for next-generation sequencing (NGS), including PCR amplification of the reporter region. However, in this case, different sets of primers and associated reporter regions are amplified in each individual eluate, resulting in amplification of precisely known subsets of SOMAmer ID sequences in each eluate.

[0100] In step 524, the individual solutions containing the amplified SOMAmer ID sequences in each aliquot are combined into a single elution solution 526. In some examples, the individual solutions can be combined again in different volumes to obtain a normalized combined solution with a desired degree of dilution of relatively abundant SOMAmer ID sequences and less overall variation in SOMAmer ID concentration, which can then be analyzed with relatively few reads.

[0101] In step 528, solution 526 is further prepared for next-generation sequencing (NGS), sequenced, the results written to a data file, and analyzed as desired. Preparation of the amplified eluate may include ligating an adapter sequence and / or barcode sequence for demultiplexing the reporter region of the trimolecular compound. The prepared solution may then be sequenced using NGS technology, and the data obtained from the sequencing may be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample, as described above.

[0102] 3. Four hybridization groups and four PCR groups Figure 6 shows the steps and byproducts of an exemplary NGS assay, generally indicated as 600, with four hybridization groups and four PCR groups, thus combining embodiments of assays 400 and 500 in Figures 4-5. Step 602 provides a SOMAmer-containing eluate 604 containing SOMAmers resulting from prior exposure to a biological sample and separation from the target molecule.

[0103] In step 606, the eluate 604 is divided into four equal parts or aliquots 608, 610, 612, and 614. These aliquots may be diluted to various degrees of choice, and in some examples, the aliquots may not be diluted at all.

[0104] In step 616, four aliquots are paired with groups of hybridization capture probes 618, 620, 622, and 624, respectively, labeled “Group 1,” “Group 2,” “Group 3,” and “Group 4,” each probe being configured to bind to a different subset of SOMAmer in the eluate, and then the aliquots are hybridized individually. Thus, the result of step 616 is four distinct solutions, each containing a set of trimolecular compounds, each containing SOMAmer and the corresponding probe hybridized to the SOMAmer. Each compound may be generally similar to compound 100 in Figure 1.

[0105] Each of the four hybridization capture probe groups may optionally contain a fixed ratio of untagged H1 probes to tagged H1 probes to further equalize the count.

[0106] In step 626, the four hybridized solutions produced in step 616 are combined into a single eluent 628, which is then sequentially captured by beads, washed, and eluted. This can be achieved, for example, as described above with respect to steps 304, 306, and 308 of method 300 shown in Figure 3A. As previously mentioned, each hybridized solution may or may not be diluted before being combined again, and differential volumes of each group can also be used to compress analyte variations before bead capture and washing.

[0107] In step 630, the eluate obtained from step 626 is divided into four equal parts or aliquots 632, 634, 636, and 638. Each aliquot may be diluted at this stage to any desired degree by choice in order to normalize the expected concentration of the SOMAmer ID sequence to be amplified in the next step. However, Figure 6 does not show any dilution in step 630.

[0108] In step 640, the individual eluates obtained from step 630 are prepared for next-generation sequencing (NGS), including PCR amplification of the reporter region. Similar to assay 500 in Figure 5, different sets of primers and associated reporter regions are amplified in each individual eluate, resulting in amplification of a subset of SOMAmer ID sequences in each eluate.

[0109] In step 642, the individual solutions containing the amplified SOMAmer ID sequences in each aliquot are recombined into a single elution solution 644. In some examples, the individual solutions can be recombined in different volumes to obtain a normalized combined solution with relatively abundant SOMAmer ID sequences diluted to the desired degree and with less overall variation in SOMAmer ID concentration, which can then be analyzed with relatively few reads.

[0110] In step 646, solution 644 is further prepared for NGS, sequenced, the results written to a data file, and analyzed as desired. Preparation of the amplified eluate may include ligating an adapter sequence and / or barcode sequence for demultiplexing the reporter region of the trimolecular compound. The prepared solution may then be sequenced using NGS technology, and the data obtained from the sequencing may be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample.

[0111] Normalization of quantitative spikes in the D-PCR group In NGS-based systems, the signal is measured as a sequenced read count, and the read counts of all analytes in a given sample are measured in the same sequencing mix having a fixed or finite total set of reads. As proportions of the same mixture, the NGS read counts of all analytes measured in a given sample influence each other, and thus the signal count observed per analyte is the "net result" of the increases and decreases of all analytes measured in a "zero-sum game" per sample with a fixed total number of reads. More specifically, in an NGS system with a fixed total number of reads, any increase in the count of one analyte results in a corresponding decrease in the count of other analytes, and the decreases are distributed according to the total number of reads in each fraction of the analyte.

[0112] Figure 7 schematically illustrates this "zero-sum game" by showing the simplified assays of two analytes from two separate samples in histogram format, where the vertical axis represents the total number of read counts for each analyte, fixed at 2 million. In Sample 1, analytes A and B have equal counts. In Sample 2, analyte A appears to have increased by 500,000 counts, while analyte B appears to have decreased proportionally by 500,000 counts. However, because the total number of reads is finite, it is impossible from Figure 7 to determine whether the difference in counts between analytes A and B in Sample 2 is due to an increase in analyte A, a decrease in analyte B, or a combination of both.

[0113] Figure 8 illustrates how the introduction of a reference reporter or quantitative spike control reporter ("qSpike") can be used to normalize complementary read counts across a sample, thereby enabling the identification of actual signal changes. In the NGS assay shown in Figure 8, the qSpike reporter is physically added (spiked) to two analyte systems containing analytes A and B at the same known concentration in all samples. In this example, the qSpike reporter happens to be at the same concentration as analytes A and B in sample 1. In sample 2, an increase in analyte A and a decrease in analyte B are observed, as before. However, a decrease in qSpike can be observed in sample 2 compared to sample 1, where the same concentration of qSpike is known to have been added. By scaling / adjusting all analytes in sample 2 to return the spike to the expected concentration, it becomes possible to clearly identify the increase in analyte A compared to analyte B, as shown by the "Sample 2 qSpike Adjusted" histogram in Figure 8.

[0114] In more practical NGS assays, a mixture of multiple qSpike reference reporters may be used to compensate for complementary changes in the count proportionality of the analyte. For example, in the assay taught in the present invention, a mixture of four unique H2 reporters may be used, i.e., four uniquely amplifiable reporters that form part of a second probe introduced after the elution of the trimolecular complex in step 308 of assay 300 shown in Figure 3A. Alternatively, a specific qSpikeSOMAmer may be introduced into the eluate, and a suitable qSpike reporter may be included in a library of SOMAmer-specific probes. The qSpike reporter or SOMAmer may be provided at different relative concentrations.

[0115] Figures 9-10 are histograms showing the raw results and qSpike-adjusted results of these assays, respectively, and the references in the explanatory text have the following meanings: QSpike-H = High-Concentration QSpike Reporter QSpike-MH = Medium-to-High Concentration QSpike Reporter QSpike-ML = Medium-to-Low Concentration QSpike Reporter QSpike-L = Low-concentration QSpike reporter • Apo E2, transferrin, kininogen HMW=SOMAmer analyte

[0116] In the assays shown in Figures 9-10, three SOMAmer ApoE2, transferrin, and kininogen HMW were titrated in buffer at concentrations ranging from 50 pM to 50 aM and measured using an NGS HC assay. Each measurement point was a separate sample in which all three SOMAmer analytes were sequenced together, and qSpike was added to all samples at the same concentration.

[0117] In graph (A) of Figure 9, qSpike exhibits complementary changes due to changes in the dose-response signal of the analyte. The NGS assay signal (read count) is compared to the known spike to generate a scaling factor. In graph (B) of Figure 10, the NGS count is scaled so that the spikes are uniform for all samples, and the actual SOMAmer dose-response is reconstructed for the three SOMAmers measured in the assay.

[0118] The use of a qSpike reporter to complement a finite read count can be incorporated into any of the aforementioned next-generation sequencing assays. For example, Figure 11 shows some of the steps and byproducts of an exemplary NGS assay, generally shown in 1100, with four PCR groups and the addition of a qSpike reporter to each group. Thus, the steps of assay 1100 can be incorporated into any NGS assay using multiple PCR groups, such as assays 500 and 600 shown in Figures 5-6.

[0119] Step 1102 provides an eluate 1104 containing SOMAmer that has already hybridized to the probe, been captured by the beads, washed, and eluted. Thus, the eluate 1104 is generally considered to be similar to, for example, the eluate obtained from step 510 of assay 500 shown in Figure 5, or the eluate obtained from step 626 of assay 600 shown in Figure 6.

[0120] In step 1106, eluate 1104 is divided into four equal portions or aliquots 1108, 1110, 1112, and 1114. Each aliquot may be diluted at this stage to any desired degree, at the option of normalizing the expected concentration of the SOMAmer ID sequence to be amplified in the next step.

[0121] In step 1116, the individual eluates obtained from step 1106 are prepared for next-generation sequencing (NGS), including PCR amplification of the reporter region. Similar to assays 500 and 600, different sets of primers and associated reporter regions are amplified in their respective individual eluates, and amplification of a subset of SOMAmer ID sequences is obtained in each eluate. However, in this example, different qSpike reporters are also added to each eluate at known concentrations before PCR amplification.

[0122] In step 1118, the individual solutions containing the amplified SOMAmer ID sequence and qSpike reporter in each aliquot are recombined into a single elution solution 1120. In some examples, the individual solutions can be recombined in different volumes to obtain a normalized combined solution with a desired degree of dilution of the relatively abundant SOMAmer ID sequence and less overall variation in SOMAmer ID concentration, which can then be analyzed with relatively few reads.

[0123] In step 1122, solution 1120 is further prepared for NGS, sequenced, the results written to a data file, and analyzed. Preparation of the amplified eluate may involve concatenating an adapter sequence and / or a barcode sequence for demultiplexing the amplified reporter sequence of the trimolecular compound. The prepared solution may then be sequenced using NGS technology, and the data obtained from the sequencing may be analyzed or otherwise processed to determine the concentration of the target analyte in the original biological sample. With the use of the qSpike reporter, the analysis may involve scaling or renormalizing the data, thereby returning the qSpike concentration to a known level, and thereby compensating for possible count errors due to the finite sequencing reads.

[0124] Determination of the stability of the E. SOMAmer probe. As described above, according to the teaching aspects of the present invention, hybridization regions H1 and H2 are configured to bind to their respective complementary portions of the corresponding SOMAmers, and similar or intentionally different melting temperatures (T) are used to achieve uniform hybridization under a given series of assay conditions. m ) may be designed to have. As described in this section, in some examples, the melting temperature of the hybridization region can be estimated by computer using experimentally determined melting profiles of SOMAmer-probe pairs.

[0125] 1. Background technology The most widely used method for predicting the stability of nucleic acid double helix is ​​known as the nearest neighbor model. The nearest neighbor model assumes that the thermodynamic properties of helix formation depend primarily on the identity of adjacent base pairs within the double helix. This model has been widely applied in predicting the stability of double helix formation in primer design required for PCR and other applications where oligonucleotide double helix formation is important. In the case of NGS assays (i.e., with SOMAmers) as taught in this invention, it is desirable to extend this method to accurately predict the stability of a double helix consisting of one strand containing one type of modified DNA base and a second strand containing native DNA bases.

[0126] Absorbance-to-temperature profiles (melting curves) measured with UV-vis spectrophotometers have traditionally been used to investigate the stability of DNA secondary structures. Hybridization is typically performed in 1.0 M NaCl, 10 mM sodium cacodylate, and 0.5 mM Na2EDTA buffer, pH 7. Oligonucleotide concentrations vary over a 100-fold range, and thermodynamic parameters are used to determine the backmelting temperature (T). M -1 ) and the plot of the natural logarithm of the total DNA concentration are obtained and applied as follows:

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[0127] According to aspects of the teachings of this invention, an extension of the nearest neighbor model is developed for SOMAmers containing the three most common modified bases: Nap-dU, 2-Nap-dU, and Benzyl-dU. As discussed below, melting profiles were experimentally obtained using fluorescence measurements of over 400 SOMAmer probe pairs. Using these data, nearest neighbor parameters necessary to predict the stability of SOMAmer probes under assay reading conditions were defined.

[0128] 2. Double-strand formation to model SOMAmer-probe binding SOMAmer-probe double-chain formation follows the following process:

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[0129] 3. SOMAmer melting model SOMAmers with internal structures can also be viewed as a simple two-state model, as follows:

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[0130] This is a unimolecule reaction, and under moderate dilution conditions, all reactions are independent, so there is no entropy contribution from the SOMAmer concentration. The simplest model assumes that the primer melts after the SOMAmer melts, or vice versa, as independent processes. Without additional experiments such as independent SOMAmer melting, there is no way to know a priori whether the two transitions are due to the melting of the SOMAmer structure or the hybridization primer. Primer melting corresponds better than 16-base pair melting, and therefore likely has higher free energy data.

[0131] 4. Experimental determination of the thermodynamics of melting The fluorescence intensity versus temperature profile (melting curve) was measured using the fluorescent dye SYBR Green I. Since the fluorescence intensity of SYBR Green I increases 100-fold when bound to double-stranded DNA compared to single-stranded DNA, the fluorescence intensity decreases as the SOMAmer-probe double-stranded structure melts.

[0132] Thermal thawing was performed in a buffer containing the components defined by the SOMAscan assay eluate (in this case, 100 mM Tris pH 8.0, 200 mM NaCl, and 0.9 M perchlorate). Perchlorate is known to reduce the stability of DNA double helix. For both SOMAmer and probe, all thermal thawing was performed in 120 μL (8.3 x 10⁻⁶). -7 The samples were obtained at a single concentration of 100 pM in M. Therefore, all thermodynamic parameters were obtained from a single fit to the individual thermal melting profiles. Melting profiles exhibiting behavior more complex than that expected in the two-state model were excluded from the analysis. A total of four plates were measured for both the H1 probe and the H2 probe. These 800 melting profiles were evaluated to produce data consistent with the assumed two-state model of double-strand formation. Of these 800 profiles, a total of 408 melting profiles from SOMAmers containing three different modified nucleotides (Nap-dU, 2-Nap-dU, and Benzyl-dU) were used in this analysis.

[0133] a. Single-phase model fit Figure 12 shows typical thermal fusion data for a SOMAmer-probe double-stranded material. The vertical axis represents fluorescence measured by RFU, and the horizontal axis represents temperature, with the following two-state model fit superimposed. First, the first and last 15 data points are used to fit high-temperature and low-temperature baselines to the data. The low-temperature baseline corresponds to the double-stranded material, and the high-temperature baseline corresponds to the single-stranded material, as shown below.

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[0134] Using nonlinear regression, six free parameters

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[0135] b. Fit of a two-phase model In many cases, the data exhibits more complex melting behavior, likely because the internal structure of the SOMAmer melts first, followed by the melting of the SOMAmer-probe double chain. Figure 13 shows data representing typical two-phase behavior, with the theoretical model fit superimposed, indicated by the red solid line. The data shows two distinct transitions, likely the first being the melting of the internal SOMAmer structure, followed by the melting of the SOMAmer-probe double chain. The two transitions are assumed to be independent. Three baselines are needed to fit the two-phase model. The first corresponds to the temperature dependence of the internal SOMAmer structure, the second to the temperature dependence of the double-stranded SOMAmer-probe, and the third to the temperature dependence of the combined single-stranded material. The latter two are the same as the baselines described above. The former is denoted as follows:

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[0136] 5. Nearest Neighbor Model Once experimental data are fitted to an appropriate model (e.g., single-phase or two-phase as discussed above) and individual thermodynamic parameters are compiled, the parameters of the nearest neighbor model can be derived from the data. The change in free energy is approximated as follows:

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[0137] parameter

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[0138] 6. Exemplary Results In an exemplary process according to the above teachings, the nearest-neighbor model parameters were extended using duplex thermal melts containing three different modified nucleotides, Nap-dU, 2-Nap-dU, and Benzyl-dU. Based on these extended nearest-neighbor parameters, Python code has been developed to calculate thermodynamics and melting temperatures.

[0139] The following table summarizes the 31 parameters required for the nearest-neighbor model, including 10 parameters for the four standard bases and 7 additional parameters for each modified base. The

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[0140] Using the parameters in the table above, estimate the melting temperature T of double helices containing modified nucleotide bases Nap-dU, 2-Nap-dU, and benzyl-dU. m This may be determined by computer. A similar procedure may be used to calculate the estimated melting temperature of any other SOMAmer-containing double chain. According to aspects of the teachings of the present invention, these melting temperatures may be used to determine where to split the hybridization complement for the SOMAmer, i.e., the splitting point between the first and second hybridization regions H1 and H2 of a trimolecular compound such as compound 100 shown in Figure 1 (and thus between the first probe 104 and the second probe 106).

[0141] F. Exemplary combinations and additional examples This section describes additional aspects and features of systems and methods for detecting and quantifying target molecules in biological samples according to aspects of the teachings of the present invention, presented without limitation as a series of paragraphs, some or all of which may be designated by alphanumeric characters for clarity and efficiency. Each of these paragraphs may be combined in any preferred manner with disclosures from elsewhere in this application, including one or more other paragraphs and / or materials incorporated by reference in cross-references. Some of the following paragraphs explicitly reference and further restrict other paragraphs and provide without limitation some examples of preferred combinations.

[0142] A. A system for quantifying the abundance of a target protein in a biological sample, comprising: a plurality of aptamers, each configured to bind to a specific target protein when a biological sample containing the target protein is exposed to the aptamer, thereby forming an aptamer-containing eluate; a plurality of capture probes, each configured to hybridize to a specific aptamer, wherein the plurality of capture probes include a first capture probe having a portion hybridized to a first portion of each aptamer, a second capture probe having a portion hybridized to a second portion of each aptamer, a DNA primer region, and an aptamer ID sequence corresponding to the aptamer; means for forming a plurality of tripolecular complexes by exposing aptamers in the eluate to the capture probes; and means for sequencing the aptamer ID sequence, thereby determining the abundance of the target protein in the biological sample.

[0143] B. A system for quantifying the abundance of two or more target proteins in a biological sample, comprising: a plurality of aptamers, each configured to capture a specific protein in the sample, thereby forming an aptamer-containing eluate when an aptamer capturing one of the target proteins in the biological sample is isolated; a plurality of first probes, each hybridizing to a corresponding first portion of a specific aptamer; a plurality of second probes, each hybridizing to a corresponding second portion of a specific aptamer, each second probe comprising at least one DNA primer region and an aptamer ID sequence corresponding to a specific aptamer; means for sequencing the aptamer ID sequences; and means for quantifying the abundance of the target protein based on the sequenced aptamer ID sequences.

[0144] C. A system for detecting a target protein in a biological sample, comprising: a plurality of aptamers, each configured to capture a specific protein; a plurality of first probes, each comprising a portion hybridized to a corresponding first portion of one of the aptamers that captured the target protein; a plurality of second probes, each comprising a portion hybridized to a corresponding second portion of one of the aptamers that captured the target protein, at least one DNA primer region, and an aptamer ID sequence corresponding to the aptamer that captured the target protein; means for amplifying the aptamer ID sequence; and means for sequencing the aptamer ID sequence to identify the aptamer ID sequence, thereby identifying the aptamer that captured the target protein and the target protein.

[0145] D. A system described in any of the preceding paragraphs, further comprising means for normalizing the complementary read counts of all samples.

[0146] E. A system described in any of the preceding paragraphs, further comprising means for performing dynamic range compression of the abundance of aptamers and / or aptamer ID sequences before sequencing and counting.

[0147] Benefits, features, and advantages The various embodiments and examples of methods and systems for detecting and quantifying the presence of target molecules in biological samples described herein offer several advantages over conventionally known solutions. For example, the exemplary embodiments and examples described herein enable the quantification of aptamer-based protein detection using next-generation sequencing by simplifying the sequencing target from aptamers to aptamer identification sequences.

[0148] Furthermore, among other benefits, the exemplary embodiments and examples described herein enable the precise detection of target molecules in quantities of several orders of magnitude by dividing the assay eluate into multiple dilution groups at one or more stages of the assay and recombining them before next-generation sequencing.

[0149] Furthermore, among other advantages, the exemplary embodiments and examples described herein allow for the addition of a quantitative spike reporter to the assay eluent to normalize the complementary read count for the entire sample, thereby correcting errors arising from finite sequencing reads.

[0150] No known system or device can perform these functions. However, not all embodiments and examples described herein offer the same or the same degree of benefits.

[0151] conclusion The above disclosure may encompass multiple distinct embodiments, each possessing independent utility. While each of these is disclosed in its preferred form(s), the specific embodiments disclosed and illustrated herein should not be considered restrictively, as numerous variations are possible. Where section headings are used within this disclosure, such headings are for structural purposes only. The subject matter of this disclosure includes all novel and non-obvious combinations and partial combinations of the various elements, features, functions, and / or characteristics disclosed herein. The following claims specifically point to certain combinations and partial combinations that are considered novel and non-obvious. Other combinations and partial combinations of features, functions, elements, and / or characteristics may be claimed in applications claiming priority from this application or related applications. Such claims, whether broader, narrower, equal to, or different from the original claims, are considered to be included within the subject matter of this disclosure.

Claims

1. A method for quantifying the abundance of a target protein in a biological sample, The biological sample is captured by exposing it to a plurality of aptamers, each configured to bind to a specific protein; Isolating the aptamer that has captured one of the target proteins in the aptamer-containing eluate; The eluate is exposed to a plurality of capture probes, each configured to hybridize to a specific aptamer, thereby forming a plurality of tripolecular complexes, wherein each tripolecular complex is One of the aptamers from the eluate, A first capture probe including a portion hybridized to the first portion of the aptamer, Forming a second capture probe comprising a portion hybridized to the second portion of the aptamer, a DNA primer region, and an aptamer ID sequence corresponding to the aptamer; The three-molecule complex is separated from the capture probe that is not bound to the aptamer; Dissociating the capture probe in the tripolecular complex from the corresponding aptamer; To amplify the aptamer ID sequence in the dissociated capture probe; The aptamer ID sequence is determined by next-generation sequencing, and: A method comprising determining the amount of the target protein in the biological sample based on data obtained by sequencing the aptamer ID sequence.

2. The method according to claim 1, wherein the aptamer is SOMAmer.

3. The method according to claim 1, further comprising dividing the aptamer-containing eluate into two or more groups before forming the tripolecular complex, wherein the plurality of capture probes comprises two or more separate sets of capture probes corresponding to the two or more groups.

4. The method according to claim 1, further comprising dividing the dissociated capture probe into a plurality of aliquots before amplifying the aptamer ID sequence, wherein the amplification of the aptamer ID sequence includes amplifying different subsets of the aptamer ID sequence in each aliquot.

5. The method according to claim 4, further comprising diluting at least one of the plurality of aliquots before amplifying the aptamer ID sequence.

6. The method according to claim 4, further comprising adding a quantitative spike reporter to each aliquot before amplifying the aptamer ID sequence.

7. The method according to claim 6, further comprising recombining the aliquots before sequencing the aptamer ID sequence.

8. A method for quantifying the abundance of two or more target proteins in a biological sample, wherein the method is The capture of the target protein is achieved by exposing the biological sample to multiple aptamers, each configured to capture a specific protein; By isolating the aptamer that has captured one of the target proteins in the biological sample, an aptamer-containing eluate is formed; This involves forming multiple tripolecular complexes, where each tripolecular complex is: A specific aptamer present in the aptamer-containing eluate, A first capture probe hybridized to the corresponding first portion of the specific aptamer, Forming a second capture probe comprising a portion hybridized to a corresponding second portion of the specific aptamer, at least one DNA primer region, and an aptamer ID sequence corresponding to the specific aptamer, To amplify the aforementioned aptamer ID sequence; Determining the sequence of the aforementioned aptamer ID sequence; A method comprising quantifying the amount of the target protein based on the sequenced aptamer ID sequence.

9. The method according to claim 8, wherein the aptamer is SOMAmer.

10. The method according to claim 8, further comprising dividing the aptamer-containing eluate into two or more dilution groups before forming the tripolecular complex, wherein each dilution group comprises a separate set of capture probes.

11. The method of claim 8, further comprising dividing the capture probe into a plurality of aliquots before amplifying the aptamer ID sequence, wherein at least one of the aliquots is diluted, and the amplification of the aptamer ID sequence includes amplifying different subsets of the aptamer ID sequence in each aliquot.

12. The method according to claim 11, further comprising adding a quantitative spike reporter to each aliquot before amplifying the aptamer ID sequence.

13. The method according to claim 12, further comprising recombining the aliquots before sequencing the aptamer ID sequence.

14. The method according to claim 8, wherein the sequencing of the aptamer ID sequence is achieved by next-generation sequencing.

15. A method for detecting a target protein in a biological sample, The biological sample is combined with multiple aptamers, each configured to capture a specific protein, thereby capturing the target protein with the aptamers; This involves forming a trimolecular complex, The aptamer that has captured the target protein, A first probe comprising a portion hybridized to the corresponding first portion of the aptamer that has captured the target protein, To form a tripolecular complex comprising: a portion hybridized to the corresponding second portion of the aptamer that has captured the target protein; at least one DNA primer region; and a second probe containing an aptamer ID sequence corresponding to the aptamer that has captured the target protein; To amplify the aforementioned aptamer ID sequence; A method comprising sequencing the aptamer ID sequence to identify the aptamer ID sequence, thereby identifying the aptamer that captured the target protein and the target protein.

16. The method of claim 15, further comprising determining a target region on the aptamer, and then determining a boundary for dividing the complement of the target region into a first probe and a second probe, thereby forming the first probe and the second probe.

17. The method according to claim 16, wherein determining the boundary is based on achieving a desired melting temperature of the hybridized portion of the first probe and the second probe.

18. The method according to claim 17, further comprising: determining the melting temperature of the complementary material in the target region by computer; and then stepwise modifying the boundary between the hybridized portion of the first probe and the second probe until a desired melting temperature of the hybridized portion of the hybridized portion is achieved.

19. The method according to claim 17, wherein the sequencing of the aptamer ID sequence is achieved by next-generation sequencing.

20. The method according to claim 15, further comprising dissociating the second probe from the aptamer before amplifying the aptamer ID sequence.

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