Improved aptamer detection techniques

Abstract

Aptamer detection techniques are described that improve detection outcomes by using a novel protease-based elution of captured aptamers relative to high-salt elution techniques to generate an aptamer eluate that, in subsequent detection workflows, has a higher signal to noise ratio. In embodiments, the protease or enzymatic elution yields higher quality detection or sequencing results, permitting more sensitive and / or specific characterization of aptamers in a particular sample.

Description

IMPROVED APTAMER DETECTION TECHNIQUESBACKGROUND

[0001] The disclosed technology relates generally to aptamer detection and / or identification techniques in conjunction with an aptamer-based assay. Tn particular, the technology disclosed relates to techniques for improving signal and reducing noise in detection steps that may include hybridization-based and / or nucleic acid sequencing for direct or indirect aptamer detection in conjunction with an aptamer-based assay.

[0002] The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, a problem mentioned in this section or associated with the subject matter provided as background should not be assumed to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which in and of themselves can also correspond to implementations of the claimed technology.

[0003] Protein expression patterns help define a cell’s identity and state. RNA transcripts are often used as a surrogate for protein expression, but the relationship between abundance of proteins and mRNA is not one-to-one. There are differences caused by regulation of posttranscriptional, translational and protein degradation. Therefore, direct nucleic acid sequencing of RNA transcripts may not provide an accurate estimation of protein expression.

[0004] Aptamers are nucleic acids that bind to molecular targets, such as proteins, with high affinity and specificity. Advancements in aptamer selection and design include Systematic Evolution of Ligands by Exponential enrichment (SELEX). In SELEX, high affinity nucleic acids for different analytes of interest can be isolated from a combinatorial library, permitting high throughput characterization of aptamer-target binding and multiplexed assays for analytes in a complex biological sample. Upon aptamer binding to an analyte target, the binding event can be detected to characterize the presence and concentration of various analytes in thebiological sample. However, complexities in assay steps may result in loss of aptamer before detection can occur.BRIEF DESCRIPTION

[0005] In one embodiment, the present disclosure provides a method of aptamer detection that includes contacting analytes of a sample with a plurality of aptamers under conditions that permit analyte-aptamer complexes to form, wherein different aptamers of the plurality of aptamers have specific affinity for respective different analytes of the analytes; tagging the analyte-aptamer complexes; capturing the analyte-aptamer complexes via respective tags; and contacting the captured analyte-aptamer complexes with a protease to elute the aptamers; and detecting the eluted aptamers

[0006] In one embodiment, the present disclosure provides a capture substrate comprising an affinity tag binder configured to capture tags of analyte-aptamer complexes; and an elution buffer comprising a protease fluidically coupled to the capture substrate.

[0007] In one embodiment, the present disclosure provides an elution buffer comprising a protease and a plurality of aptamers in the elution buffer, wherein different aptamers of the plurality of aptamers have specific affinity for respective different analytes.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] These and other features, aspects, and advantages of the disclosed embodiments will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

[0009] FIG. 1 is a schematic illustration of an aptamer detection workflow with a protease elution of aptamers, according to an embodiment;

[0010] FIG. 2 shows an experimental arrangement used for assessing protease elution in an aptamer detection workflow;

[0011] FIG. 3 shows summarized results of protease elution in an aptamer detection workflow;

[0012] FIG. 4 shows an increase in signal to background in aptamer detection for a pool of aptamers using protease elution;

[0013] FIG. 5 shows signal to background ranges for aptamers using a conventional salt-based elution;

[0014] FIG. 6 shows signal to background ranges for aptamers using protease elution;

[0015] FIG. 7 shows a comparison between salt-based elution and protease elution;

[0016] FIG. 8 shows an experimental setup to assess intraplate aptamer assay variance using salt-based elution and protease elution;

[0017] FIG. 9 shows results measuring intraplate assay variance (%CV) between four replicate assay plate;

[0018] FIG. 10 shows results measuring intraplate assay variance (%CV) between four replicate assay plate; and

[0019] FIG. 11 is a block diagram of an aptamer detection device, according to an embodiment.DETAILED DESCRIPTION

[0020] The following discussion is presented to enable any person skilled in the art to make and use the technology disclosed, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed implementations will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the spirit and scope of the technology disclosed. Thus, the technology disclosed is not intended to be limited to the implementations shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0021] Aptamers are short single stranded nucleic acid molecules (ssDNA or ssRNA) that can bind to their specific target molecules with high affinity. Accordingly, aptamers can be used for multiomic applications, such as proteome characterization of a sample in a high-throughput manner. Disclosed herein are aptamer detection techniques for detecting aptamers with positive binding results (e.g., that bind to target molecules in a sample) and that may occur before or in conjunction with one or more aptamer detections step. In certain embodiments, the disclosed techniques may provide streamlined workflows with reduced equipment burden via reduction in a number of steps (e.g., single hybridization reactions or reduced number of wash steps). The disclosed techniques may include sample preparation steps and / or sample preparations that permit improved aptamer abundance measurement.

[0022] As used herein, an aptamer may refer to a non-naturally occurring nucleic acid that has specific binding affinity for a target molecule. The binding of the aptamer to the target molecule can result in catalytically changing the target molecule, reacting with the target molecule in a way that modifies or alters the target molecule or the functional activity of the target molecule, covalently attaching to the target molecule (as in a suicide inhibitor), and facilitating the reaction between the target molecule and another molecule. In one embodiment, the target molecule is a three dimensional chemical structure, other than a polynucleotide, that binds to the aptamer through a mechanism which is predominantly independent of Watson / Crick base pairing or triple helix binding. In an embodiment, the aptamer is not a nucleic acid having the known physiological function of being bound by the target molecule.

[0023] Aptamers include nucleic acids that are identified from a candidate mixture of nucleic acids. A specific binding affinity of an aptamer for its target may refer to aptamer binding to its target generally with a much higher degree of affinity than it binds to other, non-target, components in a mixture or sample. Different aptamers may have either the same number or a different number of nucleotides. Aptamers may be DNA or RNA and may be single stranded, double stranded, or contain double stranded regions. The aptamers discussed herein can be used in any diagnostic, imaging, high throughput screening or target validation techniques orprocedures or assays for which aptamers, oligonucleotides, antibodies and ligands, without limitation can be used.

[0024] Aptamers as disclosed herein may be used in aptamer-based assays, such as those disclosed in U.S. Pat. Nos. 7,855,054 and 7,964,356 and U.S. Publication Nos. US / 2011 / 0136099 and US / 2012 / 0115752. In one example, a panel of aptamers to different target molecules is provided attached to a solid support (step 104). The attachment of the aptamers to the solid support is accomplished by contacting a first solid support with the aptamer / s and allowing the releasable first tag included on the aptamer to associate, either directly or indirectly, with an appropriate first capture agent that is attached to or part of the first solid support. A test sample 102 is then prepared and contacted with the immobilized aptamers that have a specific affinity for their respective target molecules, which may or may not be present in the sample. If the test sample contains the target molecule(s), an aptamertarget affinity complex will form in the mixture with the test sample. In addition to aptamertarget affinity complexes, uncomplexed aptamer will also be attached to the first solid support. The aptamer-target affinity complex and uncomplexed aptamer that has associated with the probe on the solid support is then partitioned from the remainder of the mixture, thereby removing free target and all other uncomplexed matter in the test sample (sample matrix); i.e., components of the mixture not associated with the first solid support. This partitioning step is referred to herein as the Catch-1 partition (see definition below). Following partitioning the aptamer-target affinity complex, along with any uncomplexed aptamer, is released from the first solid support using a method appropriate to the particular releasable first tag being employed.

[0025] In one embodiment, aptamer-target affinity complexes bound to the solid support are treated with an agent that introduces a second tag (step 106) to the target molecule component of the aptamer-target affinity complexes. In one embodiment, the target is a protein or a peptide, and the target is biotinylated by treating it with NHS-PEO4-biotin. The second tag introduced to the target molecule may be the same as or different from the aptamer capture tag. If the second tag is the same as the first tag, or the aptamer capture tag, free capture siteson the first solid support may be blocked prior to the initiation of this tagging step. In this exemplary embodiment, the first solid support is washed with free biotin prior to the initiation of target tagging. Tagging methods, and in particular, tagging of targets such as peptides and proteins are described in U.S. Pat. No. 7,855,054.

[0026] Partitioning is completed by releasing of uncomplexed aptamers and aptamer-target affinity complexes from the first solid support (step 108). In one embodiment, the first releasable tag is a photocleavable moiety that is cleaved by irradiation with a UV lamp under conditions that cleave >90% of the first releasable tag. In other embodiments, the release is accomplished by the method appropriate for the selected releasable moiety in the first releasable tag. Aptamer-target affinity complexes may be eluted and collected for further use in the assay or may be contacted to another solid support to conduct the remaining steps of the assay.

[0027] At one or more points in the workflow 100, a kinetic challenge may take place (e.g., step 110). In one embodiment, the kinetic challenge is performed after the aptamer affinity complex is released from the first solid support. In this embodiment, the kinetic challenge is performed by releasing the aptamer affinity complex into a buffer that contains a high concentration of a polyanionic competitor (e.g., dextran sulfate) and subsequently incubating the aptamer affinity complexes in the competitor solution for a time less than or equal to the dissociation half life of the aptamer affinity complex by way of example.

[0028] In one embodiment, a second partition is performed (referred to herein as the Catch-2 partition, see definition below) to remove free aptamer. As described above, in one embodiment, a second tag used in the Catch-2 partition may be added to the target (e.g., at step 106) while the aptamer-target affinity complex is still in contact with the solid support used in the Catch-0 capture. In other embodiments, the second tag may be added to the target at another point in the assay prior to initiation of Catch-2 partitioning. The mixture is contacted with a solid support, the solid support having a capture element (second) adhered to its surface which is capable of binding to the target capture tag (second tag), preferably with high affinity and specificity (step 112). In one embodiment, the solid support is magnetic beads (such asDynaBeads MyOne Streptavidin Cl) contained within a well of a microtiter plate and the capture element (second capture element) is streptavidin. The magnetic beads provide a convenient method for the separation of partitioned components of the mixture. Aptamertarget affinity complexes contained in the mixture are thereby bound to the solid support through the binding interaction of the target (second) capture tag and the second capture element on the second solid support. The aptamer-target affinity complex is then partitioned from the remainder of the mixture, e.g. by washing the support with buffered solutions, including buffers comprising organic solvents including, but not limited to glycerol.

[0029] As provided herein, aptamers are then selectively eluted or separated from aptamertarget complexes with buffers that include one or more proteases (step 114), as generally discussed herein. Elution using enzyme-based techniques provides benefits relative to conventional salt-based elution. The protease-based elution has improved performance in detection assays relative to salt-based elution, such as via decreased elution of aptamers nonspecifically retained on Catch-2 beads by virtue of aptamer / aptamer interaction. Decreased elution of nonspecific aptamers results in decreased assay background.

[0030] The eluted aptamers 120 released from the Catch-2 partition, which represent aptamers corresponding to targets or analytes present in the sample 102, are detected and optionally quantified by detection methods as discussed herein, such as via hybridization-based detection on an array (step 124) and / or next generation sequencing techniques (step 126). For example, via amplification and / or sequencing of probes that bind to the eluted aptamers. In certain embodiments, the detection includes detection results that provide relative and / or estimated absolute concentrations of detected aptamers. The detection results may include a notification or output of a positive or negative detection result or a relative concentration or estimated concentration for a particular aptamer ID or barcode or a particular target of the aptamer.

[0031] The disclosed techniques provide improved elution of nucleic acid aptamers from their cognate proteins using enzymatic methods. In conventional techniques, aptamers are eluted from proteins to form an eluate that is used as input to downstream detection steps. The convention elution may be performed using a salt-based elution buffer (e.g., 20 mM NaOH).However, salt-based elution may be inefficient at releasing aptamers from proteins and may contribute toward assay background. For example, salt-based elution may release non- specifically bound aptamers bound to streptavidin beads (SA Beads) via aptamer-aptamer interaction. It should be understood that salt-based elution may be understood to be a mild elution technique that avoids protein denaturation, in contrast to protease-based elution. Protease-based elution techniques, which may have been understood to introduce downstream workflow risks via potential protease-degradation of enzymes used in sequencing reactions, are demonstrated herein to be associated with improved elution and detection. In certain cases, protease-based elution may yield improved downstream sequencing based on structural changes induced in aptamers. Certain aptamers may include modified bases that are susceptible to protease stripping or removal. This protease removal may result in a structural change to one or more aptamers to generate nucleotides that are more amendable to downstream detection steps, including hybridization to probes. Stated another way, the aptamer modifications may enhance aptamer-analyte binding while also decreasing efficiency of later workflow steps. The disclosed techniques preserve the aptamer modifications during analyte binding while removing the modifications prior to detection steps, thus improving efficiency.

[0032] It should be understood that the protease elution buffer as provided herein may include one or more proteases in a biologically compatible buffer solution such as Tris. In certain embodiments, the buffer may include SDS (e.g., up to 2% SDS). In certain embodiments, the buffer may optionally include EDTA and / or DTT. The protease may be present in a range of O.OOlU / pL to 5U / pL in certain embodiments. However, it should be understood that the concentration of protease may be dependent on the assay conditions, e.g., the estimated aptamer concentration and / or the sample concentration in the assay.

[0033] In certain embodiments, the protease elution buffer is a low-salt buffer. For example, a salt concentration may be less than 1mm. Further, certain salts may be present in the elution buffer, such as NaCl, CaCb, SDS. In embodiments, the salt concentration of the protease elution buffer is ImM to about lOmM. It should be understood that the present techniquesmay, in embodiments, encompass a combination of chemical and enzymatic elution. In certain embodiments, the protease may be a heat-active protease that is activate at a range of temperatures.

[0034] To improve S elution efficiency and specificity, enzymatic elution using protease was explored. Compared to the conventional salt-based elution methods, use of a protease such as proteinase K improved assay signal background 3-4 fold.

[0035] As used herein, the term protease may refer to a protein, polypeptide or peptide exhibiting the ability to hydrolyze polypeptides or substrates having a polypeptide portion. The present methods can use one or more proteases. The proteases provided herein can be heat-labile and thus can be inactivated by heat. In certain embodiments, the disclosed eluates may be filtered (e.g., via filtered pipette tips) to remove proteases present in the eluate. In certain embodiments, the proteases provided herein can be inactivated at a temperature above about 40° C., 45° C., 50° C., 55° C , 60° C., 65° C., 70° C., 75° C., 80° C. or above about 85° C. The proteases provided herein include, but not limited to, serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases, and metalloproteases. Typically, aspartic, glutamic and metallo proteases activate a water molecule which performs a nucleophilic attack on the peptide bond to hydrolyze it. Serine, threonine and cysteine proteases typically use a nucleophilic residue to perform a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolyzed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme. The protease may include a proteinase, such as proteinase K (NEB, Qiagen). In an embodiment, the proteinase K may be a subtilisin-related serine protease derived from the Parengyodontium album (Tritirachium album) that is active under a wide range of reaction conditions, including elevated temperatures and the presence of Sodium Dodecyl Sulfate (SDS). Exemplary proteases used herein include a serine protease isolated from a recombinant Bacillus strain. Exemplary proteases used herein include subtilisin and variants thereof, including subtilisin Carlsberg, ALCALASE, and subtilisin S41. Subtilisins andvariants thereof are known to those of skill in the art and include, for example ALCALASE, ALCALASE 0.6L, ALCALASE 2.5L, ALK-enzyme, bacillopeptidase A, bacillopeptidase B, Bacillus subtilis alkaline proteinase bioprase, bioprase AL 15, bioprase APL 30, colistinase, subtilisin J, subtilisin S41, subtilisin Sendai, subtilisin GX, subtilisin E, subtilisin BL, GENENASE I, ESPERASE, MAXATASE, thermoase PC 10, protease XXVII, thermoase, SUPERASE, subtilisin Carlsberg subtilisin DY, subtilopeptidase, SP 266, SAVINASE 8.0L, SAVINASE 4.0T, KAZUSASE, protease VIII, OPTICLEAN, protin A 3L, SAVINASE, SAVINASE 16.0L, SAVINASE 32.0L EX, orientase 10B, protease S, serine endopeptidase.

[0036] FIG. 2 shows an experimental setup to assess protease elution relative to a salt-based elution. Aptamer assay steps between the salt-based and protease elution were the same, except the elution buffer in protease elution samples were replaced with a MIDI plate containing the six elution buffers tested as shown in FIG. 2. The elution procedure (i.e., elution time, volume, temperature, and shake speed) was otherwise maintained and consistent.

[0037] After elution, the concentration of salts and hybridization controls was normalized between elution methods. Specially, to 40uL of salt-based eluate, 40uL of lx proteinase K elution buffer (w / o Proteinase K) was added, and to 40uL of each proteinase K eluate, 40ul of salt-based elution buffer containing hybridization controls was added to control for any other composition differences relative to the salt-based elution buffer.

[0038] Array sample setup prep and hybridization followed the standard aptamer detection protocols. Namely, 25uL of 2x array hybridization buffer, 5uL of slide block, and 20uL of eluate was combined. 45uL of the 50uL mixture was added to each sub-array on a micro-array and allowed to hybridize for 19hrs at 50°C. After hybridization, the slides were processed for imaging on the array scanner.

[0039] The elution protocol was performed using six elution buffers - the standard salt-based elution buffer and five proteinase K elution buffers varying in proteinase K and SDS concentration. Elution was tested across two sample types - Plasma (PPS) and Blank. Theconcentrations of aptamers (SOMAmers) eluted using each method was measured on an Agilent Access array. Prior to hybridizing eluate on the array, the individual components of each elution buffer were normalized between conditions. Elution efficiency was compared between methods by comparing the S:B of each elution method (i.e. the concentration of SOMAmers in the plasma sample vs the blank sample).

[0040] As shown in FIG. 3, results of an array readout of eluted aptamers for the various experimental conditions of FIG. 2 show that proteinase K elution methods greatly increases signal to background ratio relative to salt-based elution, and rescue most of the worse performing aptamers. The median value is the median of all aptamers. All of the proteinase k elution methods or conditions greatly increase signal to background ratio and rescue most of the worse performing aptamers

[0041] FIG. 4 shows assay results for different aptamers demonstrating that the proteinase K elution method greatly increases signal-background relative to salt-based elution.

[0042] As shown in FIG. 5-6, the proteinase K elution method (FIG. 6) greatly increases signal-background separation relative to salt-based elution (FIG. 5).

[0043] FIG. 7 is a signal to background comparison between salt-based elution and lx proteinase K elution.

[0044] FIG. 8 is a schematic illustration of an experimental design to assay intraplate assay variant. The design used a SomaScan assay with pooled plasma vs Tecan blank to measure the signal to background using four replicates to measure intraplate % coefficient of variability (CV) of the assay. All samples were processed on the same run but with different elution strategies used, i.e., standard salt-based elution or proteinase K at different concentration and vendors. Intraplate assay CV of the SomaScan assay was significantly reduced with the proteinase K elution approach. Lower proteinase K concentrations (data not shown) and proteinase K from two vendors (data not shown) were similarly effective at eluting aptamers in the SomaScan assay.

[0045] FIG. 9 and FIG. 10 show results of the experiment referred to in FIG. 8. Proteinase K elution reduces %CV relative to salt-based elution. High %CV in salt-based elution is associated with low abundance Somamers and likely to due to inefficient elution. Proteinase K elution (reduced variance shown on the right in FIG. 10), rescues low abundance Somamers and reduced variability.

[0046] FIG. 11 is a schematic diagram of an aptamer detection device 500 that may be used in conjunction with the disclosed embodiments for one or both of conducting all or part of an aptamer-based assay and acquiring sequencing data of identification sequences and / or index sequences to characterize an aptamer present in a sample (e.g., a biological sample) as generally discussed herein. The aptamer detection device 500 may include a sequencing device configured to acquire sequence data using a sequencing library that can be implemented according to any sequencing technique, such as those incorporating sequencing-by-synthesis methods described in U.S. Patent Publication Nos. 2007 / 0166705; 2006 / 0188901 ; 2006 / 0240439; 2006 / 0281109; 2005 / 0100900; U.S. Pat. No. 7,057,026; WO 05 / 065814; WO 06 / 064199; WO 07 / 010,251, the disclosures of which are incorporated herein by reference in their entireties. Alternatively, sequencing by ligation techniques may be used in the aptamer detection device 500 . Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides and are described in U.S. Pat. No. 6,969,488; U.S. Pat. No. 6,172,218; and U.S. Pat. No. 6,306,597; the disclosures of which are incorporated herein by reference in their entireties. Some embodiments can utilize nanopore sequencing, whereby target nucleic acid strands, or nucleotides exonucleolytically removed from target nucleic acids, pass through a nanopore. As the target nucleic acids or nucleotides pass through the nanopore, each type of base can be identified by measuring fluctuations in the electrical conductance of the pore (U.S. Patent No. 7,001,792; Soni & Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); and Cockroft, et al. J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties). Yet other embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that arecommercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in US 2009 / 0026082 Al; US 2009 / 0127589 Al; US 2010 / 0137143 Al; or US 2010 / 0282617 Al, each of which is incorporated herein by reference in its entirety. Particular embodiments can utilize methods involving the real-time monitoring of DNA polymerase activity. Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate-labeled nucleotides, or with zeromode waveguides as described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties. Other suitable alternative techniques include, for example, fluorescent in situ sequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS). In particular embodiments, the aptamer detection device 500 may be a HiSeq, MiSeq, or HiScanSQ from Illumina (La Jolla, CA). In other embodiment, the aptamer detection device 500 may be configured to operate using a CMOS sensor with nanowells fabricated over photodiodes such that DNA deposition is aligned one-to-one with each photodiode.

[0047] The sequencing device of the aptamer detection device 500 may be “one-channel” a detection device, in which only two of four nucleotides are labeled and detectable for any given image. For example, thymine may have a permanent fluorescent label, while adenine uses the same fluorescent label in a detachable form. Guanine may be permanently dark, and cytosine may be initially dark but capable of having a label added during the cycle. Accordingly, each cycle may involve an initial image and a second image in which dye is cleaved from any adenines and added to any cytosines such that only thymine and adenine are detectable in the initial image but only thymine and cytosine are detectable in the second image. Any base that is dark through both images in guanine and any base that is detectable through both images is thymine. A base that is detectable in the first image but not the second is adenine, and a base that is not detectable in the first image but detectable in the second image is cytosine. By combining the information from the initial image and the second image, all four bases are able to be discriminated using one channel.

[0048] In the depicted embodiment, the aptamer detection device 500 includes a separate sample processing device 502 and an associated computer 504. However, as noted, these may be implemented as a single device. Further, the associated computer 504 may be local to or networked or otherwise in communication with the sample processing device 502. The sample processing device 502 may be a flow cell handling device. Thus, FIG. 11 may include a flow cell handling device, such as a flow through system available from Tecan (Maennedorf, Switzerland). An example is the Freedom EVO® robotic workstation from the present applicant (Tecan Schweiz AG, Seestrasse 103, CH-8708 Mannedorf, Switzerland), which enables automated liquid handling in a stand-alone instrument or in automated connection with an analytical system. Examples of such devices are discussed in U.S. Patent Nos. 7988934, 8287806, 8580197, 8841925, 9333499, 9662654, 7061608, 7964160, the disclosures of which are incorporated by reference in their entireties herein. Accordingly, the sample processing device 502 may include components that facilitate liquid handling in various steps in the workflow discussed in FIG. 1. Further, the sample processing device 502 may include one or more reagents 503 used in processing the sample at various stages in the workflow of FIG. 1.

[0049] In an embodiment, the sample processing implemented by the device 500 uses the disclosed protease (e.g., in an appropriate buffer), which is provided as part of the reagents 503. In an embodiment, the protease is provided as a protease elution buffer 505. The protease elution buffer 505 may be provided in a reservoir that is fluidically coupled to the sample substrate 510. In an embodiment, the protease elution buffer 505 is provided pre-loaded into dispensing devices (e.g., pipettes) or pre-loaded into wells or channels of the sample substrate. In an embodiment, the protease is provided as a dried or lyophilized component that is mixed or hydrated to generate the elution buffer 505 as part of sample handling by the device 500. Further, as discussed herein, the protease may include a mix of different proteases, and the generation of the elution buffer 505 may include mixing different proteases in appropriate concentrations.

[0050] In the depicted embodiment, the biological sample may be loaded into the sample processing device 502 onto the sample substrate 510, e.g., a flow cell, well plate, planarsubstrate, bead / beads (e.g., magnetic beads), or slide, that is processed as part of an aptamer detection assay. In an embodiment, the sample substrate 510 may refer to one or more sample substrates that are processed in parallel in a multiplexed reaction. Further, the sample substrate 510 may refer to one or more sample substrates 510 that are processed in series as part of separate processing steps for analysis of a particular biological sample. For example, steps of the workflow 100 of FIG. 1 may include separation steps that involve moving an eluate / supernatant from a first substrate 510 to a second substrate 510. In an embodiment, the sample substrate 510 may include a sequencing library prepared using the aptamers or detectable reporters that can be imaged to generate sequence data. For example, reagents that interact with the biological sample fluoresce at particular wavelengths in response to an excitation beam generated by an imager 512 and thereby return radiation for imaging. For instance, the fluorescent components may be generated by fluorescently tagged nucleic acids that hybridize to complementary molecules of the components or to fluorescently tagged nucleotides that are incorporated into an oligonucleotide using a polymerase. As will be appreciated by those skilled in the art, the wavelength at which the dyes of the sample are excited and the wavelength at which they fluoresce will depend upon the absorption and emission spectra of the specific dyes. Such returned radiation may propagate back through the directing optics. This retrobeam may generally be directed toward detection optics of the imager 512.

[0051] The imager detection optics may be based upon any suitable technology, and may be, for example, a charged coupled device (CCD) sensor that generates pixilated image data based upon photons impacting locations in the device. However, it will be understood that any of a variety of other detectors may also be used including, but not limited to, a detector array configured for time delay integration (TDI) operation, a complementary metal oxide semiconductor (CMOS) detector, an avalanche photodiode (APD) detector, a Geiger-mode photon counter, or any other suitable detector. TDI mode detection can be coupled with line scanning as described in U.S. Patent No. 7,329,860, which is incorporated herein by reference. Other useful detectors are described, for example, in the references provided previously herein in the context of various nucleic acid sequencing methodologies.

[0052] The imager 512 may be under processor control, e.g., via a processor 514, and the sample receiving device 502 may also include I / O controls 516, an internal bus 518, nonvolatile memory 520, RAM 522 and any other memory structure such that the memory is capable of storing executable instructions, and other suitable hardware components that may be similar to those described with regard to FIG. 11. Further, the associated computer 504 may also include a processor 524, I / O controls 526, communications circuity 527, and a memory architecture including RAM 528 and non-volatile memory 530, such that the memory architecture is capable of storing executable instructions 532. The hardware components may be linked by an internal bus, which may also link to the display 534. In embodiments in which the aptamer detection device 500 is implemented as an all-in-one device, certain redundant hardware elements may be eliminated. In embodiments in which the aptamer detection device 500 conducts all or part of the workflow of FIG. 1, the sample receiving device 502 may include a substrate handling subsystem that controls motorized movements of the substrate, sample handling, liquid transfer, pipetting, substrate washing, heating (e.g., via a heating block or heating element), or other automated steps. Accordingly, the device 500 may provide control instructions to cause activation of various components driven by the substrate handling subsystem 536.

[0053] As part of sequence data generation by the device 500, the processor 514, 524 may be programmed to assign individual sequencing reads to a sample (in a multiplexed reaction) and / or to a particular aptamer based on the associated index sequence or sequences according to the techniques provided herein. In particular embodiments, based on the image data acquired by the imager 512, the aptamer detection device 500 may be configured to generate sequencing data that includes base calls for each base of a sequencing read. Further, based on the image data, even for sequencing reads that are performed in series, the individual reads may be linked to the same location via the image data and, therefore, to the same template strand. In this manner, index sequencing reads may be associated with a sequencing read of an insert sequence before being assigned to a sample of origin. The processor 514, 524 may also be programmed to perform downstream analysis on the sequences corresponding to the inserts for a particular sample subsequent to assignment of sequencing reads to the sample.

[0054] In certain embodiments, the I / O controls 516, 526 may be configured to receive user inputs that automatically select sequencing parameters based on a selection of a sequence library preparation kit or reading a barcode or identifier of a sequence library preparation kit. However, it should be understood that the sequencing parameters may be automatically selected based on sensing or other workflow identification metrics. In an embodiment, improved NGS results as a result of protease elution of aptamers may permit the elimination of certain dynamic range compression techniques. For example, in conventional approaches, combining aptamers to high-abundancy proteins together with low-abundancy proteins in a single panel is challenging. For example, human serum / plasma contains proteins can differ in concentration by many orders of magnitude, e.g., a 10-log range. Certain aptamer detection platforms can compress the dynamic range of detected proteins. However, even after compression, the dynamic range can nonetheless be relatively large, for example 5-log dynamic range within aptamer detection results for a sample. To address complexities of dynamic range, samples may undergo pretreatment or targeted panels are used to measure proteins over a particular range. These approaches add additional complexity and opportunities for loss of low concentration proteins. The disclosed techniques permit elimination of certain dynamic range compression steps, because the yield of eluted proteins has improved (more aptamers are rescued), which in turn results in a more compressed eluted aptamer group. Accordingly, elimination of dynamic range compression may permit different operation controls, such as elimination of separating the eluted aptamers into different dilution groups, when the present techniques are implemented. The techniques preserve the aptamer binding for low-abundancy proteins that are assessed together with high-abundancy proteins. Further, because low-abundancy proteins may correspond to biomarkers that can be used for diagnostic purposes, the disclosed techniques prevent noise or false negative results of an aptamer-based assay caused by high-abundancy proteins obscuring the results. In addition, compressing the dynamic range can also reduce the amount of total sequencing data required to detect aptamers in a detection assay by reducing the amounts of reads wasted on high-abundance aptamer sequences.

[0055] In embodiments of the disclosed techniques, aptamer detection may be based on a presence of the uniquely identifying identification sequence for an individual aptamer in sequencing data generated by the aptamer detection device 500. Accordingly, in an embodiment, the aptamer detection device 500 may perform analysis of sequence reads to identify one or more identification sequences for a panel of aptamers. Based on the identified aptamers, a notification or report of positive aptamer identification may be generated. In an embodiment, the notification is provided on the display 534 or communicated via the communications circuitry 527 to a remote device or a cloud server.

[0056] One or more compositions (e.g., analyte-aptamer complexes) as discussed herein may include a tag, e.g., an affinity tag. Affinity tags can be useful for a variety of applications, for example the bulk separation of target nucleic acids hybridized to hybridization tags. As used herein, the term “affinity tag” and grammatical equivalents can refer to a component of a multicomponent complex, wherein the components of the multi-component complex specifically interact with or bind to each other. For example an affinity tag can include biotin or poly-His that can bind streptavidin or nickel, respectively. Other examples of multiple-component affinity tag complexes are listed, for example, U.S. Patent Application Pub. No. 2012 / 0208705, U.S. Patent Application Pub. No. 2012 / 0208724 and Int. Patent Application Pub. No. WO 2012 / 061832, each of which is incorporated by reference in its entirety.

[0057] The disclosed techniques are directed to aptamer detection, such as for analysis of an eluate of an aptamer-based assay. The detection may include one or more amplification steps or preparation steps that can be part of sequencing library preparation that may couple oligonucleotide adaptors to eluted aptamers or to probes bound to eluted aptamers downstream sequencing. The adaptors may be attached to the target polynucleotide in any suitable manner. In some embodiments, the adaptors are introduced in a multi-step process, such as a two-step process, involving ligation of a portion of the adaptor to the target polynucleotide having a universal primer sequence. The second step includes extension, for example by PCR amplification, using primers that include a 3' end having a sequence complementary to the attached universal primer sequence and a 5' end that contains other sequences of an adaptor.By way of example, such extension may be performed as described in U.S. Pat. No. 8,053,192, which is hereby incorporated by reference in its entirety. Additional extensions may be performed to provide additional sequences to the 5' end of the resulting previously extended polynucleotide.

[0058] In some embodiments, the adaptor may be ligated directly to the aptamer or to reporter probes that bind to at least a portion of the aptamer, as generally discussed in U.S. Application No. 18 / 571,069, which is incorporated by reference herein. Any suitable adaptor may be attached to a target polynucleotide, such as a reporter probe, via any suitable process, such as those discussed herein. The adaptor can include a library-specific index tag sequence (e.g., i5, i7). The index tag sequence may be attached to the target polynucleotides from each library before the sample is immobilized for sequencing. The index tag is not itself formed by part of the target polynucleotide, but becomes part of the template for amplification. The index tag may be a synthetic sequence of nucleotides which is added to the target as part of the template preparation step. Accordingly, a library-specific index tag is a nucleic acid sequence tag which is attached to each of the target molecules of a particular library, the presence of which is indicative of or is used to identify the library from which the target molecules were isolated. Preferably, the index tag sequence is 20 nucleotides or less in length. For example, the index tag sequence may be 1-10 nucleotides or 4-6 nucleotides in length. A four nucleotide index tag gives a possibility of multiplexing 256 samples on the same array, a six base index tag enables 4,096 samples to be processed on the same array. The adaptors may contain more than one index tag so that the multiplexing possibilities may be increased.

[0059] The adaptors may include any other suitable sequence in addition to the index tag sequence. For example, the adaptors may include universal extension primer sequences, which are typically located at the 5' or 3' end of the adaptor and the resulting polynucleotide for sequencing. The universal extension primer sequences may hybridize to complementary primers bound to a surface of a solid substrate. The complementary primers include a free 3' end from which a polymerase or other suitable enzyme may add nucleotides to extend the sequence using the hybridized library polynucleotide as a template, resulting in a reversestrand of the library polynucleotide being coupled to the solid surface. Such extension may be part of a sequencing run or cluster amplification.

[0060] In some embodiments, the adaptors include one or more universal sequencing primer sequences. The universal sequencing primer sequences may bind to sequencing primers to allow sequencing of an index tag sequence, a target sequence, or an index tag sequence and a target sequence. In some embodiments, the disclosed reporter probes, e.g., reporter probe 24, may include a “sequencing adaptor” or “sequencing adaptor site”, that is to say a region that comprises one or more sites that can hybridize to a primer. In some embodiments, a sequence can include at least a first primer site useful for amplification, sequencing, and the like.

[0061] After adaptor incorporation, the disclosed reporter probes may be sequenced. In one example, the sequencing may be via Illumina's sequencing-by-synthesis and reversible terminator-based sequencing chemistry. Illumina's sequencing technology relies on the attachment of fragmented genomic DNA to a planar, optically transparent surface on which oligonucleotide anchors are bound. Template DNA is end-repaired to generate 5'- phosphorylated blunt ends, and the polymerase activity of Klenow fragment is used to add a single A base to the 3' end of the blunt phosphorylated DNA fragments. This addition prepares the DNA fragments for ligation to oligonucleotide adaptors, which have an overhang of a single T base at their 3' end to increase ligation efficiency. The adaptor oligonucleotides are complementary to the flow-cell anchors. Under limiting-dilution conditions, adaptor- modified, single-stranded template DNA is added to the flow cell and immobilized by hybridization to the anchors. Attached DNA fragments are extended and bridge amplified to create an ultra-high density sequencing flow cell with hundreds of millions of clusters, each containing ~l,000 copies of the same template. In one embodiment, the randomly fragmented genomic DNA is amplified using PCR before it is subjected to cluster amplification. Alternatively, an amplification-free genomic library preparation is used, and the randomly fragmented genomic DNA is enriched using the cluster amplification alone. The templates are sequenced using a robust four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescent dyes. High-sensitivity fluorescencedetection is achieved using laser excitation and total internal reflection optics. Sequence are aligned against a truth table or stored correlations between aptamer identity and identification sequences using specially developed data analysis pipeline software.

[0062] The present techniques are used for analysis of proteins present in oe or more samples of interest. The sample may be any sample taken from a biological organism or environmental sample (taken from an environment, such as a water, soil, or air sample). A biological sample may be obtained from a subject (such as a human or veterinary subject) that is a biological organism. In some examples, the sample is fixed, such as an FFPE sample. In particular examples, the biological sample is a biological fluid sample from any bodily fluid, such as peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchioalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre- ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates or other lavage fluids. A biological sample may also include the blastocyl cavity, umbilical cord blood, or maternal circulation which may be of fetal or maternal origin. The biological sample may also be a tissue sample or biopsy (including a fine needle aspirate). In some examples, the biological sample is a tumor sample. Such samples can be used to in the disclosed aptamer detection.

[0063] Biological samples may include nucleic acids (DNA, RNA), proteins, peptides, or protein fragments or other biological molecules (saccharides, fats, hydrocarbons) that may be aptamer targets. The biological sample may be a complex sample, such as a cell lysate. In embodiments, the biological sample may undergo pre-processing before entering the workflow as discussed herein.

[0064] This written description uses examples to enable any person skilled in the art to practice the disclosed embodiments, including making and using any devices or systems and performing any incorporated methods. The patentable scope is defined by the claims, and mayinclude other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims

CLAIMSWhat is claimed is:

1. A method of aptamer detection, comprising: contacting analytes of a sample with a plurality of aptamers under conditions that permit analyte-aptamer complexes to form, wherein different aptamers of the plurality of aptamers have specific affinity for respective different analytes of the analytes; tagging the analyte-aptamer complexes; capturing the analyte-aptamer complexes via respective tags; and contacting the captured analyte-aptamer complexes with a protease to elute the aptamers; and detecting the eluted aptamers.

2. The method of claim 1, inactivating the protease after the eluting and before the detecting.

3. The method of claim 1, inactivating the protease using a heat inactivation.

4. The method of claim 1, wherein the protease comprises a proteinase.

5. The method of claim 4, wherein the proteinase comprises proteinase K.

6. The method of claim 5, wherein the protease comprises a mix of proteases.

7. The method of claim 1, wherein the contacting comprises using a buffer comprising the protease.

8. The method of claim 7, wherein the buffer comprises up to 2% sodium dodecyl sulfate.

9. The method of claim 1, wherein the contacting is at a temperature from 20°C to 60°C.

10. The method of claim 1, wherein the protease is present in an amount equal to or less than 1U.

11. The method of claim 1, wherein the plurality of aptamers comprise aptamers with modifications, and wherein the contacting with the protease strips at least some of the modifications such that at least some of the aptamers are structurally different after the contacting.

12. An aptamer detection device, comprising: a capture substrate comprising an affinity tag binder configured to capture tags of analyte-aptamer complexes; and an elution buffer comprising a protease fluidically coupled to the capture substrate.

13. The aptamer detection device of claim 12, wherein the capture substrate comprises a bead.

14. The aptamer detection device of claim 12, comprising a channel or pipette configured to remove the elution buffer from the substrate after contact.

15. The aptamer detection device of claim 12, wherein the elution buffer comprises eluted aptamers.

16. The aptamer detection device of claim 12, wherein the capture substrate comprises captured analyte-aptamer complexes.

17. The aptamer detection device of claim 12, comprising a second substrate comprising a plurality of aptamers.

18. The aptamer detection device of claim 12, wherein the affinity tag binder is streptavidin.

19. The aptamer detection device of claim 12, wherein the protease is proteinase K.

20. The aptamer detection device of claim 12, wherein the elution buffer is stored in a channel of the capture substrate.

21. An aptamer eluate, comprising: an elution buffer comprising a protease; and a plurality of aptamers in the elution buffer, wherein different aptamers of the plurality of aptamers have specific affinity for respective different analytes.

22. The aptamer eluate of claim 21, wherein the protease is proteinase K.

23. The aptamer eluate of claim 21, wherein the protease is heat-inactivated.

24. The aptamer eluate of claim 21, wherein the protease activity in the buffer is in a range of O.OOlU / pL to 1U / pL.

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