Aptamers for detecting variants of a target analyte, methods for their production and use - Patents.com

Abstract

The present disclosure relates to a method for identifying or producing an aptamer capable of binding to at least two variants of a target analyte, comprising: generating a mixture of aptamers selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by at least one round of SELEX using a pool of SELEX-compatible aptamers simultaneously or sequentially for at least two variants of the target analyte; sequencing the mixture of SELEX-selected aptamers that form a complex with each of the at least two variants of the target analyte by high-throughput sequencing; aligning the mixture of SELEX-selected aptamers that form a complex with each of the at least two variants of the target analyte by sequence alignment analysis; and identifying or producing an aptamer that can bind to at least two variants of the target analyte. The aptamer and the use of the aptamer are also disclosed.

Description

[Technical field]

[0001] Related Applications This disclosure claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 298,381, filed Jan. 11, 2022, which is incorporated by reference in its entirety.

[0002] Incorporating sequence tables The computer-readable format of the sequence listing, “3244-P67236PC00_SequenceListing.xml” (124,468 bytes), created on January 9, 2023, is filed herewith by electronic submission and is incorporated herein by reference.

[0003] The present disclosure relates to the field of nucleic acid aptamers, and in particular to aptamers capable of binding to variants of a target analyte, methods of making and uses thereof. [Background technology]

[0004] The COVID-19 epidemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a great burden on human society [1]. The first virus was discovered in the city of Wuhan, China in December 2019 and has since spread globally. Over the past 18 months, several Variants of Concern (VoC) have emerged, the most notable VoCs being B.1.1.7 (UK variant, alpha), B.1.351 (South African variant, beta), P.1 (Brazilian variant, gamma), B.1.429 (California variant, epsilon), and B.1.617.2 (Indian variant, delta) [2], as well as, most recently, B.1.1.529 (Omicron) [3].

[0005] Large-scale testing, contact tracing, and isolation have been implemented as one method to curb the spread of SARS-CoV-2. However, current tests using quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR), while highly sensitive and specific for detecting SARS-CoV-2, suffer from slow turnaround times and high costs, making them unsuitable as screening tools to enable rapid response to outbreaks [4]. Several commercially available antigen (Ag) tests, such as the Abbott PanBio™ COVID-19Ag test, the Abbott BINAX Now™ COVID-19Ag test, and the Ellume rapid COVID-19 test, are relatively fast and inexpensive but inherently less sensitive than RT-PCR assays [5,6]. For example, the sensitivity of the Abbott PanBio™ COVID-19 Ag test was found to be only 57.7% and 2.6% using throat and saliva swabs, respectively [7]. Furthermore, known rapid tests have been reported to be insufficiently sensitive to detect the Omicron variant until several days after it becomes infectious, indicating that rapid tests are inadequate to detect newly emerged variants.With the SARS-CoV-2 genome constantly mutating and new VoCs rapidly emerging, there is an urgent need for simpler, faster, and cost-effective large-scale testing methods that work for both current and emerging VoCs.

[0006] The background art herein is included solely to provide a context for the present disclosure and is not an admission that any of the referenced material was published, known, or part of the common general knowledge as of the priority date. Summary of the Invention

[0007] DNA aptamers, which can be selected from random sequence pools by in vitro selection (SELEX) [8, 9], offer several important advantages over antibodies for rapid test development, such as small scale, high chemical and thermal stability, easy and precise modification, scalable production, and minimal batch-to-batch variation

[10] . Moreover, they have been shown to be capable of detecting targets present in clinical samples [11-13]. Because of these attractive properties, aptamers have become important molecular recognition elements (MREs) for assay and diagnostic development [14-16].

[0008] According to one embodiment, a method is provided for identifying or generating aptamers that universally (or non-discriminatory) recognize variants of a target analyte.

[0009] Provided herein is a method for identifying or generating an aptamer capable of binding to at least two variants of a target analyte, comprising: a) generating a mixture of aptamers selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by at least one round of SELEX using a pool of SELEX-compatible aptamers simultaneously or sequentially against at least two variants of a target analyte; b) sequencing the mixture of SELEX-selected aptamers that are complexed with each of the at least two target analyte variants by high throughput sequencing; c) aligning the mixture of SELEX-selected aptamers that form complexes with each of the at least two target analyte variants by sequence alignment analysis; d) identifying or producing aptamers capable of binding to at least two variants of the target analyte.

[0010] In some embodiments, the method further comprises generating a SELEX-compatible aptamer pool prior to a). In some embodiments, the SELEX-compatible aptamer pool comprises a plurality of nucleic acid molecules comprising at least one random nucleotide domain having an aptamer-like structure, the random nucleotide domain being adjacent to a 5'-terminal region and a 3'-terminal region. In some embodiments, the 5'-terminal region hybridizes to the 3'-terminal region to form a double-stranded DNA element. In some embodiments, the 5'-terminal region comprises the nucleotide sequence of SEQ ID NO: 102 or 103, and the 3'-terminal region comprises the nucleotide sequence of SEQ ID NO: 132 or the reverse complement of SEQ ID NO: 104 or 105. In some embodiments, the SELEX-compatible aptamer pool comprises a plurality of nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 101.

[0011] In some embodiments, the target analyte variant is a microorganism, a virus, and / or a molecule present in a microorganism or a virus. In some embodiments, the target analyte variant is a protein. In some embodiments, the target analyte variant is a protein from SARS-CoV-2. In some embodiments, the target analyte variant is a SARS-CoV-2 spike protein. In some embodiments, the method includes in a) generating a mixture of SELEX-selected aptamers with at least 3, 4, 5, 6, 7, 8, or 9 target analyte variants.

[0012] In some embodiments, the high-throughput sequencing of b) comprises single molecule real-time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis sequencing, sequencing by ligation, nanopore sequencing, or GenapSys™ sequencing. In some embodiments, the high-throughput sequencing comprises sequencing by synthesis.

[0013] Several groups have isolated DNA aptamers that bind to the S1 subunit of the spike protein of the original SARS-CoV-2 virus or its receptor-binding domain (RBD) [17-25]. However, apart from the finding that two S1-binding DNA aptamers, named MSA1 and MSA5, also showed similar affinity for the spike protein of the B.1.1.7 variant of interest, there is limited information available on whether the reported aptamers can bind to the spike proteins of other important VoCs

[17] .

[0014] Also provided is an aptamer comprising a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-100, functional fragments and functional variants thereof, or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 113-117, functional fragments and functional variants thereof. In some embodiments, the 5'-terminal region comprises a nucleotide sequence of SEQ ID NO: 102 or 103, and the 3'-terminal region comprises a nucleotide sequence of SEQ ID NO: 132 or the reverse complement of SEQ ID NO: 104 or 105. In some embodiments, the aptamer comprises a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-10, functional fragments and functional variants thereof. In some embodiments, the aptamer comprises a nucleotide sequence comprising SEQ ID NO: 116 or 117.

[0015] 1. A method for detecting the presence of SARS-CoV-2 spike protein in a sample, comprising: a) contacting the sample with an aptamer as described herein, where the aptamer binds to the spike protein; b) detecting the presence of an aptamer bound to the spike protein in the sample, wherein detection of the bound aptamer indicates the presence of the SARS-CoV-2 spike protein or a variant thereof.

[0016] In some embodiments, the SARS-CoV-2 spike protein variant comprises a B.1.1.7, B.1.351, P.1, B.1.429, B.1.617.1 B.1.617.2, B.1.617.2.1, or B.1.1.529 spike protein variant. In some embodiments, the aptamer comprises a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-100, functional fragments and functional variants thereof, or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 106-117, functional fragments and functional variants thereof. In some embodiments, the aptamer comprises a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-10, functional fragments and functional variants thereof. In some embodiments, the aptamer comprises a 5' terminal region, a 3' terminal region, and the nucleotide sequence of SEQ ID NO: 1, a functional fragment, or a functional variant thereof. In some embodiments, the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 108, 112-117, a functional fragment, and a functional variant thereof. In some embodiments, the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 108, 112, 116, 117, a functional fragment, and a functional variant thereof. In some embodiments, the aptamer comprises a nucleotide sequence of SEQ ID NO: 112, a functional fragment, or a functional variant thereof.

[0017] In some embodiments, the method detects SARS-CoV-2 infection in a subject. In some embodiments, the sample is saliva, sputum, urine, blood, serum, other bodily fluids, and / or secretions.

[0018] Also provided herein is the use of the aptamers described herein for detecting SARS-CoV-2 spike protein.

[0019] This disclosure provides the first evidence that it is possible to generate high affinity aptamers against multiple variants of a single protein, including newly emerged variants, making them suitable for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.

[0020] Other features and advantages of the present disclosure will become apparent from the following detailed description of the invention. However, it should be understood that the detailed description and specific examples, while showing embodiments of the present disclosure, are shown for illustrative purposes only, and that the claims are not limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.

[0021] Certain embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. [Brief description of the drawings]

[0022] [Figure 1A] In an exemplary embodiment of the present disclosure, dot blot results of MSA1 for binding to the original (WHS) and four variants of the complete spike protein of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 1B] In an exemplary embodiment of the present disclosure, dot blot results of MSA5 for binding to the original (WHS) and four variants of the complete spike protein of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 2A] In an exemplary embodiment of the present disclosure, the binding affinity of previously reported DNA aptamers was evaluated using a dot blot assay. Figure 2A shows the binding curves of MSA1 to the spike protein of the original SARS-CoV-2 (WHS) and four variants of the spike protein (B.1.1.7S, B.1.351S, P.1S, B.1.429S). [Figure 2B]In an exemplary embodiment of the present disclosure, the binding affinity of previously reported DNA aptamers was evaluated using a dot blot assay. Figure 2B shows the binding curves of MSA5 to the original SARS-CoV-2 (WHS) spike protein and four variant spike proteins (B.1.1.7S, B.1.351S, P.1S, B.1.429S). [Figure 3A]

[0023] Figure 3 shows the discovery of MSA52 in an exemplary embodiment of the present disclosure. Figure 3A shows the sequence of the DNA library used in the original SELEX experiment. [Figure 3B] 3A shows the discovery of MSA 52 in an exemplary embodiment of the present disclosure. FIG. 3B shows a schematic diagram of the reselection used for the discovery of MSA 52. [Figure 3C] Figure 3C shows the discovery of MSA52 in an exemplary embodiment of the present disclosure. The top ten members of the MSA52 aptamer family are shown (MSA52R1 (SEQ ID NO: 1), MSA52R2 (SEQ ID NO: 2), MSA52R3 (SEQ ID NO: 3), MSA52R4 (SEQ ID NO: 4), MSA52R5 (SEQ ID NO: 5), MSA52R6 (SEQ ID NO: 6), MSA2R7 (SEQ ID NO: 7), MSA52R8 (SEQ ID NO: 8), MSA52R9 (SEQ ID NO: 9), and MSA52R10 (SEQ ID NO: 10)). Each point mutation associated with the top ranking sequence is highlighted. [Figure 3D] Figure 3D shows the discovery of MSA52 in an exemplary embodiment of the present disclosure. The increase in the rank of each of the top 10 sequences in the UK (B.1.1.7), BZ (P.1), SA (B.1.351), and CA (B.1.429) pools compared to their rank in the WH pool. [Figure 4] 1 shows a plot of the log (frequency) difference of the top 15 MSA52 cluster members in the WHS, UKS, BZS, SAS, and CAS pools compared to the round 13 pool in an exemplary embodiment of the present disclosure. [Figure 5A]In an exemplary embodiment of the present disclosure, a dot blot assay is used to assess the binding affinity of MSA52 to the trimeric spike protein of SARS-CoV-2 and its B.1.1.7, B.1.351, P.1, B.1.429, B.1.617.1, B.1.617.2, and B.1.1.529 variants. Figure 5A shows representative dot blot results showing binding of MSA52 to eight trimeric spike proteins. [Figure 5B] In an exemplary embodiment of the present disclosure, a dot blot assay is used to assess the binding affinity of MSA52 to the trimeric spike protein of SARS-CoV-2 and its B.1.1.7, B.1.351, P.1, B.1.429, B.1.617.1, B.1.617.2, and B.1.1.529 variants. Figure 5B shows the binding curve used to derive the Kd value of MSA52 for the trimeric spike protein. [Figure 6] 1 shows a competition assay for probing the binding site on the S1 protein of SARS-CoV-2 by MSA52, MSA1, and MSA5 in an exemplary embodiment of the present disclosure. [Figure 7A] In an exemplary embodiment of the present disclosure, dot blot results of a competition assay are shown. Figure 7A shows MSA1 competing with MSA52. Competing aptamers were 2.5 nM and radiolabeled (asterisk indicated). Competing aptamers were unlabeled and ranged in concentration from 1.2 to 160 nM. 5 nM of SARS-CoV-2 S1 protein was used in the assay. BA and UA: binding and non-binding aptamers. [Figure 7B] 7A-7C show dot blot results of a competition assay in an exemplary embodiment of the present disclosure. FIG. 7B shows MSA5 competing with MSA52. Competing aptamers were 2.5 nM and radiolabeled (marked with an asterisk). Competing aptamers were unlabeled and ranged in concentration from 1.2 to 160 nM. 5 nM of SARS-CoV-2 S1 protein was used in the assay. BA and UA: binding and non-binding aptamers. [Figure 7C]In an exemplary embodiment of the present disclosure, dot blot results of a competition assay are shown. FIG. 7C shows MSA52 competing with MSA1. Competing aptamers were 2.5 nM and radiolabeled (asterisk indicated). Competing aptamers were unlabeled and ranged in concentration from 1.2 to 160 nM. 5 nM of SARS-CoV-2 S1 protein was used in the assay. BA and UA: binding and non-binding aptamers. [Figure 7D] In an exemplary embodiment of the present disclosure, dot blot results of a competition assay are shown. FIG. 7D shows MSA52 competing with MSA5. Competing aptamers were 2.5 nM and radiolabeled (marked with an asterisk). Competing aptamers were unlabeled and ranged in concentration from 1.2 to 160 nM. 5 nM of SARS-CoV-2 S1 protein was used in the assay. BA and UA: binding and non-binding aptamers. [Figure 7E] In an exemplary embodiment of the present disclosure, dot blot results of a competition assay are shown. FIG. 7E shows a negative control of MSA52 competing with a mutant sequence (shuffled MSA52, i.e., mutant control). Competing aptamers were 2.5 nM and radioactively labeled (marked with an asterisk). Competing aptamers were unlabeled and ranged in concentration from 1.2 to 160 nM. 5 nM of SARS-CoV-2 S1 protein was used in the assay. BA and UA: binding and non-binding aptamers. [Figure 7F] In an exemplary embodiment of the present disclosure, dot blot results of a competition assay are shown. FIG. 7F shows self-competition of MSA52. Competing aptamers were 2.5 nM and radiolabeled (asterisk indicated). Competing aptamers were unlabeled and ranged in concentration from 1.2 to 160 nM. 5 nM of SARS-CoV-2 S1 protein was used in the assay. BA and UA: binding and non-binding aptamers. [Figure 7G]In an exemplary embodiment of the present disclosure, dot blot results of a competition assay are shown. FIG. 7G shows self-competition of MSA1. Competing aptamers were 2.5 nM and radiolabeled (asterisk indicated). Competing aptamers were unlabeled and ranged in concentration from 1.2 to 160 nM. 5 nM of S1 protein of SARS-CoV-2 was used in the assay. BA and UA: binding and non-binding aptamers. [Figure 7H] In an exemplary embodiment of the present disclosure, dot blot results of a competition assay are shown. FIG. 7H shows self-competition of MSA5. Competing aptamers were 2.5 nM and radiolabeled (asterisk indicated). Competing aptamers were unlabeled and ranged in concentration from 1.2 to 160 nM. 5 nM of S1 protein of SARS-CoV-2 was used in the assay. BA and UA: binding and non-binding aptamers. [Figure 8] In an exemplary embodiment of the present disclosure, specificity testing of MSA52 binding to the spike protein of SARS-CoV-2 and eight control proteins including BSA, human α-thrombin, and the RBD and spike (S) proteins of SARS-CoV1, the RBD protein of MERS, and the three seasonal coronaviruses 229E, NL63, and OC43. 50 nM of protein was used in the assay. [Figure 9A] 1 shows a dot blot result of MSA52 binding to the spike protein of SARS-CoV-1 in an exemplary embodiment of the present disclosure. BA: binding aptamer. UA: non-binding aptamer. [Figure 9B] 1 shows a dot blot result of MSA52 binding to the RBD protein of seasonal coronavirus 229E in an exemplary embodiment of the present disclosure. BA: binding aptamer. UA: non-binding aptamer. [Figure 9C] In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to the RBD protein of seasonal coronavirus OC43 are shown. BA: binding aptamer. UA: non-binding aptamer. [Figure 9D]In an exemplary embodiment of the present disclosure, the affinity (Kd) determination from the dot blot results of Figures 9A, 9B, and 9C is shown. BA: binding aptamer. UA: non-binding aptamer. [Figure 10A] In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to prototype (WH) SARS-CoV-2 pseudotyped viruses are shown. BA and UA: binding and non-binding aptamers. [Figure 10B] In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to pseudotyped viruses of the B.1.1.7 variant of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 10C] In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to pseudotyped viruses of the B.1.351 variant of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 10D] In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to pseudotyped viruses of the P.1 variant of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 10E] In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to pseudotyped viruses of the B.1.429 variant of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 10F] In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to pseudotyped viruses of the B.1.617.1 variant of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 10G] In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to pseudotyped viruses of the B.1.617.2 variant of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 10H]In an exemplary embodiment of the present disclosure, dot blot results of MSA52 binding to pseudotyped viruses of the B.1.617.2.1 variant of SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 10I] 1 shows an exemplary embodiment of a dot blot result of binding of MSA52 of the present disclosure to a control lentivirus (CV).BA and UA: binding and non-binding aptamer. [Figure 10J] In an exemplary embodiment of the present disclosure, dot blot results of binding of inactive mutant sequences of MSA52 (MC) to prototype (WH) SARS-CoV-2 are shown. BA and UA: binding and non-binding aptamers. [Figure 11A] Characterization of MSA52 and MSA52-T5 in exemplary embodiments of the present disclosure. Figure 11A shows an assessment of the binding affinity of MSA52 to the prototype SARS-CoV-2 virus (WH) and lentiviruses pseudotyped to express the spike protein of the B.1.1.7, B.1.351, P.1, B.1,429, B.1.617.1, B.1.617.2, and B.1.617.2.1 variants. MC: mutant control. [Figure 11B] Figure 11B shows the characterization of MSA52 and MSA52-T5 in an exemplary embodiment of the present disclosure. The proposed secondary structure of full-length MSA52 (SEQ ID NO: 112) is shown. [Figure 11C] Figure 11B shows the characterization of MSA52 and MSA52-T5 in an exemplary embodiment of the present disclosure. Figure 11B shows the proposed secondary structure of a minimized version of MSA52 (designated MSA52-T5, SEQ ID NO: 117). [Figure 12] 1 shows an investigation of the predicted secondary structure of MSA52 through sequence truncation in an exemplary embodiment of the present disclosure. Full-length MSA52 is SEQ ID NO: 112. The five truncation mutants were named MSA52-T1 to MSA52-T5 (SEQ ID NOs: 113 to 117). The Kd value represents the binding activity to SARS-CoV-2 S1 protein. [Figure 13A]FIG. 1 shows a schematic diagram of the working principle of an 11-aptamer sandwich assay for detecting prototype SARS-CoV-2 (WH) and pseudotyped lentiviruses expressing the B.1.1.7, B.1.351, P.1, and B.1.617.2 variants in an exemplary embodiment of the present disclosure. [Figure 13B] Shown is an original image of a representative 96-well microtiter plate assay of an 11-aptamer sandwich assay for detecting prototype SARS-CoV-2 (WH), and pseudotyped lentiviruses expressing the B.1.1.7, B.1.351, P.1, and B.1.617.2 variants in an exemplary embodiment of the present disclosure. [Figure 14A] In an exemplary embodiment of the present disclosure, an 11-aptamer sandwich assay for detecting prototype SARS-CoV-2 (WH) and pseudotyped lentiviruses expressing the B.1.1.7, B.1.351, P.1, and B.1.617.2 variants is shown. Figure 14A shows the absorbance at 450 nm generated by the HRP-tagged aptamer upon oxidation of TMB in the presence of H2O2 and subsequent quenching with H2SO4. [Figure 14B] In an exemplary embodiment of the present disclosure, an 11-aptamer sandwich assay for detecting prototype SARS-CoV-2 (WH) and pseudotyped lentiviruses expressing the B.1.1.7, B.1.351, P.1, and B.1.617.2 variants is shown. Figure 14B shows an image of a representative 96-well microtiter plate assay (the image has been processed in Photoshop for visual effect; the original image is shown in Figure 13). [Figure 15A] FIG. 1 shows an image of a representative 96-well microtiter plate assay of an 11-aptamer sandwich assay for detecting the trimeric spike protein (5 nM) of the B.1.1.529 (Omicron) variant of SARS-CoV-2 in an exemplary embodiment of the disclosure. [Figure 15B]In an exemplary embodiment of the present disclosure, an 11-aptamer sandwich assay for detecting the trimeric spike protein (5 nM) of the B.1.1.529 (Omicron) variant of SARS-CoV-2 is shown. Figure 15B shows the absorbance at 450 nm generated by the HRP-tagged aptamer upon oxidation of TMB in the presence of H2O2 and subsequent quenching with H2SO4. The A450 value of each reaction mixture was corrected for background by subtracting the A450 observed in the matching BSA reaction mixture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] I. Definition Unless otherwise indicated, the definitions and embodiments described in this section and other sections are intended to be applicable to all embodiments and aspects of the disclosure described herein where they are appropriate, as understood by one of ordinary skill in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0024] In understanding the scope of the present disclosure, the term "comprising" and its derivatives as used herein are intended to be open ended terms that specify the presence of the stated features, ingredients, components, groups, elements, and / or steps, but do not exclude the presence of other unstated features, ingredients, components, groups, elements, and / or steps. The above also applies to words of similar meaning, such as the terms "including," "having," and their derivatives. As used herein, "consisting" and its derivatives are intended to be closed terms that specify the presence of the stated features, ingredients, components, groups, elements, and / or steps, but exclude the presence of other unstated features, ingredients, components, groups, elements, and / or steps. As used herein, the term "consisting essentially of" is intended to specify the presence of the described features, ingredients, components, groups, elements, and / or steps, as well as things that do not materially affect the basic and novel characteristic(s) of the features, ingredients, components, groups, elements, and / or steps.

[0025] As used herein, terms of degree such as "substantially," "about," and "approximately" refer to a reasonable amount of deviation from the modified term that does not significantly change the end result. These terms of degree should be interpreted to include deviations of at least ±5% of the modified term, if this deviation does not negate the meaning of the word it modifies. Furthermore, all ranges set forth herein include the ends of the ranges, and any intermediate range points, whether or not they are explicitly stated.

[0026] As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0027] In embodiments that include an "additional" or "second" component, the second component, as used herein, is chemically distinct from the other component or the first component. A "third" component is distinct from the other component, the first component, and the second component, and further recited or "additional" components are similarly distinct.

[0028] As used herein, the term "and / or" means that the listed items may be present or utilized either individually or in any combination. In effect, the term means that "at least one" or "one or more" of the listed items are utilized or present.

[0029] The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." The word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0030] As used herein, the terms "target", "analyte", or "target analyte" refer to any agent that one desires to sense or detect, including, but not limited to, small inorganic molecules, small organic molecules, metal ions, biomolecules, toxins, biopolymers (such as nucleic acids, carbohydrates, lipids, peptides, proteins, etc.), cells, tissues, microorganisms, and viruses. Analytes can be either isolated from natural sources or synthetic. Analytes can be single compounds or classes of compounds, such as classes of compounds that share structural or functional characteristics. The term analyte also includes combinations (e.g., mixtures) of compounds or agents, including, but not limited to, combinatorial libraries and samples from organisms or natural environments.

[0031] As used herein, the term "sample" or "test sample" refers to any material in which the presence or amount of a target analyte is unknown and can be determined by an assay. A sample may be from any source, for example, any biological (e.g., human or animal sample, including clinical samples), environmental (e.g., water, soil, or air), or natural (e.g., plant) source, or any processed or synthetic source (e.g., food or beverage). A sample may consist of one or more analytes or be suspected of containing one or more analytes. A sample may be a "biological sample" that includes cellular and non-cellular material, including, but not limited to, tissue samples, saliva, sputum, urine, blood, serum, other bodily fluids, and / or secretions. In some embodiments, the sample includes saliva, sputum, oropharyngeal and / or nasopharyngeal secretions. In some embodiments, the sample includes saliva.

[0032] As used herein, the term "subject" includes all members of the animal kingdom, including mammals, such as mice, rats, dogs, and humans.

[0033] The term "virus" as used herein refers to a simple structured organism that consists of proteins and nucleic acids and can replicate using its metabolism only within certain living cells. In some embodiments, the virus is an enveloped virus, a non-enveloped virus, a DNA virus, a single-stranded RNA virus, and / or a double-stranded RNA virus. Non-limiting examples of viruses include rhinoviruses, myxoviruses (including influenza viruses), paramyxoviruses, coronaviruses such as SARS-CoV-2, noroviruses, rotaviruses, herpes simplex viruses, poxviruses (including smallpox viruses), reoviruses, adenoviruses, enteroviruses, encephalomyocarditis viruses, cytomegaloviruses, varicella zoster viruses, rabies viruses, and retroviruses (including HIV).

[0034] As used herein, the term "severe acute respiratory syndrome coronavirus 2," "coronavirus 2," or "SARS-CoV-2" refers to the coronavirus first identified in 2019 in Wuhan, China that can cause coronavirus disease (COVID-19). The term includes any variant of the SARS-CoV-2 virus that has a variant and / or mutated nucleic acid sequence from the prototype identified in Wuhan. Variants include, but are not limited to, UK B.1.1.7 (501Y.V1), South African B.1.351 (501Y.V2), Brazilian P.1 (501Y.V3), Indian B.1.617, and B.1.1.529 (Omicron).

[0035] The term "nucleic acid" as used herein refers to biopolymers comprising nucleotide monomers, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and other polynucleotides of modified nucleotides and / or nucleotide derivatives, and can be either double-stranded (ds) or single-stranded (ss). In some embodiments, modified nucleotides can contain one or more modified bases (e.g., unusual bases such as inosine, and functional modifications to bases such as amino), modified backbones (e.g., peptide nucleic acids, PNA), and / or other chemically, enzymatically, or metabolically modified forms.

[0036] The term "aptamer" as used herein refers to short chemically synthesized nucleic acid molecules or oligonucleotide sequences that can be generated by in vitro selection to fold into specific three-dimensional structures that bind to specific analytes with dissociation constants in the picomolar to nanomolar range, for example. Aptamers can be single-stranded DNA and can include RNA, modified nucleotides and / or nucleotide derivatives. Aptamers can also be naturally occurring RNA aptamers called "riboswitches." Functional aptamer sequences can also be rationally designed, truncated, conjugated, or otherwise modified from the original parent (or full-length) sequence.

[0037] The term "variant" as used herein refers to a subtype of a microorganism having a genome that includes one or more mutations from another strain, e.g., a primary strain. A variant can itself be a primary strain, as compared to the mutant genome. Thus, when referring to two variants, it can be any of the two subtypes. With respect to the SARS-CoV-2 virus, variants have mutated nucleic acid sequences from the prototype identified in Wuhan, including but not limited to B.1.1.7, B.1.351, P.1, B.1.617, and B.1.1.529, which is itself considered a variant of other subtypes. Variants may have the potential to result in increased transmissibility, increased morbidity, increased mortality, the ability to evade detection by diagnostic tests, reduced susceptibility to antiviral drugs, reduced susceptibility to neutralizing antibodies, the ability to evade natural immunity, the ability to infect vaccinated individuals, increased risk of certain conditions, such as multisystem inflammatory syndrome or long COVID, and / or increased affinity for certain demographic or clinical groups, such as children or individuals with immunodeficiencies. For any particular molecular target, such as a protein, a variant can be, for example, a mutant protein with at least 75% sequence identity with the original protein, and / or a functional variant that retains some functional aspects of the original protein. A variant can be the main, wild type, or original target analyte (e.g., strain or protein) itself, compared to the mutated target analyte. Thus, when referring to the variants of two target analytes, it can be any two subtypes of the target analyte. Those skilled in the art can recognize the variants of target analytes, for example, regardless of the level of microorganism or protein.

[0038] The term "systematic evolution of ligands by exponential enrichment" or "SELEX" as used herein refers to a method for selecting aptamers that specifically bind to a target molecule, such as those described in, for example, Ellington, AD & Szostak, JW, "In vitro selection of RNA molecules that bind specific ligands." Nature 346, 818-822 (1990), McConnell, EM et al., "Biosensors Made of Synthetic Functional Nucleic Acids Toward Better Human Health." Anal. Chem. 92, 327-344 (2020), U.S. Patent Nos. 5,475,096, and 5,270,163 (each of which is incorporated herein by reference in its entirety). SELEX involves immobilizing the selected target on a column, and a solution containing a library or combination of random DNA or RNA molecules is contacted with the target under conditions that favor binding. After a certain time, the column is flushed, so that unbound nucleic acid molecules are washed out of the column and nucleotide molecules that bind to the target remain on the column. The nucleic acid-target complex is then dissociated, and the nucleic acid molecules selected by binding to the target are amplified. This cycle can be repeated to obtain nucleic acids with higher affinity. SELEX-compatible aptamer pools have aptamers, which are fully or partially randomized oligonucleotide sequences of a certain length flanked by defined regions that allow PCR amplification (or in vitro transcription in the case of RNA SELEX) of their randomized regions. For example, a SELEX-compatible aptamer pool can have a plurality of nucleic acid molecules that include at least one random nucleotide domain with an aptamer-like structure, where the random domain is flanked by a 5'-terminal region and a 3'-terminal region, and the 5'-terminal region can hybridize to the 3'-terminal region to form a double-stranded DNA element.The random domains can have a length of about 40 nucleotides, so one example of a SELEX-compatible aptamer pool comprises a DNA library having the nucleotide sequence of SEQ ID NO:101.

[0039] II. METHODS, USES, AND APTAMERS Disclosed herein is a method for identifying or producing DNA aptamers against variants of SARS-CoV-2 spike protein subunit S1 from pre-structured random DNA libraries using parallel systematic evolution of ligands by exponential enrichment (SELEX), high-throughput sequencing, and sequence alignment analysis. We show that it is possible to generate high affinity aptamers against multiple variants of a single protein, making them suitable for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.

[0040] Accordingly, provided herein is a method for identifying or generating an aptamer capable of binding to at least two variants of a target analyte, comprising: a) generating a mixture of aptamers selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by at least one round of SELEX using a pool of SELEX-compatible aptamers simultaneously or sequentially against variants of said at least two target analytes; b) sequencing the mixture of SELEX-selected aptamers that are complexed with each of the at least two target analyte variants by high throughput sequencing; c) aligning the mixture of SELEX-selected aptamers that form complexes with each of the at least two target analyte variants by sequence alignment analysis; d) identifying or producing aptamers capable of binding to at least two variants of the target analyte.

[0041] In some embodiments, the method includes generating a SELEX-compatible aptamer pool prior to a). In some embodiments, the SELEX-compatible aptamer pool includes a plurality of nucleic acid molecules including at least one random nucleotide domain having an aptamer-like structure, the random nucleotide domain being adjacent to a 5'-terminal region and a 3'-terminal region. In some embodiments, the 5'-terminal region hybridizes to the 3'-terminal region to form a double-stranded DNA element. In some embodiments, the 5'-terminal region includes the nucleotide sequence of SEQ ID NO: 102 or 103, and the 3'-terminal region includes the nucleotide sequence of SEQ ID NO: 132 or the reverse complement of SEQ ID NO: 104 or 105. In some embodiments, the SELEX-compatible aptamer pool includes a plurality of nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 101. In some embodiments, the SELEX-compatible aptamer pool comprises at least one aptamer comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 108, 112-117, functional fragments and functional variants thereof, and / or comprises at least one aptamer comprising a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-100, functional fragments and functional variants thereof.

[0042] In some embodiments, SELEX involves contacting a pool of aptamers with a target analyte variant under conditions favoring binding, segregating unbound aptamers from aptamers bound to the target analyte variant, dissociating the aptamer-target analyte variant complexes, and amplifying the aptamers dissociated from the aptamer-target analyte variant to obtain a mixture of aptamers enriched for the target analyte variant. In some embodiments, the target analyte variant is a microorganism, a virus, and / or a molecule present in a microorganism or virus. In some embodiments, the target analyte variant is a protein. In some embodiments, the target analyte variant is a protein from SARS-CoV-2. In some embodiments, the target analyte variant is a SARS-CoV-2 spike protein.

[0043] In some embodiments, the method comprises in a) generating a mixture of SELEX-selected aptamers with at least 3, 4, 5, 6, 7, 8, or 9 variants of the target analyte. In some embodiments, the method comprises in a) generating a mixture of SELEX-selected aptamers with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 variants of the target analyte. In some embodiments, the method comprises in a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 rounds of SELEX using a SELEX-compatible aptamer pool.

[0044] In some embodiments, high throughput sequencing comprises single molecule real-time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis sequencing, sequencing by ligation, nanopore sequencing, or GenapSys™ sequencing. In some embodiments, high throughput sequencing comprises sequencing by synthesis.

[0045] In some embodiments, the method includes at least one of merging sequences into a consensus sense read, deduplication, clustering at 90% identity, multiple sequence alignment, conversion to a sequence logo, identifying sequence copy number, identifying sequence frequency, and identifying cluster linkages.

[0046] In some embodiments, the method includes steps or methods described in the Examples.

[0047] In another aspect, a DNA aptamer, designated MSA52, is provided that exhibits universally high affinity for wild-type SARS-CoV-2 and alpha, beta, gamma, epsilon, kappa delta, and omicron variant spike proteins. Using the aptamer pool generated from round 13 of selection against the S1 domain of the wild-type spike protein, one round of SELEX experiments was performed with five different trimeric spike proteins from the variants, followed by high-throughput sequencing and sequence alignment analysis of the aptamers complexed with all proteins. MSA52 exhibits a K in the range of 2-10 nM for all variant spike proteins. d The aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with Kd values ​​of 20–50 pM.

[0048] In some embodiments, the aptamer comprises a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-100, functional fragments and functional variants thereof, or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 108, 112-117, functional fragments and functional variants thereof. In some embodiments, the aptamer comprises a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-100, functional fragments and functional variants thereof, or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 113-117, functional fragments and functional variants thereof. In some embodiments, the aptamer is provided comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 106-117, functional fragments and functional variants thereof. In some embodiments, the 5'-terminal region comprises a nucleotide sequence of SEQ ID NO: 102 or 103, and the 3'-terminal region comprises a nucleotide sequence of SEQ ID NO: 132 or the reverse complement of SEQ ID NO: 104 or 105. In some embodiments, the aptamer comprises a 5' terminal region, a 3' terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-10, functional fragments and functional variants thereof. In some embodiments, the aptamer comprises a nucleotide sequence comprising SEQ ID NO: 116 or 117.

[0049] 1. A method for detecting the presence of SARS-CoV-2 spike protein in a sample, comprising: a) contacting the sample with an aptamer as described herein, where the aptamer binds to the spike protein; b) detecting the presence of an aptamer bound to the spike protein in the sample, wherein detection of the bound aptamer indicates the presence of the SARS-CoV-2 spike protein or a variant thereof.

[0050] In some embodiments, the SARS-CoV-2 spike protein variant comprises a B.1.1.7, B.1.351, P.1, B.1.429, B.1.617.1 B.1.617.2, B.1.617.2.1, or B.1.1.529 spike protein variant. In some embodiments, the aptamer comprises a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-100, functional fragments and functional variants thereof, or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 106-117, functional fragments and functional variants thereof. In some embodiments, the aptamer comprises a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-10, functional fragments and functional variants thereof. In some embodiments, the aptamer comprises a 5' terminal region, a 3' terminal region, and a nucleotide sequence of SEQ ID NO: 1, a functional fragment, and a functional variant thereof. In some embodiments, the aptamer comprises a 5' terminal region, a 3' terminal region, and a nucleotide sequence of SEQ ID NO: 1. In some embodiments, the 5' terminal region comprises a nucleotide sequence of SEQ ID NO: 102 or 103, and the 3' terminal region comprises a nucleotide sequence of SEQ ID NO: 132 or a reverse complement of SEQ ID NO: 104 or 105. In some embodiments, the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 108, 112-117, functional fragments, and functional variants thereof. In some embodiments, the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 108, 112, 116, 117, functional fragments, and functional variants thereof. In some embodiments, the aptamer comprises a nucleotide sequence comprising SEQ ID NO: 108, 112, 116, or 117. In some embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 108, a functional fragment or a functional variant thereof. In some embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 108. In some embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 112, a functional fragment or a functional variant thereof.In some embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 112. In some embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 116, a functional fragment or a functional variant thereof. In some embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 116. In some embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 117, a functional fragment or a functional variant thereof. In some embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 117. In some embodiments, the method detects SARS-CoV-2 infection in a subject. In some embodiments, the sample is saliva, sputum, urine, blood, serum, other bodily fluids, and / or secretions.

[0051] Also provided is the use of the aptamers described herein for detecting SARS-CoV-2 spike protein.

[0052] Also provided is a nucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-132.

[0053] It will be understood that any component defined as included herein may be expressly excluded by a disclaimer or negative limitation, such as any particular compound or method step, whether implicitly or explicitly defined herein. EXAMPLES

[0054] The following non-limiting examples illustrate the present disclosure.

[0055] method

[0056] Materials and Reagents: DNA oligonucleotides were obtained from Integrated DNA Technologies (IDT) and purified by standard 10% denaturing (8M urea) polyacrylamide gel electrophoresis (dPAGE) before use. Sequences are listed in Table 1A and Table 3. Wuhan SARS-CoV-2 spike protein subunit S1 (catalog number: 40591-V08B1) was purchased from Sino Biological Inc. Wuhan SARS-CoV-2 full spike protein (WHS, molecular weight 140 kDa), spike pseudotyped lentivirus of Wuhan SARS-CoV-2 (WH), variant B.1.351, variant P.1, and control lentivirus were prepared using standard methods. Intact spike proteins of variants B.1.1.7 (catalog number: SPN-C52H6), B.1.617.1 (SPN-C52Hr), B.1.617.2 (SPN-C52He), and B.1.1.529 (SPN-C52Hz) were all expressed from human 293 cells (HEK293) and obtained from Acro Biosystems. Spike proteins of variants B.1.351 (510333-1), P.1 (100989-1), and B.1.429 (101057) were all expressed from human 293 cells (HEK293) and obtained from BPS Biosciences Inc. Spike and RBD proteins of SARS-CoV-1, MERS, and seasonal coronaviruses 229E, NL63, and OC43 were provided by the Miller lab at McMaster University. Protein concentrations were quantified using the Thermo Scientific Bicinchoninic Acid (BCA) Protein Assay Kit (catalog number: 23225). SARS-CoV-2 spike pseudotyped lentiviruses of variants B.1.1.7 (catalog number: 78112-1), B.1.429 (78172-1), B.1.617.1 (78205-1), B.1.617.2 (78216-1), and B.1.617.2.1 (78219-1) were obtained from BPS Bioscience.Nitrocellulose blotting membrane (catalog number 10600125) was purchased from GE Healthcare Inc. Nylon hybridization transfer membrane (NEF994001PK) was purchased from PerkinElmer Inc. (Woodbridge, ON, Canada). T4 polynucleotide kinase (PNK), adenosine triphosphate (ATP), and deoxyribonucleoside 5'-triphosphate (dNTP) were purchased from Thermo Scientific (Ottawa, ON, Canada). γ-[. 32 [P]-ATP was obtained from PerkinElmer. Bovine serum albumin (BSA) and human thrombin were purchased from Sigma-Aldrich (Oakville, Canada). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride, magnesium chloride, Tween-20, and all other chemicals were purchased from Sigma-Aldrich (Oakville, Canada) and used without further purification. Milli-Q water was used for all experiments.

[0057] Radiolabeling of DNA aptamers: DNA aptamers were radiolabeled at the 5' end using the PNK reaction according to the manufacturer's protocol: 32 Briefly, 2 μL of 1 μM DNA aptamer was added to 2 μL of γ-[ 32 P]ATP, 1 μL of 10×PNK reaction buffer A, 10 U (U: unit) of PNK, and 4 μL of water were mixed. The mixture was incubated at 37° C. for 20 minutes and then purified by 10% dPAGE.

[0058] Preparation of recombinant full trimeric spike protein: A detailed protocol outlining the production of the protein can be found in Stadlbauer et al. 2020

[33] . The plasmid encoding the mammalian cell codon-optimized sequence of the Wuhan SARS-CoV-2 full-length spike protein is described in Amanat, F. et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat. Med. 26, 1033-1036 (2020)

[34] . Briefly, the protein was produced in Expi293 cells (ThermoFisher Scientific) using the manufacturer's instructions. The supernatant was harvested when the culture viability reached 40% and centrifuged at 500 g for 5 min. The supernatant was then incubated overnight with shaking at 4 °C with 1 ml of Ni-NTA agarose (Qiagen) per 25 ml of transfected cell supernatant. The next day, proteins were eluted using 10 ml polypropylene gravity-flow columns (Qiagen).Spiked proteins were concentrated with 50 kDa Amicon centrifugation units (Millipore) and then resuspended in phosphate-buffered saline (PBS).

[0059] Lentivirus preparation: Wuhan SARS-CoV-2 S protein pseudotyped lentivirus was produced as described by Crawford et al.

[35] . The SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit (BEI catalog number NR-52948) was obtained through the BEI source National Institute of Allergy and Infectious Diseases, National Institutes of Health. Briefly, HEK293T cells were cultured in 15 mL of standard Dulbecco's Modified Eagle Medium (DMEM) in a 15 cm dish at 1.1 × 10 7Cells were seeded at 1000 cells / mL. 16–24 hours after seeding, cells were co-transfected with HDM-nCoV-Spike-IDTopt-ALAYT (BEI Catalog No. NR-52515), pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI Catalog No. NR-52516), HDM-Hgpm2 (BEI Catalog No. NR-52517), HDM-tat1b (BEI Catalog No. NR-52518), and pRC-CMV-Rev1b (BEI Catalog No. NR-52519). 18–24 hours after transfection, the medium was replaced with complete DMEM, and 60 hours after transfection, the supernatant was collected, filtered through a 0.45 μm filter, and stored at -80 °C until future use. For purification, 40 mL of the supernatant was concentrated by spinning at 19,400 rpm for 2 hours. The resulting pellet was resuspended in 400 μl of HBSS followed by continuous vortexing for 15 min at room temperature. Protein concentration was confirmed by BCA assay.

[0060] Dot blot binding assay with spiked proteins: Dot blot assays were performed using a Whatman Minifold-1 96-well apparatus and a vacuum pump. Nitrocellulose and nylon membranes were incubated in 1xSB buffer for 1 hour prior to the experiment. γ-[ 32[P]-labeled DNA aptamer (1 nM) was dissolved in binding buffer and heated at 90 °C for 5 min, then cooled at room temperature for 20 min. Spike protein was dissolved and diluted in the same buffer. 5 μL of the above aptamer solution was mixed with 15 μL of spike protein at different concentrations. The mixture was incubated at room temperature for 1 h. A dot blot apparatus was assembled with a nitrocellulose membrane on top, a nylon membrane in the middle, and moistened Whatman paper on the bottom. After washing each well with 100 μL of binding buffer, the binding mixture was loaded and drained by a vacuum pump (force: 550 mmHg for 8 s). The wells were then washed twice with 100 μL of binding buffer. The membranes were imaged using a Typhoon 9200 imager (GE Healthcare) and analyzed using Image J software

[28] . Each binding assay was performed in triplicate. The bound fraction was quantified and plotted against the protein concentration. K d The value is expressed as Y=B max X / (K d +X) and derived by curve fitting using Origin 8.0 (Y is the bound fraction of the aptamer to the protein, and B max is the maximum binding fraction of the aptamer and X is the protein concentration).

[0061] Dot blot binding assays with SARS-CoV-2 spike-pseudotyped lentiviruses: Dot blot assays with SARS-CoV-2 spike-pseudotyped lentiviruses and control lentiviruses without spike protein were performed by binding aptamer solutions to various concentrations of virus (0–600 pM viral particles, 0–3 × 10 11 cp mL -1 The same procedure as above was used, except that the dot blot assay was performed after incubation with 10 min of 0.1% PBS (corresponding to 0.1% PBS) for 10 min.

[0062] Rapid selection of aptamers for spike proteins of SARS-CoV-2 variants: A one-round selection process was performed by a native gel-based method using a pre-enriched DNA pool against the wild-type SARS-CoV-2 spike protein after 13 rounds of selection

[17] . Briefly, the pre-enriched DNA pool labeled with FAM was diluted to 100 nM in 10 μL of selection buffer (1xSB; 50 mM HEPES, 6 mM KCl, 150 mM NaCl, 2.5 mM CaCl2, 2.5 mM MgCl2, 0.01% v / v Tween-20, pH 7.4), followed by heating at 90 °C for 5 min and annealing at room temperature for 10 min. Then, spike proteins of various SARS-CoV-2 variants (10 μL, 800 nM, in 1xSB) were mixed with the DNA pool and incubated at 23 °C for 30 min. Spike protein-bound DNA was separated from unbound DNA using 10% (v / v) native PAGE, which was imaged using a Typhoon imaging system (Typhoon™ FLA9500, GE Healthcare, USA). Bound DNA was then sheared and eluted from the gel by incubation for 20 min in 1×Taq buffer (200 μL, 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, 1% v / v Triton X-100, pH 9.0) at 23° C. DNA in the supernatant was amplified by PCR after adding FP1 (10 μL, 10 μM), RP1 (10 μL, 10 μM), Taq DNA polymerase (2 μL, 5 U / μL), and dNTPs (20 μL, 2 mM). The PCR temperature profile was set as follows: preheating at 94°C for 30 seconds; temperature cycles at 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds; annealing at 72°C for 5 minutes. The PCR products were then further amplified by PCR using sequencing primers and subsequently analyzed using the MiSeq (Illumina) sequencing platform. The selection process was performed simultaneously for WHS, UKS, SAS, BZS, and CAS.

[0063] Sequencing data analysis: Sequencing samples were prepared from each parallel SELEX experiment by PCR tagging with Illumina sequencing primers. Samples were size purified by agarose gel electrophoresis and then quantified by measuring absorbance at 260 nm. Tagged samples were pooled and paired-end sequenced on an Illumina MiSeq high-throughput DNA sequencer. Processing of sequence data was performed on a Windows 10 computer running Ubuntu 20.04 under WSL2. Raw paired-end reads were trimmed from sequencing and library primers using cutadapt 3.4

[29] . Trimmed paired-end reads were then 1) merged into consensus sense reads, 2) dereplicated, and 3) clustered at 90% identity using USEARCH v11.0.667_i86linux32

[30] . Sequence frequency and rank lists were generated using custom Python scripts. Multiple sequence alignments were performed using MUSCLE v3.8.1551 and converted to sequence logos using WebLogo 3.7.8 [31, 32]. Processed sequencing and cluster association data were stored in a MySQL 8.0.22 database. Analysis of sequence copy number, frequency, cluster association, and data plots were performed using the database and Microsoft Excel.

[0064] ELABA against pseudotyped lentiviruses and spike protein of B.1.1.529 (Omicron): ELABA (enzyme-linked aptamer binding assay) against different pseudotyped lentiviruses and B.1.1.529 spike protein (B.1.1.529S) was performed on Pierce™ streptavidin-coated microtiter plates (Thermo Scientific™, catalog number 15121). After binding each reagent, plate wells were washed three times with 200 μL of 1×SB buffer (50 mM HEPES, 6 mM KCl, 150 mM NaCl, 2.5 mM CaCl2, 2.5 mM MgCl2, 0.01% v / v Tween-20, pH 7.4). Plate wells were first blocked with blocking buffer (300 μL, 1×SB buffer, 10% w / v BSA) by incubating at 37° C. for 1 hour. Biotinylated aptamer (100 μL, 200 nM) in dilution buffer (1×SB with 0.1% w / v BSA) was then added to the wells and incubated for 30 min at 22°C. Pseudotyped lentivirus (100 μL, 2 pM) or B.1.1.529S (100 μL, 5 nM) in dilution buffer was then introduced and allowed to be captured by the aptamer on the plate, followed by incubation at 22°C for 1 h. The same biotinylated aptamer (100 μL, 200 nM) was then added again and incubated at 22°C for 30 min to serve as the reporter aptamer for binding to the virus. Streptavidin-HRP (100 μL, 1:2000 dilution, New England Biolabs, Cat. No. 3999S) in dilution buffer was then introduced and allowed to bind to the reporter aptamer by incubation at 22°C for 30 min. Finally, 1-Step™ Ultra TMB-ELISA substrate solution (100 μL, Thermo Scientific™, Cat. No. 34028) was introduced and reacted for 20 min at 22° C. H2SO4 (20 μL, 2 M) was used to terminate the catalytic reaction and convert TMB to its oxidized product, which was measured at 450 nm by a plate reader (Tecan, Switzerland).

[0065] Results and Discussion

[0066] Assessment of binding affinity of previously reported aptamers against SARS-CoV-2 variants: As a starting point to investigate aptamers for recognizing variant spike proteins, a dot blot assay (Figure 1) was used to examine the binding affinity of MSA1 (Figure 2A) and MSA5 (Figure 2B) against the full trimeric spike protein of the original SARS-CoV-2 (WHS), as well as four VoCs: B.1.1.7S (UKS), B.1.429S (CAS), B.1.351S (SAS), and P.1S (BZS; the "S" in the name stands for the full spike protein). At the time of these experiments, neither S1 nor the full trimeric spike protein was available for the delta and omicron variants. MSA1 showed a K aptamer binding affinity against the original Wuhan virus. d Compared with 19.8 nM for B.1.1.7S (K d = 1.2 nM) and B.1.429S (K d = 1.3 nM), but P.1S (K d = 75 nM), whereas B.1.351S (K d >200 nM). MSA5 has a very weak affinity for WHS (K d =5.6nM), B.1.1.7S(K d = 4.2nM), B.1.429S (K d = 5.0 nM), and B.1.351S (K d = 8.2 nM). However, MSA5 showed a markedly reduced affinity for P.1S (K d =52nM).

[0067] The results presented above indicate that the epitopes of MSA1 and MSA5 are not identical, and more importantly, the epitopes recognized by both aptamers are sensitive to conformational changes in the spike protein caused by mutations associated with some of the VoCs. Specifically, MSA5 is sensitive to conformational changes in P.1S, and MSA1 is sensitive to conformational changes in both P.1S and B.1.351S. Several other published aptamers were also investigated for binding to the spike protein of six different variants of SARS-CoV-2, but none of them were able to recognize WHS, B.1.1.7S, B.1.429S, B.1.351S, P.1S, and B.1.1.529S with uniformly good affinity. These findings indicate that these aptamers cannot serve as universal affinity agents targeting all variant spike proteins for either therapeutic or diagnostic applications.

[0068] Selection of DNA aptamers against various SARS-CoV-2 variants: Recently published aptamers against SARS-CoV-2 were selected using a pre-constructed DNA library that placed a 40 nt random region (Figure 3A) in the hairpin structure sequence often observed in many published aptamers, resulting in the isolation of the top and fifth ranked aptamer candidates, MSA1 and MSA5

[17] . However, two low-ranking aptamers, MSA50 (the 50th ranked aptamer candidate, K d = 10.2 nM) and MSA439 (aptamer candidate at position 439, K d Further investigation of the binding affinity of other aptamers, including the aptamer 1000 (p<0.01), showed that even low-ranked candidates in the final pool still exhibited excellent affinity for WHS. Based on these results, it is reasonable to assume that the enriched aptamer pool may also contain aptamer candidates that can universally recognize the spike proteins of current VoCs and even emerging VoCs.

[0069] To test this idea and rapidly generate aptamer candidates for recognizing the spike proteins of diverse VoCs, five parallel single-round SELEX experiments were performed using the 13th generation pool and each of the five spike proteins commercially available at the time, including the original SARS-CoV-2, as well as its B.1.1.7, B.1.351, P.1, and B.1.429 variants (Figure 3B). The spike proteins of the B.1.617.2 delta and B.1.1.529S omicron variants were not available at the time this experiment was performed. SELEX was performed using an electrophoretic mobility shift assay (EMSA), in which each spike protein was first incubated with the DNA pool, followed by separation of the spike protein-DNA complex from unbound DNA using native polyacrylamide gel electrophoresis. After elution from the gel, the bound DNA was amplified by PCR. The amplified DNA samples were then subjected to high-throughput sequencing analysis.

[0070] Next, we compared the rankings of the top 100 aptamer sequences discovered so far (designated MSA1-MSA100) for each pool, and found that the ranking of a particular aptamer, designated MSA52, increased significantly after reselection. MSA52 was ranked 52nd in our initial selection performed with the S1 subunit of WHS. However, the ranking increased to 46th, 24th, 19th, 17th, and 14th in the pools established with WHS, UKS, BZS, SAS, and CAS, respectively, indicating that MSA52 competed favorably with the top sequences.

[0071] There are a total of 321 members in the MSA52 aptamer family. The sequences of the top 100 members, their ranks within each pool, and frequency values ​​are shown in Table 1A and Table 1B. Figure 3C lists the top 10 sequences of the family (designated MSA52R1-10, SEQ ID NOs: 1-10, respectively). The rank increase of each of the top 10 sequences in the UK, BZ, SA, and CA pools was then calculated relative to their rank in the WH pool. Interestingly, these top 10 sequences all showed a rank increase of about 50% in each variant pool (Figure 3D), again confirming that members of this aptamer family competed well for binding to all four variants used in the SELEX experiment.

[0072] A t-test was performed to compare the ratio of the frequency of the top 15 members of the MSA52 cluster in each of the five selections to the initial (round 13) frequency (Figure 4 and Table 2). These sequences were indeed significantly enriched in the pool selected with the variant protein target (P<0.0001). In contrast, the difference between the reference population and the WHS pool in round 13 was not significant (P=0.7718; the geometric mean and 95% CI of the ratios can be seen in Table 2). Taken together, the sequencing data analysis indicates that MSA52 can function as a universal spike protein-binding aptamer that is insensitive to the mutations observed in the spike proteins of the B.1.1.7, B.1.351, P.1, and B.1.429 variants.

[0073] Binding of variant spike proteins by MSA52: The binding affinity of MSA52 (also referred to as MSA52R1 in Figure 3C) to the full trimeric spike (TS) protein of SARS-CoV-2 and its B.1.1.7, B.1.351, P.1, and B.1.429 variants was assessed using a dot blot assay. Representative dot blots from these experiments are shown in Figure 5A and K dThe binding curves of bound fraction versus protein concentration to derive K values ​​are plotted in Figure 5B. As expected, MSA52 showed strong binding to all five TS proteins and significantly increased the K values ​​of the five TS variants. d The values ​​varied only from 3.6 to 10.2 nM. The aptamer showed nearly identical affinities for WHS (3.6 nM), UKS (B.1.1.7S; 3.8 nM), and CAS (B.1.429S; 3.8 nM), and slightly reduced but still excellent affinities for SAS (B.1.351S; 8.5 nM) and BZS (P.1S; 10.2 nM). The binding data were consistent with the selection results, indicating that the increased frequency of MSA52 and its related family members was the result of its higher affinity for the variant spike protein.

[0074] As mentioned above, the spike proteins of the newly emerged VoCs were not available at the time the first round of SELEX experiments were performed. Subsequently, the spike proteins of the kappa (B.1.617.1), delta (B.1.617.2), and omicron (B.1.1.529) variants became available, providing an opportunity to evaluate whether the MSA52 aptamer could detect the newly emerged variants that had not been used in the selection experiments. Using a dot blot assay (Figure 5A), we tested the binding of MSA52 to the TS proteins of these three novel variants (designated B.1.617.1S, B.1.617.2S, and B.1.1.529S). Binding curves (FIG. 5B) show that the aptamers bind to B.1.617.1S, B.1.617.2S, and B.1.1.529S with K of 2.8 nM, 3.7 nM, and 6.2 nM, respectively. d values, and these are the K values ​​observed in WHS. d Value (K d= 3.6 nM). This observation further supports that MSA52 is a "universal" aptamer for recognizing spike protein, since it can recognize all seven spike protein variants of SARS-CoV-2 that we have tested so far, including those that were not included in the first round of reselection experiments.

[0075] Study of the binding site on the spike protein: To better understand the differences in the binding properties of MSA1, MSA5, and MSA52, we performed a competition assay to determine whether the binding site on the S1 protein of SARS-CoV-2 overlaps with all three aptamers. In this experiment, one aptamer (the competitor aptamer) was radioactively labeled (Figure 6). * The aptamer (labeled with ), was used at 2.5 nM, and the concentration of the other aptamer (the competing aptamer) was varied from 0 to 160 nM. S1 protein was used at 5 nM. Representative dot blot assays from these competitions are shown in Figure 7.

[0076] Two controls were run to verify competition: an inactive aptamer control that did not compete with any aptamer (mutation control, FIG. 6; the sequences of all DNA molecules used in this study are shown in Table 3), and self-competition between radioactive and non-radioactive versions of the same aptamer ( * MSA1+MSA1, * MSA5+MSA5, * MSA52+MSA52) was performed. Indeed, the inactive aptamer was not able to compete with MSA52, even at high concentrations (Figures 6 and 7). In contrast, each radioactive aptamer could be successfully competed by the non-radioactive aptamer in each self-competition (Figure 7), where the EC 50 The values ​​vary between 1 and 2 nM (Figure 6).

[0077] Based on these control experiments and the competition between MSA1 and MSA52 or between MSA5 and MSA52, we can conclude that: (1) MSA5 competes very well with MSA52 ( *MSA5+MSA52 and * In the case of MSA52+MSA5, EC 50 (2) MSA1 and MSA52 do not compete well with each other ( * MSA1+MSA52 and * In the case of MSA52+MSA1, EC 50 12.6 and 9.8, respectively). Considering the data shown in Figure 2, the competition results can be interpreted as inferring that although MSA52 appears to bind to amino acids of the S1 protein that are unmutated in the current variants of interest tested, the binding sites of MSA52 and MSA1 do not overlap significantly, whereas the binding sites of MSA52 and MSA5 show significant overlap.

[0078] Assessment of binding specificity of MSA52: We next investigated the specificity of MSA52 by assessing its binding to bovine serum albumin (BSA), human-α-thrombin, the RBD and spike (S) proteins of SARS-CoV1, and the RBD protein of MERS, as well as the three seasonal coronaviruses 229E, NL63, and OC43. The data are shown in Figure 8. MSA52 shows no binding to BSA and thrombin, SARS1-RBD, MERS-RBD, or NL63-RBD, but very weak binding to the spike proteins of SARS-CoV-1, 229E-RBD, and OC43-RBD (Figure 8). However, the binding affinity of MSA52 for these targets is very low, with a K d The values ​​were over 150 nM for the spike protein of SARS-CoV-1 and over 200 nM for 229E-RBD and OC43-RBD (Figure 9). Considering that SARS-CoV-1 is no longer circulating and the aptamer does not efficiently recognize 229E and OC43, it is clear that MSA52 shows sufficient selectivity for SARS-CoV-2.

[0079] Binding affinity of MSA52 to pseudotyped lentiviruses of SARS-CoV-2 and variants: We next tested the binding of MSA52 to several pseudotyped SARS-CoV-2 lentiviruses (PL) engineered to display the full trimeric S protein of SARS-CoV-2 variants within the viral envelope. These pseudotyped viruses mimic SARS-CoV-2 but are unable to replicate themselves in human cells and therefore can be handled in biosafety level 2 laboratories. Dot blot assays were performed with eight different PLs, including the spike proteins of the original SARS-CoV-2, as well as the B.1.1.7, B.1.351, P.1, B.1,429, B.1.617.1, B.1.617.2, and B.1.617.2.1 (delta plus) variants (Figure 10). The PL of the B.1.1.529 (Omicron) variant was not tested, as it is currently unavailable. The same lentivirus without the S protein was used as the control virus (CV) in this experiment. MSA52 was found to recognize all of these PVs with similar affinity, but not the CVs (Figure 11A). Excellent K values ​​ranging from 18.4 pM (WH-PL) to 49.0 pM (B.1.617.1-PL) were observed. d values ​​were observed for all eight PLs. The increased affinity compared to the purified spike protein can be explained by the fact that each virus particle carries more copies of the S protein. The copy number of the spike protein on the SARS-CoV-2 virus surface has been reported to be approximately 30 [26, 27]. However, the copy number on the virus particles of the PLs used in this experiment was not reported. Assuming that each PL virus carries 100 copies of the S protein, the protein equivalent K d The values ​​are estimated to be in the range of 1.84 nM to 4.9 nM, which corresponds to the K d More importantly, MSA52 is able to recognize the fully functional spike proteins of original SARS-CoV-2 and multiple VoCs, even though this aptamer was selected using spike proteins purified from the original viruses.

[0080] Structural analysis of MSA52 and truncations: Next, the secondary structure of MSA52 was predicted (Figure 11B). Overall, the structure contains four pairing elements (9-bp P1, 3-bp P2, 3-bp P3, and 6-bp P4) and five unpaired elements (4-nt SS12, 2-nt SS23, 2-nt SS34, 4-nt SS41, 6-nt L2, 5-nt L3, and 11-nt L4). By dot blot assay, five truncation mutants (Figure 12) were examined for their binding activity to the S1 protein of the original SARS-CoV-2 virus (referred to as MSA5-T1 to MSA5-T5, respectively). First, the K of full-length MSA52 was d The K value was determined to be 6.6 nM. P2-L2 (MSA52-T1; loss of 10 nucleotides; K of 61.8 nM) d ), P3-L3 (MSA52-T2; loss of 9 nucleotides; K of 53.4 nM d ), and P4-L4 (MSA52-T3; loss of 21 nucleotides; K of 94.8 nM d ) resulted in mutants with significantly reduced binding activity. However, L4 can be reduced to four nucleotides without reducing activity (MSA52-T4; loss of seven nucleotides; K of 6.0 nM). d Finally, the three unpaired nucleotides adjacent to P1 can be removed and the T·G and G·T wobble pairs converted to CG and GC Watson-Crick pairs without loss of activity (MSA52-T5, Figure S1C; loss of 3 nucleotides and change of 2 nucleotides; K of 5.5 nM). d The above results support the proposed four-way junction structure of MSA52.

[0081] Low levels of random mutations typically occur during the PCR step of the selection process, however, nucleotides whose bases play an important role in the structure and / or binding function of the aptamer exhibit less mutation than bases that are not structurally and / or functionally important. The nucleotides in the original random sequence domain were classified after performing a sequence alignment of the top 100 members of the MSA52 family (Figure 11B): (i) absolutely conserved nucleotides (0 mutations observed): 24-26, 30-32, 37, 40; (ii) highly conserved nucleotides (only 1 mutation): 28, 29, 38, 41, 43-45; (iii) somewhat conserved nucleotides (2-3 mutations): 27, 33-36, 39, 42, 46-51, 53, 55-56, 58; (iv) less conserved nucleotides (4-7 mutations): 52, 54, 57, 59; (v) least conserved nucleotides (9-29 mutations): 21-23, 60. The pattern of conservation seen in the sequences of MSA52 was consistent with the truncation data, with all truncation mutants losing highly conserved nucleotides experiencing a significant reduction in binding affinity. Furthermore, it is reasonably certain that the nucleotides that directly bind to the spike protein for molecular recognition are located within a 22-nt segment starting from the 24th G residue in P2-L2 and ending at the 45th G residue in P4 (Figure 11). A more detailed structural and functional analysis of these nucleotides will be of interest for future studies by testing more mutant sequence constructs.

[0082] Comparison of binding affinity of MSA52 with 10 published aptamers to spike proteins of selected variants of SARS-CoV-2: To demonstrate the uniqueness of the recognition specificity of MSA52, a comparative study was performed to determine the binding affinity of 10 published spike-binding DNA aptamers to the spike proteins of the original SARS-CoV-2, as well as the B.1.1.7, B.1.351, P.1, B.1.617.2, and the latest B.1.1.529 (Omicron) variants using a dot blot assay (Table 4). Five of these aptamers are from the inventors, and the remaining five were randomly selected from five other aptamer studies. MSA52 binds all these spike proteins with a K d It was found that the aptamers recognize B.1.1.7S (1.2 nM) and B.1.617.2S (3.2 nM) with similar affinities ranging from 3.6 to 10.2 nM. In contrast, none of the other aptamers showed universally good affinity for all five variants. For example, MSA1 showed good affinity for B.1.1.7S (1.2 nM) and B.1.617.2S (3.2 nM), but reduced affinity for WHS (19.8 nM) and weak affinity for P.1S (75.2 nM) and B.1.351S (>200 nM). The second best aptamer in terms of universal affinity was MSA3, which had a K ranging from 2.0 to 36.4 nM. dThe values ​​are shown in Table 4. The top three aptamers for each variant are also shown in Table 4. MSA52 ranks first for P.1S, B.1.617.2S, and B.1.1.529S, a close second for two variants: B.1.351S (8.5nM for MSA52 vs. 8.2nM for MSA5) and WHS (3.6nM for MSA52 vs. 3.3nM for SP6), and a close third for B.1.1.7S (3.8nM for MSA52 vs. 1.2nM for MSA1 and 2.0nM for MSA3). This head-to-head study further demonstrates two important properties of MSA52: (1) its affinity for spike protein is among the highest of any aptamer, and (2) it is highly unique in that it exhibits uniformly excellent affinity for five variants of the spike protein.

[0083] Comparison of analytical performance of MSA52 with 10 published aptamers in a sandwich assay for detection of pseudotyped lentiviruses of SARS-CoV-2 variants: Another comparative study was performed to demonstrate the analytical utility of MSA52 for detecting pseudotyped lentiviruses of prototype SARS-CoV-2 (WH), as well as B.1.1.7, B.1.351, P.1, and B.1.617.2 variants. Because each viral particle carries multiple spike proteins on its surface, we designed a sandwich assay that uses two identical biotinylated aptamers to bind to a single viral particle (Figure 13). Each of the 11 aptamers in Table 4 was biotinylated at the 5' end and immobilized on a 96-well microtiter plate coated with streptavidin. The second biotinylated aptamer was tagged with horseradish peroxidase (HRP) conjugated to streptavidin. The presence of viral particles immobilized HRP on the plate, which oxidized 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Three controls were included in the experiment: BSA as a general protein target, a lentivirus not expressing the spike protein, and polyA25 as a DNA control. After quenching the reaction with H2SO4, the blue oxidized TMB turned yellow, which was measured at 450 nm. Figure 14A shows the absorbance (A) at 450 nm in the binding reaction mixture containing a specific aptamer and PL (2 pM). 450 ) is plotted for each reaction mixture. 450 Values ​​are the A observed in the matching BSA reaction mixtures. 450 The images were corrected for background by subtracting . Figure 14B shows a representative image of a 96-well microtiter plate after the quenching reaction with H2SO4 (the image was processed in Photoshop to improve the visual effect. The original image is shown in Figure 13B).

[0084] The data in Figure 14A show that of all five variants, only MSA52 was able to generate a high signal (A > 0.05). 450), clearly showing that almost all aptamers were able to detect the B.1.1.7 variant under the test conditions. The next best aptamer was MSA5, which was able to detect four of the five variants, but was unable to detect the P.1 variant. Indeed, with the exception of MSA52, almost all of the other aptamers produced either very weak signals (MSA1, MSA5, MSA10, CoV2-RBD-1, S1P, and nCoV-S1-A1) or significantly lower signals (MSA3, MSA7, SP6, and SARS2-AR10) for the P.1 variant. B.1.351 was another variant that was difficult to recognize for most aptamers, with only MSA5 and MSA52 producing high levels of signal, while the others produced very weak signals. Another interesting observation was that the K d All aptamers that were ≦11.5 nM were able to generate high levels of signal. MSA52 met this criterion for all five variants, MSA5 met this criterion for four variants, while the remaining aptamers met this criterion only for 0 (MSA10 and nCoV-S1-A1), 1 (MSA7, CoV2-RBD-1, and S1P), and 2 (MSA1, MSA3, SP6, and SARS2-AR10) variants. We also tested this sandwich assay against the B.1.1.529 (Omicron) variant using the trimeric spike protein, since pseudotyped viruses for the Omicron variant are not currently available. Of the four aptamers (MSA52, MSA1, MSA3, and MSA5) that showed high levels of signal, MSA52 was the best (Figures 15A and 15B). In contrast, the other aptamers gave either very weak or no signals. These observations are consistent with the dot blot results in Table 4 (MSA52 ranked first for Omicron).

[0085] In summary, we performed parallel selection using aptamer pools from previous SELEX experiments with the wild-type S1 protein of SARS-CoV-2, followed by high-throughput DNA sequencing and bioinformatics analysis to search for DNA aptamers that universally (i.e., non-discriminatory) recognize the spike protein of SARS-CoV-2 variants. This effort resulted in the discovery of a unique DNA aptamer, designated MSA52, capable of binding to the spike protein of wild-type SARS-CoV-2, as well as its eight current variants of interest (including B.1.1.7, B.1.351, P.1, B.1.429, B.1.617.1 B.1.617.2, B.1.617.2.1, and B.1.1.529), while still retaining selectivity for non-SARS-CoV-2 spike proteins. This aptamer was discovered using a rapid one-round reselection against four variants of the SARS-CoV-2 spike protein and was observed to exhibit high affinity binding not only to these four variants but also to three other variants that were not present during the reselection (including the latest Omicron variant), demonstrating universal high affinity to both known and newly emerged VoCs. The entire discovery process (SELEX, sequencing, and bioinformatics analysis) was very rapid, taking less than one week. Competitive assays showed that this unique aptamer binds to key amino acids present in all variants, thus allowing universal recognition of the spike protein of current and newly emerged variants of interest, making it suitable as a molecular recognition element for developing diagnostic and therapeutic solutions against this constantly evolving coronavirus.

[0086] Although the present disclosure has been described with reference to examples, it should be understood that the claims should not be limited by the embodiments set forth in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

[0087] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event that a term in this disclosure is found to be defined differently in a document incorporated herein by reference, the definition provided herein shall serve as the definition of such term. [Table 1] TIFF2025503670000003.tif227159TIFF2025503670000004.tif55159 [Table 2] TIFF2025503670000006.tif240159TIFF2025503670000007.tif77159 [Table 3] [Table 4] TIFF2025503670000010.tif229159 [Table 5] quotation 1.Cutler, DM & Summers, LH The COVID-19 Pandemic and the $16 Trillion Virus.JAMA 324, 1495-1496 (2020). 2. Lazarevic, I., Pravica, V., Miljanovic, D. & Cupic, M. Immune Evasion of SARS-CoV-2 Emerging Variants: What Have We Learnt So Far? Viruses 13, 1192 (2021). 3.CDC COVID-19 Response Team. 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Soc. 131, 4262-4266 (2009). 14.Liu, R., McConnell, E. M., Li, J. & Li, Y. Advances in functional nucleic acid based paper sensors.J. Mater. Chem. B 8, 3213-3230 (2020). 15.Stanciu, L. A., Wei, Q., Barui, A. K. & Mohammad, N. Recent Advances in Aptamer-Based Biosensors for Global Health Applications.Annu. Rev. Biomed. Eng. (2021) doi:10.1146 / annurev-bioeng-082020-035644. 16.Wang, T. et al. Development of nucleic acid aptamer-based lateral flow assays: A robust platform for cost-effective point-of-care diagnosis.Theranostics 11, 5174-5196 (2021). 17.Li, J. et al. Diverse high-affinity DNA aptamers for wild-type and B.1.1.7 SARS-CoV-2 spike proteins from a pre-structured DNA library.Nucleic Acids Res. 49, 7267-7279 (2021). 18.Song, Y. et al. Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein.Anal. Chem. 92, 9895-9900 (2020). 19.Liu, X. et al. Neutralizing Aptamers Block S / RBD-ACE2 Interactions and Prevent Host Cell Infection.Angew. Chem. Int. Ed. 60, 10273-10278 (2021). 20.Schmitz, A. et al. A SARS-CoV-2 Spike Binding DNA Aptamer that Inhibits Pseudovirus Infection by an RBD-Independent Mechanism.Angew. Chem. Int. Ed. 60, 10279-10285 (2021). 21.Sun, M. et al. Aptamer Blocking Strategy Inhibits SARS-CoV-2 Virus Infection.Angew. Chem. Int. Ed. 60, 10266-10272 (2021). 22.Gupta, A. et al. A novel G-quadruplex aptamer-based spike trimeric antigen test for the detection of SARS-CoV-2.Mol. Ther. - Nucleic Acids 26, 321-332 (2021). 23.Yang, G. et al. Identification of SARS-CoV-2-against aptamer with high neutralization activity by blocking the RBD domain of spike protein 1. Signal Transduct. Target. Ther. 6, 1-4 (2021). 24.Peinetti, A. S. et al. Direct detection of human adenovirus or SARS-CoV-2 with ability to inform infectivity using DNA aptamer-nanopore sensors.Sci. Adv. 7, eabh2848 (2021). 25.Kacherovsky, N. et al. Discovery and Characterization of Spike N-Terminal Domain-Binding Aptamers for Rapid SARS-CoV-2 Detection.Angew. Chem. Int. Ed. 60, 21211-21215 (2021). 26.Ke, Z. et al. Structures and distributions of SARS-CoV-2 spike proteins on intact virions.Nature 588, 498-502 (2020). 27.Yao, H. et al. Molecular Architecture of the SARS-CoV-2 Virus.Cell 183, 730-738 (2020). 28.Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis.Nat. Methods 9, 671-675 (2012). 29.Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads.EMBnet.journal 17, 10-12 (2011). 30.Edgar, R. C. Search and clustering orders of magnitude faster than BLAST.Bioinformatics 26, 2460-2461 (2010). 31.Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity.BMC Bioinformatics 5, 113 (2004). 32.Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. 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Claims

1. 1. A method for identifying or producing an aptamer capable of binding to variants of at least two target analytes, comprising: a) generating a mixture of aptamers selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by at least one round of SELEX using a SELEX-compatible aptamer pool, either simultaneously or sequentially, against variants of said at least two target analytes; b) sequencing the mixture of SELEX selected aptamers that form a complex with each of the at least two target analyte variants by high throughput sequencing; c) aligning the mixture of SELEX selected aptamers that form complexes with each of the at least two target analyte variants by sequence alignment analysis; d) identifying or producing said aptamers capable of binding to variants of said at least two target analytes.

2. The method of claim 1, further comprising generating a SELEX-compatible aptamer pool prior to a).

3. The method of claim 1 or 2, wherein the SELEX-compatible aptamer pool comprises a plurality of nucleic acid molecules comprising at least one random nucleotide domain having an aptamer-like structure, the random nucleotide domain being flanked by a 5'-terminal region and a 3'-terminal region.

4. The method of any one of claims 1 to 3, wherein the 5' terminal region comprises the nucleotide sequence of SEQ ID NO: 102 or 103, and the 3' terminal region comprises the nucleotide sequence of SEQ ID NO: 132 or the reverse complement of SEQ ID NO: 104 or 105.

5. The method of any one of claims 1 to 4, wherein the SELEX-compatible aptamer pool comprises a plurality of nucleic acid molecules having the nucleotide sequence of SEQ ID NO:

101.

6. The method of any one of claims 1 to 5, wherein the variant of the target analyte is a microorganism, a virus, and / or a molecule present in a microorganism or a virus.

7. The method of any one of claims 1 to 6, wherein the target analyte variant is a protein.

8. The method of any one of claims 1 to 7, wherein the target analyte variant is a protein from SARS-CoV-2.

9. The method of any one of claims 1 to 8, wherein the target analyte variant is SARS-CoV-2 spike protein.

10. The method of any one of claims 1 to 9, wherein a) comprises generating a mixture of SELEX selected aptamers with at least 3, 4, 5, 6, 7, 8, or 9 variants of the target analyte.

11. 11. The method of any one of claims 1 to 10, wherein the high throughput sequencing comprises single molecule real-time sequencing, ion semiconductor sequencing, pyrosequencing, sequencing-by-synthesis, combinatorial probe anchor synthetic sequencing, sequencing-by-ligation, nanopore sequencing, or GenapSys™ sequencing.

12. The method of any one of claims 1 to 11, wherein said high throughput sequencing comprises sequencing-by-synthesis.

13. An aptamer comprising a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-100, functional fragments and functional variants thereof, or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 113-117, functional fragments and functional variants thereof.

14. The aptamer of claim 13, comprising a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2 to 10, functional fragments and functional variants thereof.

15. 15. The aptamer of claim 13 or 14, comprising a nucleotide sequence comprising SEQ ID NO: 116 or 117.

16. The aptamer according to any one of claims 13 to 15, wherein the 5' terminal region comprises the nucleotide sequence of SEQ ID NO: 102 or 103, and the 3' terminal region comprises the nucleotide sequence of SEQ ID NO: 104 or 105.

17. 1. A method for detecting the presence of SARS-CoV-2 spike protein in a sample, comprising: a) contacting the sample with an aptamer comprising a 5' terminal region, a 3' terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-100, functional fragments and functional variants thereof, or comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 106-117, functional fragments and functional variants thereof, wherein the aptamer binds to the spike protein; and b) detecting the presence of the aptamer bound to the spike protein in the sample, wherein detection of the bound aptamer indicates the presence of SARS-CoV-2 spike protein or a variant thereof.

18. 18. The method of claim 17, wherein the SARS-CoV-2 spike protein variant comprises a B.1.1.7, B.1.351, P.1, B.1.429, B.1.617.1 B.1.617.2, B.1.617.2.1, or B.1.1.529 spike protein variant.

19. The method according to claim 17 or 18, wherein the aptamer comprises a 5'-terminal region, a 3'-terminal region, and a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 10, functional fragments and functional variants thereof.

20. The method of claim 17 or 18, wherein the aptamer comprises a 5'-terminal region, a 3'-terminal region, and the nucleotide sequence of SEQ ID NO: 1, a functional fragment or a functional variant thereof.

21. The method of claim 17 or 18, wherein the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 108, 112-117, functional fragments and functional variants thereof.

22. 19. The method of claim 17 or 18, wherein the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 108, 112, 116, 117, functional fragments and functional variants thereof.

23. The method of claim 17 or 18, wherein the aptamer comprises the nucleotide sequence of SEQ ID NO: 112, a functional fragment or a functional variant thereof.

24. The method of any one of claims 17 to 23, wherein the method detects SARS-CoV-2 infection in a subject.

25. The method according to any one of claims 17 to 24, wherein the sample is saliva, sputum, urine, blood, serum, other body fluids and / or secretions.

26. Use of the aptamer according to any one of claims 13 to 16 for detecting SARS-CoV-2 spike protein.

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