Compositions and methods related to multi-complimentary aptamers

Abstract

The present disclosure provides compositions and methods related to multi-complimentary aptamers. In particular, the present disclosure provides nucleic acid aptamers capable of binding to a plurality of different epitopes on a target biomolecule, as well as corresponding methods of generating and characterizing the multi-complimentary aptamers.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 386,570, filed on December 8, 2022, which is incorporated herein by reference in its entirety and for all purposes. Technical field

[0003] The present disclosure provides compositions and methods related to multi - complementary - site aptamers. In particular, the present disclosure provides nucleic acid aptamers capable of binding to multiple different epitopes on a target biomolecule, as well as corresponding methods for generating and characterizing such multi - complementary - site aptamers. Background art

[0004] There are many chemotherapeutic agents for treating cancer, which generally target important processes for cancer cell survival, proliferation, and metastasis, such as the cell cycle, growth signaling pathways, or DNA synthesis and repair. Traditional chemotherapeutic drugs are very effective, but they have severe off - target effects because they cannot distinguish cancer cells from healthy cells. Newer targeted therapies, such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies, are now widely used as monotherapies or as adjuvant therapies in combination with traditional chemotherapy to improve treatment outcomes by enhancing selectivity. Many targeted drugs inhibit overexpressed or mutated cell signaling proteins that drive tumor development and / or growth. This allows for the selective destruction of diseased cells while largely sparing healthy cells. However, targeted therapies are highly prone to acquired drug resistance. This drug resistance is particularly common in therapies targeting the epidermal growth factor receptor (EGFR) and may be caused by overexpression and / or mutation of EGFR, mutant activation of downstream signaling proteins (e.g., KRAS G12C), or utilization of alternative signaling pathways to maintain cell proliferation and survival (e.g., overexpression of HER2). Some of these drug resistance mechanisms can be overcome with new alternative inhibitors (e.g., sotorasib for KRAS G12C) or with known drugs targeting upregulated pathways (e.g., trastuzumab for HER2 overexpression). However, target - site mutations that impair drug - receptor binding remain a challenging drug resistance mechanism that needs to be persistently overcome.

[0005] For example, the TKI gefitinib is highly effective in patients with the EGFR variant L858R, but the acquisition of a mutation at the target binding site (T790M) renders the drug completely ineffective. To date, three generations of anti-EGFR TKIs have been developed to address this resistance mechanism, but even the latest generation (i.e., osimertinib) encounters resistance due to a new target mutation (C797X). Antibody therapies similarly become ineffective due to target site mutations. For example, 16% of patients treated with the anti-EGFR antibody cetuximab develop the EGFR mutation S492R, which completely impairs cetuximab binding. Administering another antibody that binds to a different epitope on EGFR only temporarily solves this problem, as even a single point mutation at its epitope can lead to the re-emergence of resistance, as observed, for example, in the case of TKIs.

[0006] Multiple efforts are currently underway to combat mutation-based resistance to antibody therapies. One approach is to simultaneously target multiple non-overlapping epitopes of the target, on the basis that individual mutations at one site will not affect the binding affinity of drugs that recognize different epitopes on the same target and, therefore, will not affect the therapeutic efficacy of the drug. For example, the novel "oligoclonal" antibody regimens currently in clinical trials employ a mixture of three antibodies that bind to different epitopes of the extracellular domain of EGFR and have demonstrated potent and similar inhibitory activity against both wild-type and mutant EGFR. However, oligoclonal antibody therapies face difficulties in formulation due to differences in the pharmacokinetics of their constituent antibodies and their combined toxicity profiles. To address this issue, several affinity elements that bind to different epitopes on the same target can be integrated into a single construct (i.e., a "multi-epitope" binder). This not only synchronizes the pharmacokinetics but also enhances the binding affinity. For example, by linking the N-terminus of a monoclonal antibody heavy chain to another affinity element (e.g., a Fab or a scaffold-based binding protein) via a flexible linker, bispecific antibodies that bind to two different epitopes on a single target have been developed. These antibodies typically have a 10- to 100-fold lower k off than standard antibodies, while also retaining the pharmacokinetic benefits of intact antibodies. Multi-epitope constructs can also be made by covalently linking multiple scaffold-based binding proteins (e.g., affimers or nanobodies), and these constructs typically achieve a 10-fold reduction in K D and k off relative to their individual constituent subunits.

[0007] However, protein-based multi-complementary site constructs, whether based on antibodies, affibodies, or nanobodies, have limited clinical applications due to the need for a large amount of labor-intensive trial-and-error processes to select the best combination of affinity elements and optimal linker domains. They also have high production costs, are prone to batch-to-batch variations, are immunogenic, and have a short shelf life. Therefore, there is a great need for new and generally applicable methods for directly generating high-affinity multi-complementary site biomolecules. Summary of the Invention

[0008] Embodiments of the present disclosure include a single-stranded nucleic acid molecule comprising an anchor aptamer capable of binding to a target biomolecule and at least one candidate aptamer coupled to the anchor aptamer. According to these embodiments, the at least one candidate aptamer comprises a random nucleic acid sequence.

[0009] In some embodiments, the anchor aptamer comprises a predetermined nucleic acid sequence that binds to an epitope on the target biomolecule. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence of a reference aptamer sequence. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having at least one modification as compared to the reference aptamer sequence. In some embodiments, the at least one modification comprises one or more of the following: (i) substitution of one or more nucleotides; (ii) deletion of one or more nucleotides; and / or (iii) insertion of one or more nucleotides as compared to the reference aptamer sequence. In some embodiments, the anchor aptamer binds to the target biomolecule with a K of from about 10 nM to about 10 μM. D Binds to the target biomolecule.

[0010] In some embodiments, the nucleic acid molecule comprises two candidate aptamers flanking the anchor aptamer. In some embodiments, the at least one candidate aptamer comprises a random nucleic acid sequence that has been determined to bind to the target biomolecule. In some embodiments, the at least one candidate aptamer binds to an epitope on the target biomolecule that is different from the epitope bound by the anchor aptamer. In some embodiments, the at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 50 nucleotides.

[0011] In some embodiments, the nucleic acid molecule binds to the target biomolecule with a higher affinity than a reference anchor aptamer. In some embodiments, the nucleic acid molecule binds to the target biomolecule with a K of from about 1 fM to about 1 nM. D Binds to the target biomolecule. In some embodiments, the length of the nucleic acid molecule is from about 30 nucleotides to about 150 nucleotides.

[0012] In some embodiments, the nucleic acid molecule is RNA, DNA, or a combination thereof. In some embodiments, the nucleic acid molecule comprises 2'-deoxy-2'-fluoro-ribonucleotides (2'-F RNA), phosphorothioate modifications, 2'-O-methyl sugars, LNA (locked nucleic acid), and threose nucleic acid (TNA), and any combination thereof.

[0013] In some embodiments, the nucleic acid molecule further comprises a linker. In some embodiments, the nucleic acid molecule further comprises a linker, and wherein the linker facilitates the binding of the nucleic acid molecule to a solid support. In some embodiments, the nucleic acid molecule further comprises a fluorescent tag or an electroactive tag.

[0014] In some embodiments, the target biomolecule is a protein or polypeptide. In some embodiments, the target biomolecule is selected from the group consisting of: cell surface receptors, endogenous biomarkers, cardiac proteins, oncoproteins, endocrine polypeptide hormones, gastrointestinal peptide hormones, cytokines, viral proteins, bacterial proteins, fungal proteins, and plant proteins.

[0015] In some embodiments, the target biomolecule is a protein or polypeptide present in solution and not bound to a solid support.

[0016] Embodiments of the present disclosure also include a library comprising a plurality of any of the nucleic acid molecules described herein. In some embodiments, the plurality of nucleic acid molecules comprises at least 10 10 individual nucleic acid molecules, and each sequence of at least one candidate aptamer is different.

[0017] Embodiments of the present disclosure also include a method for identifying a multi-complementary site aptamer, the method comprising exposing a library of candidate multi-complementary site aptamers to a target biomolecule, wherein each candidate multi-complementary site aptamer in the library comprises an anchor aptamer capable of binding to the target biomolecule and at least one candidate aptamer coupled to the anchor aptamer, wherein the at least one candidate aptamer comprises a random nucleic acid sequence; and evaluating the binding of the candidate multi-complementary site aptamer to the target biomolecule.

[0018] In some embodiments, the library of candidate multi-complementary site aptamers is exposed to the target biomolecule in multiple rounds of selection. In some embodiments, the multiple rounds of selection include exposing the library of candidate multi-complementary site aptamers to variants of the target biomolecule in each round.

[0019] In some embodiments, the target biomolecule is a protein or polypeptide. In some embodiments, the protein or polypeptide is expressed by a cell. In some embodiments, the protein or polypeptide is conjugated to a solid support.

[0020] In some embodiments, evaluating the binding of the candidate multi-complementary site aptamer to the target biomolecule comprises determining the KD whether it is below a threshold. In some embodiments, the threshold K D is at least 10-fold lower than the K of the anchor aptamer D is at least 10-fold lower.

[0021] In some embodiments, evaluating the binding of a candidate multi-complementary-site aptamer to a target biomolecule includes performing surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis, biolayer interferometry, strand displacement fluorometry, and intrinsic tryptophan fluorescence quenching, and any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Representative schematic diagrams showing the structures and affinities of the thrombin aptamers Tasset-29 and Bock-15 and the dual-complementary-site aptamer TBV-08.

[0023] Figure 2 : Advantages of the multi-complementary-site aptamers of the present disclosure: Different from the single-complementary-site conjugates (left), the multi-complementary-site conjugates have ultra-high affinity and can tolerate single-site mutations due to binding to multiple epitopes (right).

[0024] Figures 3A - 3C : Isolation of multi-complementary-site binding aptamers for the extracellular domain of cell surface hEGFR using a structured 2'F-RNA library. The three-dimensional structure of monomeric hEGFR as determined by X-ray crystallography (PDB: 3QWQ), which is embedded in a phospholipid bilayer (gray) through its transmembrane domain (PDB: 2KS1) ( Figure 3A ). The side view and top view are shown on the left and right, respectively. Schematic diagram of the organization of the hEGFR extracellular domain ( Figure 3B ). In A and B, domains I, II, III, and IV are colored gold, magenta, green, and blue, respectively. The reported hEGFR aptamer (red anchor) binds to domain III and is flanked by binding domains (black lines) at both ends. The sequence and structure of the proposed 2'-F RNA anchor-SELEX library ( Figure 3C ). Region 1 (R1) is the anchor aptamer, which will be doped at a ratio of 2:1 of native nucleotides to substituted nucleobases. Regions 2 and 3 (R2 and R3) are 20-nt random regions at the 5' and 3' ends, respectively, which act as additional binding domains for other epitopes on hEGFR.

[0025] Figures 4A - 4B : Strategy of anchor-SELEX. The anchor-SELEX process, including the target cell line and hEGFR variants used in each round of selection ( Figure 4A ). The structure of hEGFR, with various mutants highlighted in red ( Figure 4B ).

[0026] Figure 5 : The binding affinities of multivalent aptamers were compared to those of the monoclonal antibody cetuximab and the anchor aptamer, and the unit-site hEGFR mutations that rank the binding affinities from highest to lowest may impair the binding of trimeric aptamers, but they should retain an affinity that is superior to or equal to that of cetuximab.

[0027] Figure 6 : Monomeric and multimeric constructs derived from trimeric aptamers for affinity testing. Detailed Description

[0028] Embodiments of the present disclosure have a systematic method for selecting aptamers (referred to as "multimeric" aptamers) that simultaneously bind to multiple independent epitopes on a target protein. Such aptamers are of great value for both clinical chemistry and the development of new, highly targeted therapies. For the former, the greatly enhanced affinity of such aptamers will translate into improved detection limits for pathogens and other low-concentration analytes. In addition, multimeric aptamers can also be important for the development of new therapies, such as new cancer therapies.

[0029] In this regard, aptamers are promising candidates. Generally, aptamers are single-stranded nucleic acid-based affinity reagents isolated from in vitro randomized libraries by various methods, including but not limited to the use of systematic evolution of ligands by exponential enrichment (i.e., SELEX) to bind specifically to a target with high affinity. Aptamers have several desirable properties as therapeutic agents, including high affinity and specificity, low toxicity, low immunogenicity, effective tissue penetration, and the ease with which they can be chemically modified to achieve an improved half-life and high chemical stability in vivo.

[0030] In particular, two factors make aptamers an ideal choice for the development of multimeric agents. First, aptamers are modular and highly amenable to sequence engineering, allowing multiple aptamer domains to be conjugated into a single construct that binds to multiple epitopes on a single target. A dimeric aptamer in which two different aptamers are linked by a polynucleotide linker has been shown to exhibit a 10 - 50-fold increase in target affinity. The second advantage is that SELEX is capable of screening large, highly diverse oligonucleotide libraries (about 10 15 unique sequences), and selection pressure can be applied to isolate the best multimeric aptamers with the desired binding properties. This stands in sharp contrast to the engineering of dimeric antibodies or aptamers, which requires trial and error on a few constructs with an almost infinite number of linker and binding domain combinations.

[0031] As further described herein, by using SELEX to optimize only the linker domain of the dual - complementary - site thrombin - binding aptamer, the target affinity was increased by at least 200 - fold. Given these advantages, experiments can be conducted to develop a new selection technique called "anchor - SELEX" to generate multi - complementary - site aptamers that bind to specific targets with ultra - high affinity and remain tightly bound even when a single target - site mutation occurs. This method utilizes a structured library in which a known target - binding aptamer (e.g., a reference aptamer) that acts as an "anchor" flanks random sequences that act as additional binding domains. Multiple rounds of selection are performed on this library to isolate high - affinity aptamers with multi - complementary - site binding ability to the target of interest.

[0032] The section headings used in this chapter and the entire disclosure herein are for organizational purposes only and are not intended to be limiting.

[0033] 1. Definitions

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document (including definitions) will prevail. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0035] As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)" and variations thereof are intended to be open - ended conjunctions, terms, or words that do not preclude the possibility of additional acts or structures. Unless otherwise expressly specified in the context, the singular forms "a", "an", and "the" include plural referents. The present disclosure also contemplates other embodiments "comprising the embodiments or elements presented herein", "consisting of the embodiments or elements presented herein", and "consisting essentially of the embodiments or elements presented herein", whether or not expressly set forth.

[0036] For the recitation of numerical ranges herein, each intermediate value therebetween having the same degree of precision is explicitly contemplated. For example, for the range of 6 - 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and for the range of 6.0 - 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0037] "Related thereto" as used herein refers to a comparison therewith.

[0038] The term "aptamer" generally refers to an oligonucleotide of a single defined sequence or a mixture of such oligonucleotides, wherein the mixture retains the property of specifically binding to a target molecule. Thus, as used herein, "aptamer" refers to both singular and plural sequences of oligonucleotides. The term "aptamer" generally refers to a single strand that is capable of binding to a protein or other molecule and thereby modulating function.

[0039] The term "single-stranded" oligonucleotide generally refers to those oligonucleotides that contain a single series of covalently linked nucleotide residues.

[0040] The terms "oligomer" or "oligonucleotide" include RNA or DNA sequences of more than one nucleotide in single-stranded or duplex form, and specifically include short sequences in single-stranded or duplex form, such as dimers and trimers, which can be intermediates in the production of specifically binding oligonucleotides. The "modified" forms used in the candidate pool contain at least one unnatural residue. "Oligonucleotide" or "oligomer" generally refers to polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or a modified form thereof), such as DNA, to polyribonucleotides (containing D-ribose or a modified form thereof), such as RNA, and to any other type of polynucleotide, which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or a modified purine or pyrimidine base or abasic nucleotide. "Oligonucleotide" or "oligomer" can also be used to describe synthetic polymers that are analogous to oligomers having RNA and DNA, including but not limited to peptide nucleic acids (PNA).

[0041] The terms "binding activity" and "binding affinity" generally refer to the tendency of a ligand molecule to bind or not bind to a target. The energetics of these interactions are significant in "binding activity" and "binding affinity" as they can include the definition of the interacting partner concentrations, the rate at which these partners can associate, and the relative concentrations of bound and free molecules in solution.

[0042] "Complementary" refers to the characteristic that two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) are capable of hybridizing, dimerizing, or otherwise forming a complex with each other. For example, "complementary peptides and polypeptides" are capable of aggregating together to form a complex. Complementary elements may require assistance to form a complex (e.g., from interacting elements), such as placing the elements in the correct conformation for complementarity, co-localizing the complementary elements, reducing the interaction energy of complementarity, etc.

[0043] As used herein, the term "nucleotide sequence identity" or "nucleic acid sequence identity" refers to the presence of the same nucleotide at corresponding positions in two polynucleotides. A polynucleotide has a "same" sequence if the nucleotide sequences in the two polynucleotides are the same when aligned for maximum correspondence (e.g., in a comparison window). Sequence comparison between two or more polynucleotides is typically carried out by comparing portions of the two sequences in a comparison window to identify and compare local regions of sequence similarity. The comparison window is typically about 20 to 200 consecutive nucleotides. The "percent sequence identity" of a polynucleotide, such as about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity, can be determined by comparing the two best-aligned sequences in a comparison window, where for the best alignment of the two sequences, the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence. In some embodiments, the percentage is calculated by: (a) determining the number of positions at which the same nucleic acid base occurs in the two sequences; (b) dividing the number of matching positions by the total number of positions in the comparison window; and (c) multiplying the result by 100. The best alignment of the sequences for comparison can also be carried out by computerized implementations of known algorithms or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are the Basic Local Alignment Search Tool (BLAST) and the ClustalW / ClustalW2 / Clustal Omega programs, which are available on the Internet (e.g., the EMBL-EBI website). Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA, which are part of the Accelrys GCG software package available from Accelrys, Inc., San Diego, California, USA. See also Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; Ausubel et al., 1988; and Sambrook and Russell, 2001.

[0044] 2. Multicomponent aptamers

[0045] According to various embodiments of the present disclosure, methods and compositions related to identifying multi-complementary-site nucleic acid molecules capable of binding to multiple epitopes on a given target biomolecule are described herein. As a proof of concept, previous experiments using SELEX identified the optimal linker sequences for a dual-complementary-site DNA aptamer from an oligonucleotide library containing two previously reported single-complementary-site thrombin-binding aptamers (Tasset-29 and Bock-15) linked by a 35 nt random region. After five rounds of selection, the isolated aptamer (TBV-08) had a K D , which was 200-fold and 300-fold lower than the original Tasset-29 and Bock-15 aptamers, respectively ( Figure 1 ). In contrast, designing and testing 10 21 linker domain sequences would be required to identify such an aptamer through brute-force engineering and characterization. Notably, the affinity of TBV-08 was improved by several orders of magnitude relative to other dual-complementary aptamers engineered from Tasset-29 and Bock-15. Additionally, the potency of TBV-08 to inhibit thrombin in a fibrinogen cleavage activity assay was 80-fold higher than that of Bock-15, which may be due to the increased affinity of its dual-complementary-site binding. These results support the feasibility of using anchor-SELEX to isolate (rather than engineer) high-affinity multi-complementary-site aptamers against protein targets.

[0046] According to the above, embodiments of the present disclosure relate to the development of a generalizable SELEX method for isolating high-affinity multi-complementary-site aptamers. For example, multi-complementary-site aptamers can be identified that serve as novel targeted cancer therapeutics to circumvent drug resistance through target mutations. In one embodiment, the therapeutic agent can be based on a multi-complementary-site 2'-fluoro (2'-F) RNA aptamer that binds to the extracellular domain of human epidermal growth factor receptor (hEGFR) on cells. hEGFR, which is aberrantly activated in many tumors, is a well-established target for colorectal, lung, skin, and head and neck cancers. A previously reported single-complementary-site 2'-F-modified RNA aptamer (e.g., reference anchor aptamer sequence) that binds cell surface hEGFR with a K D of 50 nM can be used as a scaffold to construct a library containing randomized domains at both ends as putative binding sites for recognizing additional epitopes on hEGFR. Thus, the core aptamer scaffold serves as an "anchor" for two additional aptamer domains that surround the periphery of the target to further stabilize and enhance binding. By being able to recognize multiple target domains, this multi-complementary-site binding confers robust tolerance to individual mutations relative to conventional aptamers ( Figure 2 ). Quantitatively, the isolated multi-complementary-site aptamer can have a K D, which is approximately 1,000-fold lower than the original single-complementary-site aptamer. Even with a single target-site mutation, the remaining two aptamer domains still bind the target with an affinity similar to that of the FDA-approved drug cetuximab (K D < 1 nM).

[0047] Multicomponent aptamer sequences can also be identified and their binding capabilities, stoichiometry, affinity, and kinetics for binding to free extracellular hEGFR characterized. Similar analyses can be performed on various cancer cell lines with hEGFR or its mutant isotypes. Experiments can evaluate the potential therapeutic efficacy of the optimal aptamer in vitro using various cancer cell lines, conduct mechanistic studies on how aptamer-EGFR binding inhibits cell proliferation and survival, and compare aptamer performance to standard EGFR-targeted antibody therapies. These data can be the basis for generating multicomponent aptamers against other protein biomarkers, preparing these aptamers for in vivo use by modulating their pharmacokinetics (e.g., binding to polyethylene glycol, gold nanoparticles, or Fc proteins to increase the biological half-life), and evaluating their safety and efficacy as in vivo therapeutic agents. Importantly, such high-affinity receptors can also be used as novel diagnostic reagents capable of detecting ultra-low levels of medically important analytes (e.g., pathogens or endogenous biomarkers).

[0048] As described herein, embodiments of the present disclosure include using cell-SELEX technology with a structured 2'-F RNA oligonucleotide library to isolate high-affinity multicomponent aptamers against cell surface hEGFR. 2'-F RNA is highly resistant to nuclease digestion, and such aptamers have been successfully applied to in vivo applications. To ensure that the isolated aptamers are tolerant to individual target-site mutations, anchor-SELEX can be used to isolate aptamers that bind hEGFR at multiple sites simultaneously, and a "switching" selection strategy against different cell lines expressing wild-type hEGFR or its mutants. However, as will be recognized by those of ordinary skill in the art, other types of SELEX methods can also be used in conjunction with various embodiments of the present disclosure, including target immobilized SELEX, library immobilized SELEX, nuclease-assisted SELEX, and filtration SELEX.

[0049] For example, the extracellular region of hEGFR contains four distinct domains. In the Figure 3A inactive "tethered" conformation shown, domains I and III serve as binding sites for human epidermal growth factor (hEGF) and other ligands. Domains II and IV form intermolecular interactions (or tethers) in the unliganded monomer ( Figure 3A)。In the ligand-bound dimer, the receptor adopts a very different conformation, and Domain II forms the primary dimerization interface. The library design of Anchor-SELEX incorporates a well-characterized aptamer that binds to target Domain III with a K of approximately 50 nM D binds to hEGFR on cancer cells as the core "anchor", flanked by two randomized regions that form additional interactions with hEGFR ( Figure 3B )。Previous studies have shown that the functional binding region of this aptamer is a 22-nucleotide (nt) segment located in the middle of the sequence. Therefore, to minimize the length of the final multi-complementary-site aptamer, this 22-nt motif was used as the anchor. Each RNA library molecule contains three regions, flanked by PCR primer sites at both ends ( Figure 3C )。Region 1 (black) consists of the 22-nt anchor aptamer sequence, where each nucleotide position is "doped" (i.e., partially randomized) such that the probability of retaining the original nucleobase is 67%. This is done to ensure that the final aptamer can recognize hEGFR with mutations at Domain III. Regions 2 (pink) and 3 (blue) are fully randomized 20-nt sequences that serve as additional linkers and binding sites for other epitopes on hEGFR. The length of the random regions should be sufficient to form high-affinity binding domains with optimal spacing, as most high-affinity protein-binding aptamers have only 3-8 nt interacting with the target on average. Since the anchor targets Domain III, the two surrounding sites can enable binding to adjacent regions on Domain III or Domains II and IV.

[0050] According to the above description, embodiments of the present disclosure include a single-stranded nucleic acid molecule comprising an anchor aptamer capable of binding to a target biomolecule and at least one candidate aptamer coupled to the anchor aptamer. In some embodiments, the at least one candidate aptamer comprises a random nucleic acid sequence.

[0051] In some embodiments, the anchor aptamer comprises a predetermined nucleic acid sequence (e.g., a reference aptamer sequence) that binds to an epitope on a target biomolecule. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 45 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 40 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 35 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 30 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 25 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 20 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 15 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 15 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 20 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 25 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 30 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 35 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 40 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 45 nucleotides to about 50 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 20 nucleotides to about 40 nucleotides. In some embodiments, the anchor aptamer comprises a nucleic acid sequence having a length of from about 15 nucleotides to about 30 nucleotides.

[0052] In some embodiments, the anchor aptamer comprises a nucleic acid sequence of a reference aptamer sequence. In some embodiments, the anchor aptamer comprises a nucleic acid sequence that is at least 90% identical to the reference aptamer sequence. In some embodiments, the anchor aptamer comprises a nucleic acid sequence that is at least 95% identical to the reference aptamer sequence. In some embodiments, the anchor aptamer comprises a nucleic acid sequence that is at least 98% identical to the reference aptamer sequence. In some embodiments, the anchor aptamer comprises a nucleic acid sequence that is 100% identical to the reference aptamer sequence. In some embodiments, the anchor aptamer comprises a nucleic acid sequence that comprises at least one modification as compared to the reference aptamer sequence. In some embodiments, the at least one modification comprises one or more of the following: (i) substitution of one or more nucleotides; (ii) deletion of one or more nucleotides; and / or (iii) insertion of one or more nucleotides (and any combination thereof) as compared to the reference aptamer sequence. In some embodiments, the modification comprises substitution of one or more nucleotides as compared to the reference aptamer sequence. In some embodiments, the modification comprises deletion of one or more nucleotides as compared to the reference aptamer sequence. In some embodiments, the modification comprises insertion of one or more nucleotides as compared to the reference aptamer sequence.

[0053] In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 10 nM to about 10 μM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 10 nM to about 5 μM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 10 nM to about 1 μM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 10 nM to about 500 nM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 10 nM to about 250 nM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 10 nM to about 100 nM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 10 nM to about 50 nM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 20 nM to about 10 μM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 100 nM to about 10 μM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 250 nM to about 10 μM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 500 nM to about 10 μM. D In some embodiments, the anchor aptamer binds to the target biomolecule with a K of about 1 µM to about 10 μM.D Bind to the target biomolecule. In some embodiments, the anchor aptamer has a K of about 5 µM to about 10 μM D Bind to the target biomolecule. In some embodiments, the anchor aptamer has a K of about 500 nM to about 5 μM D Bind to the target biomolecule. In some embodiments, the anchor aptamer has a K of about 100 nM to about 1 μM D Bind to the target biomolecule. In some embodiments, the anchor aptamer has a K of about 250 nM to about 2.5 μM D Bind to the target biomolecule. In some embodiments, the anchor aptamer has a K of about 750 nM to about 5 μM D Bind to the target biomolecule.

[0054] In some embodiments, the nucleic acid molecule comprises two candidate aptamers flanking the anchor aptamer. In some embodiments, at least one candidate aptamer comprises a random nucleic acid sequence determined to bind to the target biomolecule. In some embodiments, at least one candidate aptamer binds to an epitope on the target biomolecule that is different from the epitope bound by the anchor aptamer.

[0055] In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 50 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 45 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 40 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 35 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 30 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 25 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 20 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 10 nucleotides to about 15 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 15 nucleotides to about 50 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 20 nucleotides to about 50 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 25 nucleotides to about 50 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 30 nucleotides to about 50 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 35 nucleotides to about 50 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 40 nucleotides to about 50 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 45 nucleotides to about 50 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 20 nucleotides to about 40 nucleotides. In some embodiments, at least one candidate aptamer comprises a nucleic acid sequence having a length of from about 15 nucleotides to about 30 nucleotides.

[0056] In some embodiments, the nucleic acid molecule binds to the target biomolecule with a higher affinity than a reference anchor aptamer. In some embodiments, the nucleic acid molecule has a K D for binding to the target biomolecule. In some embodiments, the nucleic acid molecule has a K D for binding to the target biomolecule. In some embodiments, the nucleic acid molecule has a K D for binding to the target biomolecule. In some embodiments, the nucleic acid molecule has a K DBind to a target biomolecule. In some embodiments, the nucleic acid molecule has a K of from about 1 fM to about 500 pM D Bind to a target biomolecule. In some embodiments, the nucleic acid molecule has a K of from about 1 fM to about 1 pM D Bind to a target biomolecule. In some embodiments, the nucleic acid molecule has a K of from about 1 fM to about 500 fM D Bind to a target biomolecule. In some embodiments, the nucleic acid molecule has a K of from about 500 fM to about 500 pM D Bind to a target biomolecule.

[0057] In some embodiments, the length of the nucleic acid molecule is from about 30 nucleotides to about 150 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 30 nucleotides to about 125 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 30 nucleotides to about 100 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 30 nucleotides to about 75 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 30 nucleotides to about 50 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 50 nucleotides to about 150 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 75 nucleotides to about 150 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 100 nucleotides to about 150 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 125 nucleotides to about 150 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 75 nucleotides to about 125 nucleotides. In some embodiments, the length of the nucleic acid molecule is from about 50 nucleotides to about 100 nucleotides.

[0058] In some embodiments, the nucleic acid molecule is RNA, DNA, or a combination thereof. In some embodiments, the nucleic acid molecule comprises 2'-deoxy-2'-fluoro-ribonucleotides (2'-F RNA), phosphorothioate modifications, 2'-O-methyl sugars, LNA (locked nucleic acid), and threose nucleic acid (TNA), and any combination thereof. In some embodiments, the nucleic acid molecule includes 2'-deoxy-2'-fluoro-ribonucleotides (2'-F RNA). In some embodiments, the nucleic acid molecule comprises a phosphorothioate modification. In some embodiments, the nucleic acid molecule comprises 2'-O-methyl sugars. In some embodiments, the nucleic acid molecule comprises LNA (locked nucleic acid). In some embodiments, the nucleic acid molecule comprises threose nucleic acid (TNA).

[0059] In some embodiments, the nucleic acid molecule further comprises a linker. In some embodiments, the nucleic acid molecule further comprises a linker, and the linker facilitates the binding of the nucleic acid molecule to a solid support. The solid support can be any solid material to which the multi-complementary site aptamers of the present disclosure can be grafted, and can optionally include pores (e.g., those that can be used as the stationary phase / packing material for chromatography). In one example, the solid support comprises an inorganic (e.g., silica) material. In another example, the solid support comprises an organic (e.g., polymer) material (e.g., synthetic resin or agarose). In another example, the solid support comprises a hybrid inorganic-organic material. In one embodiment, the solid support comprises a metal oxide, a metalloid oxide, or a magnetic iron oxide (e.g., magnetic particles). Exemplary matrices include silica-based (e.g., silica, SiO2), titanium dioxide-based (e.g., titanium oxide, TiO2), germanium-based (e.g., germanium oxide), zirconium oxide-based (e.g., zirconium oxide, ZrO2), alumina-based (e.g., alumina, Al2O3) materials or mixtures thereof. Other matrices include crosslinked and uncrosslinked polymers, carbonized materials, and metals. The matrix can also be incorporated with a polymeric network, a sol-gel network, or a hybrid form thereof. In one embodiment, the matrix is a silica-based matrix. Exemplary silica-based matrices include silica gel, glass, sol-gel, polymer / sol-gel hybrids, core-shell structures, and silica monolithic materials.

[0060] In some embodiments, the nucleic acid molecule further comprises a fluorescent tag or an electroactive tag. The fluorescent tag or electroactive tag can be any tag known in the art. In some embodiments, the fluorescent tag can include, but is not limited to, green fluorescent protein (GFP), GFP derivatives (such as red (RFP), blue (BFP), cyan (CFP), yellow (YFP)), coumarin dyes, Cascade Blue, CyDyes, fluorescein, Bodipy dyes, rhodamine (and its derivatives), allophycocyanin, Alexa Fluor dyes, Texas Red, Lucifer Yellow, TruRed, or peridinin-chlorophyll protein. In other embodiments where fluorescence detection is not used, other types of tags or reporter moieties can be used, such as tags that facilitate electronic detection or radiographic imaging. In some embodiments, the tag includes a redox tag (e.g., methylene blue), which can be used in electrochemical-based sensors.

[0061] In some embodiments, the target biomolecule is a protein or polypeptide. In some embodiments, the target biomolecule is selected from the group consisting of: cell surface receptors, endogenous biomarkers, cardiac proteins, oncoproteins, endocrine polypeptide hormones, gastrointestinal peptide hormones, cytokines, viral proteins, bacterial proteins, fungal proteins, and plant proteins. Based on the present disclosure, those skilled in the art will recognize that the target molecule can include any peptide, polypeptide, or protein of interest, as the multi-complementary site aptamers described herein can be selected to bind to any epitope.

[0062] According to these embodiments, the present disclosure also includes a library comprising a plurality of any of the nucleic acid molecules described herein. In some embodiments, the plurality of nucleic acid molecules comprises at least 10 10 individual nucleic acid molecules, and each sequence of at least one candidate aptamer is different. In some embodiments, the multi-complementary site aptamer library of the present disclosure comprises a plurality of the above-mentioned aptamers. In some embodiments, the size of the nucleic acid aptamer library of the present invention is 10 10 or more, 10 11 or more, 10 12 or more, 10 13 or more, 10 14 or more or 10 15 or more.

[0063] 3. Methods for Identifying Multi-Complementary Site Aptamers

[0064] Embodiments of the present disclosure also include methods for isolating multi-complementary site aptamers that bind to different epitopes of monomeric cell surface hEGFR in their native conformation. As described above, well-established cell-SELEX techniques and designed libraries can be used, with cancer cell lines expressing hEGFR as the target for experimentation. However, as those of ordinary skill in the art will recognize, other types of SELEX methods can also be used in conjunction with various embodiments of the present disclosure, including target immobilized SELEX, library immobilized SELEX, nuclease-assisted SELEX, and filtration SELEX.

[0065] In one embodiment, A549 EGFR knockout cells are first used for negative selection to remove non-specific binders from the library. Then, the collected pool is used for five rounds of "switching" selection using multiple cell lines expressing wild-type or mutant hEGFR ( Figure 4B ) ( Figure 4A)To ensure that the isolated aptamers can cross-react with all hEGFR variants. In the first round, the library was incubated with A549 cells overexpressing wild-type hEGFR. Since each library molecule contains an anchor for binding to domain III of hEGFR, the library can evolve mainly towards enrichment of aptamers that bind this target rather than other cell surface receptors. In subsequent rounds, four different cell lines expressing hEGFR point mutants were used as targets: SW48 cells with S492R in domain III (round 2); SW48 cells with L65V in domain I (round 3); NCI-H1975 with intracellular L858R, which promotes ligand-independent receptor dimerization (round 4); and RKO cells with C240Y in domain II (round 5). To enrich the aptamers with the highest affinity, in each subsequent round, a cell line expressing a reduced level of hEGFR was used (i.e., A549 and RKO have the highest and lowest EGFR expression, respectively). This process ultimately enriched aptamers that can bind both wild-type and mutant hEGFR. After five rounds, the aptamer pool was evaluated for affinity and cross-reactivity against all target cell lines, and the pool showed high affinity for all cell lines.

[0066] Anchor-SELEX was performed using a modified version of a previously reported protocol. For example, the targets selected could include five different human cancer cell lines overexpressing wild-type or mutant hEGFR. The targets were alternated in each round of SELEX to isolate multi-complementary-site aptamers that cross-react with both wild-type and mutant hEGFR. First, a DNA library containing a 5’ T7 promoter site was in vitro transcribed using the DuraScribe T7 Transcription Kit to generate a 2'-F RNA library ( Figure 3C ). Then, 10 nmol of the 2'-F RNA library (approximately 10 15 unique sequences) was heated to 85 °C, cooled on ice to allow the library strands to fold correctly, and then resuspended in 800 µL of binding buffer (Dulbecco's phosphate-buffered saline (DPBS; pH 7.4), 5 mM MgCl2, 25 mM glucose, 0.1 mg / ml yeast tRNA, and 1 mg / ml BSA). Excess tRNA was used to prevent non-specific adsorption of the library strands to the cells. Each cell line was cultured in a 10-cm culture dish to 90% confluence (approximately 10 7cells). Prior to adding the library, the cell culture medium was removed and the cells were washed with wash buffer (binding buffer without tRNA and BSA). The library was then incubated with the cells at 37 °C for 1 hour in the absence of hEGF. After incubation, the supernatant containing the unbound library strands was removed and discarded; this also included oligomers that misfolded and disrupted the anchor domain. The cells were washed and then collected by scraping the dish and rinsing with 2 mL of DPBS. The collected solution was heated at 85 °C for 10 minutes to release the target-bound strands into solution and then centrifuged at 13,000 rcf. The supernatant containing the library strands bound to hEGFR was collected and reverse transcribed to complementary DNA (cDNA) using a kit from Applied Biosciences and then amplified by PCR. These amplicons were then used as templates to create a new pool of 2'-F RNAs for the next round of selection. This process was repeated for five rounds. Using the previously described flow cytometry protocol, the pool affinity was monitored in each round by transcribing the RNA sequences and hybridizing them to reverse primers labeled with fluorescein at the 5' end. Hybridization pools of different concentrations (0–500 nM) of the probe were mixed with 10 5 target cells in binding buffer and incubated at 37 °C for 1 hour. The cells were then washed to remove unbound library strands and analyzed by flow cytometry using a BD Melody instrument, acquiring 10 4 events per sample. The proportion of cells bound to the fluorescent library construct was used to determine the K D of the pool. For the final enriched pool, the cross-reactivity to various cell lines was determined.

[0067] After completion of anchor-SELEX, the binding properties of the isolated hEGFR-binding aptamers were identified and characterized. First, high-throughput sequencing was performed to identify highly enriched aptamer candidates from the final SELEX pool. Then the binding affinities of these aptamers to different cell lines expressing wild-type and mutant isoforms of hEGFR were determined to evaluate the ability of the aptamers to tolerate target mutations. Then, for example, surface plasmon resonance (SPR) was used to verify the binding affinity, kinetics, and multi-complementary site binding ability of the aptamers.

[0068] In addition, high-throughput sequencing was performed on the finally enriched library pool. Five of the most abundant sequences were identified and synthesized, which did not contain the PCR primer binding region and had a 5'-FAM label (TriLink Biotechnologies). First, the binding affinities of these aptamer candidates were tested against five cancer cell lines expressing hEGFR variants used in the anchor-SELEX and a negative control cell line (A549 EGFR knockout). Each cell line was grown to 90% confluence in a culture plate, washed with binding buffer, and then incubated with 0 - 100 nM of the 5' FAM-labeled aptamer at 37 °C for 1 hour. The cells were washed twice with DPBS to remove unbound aptamers. Flow cytometry was used to determine the proportion of FAM-labeled aptamers bound to the cells, which was then used to determine K D . As a comparison, the same procedure was repeated using a 5' FAM-labeled anchor aptamer and Cy5.5-labeled cetuximab. Based on a 200-fold increase in affinity observed in the case of the dual-compleX thrombin aptamer, the binding affinity of the triple-compleX aptamer for these cell lines was predicted to be at least 50 pM, which was 1,000-fold higher than that of the anchor aptamer and 10-fold higher compared to cetuximab ( Figure 5 ). To demonstrate the robust tolerance of the aptamer to point mutations, flow cytometry was used to determine the affinity of the aptamer for SW48 cells expressing hEGFR mutants (including I491M, S464L, and G465M) not used during the anchor-SELEX and HCC827 cells carrying an E746 - A750 deletion. If these mutations were in or near the anchor aptamer binding site, a decrease in aptamer binding was expected, but the aptamer should retain an affinity equivalent to that of cetuximab due to the retention of binding by the other two aptamer domains ( Figure 5 ).

[0069] The binding kinetics of the two triple-compleX aptamers with the greatest affinity for all cell lines were further evaluated using a Biacore 3000 SPR instrument. hEGFR was modified onto a CM5 dextran chip (Cytiva) by EDC-NHS chemistry. Unlabeled 2'-F aptamer (TriLink Biotechnologies) dissolved in binding buffer was flowed over the chip; after equilibrium was reached, the aptamer was washed away with binding buffer. The instrument recorded the change in the SPR resonance angle associated with aptamer-target binding over time, and the resulting data could be used to determine the binding stoichiometry, K D , k on and k off . SPR was used to measure the binding characteristics of the original anchor aptamer and cetuximab. The optimal triple-compleX aptamer was expected to exhibit a 1,000-fold increase in K Dand k off .

[0070] SPR will also be used to evaluate the binding contributions of the individual regions of the best-performing trimeric aptamer. R1 is the core anchor aptamer, while R2 and R3 are the putative binding regions at the 5'- and 3'-ends, respectively. Nine constructs were derived from the trimeric aptamer ( Figure 6 ): the monomeric aptamers R1, R2, and R3, three dimeric aptamers made by removing one of the binding domains from the trimeric aptamer, and three dimeric aptamers with a point mutation that impairs the affinity of a specific domain at one of the three domains. To confirm that all three regions can bind hEGFR independently, saturated concentrations of R1, R2, and R3 were flowed over the hEGFR-modified chip in sequence. If these aptamers can bind hEGFR simultaneously, the SPR resonance angle should increase continuously upon addition of each aptamer. Then, the K D and k off of each construct were measured using the hEGFR-modified chip. The truncated and mutated dimeric aptamers may have worse K D and k off than the native trimeric aptamer, although they should still have better K D and k off .

[0071] To evaluate the therapeutic efficacy of the aptamer and study its mechanism of inhibiting cell proliferation, a series of in vitro experiments were performed. For example, an experiment can first be conducted to confirm whether the aptamer can compete with hEGF for binding to cell surface hEGFR. Given that it has been previously demonstrated that the anchor aptamer blocks the binding of hEGF to hEGFR, this is expected to occur. Approximately 10 5 A549 cells expressing wild-type hEGFR were incubated with 1 nM 5'-FAM-labeled aptamer in binding buffer at 37 °C for 1 hour. Then, 0 - 100 nM TRITC-labeled hEGF was added to the cells and incubated at 37 °C for 1 hour to allow hEGF to displace the aptamer. The cells were then washed with DPBS to remove unbound aptamer and hEGF, and the cells bound to TRITC-hEGF or FAM-aptamer were counted using flow cytometry. As a control, the same protocol can be performed using a scrambled aptamer sequence labeled with 5'-FAM, which should have no affinity for hEGFR and thus not inhibit hEGF binding. Since the trimeric aptamer has ultra-high affinity for hEGFR, the trimeric aptamer can inhibit hEGF binding even at high concentrations.

[0072] In addition, experiments can be conducted to test the effect of the three-complementary-site aptamer on cell proliferation. Five cell lines expressing hEGFR variants used as SELEX targets were cultured; the medium was replenished every other day, and the cells were passaged after reaching confluence. To evaluate the anti-proliferative effect of the aptamer, the cells were seeded at a density of 2,000 cells / well in the corresponding medium in a 96-well plate and treated with 10 nM hEGF and 0 - 100 nM aptamer at 37 °C for 72 hours. Proliferation was measured using a 5-bromo-2'-deoxyuridine labeling and detection assay. A scrambled three-complementary-site aptamer mutant was used as a negative control and the same test was performed with cetuximab to compare aptamer efficacy. Proliferation of cancer cells expressing wild-type or mutant hEGFR was significantly reduced even at low aptamer concentrations.

[0073] Next, the efficacy of the three-complementary-site aptamer as a potential targeted therapeutic agent to be used in conjunction with traditional cytotoxic chemotherapy was evaluated. The chemotherapeutic agent 5-fluorouracil was used with or without the multi-complementary-site aptamer, and experiments were performed on five cancer cell lines used as SELEX targets using the standard methyl thiazolyl tetrazolium (MTT) assay (Roche). The cells were transferred to a 96-well plate at a seeding density of 5,000 cells / well in their respective media for one day to allow them to adhere. After rinsing with DPBS, the cells were incubated with a fixed concentration of aptamer (or, for the control, DPBS) that was determined to have a significant effect on cell proliferation and 0–1,000 nM 5-fluorouracil in cell culture medium at 37 °C for 72 hours. The LC 50 (5-fluorouracil concentration resulting in 50% cell viability) of cancer cells treated in combination with the aptamer was expected to be much lower than that of cancer cells treated with 5-fluorouracil alone, regardless of whether the cells expressed wild-type or mutant hEGFR.

[0074] According to these embodiments, the present disclosure includes a method for identifying a multi-complementary-site aptamer, the method comprising exposing a library of candidate multi-complementary-site aptamers to a target biomolecule, wherein each candidate multi-complementary-site aptamer in the library comprises an anchor aptamer capable of binding the target biomolecule and at least one candidate aptamer coupled to the anchor aptamer, and wherein the at least one candidate aptamer comprises a random nucleic acid sequence. The method further comprises evaluating the binding of the candidate multi-complementary-site aptamer to the target biomolecule.

[0075] In some embodiments, the library of candidate multi-complementary-site aptamers is exposed to the target biomolecule in multiple rounds of selection. In some embodiments, the multiple rounds of selection comprise exposing the library of candidate multi-complementary-site aptamers to variants of the target biomolecule in each round.

[0076] In some embodiments, the target biomolecule is a protein or polypeptide. In some embodiments, the protein or polypeptide is expressed by a cell (e.g., cell-SELEX method). In some embodiments, the protein or polypeptide is conjugated to a solid support. In other embodiments, the target biomolecule is a protein or polypeptide that is present in solution and not bound to a solid support. In some embodiments, the target protein may be conjugated to or immobilized on a solid support such as a bead (e.g., target immobilized SELEX), including streptavidin-linked beads, where hybridization of the complementary DNA is biotinylated (e.g., library immobilized SELEX). In some embodiments, the target biomolecule is selected from the group consisting of: cell surface receptors, endogenous biomarkers, cardiac proteins, oncoproteins, endocrine polypeptide hormones, gastrointestinal peptide hormones, cytokines, viral proteins, bacterial proteins, fungal proteins, and plant proteins. Based on the present disclosure, one of ordinary skill in the art will recognize that the target molecule can include any peptide, polypeptide, or protein of interest, as the multi-complementary aptamers described herein can be selected to bind any epitope.

[0077] In some embodiments, evaluating the binding of a candidate multi-complementary aptamer to a target biomolecule includes determining the K D of the candidate multi-complementary aptamer to see if it is below a threshold. In some embodiments, the threshold K D is at least 10-fold lower than the K D of the anchor aptamer. In some embodiments, the threshold K D is at least 11-fold lower than the K D of the anchor aptamer. In some embodiments, the threshold K D is at least 12-fold lower than the K D of the anchor aptamer. In some embodiments, the threshold K D is at least 13-fold lower than the K D of the anchor aptamer. In some embodiments, the threshold K D is at least 14-fold lower than the K D of the anchor aptamer. In some embodiments, the threshold K D is at least 15-fold lower than the K D of the anchor aptamer. In some embodiments, the threshold K D is at least 20-fold lower than the K D of the anchor aptamer. In some embodiments, the threshold K D is at least 50-fold lower than the K D of the anchor aptamer. In some embodiments, the threshold K D is at least 100-fold lower than the K D [[ID=D41]]of the anchor aptamer. In some embodiments, the threshold K D is 100-fold or more lower than the K D of the anchor aptamer.

[0078] In some embodiments, assessing the binding of a candidate multi-complementary-site aptamer to a target biomolecule includes performing surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis, biolayer interferometry, strand displacement fluorescence assay, and intrinsic tryptophan fluorescence quenching, and any combination thereof. Based on the present disclosure, one of ordinary skill in the art will recognize that other methods and assays can be used to evaluate the binding affinity of one or more candidate multi-complementary-site aptamers to a target biomolecule.

Claims

1. A single-stranded nucleic acid molecule comprising: An anchor aptamer capable of binding to a target biomolecule; and At least one candidate aptamer coupled to the anchor aptamer, wherein the at least one candidate aptamer comprises a random nucleic acid sequence.

2. The nucleic acid molecule according to claim 1, wherein the anchor aptamer comprises a predetermined nucleic acid sequence that binds to an epitope on the target biomolecule.

3. The nucleic acid molecule according to claim 1 or claim 2, wherein the anchor aptamer comprises a nucleic acid sequence having a length of about 10 nucleotides to about 50 nucleotides.

4. The nucleic acid molecule according to any one of claims 1 to 3, wherein the anchor aptamer comprises a nucleic acid sequence of a reference aptamer sequence.

5. The nucleic acid molecule according to any one of claims 1 to 4, wherein the anchor aptamer comprises a nucleic acid sequence comprising at least one modification as compared to the reference aptamer sequence.

6. The nucleic acid molecule according to claim 5, wherein the at least one modification comprises one or more of the following: (i) substitution of one or more nucleotides; (ii) deletion of one or more nucleotides; and / or (iii) insertion of one or more nucleotides as compared to the reference aptamer sequence.

7. The nucleic acid molecule according to claim 6, wherein the anchor aptamer has a K of about 10 nM to about 10 μM D for binding to the target biomolecule.

8. The nucleic acid molecule according to any one of claims 1 to 7, wherein the nucleic acid molecule comprises two candidate aptamers flanking the anchor aptamer.

9. The nucleic acid molecule according to any one of claims 1 to 8, wherein the at least one candidate aptamer comprises a random nucleic acid sequence determined to bind to the target biomolecule.

10. The nucleic acid molecule according to claim 9, wherein the at least one candidate aptamer binds to an epitope on the target biomolecule that is different from the epitope bound by the anchor aptamer.

11. The nucleic acid molecule according to any one of claims 1 to 10, wherein the nucleic acid molecule binds to the target biomolecule with a higher affinity than a reference anchor aptamer.

12. The nucleic acid molecule according to any one of claims 1 to 11, wherein the nucleic acid molecule binds to the target biomolecule with a K of about 1 fM to about 1 nM D to bind to the target biomolecule.

13. The nucleic acid molecule according to any one of claims 1 to 12, wherein the at least one candidate aptamer comprises a nucleic acid sequence having a length of about 10 nucleotides to about 50 nucleotides.

14. The nucleic acid molecule according to any one of claims 1 to 13, wherein the length of the nucleic acid molecule is about 30 nucleotides to about 150 nucleotides.

15. The nucleic acid molecule according to any one of claims 1 to 14, wherein the nucleic acid molecule is RNA, DNA, or a combination thereof.

16. The nucleic acid molecule according to any one of claims 1 to 15, wherein the nucleic acid molecule comprises 2'-deoxy-2'-fluoro-ribonucleotide (2'-F RNA), phosphorothioate modification, 2'-O-methyl sugar, LNA (locked nucleic acid), and threose nucleic acid (TNA), and any combination thereof.

17. The nucleic acid molecule according to any one of claims 1 to 16, wherein the nucleic acid molecule further comprises a linker.

18. The nucleic acid molecule according to claim 17, wherein the nucleic acid molecule further comprises a linker, and wherein the linker facilitates the binding of the nucleic acid molecule to a solid support.

19. The nucleic acid molecule according to any one of claims 1 to 18, wherein the nucleic acid molecule further comprises a fluorescent tag or an electroactive tag.

20. The nucleic acid molecule according to any one of claims 1 to 19, wherein the target biomolecule is a protein or polypeptide.

21. The nucleic acid molecule according to any one of claims 1 to 20, wherein the target biomolecule is selected from the group consisting of: cell surface receptors, endogenous biomarkers, cardiac proteins, oncoproteins, endocrine polypeptide hormones, gastrointestinal peptide hormones, cytokines, viral proteins, bacterial proteins, fungal proteins, and plant proteins.

22. A library comprising a plurality of any of the nucleic acid molecules of claims 1 to 21.

23. The library according to claim 22, wherein the plurality of nucleic acid molecules comprises at least 10 10 individual nucleic acid molecules, and wherein each sequence of the at least one candidate aptamer is different.

24. A method for identifying multi-complementary aptamers, the method comprising: exposing a library of candidate multi-complementary aptamers to a target biomolecule, wherein each candidate multi-complementary aptamer in the library comprises an anchor aptamer capable of binding to the target biomolecule and at least one candidate aptamer coupled to the anchor aptamer, wherein the at least one candidate aptamer comprises a random nucleic acid sequence; and evaluating the binding of the candidate multi-complementary aptamer to the target biomolecule.

25. The method according to claim 24, wherein the library of candidate multi-complementary aptamers is exposed to the target biomolecule in multiple rounds of selection.

26. The method according to claim 25, wherein the multiple rounds of selection comprise exposing the library of candidate multi-complementary aptamers to variants of the target biomolecule in each round.

27. The method according to any one of claims 24 to 26, wherein the target biomolecule is a protein or polypeptide.

28. The method according to claim 26, wherein the protein or polypeptide is expressed by a cell.

29. The method according to claim 26, wherein the protein or polypeptide is conjugated to a solid support.

30. The method according to claim 26, wherein the protein or polypeptide is present in solution and not bound to a solid support.

31. The method according to any one of claims 24 to 30, wherein evaluating the binding of the candidate multi-complementary aptamer to the target biomolecule comprises determining the K of the candidate multi-complementary aptamer D whether it is below a threshold value.

32. The method of claim 31, wherein the threshold K D than the K of the anchor aptamer D At least 10 times lower.

33. The method according to any one of claims 24 to 30, wherein evaluating the binding of the candidate multi-complementary aptamer to the target biomolecule comprises performing surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis, biolayer interferometry, strand displacement fluorescence assay, and intrinsic tryptophan fluorescence quenching, and any combination thereof.

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