Multivalent tetrameric or octameric aptamers for molecular recognition, methods of making and uses thereof

Abstract

Multivalent tetrameric and octameric aptamers comprising a G-quadruplex comprised of monomeric aptamer subunits having the general formula A – SA – [G]n – SB – B in which self-assembly of a G-tetrad linkage structure connects the aptameric arms in parallel orientation to provide enhanced affinity and / or avidity with a target are described, as well as methods of making and using the multivalent aptamers are provided.

Description

MULTIVALENT TETRAMERIC OR OCTAMERIC APTAMERS FOR MOLECULAR RECOGNITION, METHODS OF MAKING AND USES THEREOFFIELD OF THE INVENTION

[0001] The present invention relates to the field of aptamers and, in particular, to multivalent aptamer constructs exhibiting tetrameric and / or octameric structure, and methods of making and using thereof.BACKGROUND OF THE INVENTION

[0002] Aptamers are short single-stranded RNA or DNA molecules capable of specific binding to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells and tissues, due to the formation of characteristic spatial structures. Aptamers have been described as the chemical equivalent of antibodies, however, aptamers have the advantage of being highly specific, rapid selective generation, relatively small in size, non- immunogenic, and easily synthesized in quantity with high purity. For these reasons, it is believed that aptamers will find broad application in biosensing, bioanalysis, biotechnology, and biomedicine.

[0003] A number of approaches have been taken for generating aptamers designed to acquire a range of desired features, such as for example improved stability, target binding affinity, and conjugation to reporter groups, cell-toxic molecules, nanoparticles, etc.

[0004] Among the large variety of aptamer-based constructs, multimeric aptamers have been described that comprise two or more identical or different aptamer motifs, with or without additional structural elements or functional groups. It has been described, for example, that simple concatenation of the same aptamer motif can significantly improve the avidity of a construct due to multiple target binding sites. Other combinations of different aptamer motifs are also believed to offer opportunities to build multifunctional molecules.

[0005] Nonetheless, it remains a current objective in aptamer research and development to obtain aptamer constructs that are effective molecular recognition elements (MREs) that can be used for different bioanalytical and therapeutic applications.

[0006] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.SUMMARY OF THE INVENTION

[0007] An object of the present invention is to provide multivalent tetrameric and octameric aptamers for molecular recognition, and methods of making and uses thereof. In accordance with one aspect of the invention, there is provided a multivalent aptamer comprising a G-quadruplex comprised of four monomeric aptamer subunits, each monomeric aptamer subunit having the general formula: A - SA - [G]n- SB - B, wherein: A is an aptamer specific for one or more epitopes of a target molecule or protein, ranging from 5 to 150 nucleotides in length; G is a guanine-rich sequence that forms a guanine tetrad (G- tetrad) with a corresponding G of associating monomeric aptamer subunits to form the G- quadruplex; SA is a spacer molecule that separates A from G at a distance of up to 20.5 nm; B is a functional molecule selected from an aptamer, a reporter molecule, or a crosslinker that is linked to G at an end opposite to A; and SB is a spacer molecule that separates B from G at a distance of up to 20.5 nm.

[0008] In some embodiments, the [G]ncomprises 2 or more guanine bases. In other embodiments, the [G]ncomprises 2 to 7 guanine bases. In further embodiments, the [G]ncomprises a sequence of guanine molecules that may be consecutive or have other single bases interspersed therein to form the G-quadruplex. In other embodiments, the G-tetrad formed by the associating monomeric aptamer subunits is stabilized by a cation situated in a central channel between the associating monomeric aptamer subunits.

[0009] According to certain embodiments, the G-quadruplex is configured with the As oriented in parallel alignment to form a tetrameric aptamer. In other embodiments, the G- quadruplex is configured with the Bs oriented in parallel alignment opposite from the As to form an octameric aptamer.

[0010] In some embodiments, SA comprises a single stranded nucleic acid sequence of 0 to 30 nucleotides. In certain embodiments, SA is a single stranded thymine-rich sequence of 12 nucleotides. In other embodiments, SA comprises up to 15 linear alkane chains each comprising up to 12 carbon atoms. In further embodiments, SA comprises a linear polyethylene glycol chain of up to 55 ethylene glycol units.

[0011] In some embodiments, B is an aptamer identical to A. In other embodiments, B is an aptamer different than A. In other embodiments, B is a reporter molecule selected from an antigen, an enzyme, and a fluorescent molecule. In further embodiments, B is a crosslinker selected from thiol, amide, biotin, digoxigenein, azide, alkyne, carboxyl, and a click-chemi stry-b ased crosslinker.

[0012] In some embodiments, SB comprises a single stranded nucleic acid sequence of 0 to 30 nucleotides. In other embodiments, SB is a single stranded thymine sequence of 5 nucleotides. In further embodiments, SB comprises up to 15 linear alkane chains each comprising up to 12 carbon atoms. In other embodiments, SB comprises a linear polyethylene glycol chain of up to 55 ethylene glycol units.

[0013] In some embodiments, the aptamer is a homomeric aptamer comprising identical As. In other embodiments, the aptamer is a heteromeric aptamer comprising 2 or more As that are each specific to a different epitope of a target molecule or protein. In certain embodiments, the [G]nis linked to the 3’ end of the A. In other embodiments, the [G]nis linked to the 5’ end of the A.

[0014] In certain embodiments, the As are in the same 5’ to 3’ orientation relative to G. In other embodiments, the As are in the same 3’ to 5’ orientation relative to G.

[0015] In some embodiments, A is in a range of 20 to 90 nucleotides in length. In certain embodiments, the target molecule is SARS-CoV-2 spike protein and A is MSA52.

[0016] In accordance with a further aspect of the invention, there is provided a tetrameric aptamer comprising a multivalent aptamer comprising a G-quadruplex comprised of four monomeric aptamer subunits, each monomeric aptamer subunit having the general formula: A - SA- [G]n- SB - B, wherein: A is an aptamer specific for one or more epitopes of a target molecule or protein, ranging from 5 to 150 nucleotides in length; G is a guanine-rich sequence that forms a guanine tetrad (G-tetrad) with a corresponding G of associating monomeric aptamer subunits to form the G-quadruplex; SA is a spacer molecule that separates A from G at a distance of up to 20.5 nm; B is a functional molecule selected from an aptamer, a reporter molecule, or a crosslinker that is linked to G at an end opposite to A; and SB is a spacer molecule that separates B from G at a distance of up to 20.5 nm.

[0017] In another aspect, there is provided an octameric aptamer comprising a multivalent aptamer comprising a G-quadruplex comprised of four monomeric aptamer subunits, each monomeric aptamer subunit having the general formula: A - SA - [G]n- SB - B, wherein: A is an aptamer specific for one or more epitopes of a target molecule or protein, ranging from 5 to 150 nucleotides in length; G is a guanine-rich sequence that forms a guanine tetrad (G- tetrad) with a corresponding G of associating monomeric aptamer subunits to form the G- quadruplex; SA is a spacer molecule that separates A from G at a distance of up to 20.5 nm; B is an aptamer that is linked to G at an end opposite to A; and SB is a spacer molecule that separates B from G at a distance of up to 20.5 nm.

[0018] In accordance with another aspect of the invention, there is provided a pharmaceutical composition comprising a multivalent aptamer comprising a G-quadruplex comprised of four monomeric aptamer subunits, each monomeric aptamer subunit having the general formula: A - SA - [G]n- SB - B, wherein: A is an aptamer specific for one or more epitopes of a target molecule or protein, ranging from 5 to 150 nucleotides in length; G is a guanine-rich sequence that forms a guanine tetrad (G-tetrad) with a corresponding G of associating monomeric aptamer subunits to form the G-quadruplex; SA is a spacermolecule that separates A from G at a distance of up to 20.5 nm; B is a functional molecule selected from an aptamer, a reporter molecule, or a crosslinker that is linked to G at an end opposite to A; and SB is a spacer molecule that separates B from G at a distance of up to 20.5 nm; and one or more pharmaceutically acceptable excipients.

[0019] In accordance with another aspect of the invention, there is provided a use of a multivalent aptamer comprising a G-quadruplex comprised of four monomeric aptamer subunits, each monomeric aptamer subunit having the general formula: A - SA - [G]n- SB - B, wherein: A is an aptamer specific for one or more epitopes of a target molecule or protein, ranging from 5 to 150 nucleotides in length; G is a guanine-rich sequence that forms a guanine tetrad (G-tetrad) with a corresponding G of associating monomeric aptamer subunits to form the G-quadruplex; SA is a spacer molecule that separates A from G at a distance of up to 20.5 nm; B is a functional molecule selected from an aptamer, a reporter molecule, or a crosslinker that is linked to G at an end opposite to A; and SB is a spacer molecule that separates B from G at a distance of up to 20.5 nm; in the preparation of a medicament for the neutralization of a disease-causing target in a subject.

[0020] In accordance with another aspect of the invention, there is provided a biosensor comprising a multivalent aptamer comprised of four monomeric aptamer subunits, each monomeric aptamer subunit having the general formula: A - SA - [G]n- SB - B, wherein: A is an aptamer specific for one or more epitopes of a target molecule or protein, ranging from 5 to 150 nucleotides in length; G is a guanine-rich sequence that forms a guanine tetrad (G- tetrad) with a corresponding G of associating monomeric aptamer subunits to form the G- quadruplex; SA is a spacer molecule that separates A from G at a distance of up to 20.5 nm; B is a functional molecule selected from an aptamer, a reporter molecule, or a crosslinker that is linked to G at an end opposite to A; and SB is a spacer molecule that separates B from G at a distance of up to 20.5 nm; immobilized on and / or in a material.

[0021] In accordance with another aspect of the invention, there is provided a method for detecting the presence of a target molecule in a sample, the method comprising: a. contacting the sample with a multivalent aptamer comprising a G-quadruplex comprised of fourmonomeric aptamer subunits, each monomeric aptamer subunit having the general formula: A - SA- [G]n- SB - B, wherein: A is an aptamer specific for one or more epitopes of a target molecule or protein, ranging from 5 to 150 nucleotides in length; G is a guanine-rich sequence that forms a guanine tetrad (G-tetrad) with a corresponding G of associating monomeric aptamer subunits to form the G-quadruplex; SA is a spacer molecule that separates A from G at a distance of up to 20.5 nm; B is a functional molecule selected from an aptamer, a reporter molecule, or a crosslinker that is linked to G at an end opposite to A; and SB is a spacer molecule that separates B from G at a distance of up to 20.5 nm, wherein the multivalent aptamer binds the target molecule; and b. detecting the binding of the multivalent aptamer with the target molecule.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.

[0023] Figure 1 shows an illustration of the formation of G-quadruplex tetrameric aptamers (GTA).

[0024] Figure 2 shows an analysis of formation of tetramers wherein (A) shows agarose gel analysis of the formation of G-quadruplex tetrameric aptamers using GTA1, GTA2 and GTA3 sequences with different length of linkers, G4 and MSA52 sequences were also added to show the G4 tetramer and monomeric aptamer on gels; and (B) shows Native-page gel analysis of the formation of G4.

[0025] Figure 3 shows the analysis of the tetrameric aptamer structures with circular dichroic (CD) spectroscopy. Specifically, CD spectra of the Ctrl, G4, GTA2, GTA4 and GTA5 are shown. The G4 sequence forming parallel quadruplex tetramer (the sign of CD at -240 nm is negative, the sign of CD at -260 nm is positive) and the control sequence unable to form quadruplex were used as controls. The GTA2, GTA4 and GTA5 tetramer exhibited a negative sign at -243 nm, which is consistent with the results of G4. The positive signs ofGTA2, GTA4 and GTA5 exhibit a varying degree of shift (5—14 nm), may be attributing to different aptamers (MSA52 or / and Apt23) being plugged.

[0026] Figure 4 shows an assessment of the binding affinity of GTA2 tetramer against the BA5, XBB and BF.7 variant S-proteins of SARS-CoV-2 using dot blot assays wherein (A) shows Dot blot results and (B) shows binding curves used to derive the Kd values.

[0027] Figure 5 shows an assessment of the binding affinity of GTA2 tetramer, TMSA52 trimer and MSA52 monomer aptamers for S-protein of SARS-CoV-2 using dot blot assays. (A) Representative dot blot results and (B) Binding curves used to derive the Kd values.

[0028] Figure 6 shows a cooperativity assessment of binding S-protein by GTA2 using antisense sequence (AS) of MSA52.

[0029] Figure 7 shows a BLI kinetic analysis of GTA2 binding with the BA5 variant of the S-protein of SARS-CoV-2. Kd values consistent with the dot blot results, kon an koff indicated the association and dissociation rates.

[0030] Figure 8 shows a BLI kinetic analysis of the MSA52 monomer binding with the BA5 variant of the S-protein of SARS-CoV-2. Kd values consistent with the dot blot results, kon an koff indicated the association and dissociation rates.

[0031] Figure 9 shows a BLI kinetic analysis of the TMSA52 trimeric aptamer binding with the BA5 variant of the S-protein of SARS-CoV-2. Kd values consistent with the dot blot results, kon an koff indicated the association and dissociation rates.

[0032] Figure 10 shows the stability assessment of the GTA2 aptamer stored under various conditions for 1, 4, 7, 14, 21, 28, 35, 42, 49 and 60 days wherein (A) shows GTA2 solution stored at -20C, (B) freeze dried GTA2 at room temperature, (C) rotary-dried GTA2 at room temperature and (D) GTA2 in solution stored at room temperature.

[0033] Figure 11 shows the stability of (A) GTA2 and (B) MSA52 in a 25% pooled saliva (COV Pool 5 (25%, negative)) at different incubation times (0, 5, 10, 20, 30, 60, 90 and 120 min). The results were obtained by 10% d-PAGE gel (35 W, 60 min).

[0034] Figure 12 shows percent neutralization of SARS-CoV-2 Omicron BA.1 by trimeric (TMSA52) and tetrameric (GTA2) aptamer constructs in vitro on Vero E6 cells at 3 days post-infection to comparatively assess the aptamers ability to neutralize SARS-CoV-2 in vitro.DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention provides multivalent aptamer constructs having a tetrameric or octameric formation, and their use as molecular recognition elements (MREs) that can be used for different bioanalytical and therapeutic applications.

[0036] According to embodiments, the multivalent aptamer comprises a G-quadruplex from which individual DNA or RNA aptamers are oriented in parallel alignment for the purposes of increasing target binding affinity and avidity via the arrangement of the homomeric or heteromeric aptamers with controlled orientation (5'-3' or 3'-5') and spacing from the G-quadruplex molecule to bind homomeric or heteromeric epitopes on a target.

[0037] According to embodiments, the multivalent aptamer comprises four monomeric aptamer subunits that associate to form a G-quadruplex construct. Each monomeric aptamer subunit possesses the general structure A - SA- [G]n- SB - B, wherein association between the guanine-rich sequence ([G]n) of each monomeric subunit results in the formation of a G- tetrad, in the G-quadruplex construct, with each individual aptamer forming an aptameric arm. In such embodiments, the G-quadruplex construct allows each aptameric arm (A) to be oriented in parallel alignment to form a tetrameric aptamer. In other embodiments, the monomeric aptamer subunit can include an additional aptamer (B) at an opposing end of the guanine-rich sequence ([G]n). In such embodiments, formation of the G-quadruplex construct allows the additional aptamers (Bs) to be oriented in parallel alignment oppositefrom the As to form an octameric aptamer. In this way, the multivalent aptamer of the present invention provides versatility.

[0038] According to such embodiments, the present invention provides high-binding affinity multi-armed aptamers that allow for unimpeded folding of individual aptamer subunits, optimizable direction, and alignment of arms that produces a high concentration, reducing dissociation off-rate. According to embodiments, the high concentration of spatially aligned aptamer subunits ensures target binding by at least one of the individual aptamer subunits (A) at each of the aptameric arms. In certain embodiments, detachment of an individual aptamer subunit can be replaced by one or more of the cooperating aptamer subunits (A) of the multivalent construct. In this way, the target is bound by at least one aptamer unit (A) of the multivalent construct at any given time. In some embodiments, the target is bound by two aptamer units (A) of the multivalent construct. In further embodiments, the target is bound by three aptamer units (A) of the multivalent construct.

[0039] Some embodiments of the invention provide for enhanced affinity and avidity. According to such embodiments, the multivalent aptamer of the present invention orients the individual homomeric or heteromeric aptamer subunits to enhance the avidity of the binding interaction by simultaneously binding multiple epitopes of a target. In certain embodiments, the multivalent G-quadruplex construct design uses homomeric aptamer subunits to bind targets with a single epitope. In other embodiments, multiple identical epitopes or heteromeric aptamers are presented on the arms of the G-quadruplex construct to bind targets with different epitopes.

[0040] Other embodiments of the invention provide for enhanced folding consistency. As compared to concatemers (consecutive aptamer units on a single strand), for example, multivalent aptamers of the present invention may be more likely to fold consistently due to each aptamer element being immobilized at a single terminal. In contrast, internal aptamers of concatemers may be more restricted in folding due to the additional bulk of aptamer units up and downstream.

[0041] Further embodiments of the present invention provide for orientation control. Given that aptamers and their targets are known to bind in a preferred relative orientation, multivalent aptamer constructs of the present invention allow for the attachment of homomeric or heteromeric aptamer units in either the 5' -3' or 3' -5' orientation relative to the G-tetrad formed by the associating G-rich sequences ([G]n). This allows for the aptamer subunits to be arranged in an optimal orientation for epitope binding.

[0042] In other embodiments, the present invention provides for optimizable aptamer spacing. In such embodiments, the spacing of the aptamer from the target epitope can be optimized by the length of the guanine-rich sequence ([G]n) in each monomeric subunit. The length of the guanine-rich sequence ([G]n) determines G-tetrad formation and the consequent length of the G-quadruplex. In this way, G-quadruplex lengths can be designed to allow sufficient reach such that each aptamer subunit can reach its respective epitope on the target, without being excessively long such that aptamer folding is inhibited or steric inhibition is observed. As well, for applications where binding clusters of epitopes on a target is desired, G-quadruplex lengths can be restricted to limit binding to a local area (i.e. a single target molecule on a cell surface with multiple targets).

[0043] According to further embodiments, the present invention provides multipurpose attachment sites on the surface of the G-quadruplex opposite to the surface to which the aptamer subunits (As) are attached. In such embodiments, these attachment sites may be functionalized with a variety of other molecules to enable i) surface immobilization or crosslinking via biotin, amine, thiol or digoxigenin molecules, ii) attachment of reporter elements such as fluorophores, enzymes (HRP), nucleic acid amplification primers (RCA, PCR, LAMP, etc.) or iii) drug delivery such as small molecule drugs, antimicrobial agents, gene therapeutics.Definitions

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0045] 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, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0046] Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

[0047] As used in this disclosure, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

[0048] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0049] The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0050] The term “target”, “analyte” or “target analyte” as used herein refer to any agent, including, but not limited to, a small inorganic molecule, small organic molecule, metal ion, biomolecule, toxin, biopolymer (such as a nucleic acid, carbohydrate, lipid, peptide, protein), cell, tissue, microorganism and virus, for which one would like to sense or detect. The analyte can be either isolated from a natural source or synthetic. The analyte can be a single compound or a class of compounds, such as a class of compounds that share structural or functional features. The term analyte also includes combinations (e.g. mixtures) of compounds or agents such as, but not limited, to combinatorial libraries and samples from an organism or a natural environment.

[0051] The term “subject” as used herein includes all members of the animal kingdom including mammals such as a mouse, a rat, a dog and a human.

[0052] The term “nucleic acid” as used herein refers to a biopolymer comprising monomers of nucleotides, 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 contain one or more modified bases (e.g. unusual bases such as inosine, and functional modifications to the bases such as amino), modified backbones (e.g. peptide nucleic acid, PNA) and / or other chemically, enzymatically, or metabolically modified forms.

[0053] The term “aptamer” as used herein refers to a short, chemically synthesized nucleic acid molecule or oligonucleotide sequence which can be generated by in vitro selection tofold into specific three-dimensional structures that bind to a specific analyte with dissociation constants, for example, in the pico- to nano-molar range. Aptamers can be single- stranded DNA, RNA, modified nucleotides and / or nucleotide derivatives. Aptamers can also be naturally occurring RNA aptamers termed “riboswitches”. Functional aptamer sequences can also be rationally designed, truncated, conjugated or otherwise modified from original parent (or full length) sequences. A functional fragment of an aptamer is the portion of an aptamer that retains aptameric function, for example, function in binding to molecules such as protein, lipid, carbohydrate, and nucleic acid. A functional variant of an aptamer refers to an aptamer that has been modified, with nucleotide derivates or otherwise, elongated or truncated, and still retains aptameric function.

[0054] The term “hybridizes”, “hybridized” or “hybridization” as used herein refers to the sequence specific non-covalent binding interaction with a complementary, or partially complementary, nucleic acid sequence. When, for example, the 5 '-end region of an aptamer hybridizes to the 3 '-end region, it can form a duplex DNA element.

[0055] The term “affinity” as used herein refers to an interaction between one aptamer domain with its binding site that may be assessed by corresponding dissociation constant KD. The term “avidity” as used herein refers to the overall strength of multiple binding interactions and can be described by the KD of the completely associated aptamer-target complex.

[0056] The term "G-tetrad", or “guanine tetrad”, as used herein, refers to the hydrogen bonded association of four guanine bases to form a square planar structure. Stacking of at least two G-tetrads, and further stabilized by a central metal ion, is referred to herein by the term “G-quadruplex”.

[0057] The term “arm(s)” and “branch(es)” are used interchangeably herein when referring to the aptameric arms or branches extending from the G-quadruplex formed by the association of monomeric aptamer subunits.

[0058] The terms “attenuate”, “inhibit”, “prevent”, “treat”, and grammatical variations thereof, as used herein, refer to a measurable decrease in a given parameter or event.

[0059] The term “treatment method”, or “method for the treatment of a pathology or disorder”, means therapy aimed at restoring the health condition of a subject, maintaining the existing condition and / or preventing the worsening of said health condition.MULTIVALENT TETRAMERIC OR OCTAMERIC APTAMERS

[0060] Multivalent aptamers are provided that have a tetrameric or octameric structure. The multivalent aptamers comprise a G-quadruplex construct from which individual DNA or RNA aptamers are oriented in parallel alignment as aptameric arms extending from the G-quadruplex. The G-quadruplex construct self-assembles from four monomeric aptamer subunits that possess the general formula A - SA - [G]n- SB - B in which four linear aptameric strands (A) are coupled to their respective G-rich sequence ([G]n) by a spacer molecule (SA).

[0061] According to embodiments, the G-rich sequence ([G]n) of each monomeric aptamer subunit comprises 1, 2, 3, 4, 5, 6, or 7 guanine bases. In particular embodiments, the G-rich sequence ([G]n) of each monomeric aptamer subunit comprises 2 or more guanine bases. In other embodiments, the G-rich sequence ([G]n) of each monomeric aptamer subunit comprises 2 to 7 guanine bases.

[0062] According to further embodiments, the monomeric aptamer subunit comprises an additional aptamer (B) at an opposing end of the guanine-rich sequence ([G]n) to the aptameric sequence (A). In such embodiments, self-assembly of such monomeric aptamer subunits to form the G-quadruplex construct results in the orientation of the additional aptamers (Bs) in parallel alignment opposite from the As to form an octameric aptamer. In certain embodiments, the monomeric aptamer subunit comprises another aptamer (B) that may be identical or different to the ‘A’ aptamer element.Aptamer Elements (A) and / or (B)

[0063] According to embodiments, the individual ‘A’ and / or ‘B’ units or elements are aptamers specific for one or more epitopes of a target molecule or protein. Aptamer elements (A and or B) suitable for the multivalent aptamer construct, may be aptamers specific for epitopes of a target molecule or protein associated with a disease or disorder. A wide variety of aptamers that are specific to such target molecules or proteins are known in the art. Appropriate aptamers can be readily selected by one skilled in the art based on, for example, the desired end use of the multivalent aptamer, such as the disease or disorder against which it is to be directed and / or the subject to which it is to be administered.

[0064] For example, the aptamer can be specific for a target molecule or protein associated with a disease or disorder in an animal, such as a cancer, infectious disease, allergic reaction, or autoimmune disease. The aptamer may be specific for one or more epitopes associated with a pathogen known in the art, such as, for example, a bacterium, virus, protozoan, fungus, parasite, or infectious particle, such as a prion, or it may be a tumour-associated epitope or other biomarker.

[0065] According to embodiments, aptamers specific for one or more epitopes associated with viral targets include, for example, aptamers specific for epitopes associated with members of the families Adenoviradae; Arenaviridae (for example, Ippy virus and Lassa virus); Birnaviridae; Bunyaviridae; Caliciviridae; Coronaviridae; Filoviridae; Flaviviridae (for example, yellow fever virus, dengue fever virus and hepatitis C virus); Hepadnaviradae (for example, hepatitis B virus); Herpesviradae (for example, human herpes simplex virus 1); Orthomyxoviridae (for example, influenza virus A, B and C); Paramyxoviridae (for example, mumps virus, measles virus and respiratory syncytial virus); Picomaviridae (for example, poliovirus and hepatitis A virus); Poxyiridae; Reoviridae; Retroviradae (for example, BLV-HTLV retrovirus, HIV-1, HIV-2, bovine immunodeficiency virus and feline immunodeficiency virus); Rhabodoviridae (for example, rabies virus), and Togaviridae (for example, rubella virus).

[0066] In one embodiment, the multivalent aptamer comprises one or more aptamers (A and / or B) specific for the target molecule or protein associated with a major viral pathogen such as the dengue virus, various hepatitis viruses, human immunodeficiency virus (HIV), various influenza viruses, West Nile virus, respiratory syncytial virus, influenza virus, rabies virus, human papilloma virus (HPV), Epstein Barr virus (EBV), polyoma virus, or SARS coronavirus.

[0067] According to certain embodiments, the multivalent aptamer comprises one or more aptamers (A and / or B) specific for the target molecule SARS-CoV-2 S protein. In further embodiments, the multivalent aptamer comprises one or more MSA52 aptamers. In other embodiments, the multivalent aptamer comprises one or more aptamers (A and / or B) specific for the target molecule Influenza HA protein. In further embodiments, the multivalent aptamer comprises one or more RHA06 aptamers.

[0068] According to embodiments, aptamers specific for one or more epitopes associated with bacterial targets include, for example, aptamers specific for epitopes associated with known causative agents responsible for diseases such as diptheria, pertussis, tetanus, tuberculosis, bacterial pneumonia, fungal pneumonia, cholera, typhoid, plague, shigellosis, salmonellosis, Legionnaire's disease, lyme disease, leprosy, malaria, hookworm, onchocerciasis, schistosomiasis, trypamasomialsis, lehmaniasis, giardia, amoebiasis, filariasis, borrelia, and trichinosis.

[0069] According to embodiments, aptamers specific for one or more epitopes associated with tumour-associated targets include, for example, aptamers specific for epitopes associated with Her2 (breast cancer); GD2 (neuroblastoma); EGF-R (malignant glioblastoma); CEA (medullary thyroid cancer); CD52 (leukemia); human melanoma protein gplOO; human melanoma protein melan-A / MART-1; NA17-A nt protein; p53 protein; various MAGEs (melanoma associated antigen E), including MAGE 1, MAGE 2, MAGE 3 (HLA-A1 peptide) and MAGE 4; various tyrosinases (HLA-A2 peptide); mutant ras; p97 melanoma antigen; Ras peptide and p53 peptide associated with advanced cancers; the HPV 16 / 18 and E6ZE7 antigens associated with cervical cancers; MUC1-KLH antigenassociated with breast carcinoma; CEA (carcinoembryonic antigen) associated with colorectal cancer, DKK-1 (Dickkopf-1 protein) associated with lung cancer and the PSA antigen associated with prostate cancer.

[0070] According to other embodiments, the multivalent trident aptamer comprises one or more aptamers (A and / or B) specific for a biomarker that is predictive, prognostic, or diagnostic of a clinical condition.

[0071] According to certain embodiments, the multivalent aptamer comprises one or more aptamers (A and / or B) specific for the target molecule SARS-CoV-2 spike protein, Influenza hemagglutinin (HA) and neuraminidase (NA) proteins, Alkaline Phosphatase, other VEGF family proteins (VEGF121, VEGF145, VEGF-A, PGF), Platelet-derived growth factor (PDGF), norovirus capsid protein (VP1).

[0072] In certain embodiments, the aptamer (A and / or B) element may be up to 150 nucleotides in length. In other embodiments, the aptamer (A and / or B) element may be from 5 to 150 nucleotides in length. In further embodiments, the aptamer (A and / or B) element may be 10 to 100 nucleotides in length. In other embodiments the aptamer (A and / or B) element may be 15 to 90 nucleotides in length. In further embodiments, the aptamer (A and / or B) element may be 20 to 90 nucleotides in length.

[0073] In some embodiments, the multivalent aptamer is a homomeric aptamer construct where each of the aptameric arms / branches comprise an identical aptamer element (A and / or B). According to other embodiments, the multivalent aptamer is a heteromeric aptamer construct where two of the four or eight aptameric arms / branches comprise an identical aptamer element (A and / or B). In a further embodiment, the multivalent aptamer is a heteromeric aptamer construct where each of the aptameric arms / branches comprise an aptamer element (A and / or B) that is specific to a different epitope of a target molecule or protein.

[0074] In some embodiments, the one or more aptamer element (A and / or B) is modified to be more resistant to denaturation according to methods known in the art. For example,aptamer elements (A and / or B) may be modified to include a locked nucleic acid (LNA), 2’- O-methyl nucleotides, or 2’ -fluoro-deoxyribonucleotides. In certain embodiments, the one or more aptamer element (A and / or B) is modified to comprise a locked nucleic acid (LNA), 2’-O-methyl nucleotides, or 2’ -fluoro-deoxyribonucleotides.Spacer s / Linkers CS[A,B] ’)

[0075] Each aptamer element (A and / or B), according to embodiments, will interact with target epitopes via non-covalent forces (i.e. hydrogen bonding, hydrophobic interactions and electrostatic interactions) based on the unique three-dimensional structure of the aptamer element (A and / or B). Alternatively, binding elements may interact with target proteins via sequence specific nucleic acid binding properties of the target protein.

[0076] According to embodiments, the spacing of the aptamer element (A and / or B) from the target epitope is optimized to allow sufficient reach such that each aptamer (A and / or B) can reach its respective epitope on the target, without being excessively long such that aptamer folding is inhibited or steric inhibition is observed. In certain embodiments, for example, binding clusters of epitopes on a target is desired, and in such embodiments, the spacing of the aptamer (A and / or B) is restricted to limit binding to a local area (i.e. a single target molecule on a cell surface with multiple targets). According to further embodiments, the spacer molecule (SA and / or SB) affords flexibility to allow for a fluctuation of aptamer (A and / or B) distance to cover the distance ranges of the target molecule.

[0077] In certain embodiments, the SA and / or SB is a spacer molecule that separates an aptamer (A and / or B) element from the respective terminal end of the G-rich sequence at a distance of up to 25 nm. In other embodiments, the spacer (SA and / or SB) has a span of up to 25 nm, 20.5 nm, 18 nm, 15 nm, 12 nm, 10 nm, 8 nm, 5 nm, or 0 nm. In other embodiments, the spacer (SA and / or SB) has a span of 5 nm to 25 nm, 10 nm to 20.5 nm, or 10 nm to 18 nm. In some embodiments, the spacer SA and / or SB has the same span length for each aptamer (A and / or B) on its respective terminal end of the G-rich sequence. In otherembodiments, the span length of each spacer SA and / or SB is different for each aptamer (A and / or B).

[0078] According to embodiments, the spacer (SA and / or SB) is a linear carbon molecule. In such embodiments, the linear carbon molecule comprises up to 15 linear alkane chains each possessing up to 12 carbon atoms. In other embodiments, the spacer (SA and / or SB) is a linear polyethylene glycol chain possessing up to 55 ethylene glycol units. In further embodiments, the spacer (SA and / or SB) is an unstructured flexible single stranded nucleic acid sequence of up to 30 deoxythymidine or other bases. In other embodiments, the spacer (SA and / or SB) is an unstructured flexible single stranded nucleic acid sequence of 10 to 30, 15 to 30, 20 to 30, or 25 to 30 deoxythymidine or other bases.

[0079] Persons of skill in the art will readily appreciate the spacers that can be used. For example, in certain embodiments, the spacer (SA and / or (the spacer (SB) is Spacer Phosphoramidite 18 (https: / / www.glenresearch.com / spacer-modifiers / 10-1918.html) or Spacer C12 CE Phosphoramidite (https: / / www.glenresearch.com / spacer-modifiers / 10- 1928.html).Multipurpose Attachment Site

[0080] According to embodiments, the multivalent aptamer construct provides a multipurpose attachment site that may be functionalized with a variety of other molecules to enable i) surface immobilization or crosslinking via biotin, amine, thiol or digoxigenin molecules, ii) attachment of reporter elements such as fluorophores, enzymes (HRP), nucleic acid amplification primers (RCA, PCR, LAMP, etc.) or iii) drug delivery such as small molecule drugs, antimicrobial agents, gene therapeutics.

[0081] In certain embodiments, the monomeric aptamer subunits comprise a functional molecule at an opposing end of the guanine-rich sequence ([G]n) to the aptameric sequence (A). In such embodiments, self-assembly of such monomeric aptamer subunits to form the G-quadruplex construct results in a tetrameric aptamer construct comprising functional molecules coupled to the G-quadruplex surface opposite from the As. According toembodiments, the G-quadruplex is attached to a functional molecule (B) selected from a reporter molecule, or a crosslinker. In certain embodiments, the functional molecule (B) is a reporter molecule selected from an antigen, an enzyme, and a fluorescent molecule. In further embodiments, the functional molecule (B) is a crosslinker selected from thiol, amide, biotin, digoxigenein, azide, alkyne, carboxyl, and a click-chemistry-based crosslinker.BINDING AFFINITY & A VIDITY

[0082] In various embodiments, the multivalent aptamer described herein, and / or compositions or formulations comprising these multivalent aptamers, exhibit enhanced affinity and / or avidity for a target molecule. According to certain embodiments, the multivalent aptamers described herein exhibit a synergistic binding affinity and / or avidity to a target. In particular, it was unexpectedly found that the multivalent aptamers described herein exhibit an enhanced or synergistic affinity / avidity for a target molecule of up to 537.5- fold over its corresponding monomeric form. The unexpectedly enhanced affinity / avidity of the tetrameric aptamer was observed in its binding with target molecules of various forms, including monomeric, dimeric, and trimeric protein targets. Moreover, the G-quadruplex construct displayed synergistic cooperativity between each branch in binding the target molecule.

[0083] In certain embodiments, the multivalent aptamers described herein cooperatively bind to a trimeric protein target. In other embodiments, the multivalent aptamers described herein cooperatively bind to a dimeric protein target. In further embodiments, the multivalent aptamers described herein cooperatively bind to a monomeric protein target.

[0084] According to certain embodiments, the multivalent tetrameric / octameric aptamers described herein exhibit up to a 537.5-fold improvement in affinity / avidity with its target molecule over the affinity of the corresponding monomeric aptamer (A and / or B). In other embodiments, the multivalent tetrameric / octameric aptamers described herein exhibit up to a 425-fold improvement in affinity / avidity with its target molecule over the affinity of the corresponding monomeric aptamer (A and / or B). In further embodiments, the multivalenttetrameric / octameric aptamers described herein exhibit up to a 500-fold improvement in affinity / avidity with its target molecule over the affinity of the corresponding monomeric aptamer (A and / or B). According to other embodiments, the multivalent tetrameric / octameric aptamers described herein exhibit up to a 450-fold improvement in affinity / avidity with its target molecule over the affinity of the corresponding monomeric aptamer (A and / or B). According to other embodiments, the multivalent tetrameric / octameric aptamers described herein exhibit up to a 400-fold improvement in affinity / avidity with its target molecule over the affinity of the corresponding monomeric aptamer (A and / or B). According to certain embodiments, the multivalent tetrameric / octameric aptamers described herein exhibit up to a 300-fold improvement in affinity / avidity with its target molecule over the affinity of the corresponding monomeric aptamer (A and / or B).METHODSAND USES

[0085] The enhanced, cooperative binding affinity of the multivalent aptamer constructs described herein offers a range of applications. Moreover, the multivalent aptamer construct provides a multipurpose attachment site on the G-quadruplex structure ([SB-B]) to allow for versatile functionalization with a variety of other molecules to enable i) surface immobilization or crosslinking via biotin, amine, thiol or digoxigenin molecules, for example, ii) attachment of reporter elements such as fluorophores, enzymes (HRP), nucleic acid amplification primers (RCA, PCR, LAMP, etc.) or iii) drug delivery such as small molecule drugs, antimicrobial agents, gene therapeutics.

[0086] In certain embodiments, the branch-quadruplex can comprise another aptamer (‘B’) that may be identical or different to the ‘A’ aptamer element. Such embodiments, may provide additional multivalency for cross-reactivity of the multivalent aptamer. In futher embodiments, for example for reporter constructs, the ‘B’ element may possess a fluorescent reporter molecule, antigen or enzyme. In other embodiments, for example for immobilization constructs, the ‘B’ element may possess functional molecules to enablecrosslinking such as thiol, amide, biotin, digoxigenin, azide, alkyne, carboxyl or crosslinkers compatible with Click-Chemistry methodologies.Methods of Treatment

[0087] The multivalent aptamers of the present disclosure are useful in a variety of applications including, but not limited to, methods for the treatment of a disorder or disease. In particular, the multivalent aptamers described herein provide a method for targeted drug delivery to one or more targets, comprising administering to a cell or a subject multivalent aptamers of the invention that exhibits a therapeutic effect, or that is loaded with a drug, to target the cell or tissue for treatment. In other embodiments, the multivalent aptamers described herein provide a method for targeted binding and neutralization of a pathogen.

[0088] Further provided are pharmaceutical compositions, comprising a multivalent aptamer of the invention and one or more pharmaceutically acceptable excipients. In certain embodiments, the present invention provides for pharmaceutical compositions comprising an effective amount of a multivalent aptamer and one or more pharmaceutically acceptable carriers, diluents and / or excipients. If desired, other active ingredients may be included in the compositions, for example, additional immune stimulating compounds, standard therapeutics, vaccines or the like.

[0089] The pharmaceutical compositions can be formulated for administration by a variety of routes. For example, the compositions can be formulated for oral, topical, rectal, nasal or parenteral administration or for administration by inhalation or spray. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrathecal, intrasternal injection or infusion techniques. Intranasal administration to the subject includes administering the composition to the mucous membranes of the nasal passage or nasal cavity of the subject.

[0090] In some embodiments, the pharmaceutical compositions are formulated for mucosal administration. Mucosal administration may include, for example, oral, intranasal, aerosol, rectal or vaginal administration. The preparations for mucosal administrationinclude transdermal devices, aerosols, creams, lotions or powders pending on the mucosal site. In certain embodiments, the pharmaceutical compositions are formulated for intranasal or pulmonary administration. In some embodiments, the pharmaceutical compositions are formulated for rectal or vaginal administration.

[0091] Compositions formulated as aqueous suspensions may contain the multivalent aptamer in admixture with one or more suitable excipients, for example, with suspending agents, such as sodium carboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, hydroxypropyl -P- cyclodextrin, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethyene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, hepta- decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxy-benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.

[0092] In certain embodiments, the pharmaceutical compositions may be formulated as oily suspensions by suspending the drug-loaded multivalent aptamer in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.

[0093] In certain embodiments, the pharmaceutical compositions may be formulated as a dispersible powder or granules, which can subsequently be used to prepare an aqueous suspension by the addition of water. Such dispersible powders or granules provide themultivalent aptamer in admixture with one or more dispersing or wetting agents, suspending agents and / or preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, colouring agents, can also be included in these compositions.

[0094] Pharmaceutical compositions of the invention may also be formulated as oil-in- water emulsions in some embodiments. The oil phase can be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example, liquid paraffin, or it may be a mixture of these oils. Suitable emulsifying agents for inclusion in these compositions include naturally-occurring gums, for example, gum acacia or gum tragacanth; naturally-occurring phosphatides, for example, soy bean, lecithin; or esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monoleate.

[0095] In certain embodiments, the pharmaceutical compositions may be formulated as a sterile injectable aqueous or oleaginous suspension according to methods known in the art and using suitable one or more dispersing or wetting agents and / or suspending agents, such as those mentioned above. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Acceptable vehicles and solvents that can be employed include, but are not limited to, water, Ringer's solution, lactated Ringer's solution and isotonic sodium chloride solution. Other examples include, sterile, fixed oils, which are conventionally employed as a solvent or suspending medium, and a variety of bland fixed oils including, for example, synthetic mono- or diglycerides. Fatty acids such as oleic acid can also be used in the preparation of injectables.

[0096] Optionally the pharmaceutical compositions may contain preservatives such as antimicrobial agents, anti-oxidants, chelating agents, and inert gases, and / or stabilizers such as a carbohydrate (e.g. sorbitol, mannitol, starch, sucrose, glucose, or dextran), a protein (e.g. albumin or casein), or a protein-containing agent (e.g. bovine serum or skimmed milk)together with a suitable buffer (e.g. phosphate buffer). The pH and exact concentration of the various components of the composition may be adjusted according to well-known parameters.

[0097] Sterile compositions can be prepared for example by incorporating the multivalent aptamer in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile compositions, some exemplary methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0098] Contemplated for use in certain embodiments of the invention are various mechanical devices designed for pulmonary or intranasal delivery of therapeutic products, including but not limited to, nebulizers, metered dose inhalers, powder inhalers and nasal spray devices, all of which are familiar to those skilled in the art.

[0099] Metered dose inhalers typically use a propellant gas and require actuation during inspiration. Dry powder inhalers use breath-actuation of a mixed powder. Nebulizers produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, and the like generate small particle aerosols.

[0100] Some specific examples of commercially available mechanical devices include the ULTRA VENT® nebulizer (Mallinckrodt, Inc., St. Louis, Mo.), the ACORN II® nebulizer (Marquest Medical Products, Englewood, Colo.), the MISTY-NEB® nebulizer (Allegiance, McGraw Park, Ill.), the AEROECLIPSE® nebulizer (Trudell Medical International, Canada), the Accuspray™ nasal spray device (Becton Dickinson), the Mucosal Atomization Device (MAD300) (Wolfe Tory Medical), the OptiNose device (OptiNose, Oslo, Norway), the Nektar DPI system (Nektar Therapeutics, Inc., San Carlos, Calif.), the AERx pulmonarydrug delivery system (Aradigm Corporation, Hayward, Calif.), the Spiros® device (Dura Pharmaceuticals), and the Respimat® device (Boehringer Ingelheim).

[0101] All such devices require the use of formulations suitable for the dispensing of the multivalent aptamer. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and / or carriers useful in therapy as would be understood by a worker skilled in the art. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

[0102] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remington Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, Pa. (2000).Methods of Detection and Diagnosis

[0103] The multivalent aptamers of the present disclosure are useful in a variety of applications including, but not limited to, methods for detecting the presence of a target molecule. In one aspect, the multivalent aptamers are useful for detecting the presence of a target molecule in a biological sample. The term “detecting” as used herein includes quantitative or qualitative detection.

[0104] In one aspect, the present disclosure provides a method of detecting the presence of a target molecule in a biological sample. In certain aspects, the method comprises contacting the biological sample with a multivalent aptamer specific for the target molecule under conditions permissive for binding of the multivalent aptamer and detecting whether a complex is formed between the multivalent aptamer and the target. The biological sample can include, without limitation, urine or blood samples.

[0105] In some embodiments, detection of the target molecule is indicated by a fluorescent, colorimetric, electrochemical, surface plasmon resonance, spectroscopic, or radioactivesignal. In some embodiments, detection of the target molecule is indicated by a fluorescent signal. In some embodiments, an increase in the fluorescence signal indicates presence of the target in the sample.KITS

[0106] In certain aspects of the invention, kits are provided comprising a container housing a composition comprising the multivalent aptamers.Pharmaceutical Kits

[0107] Certain embodiments of the invention provide for pharmaceutical kits comprising multivalent aptamers for use as a therapeutic. Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale. The kit may optionally contain instructions or directions outlining the method of use or administration regimen for the multivalent aptamers.

[0108] When the kit comprises multivalent aptamers for use as a drug delivery system, the kit may further comprise one or more drugs for use in combination with the multivalent aptamers.

[0109] When one or more components of the kit are provided as solutions, for example an aqueous solution, or a sterile aqueous solution, the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the solution may be administered to a subject or applied to and mixed with the other components of the kit.

[0110] The components of the kit may also be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components. Irrespective of the number or type of containers, the kits of the invention also may comprise an instrument for assisting with the administration of the composition to apatient. Such an instrument may be an inhalant, nasal spray device, nebulizer, syringe, pipette, forceps, measured spoon, eye dropper or similar medically approved delivery vehicle.Target Detection Kits

[0111] According to certain embodiments, the multivalent aptamer can be combined into a test kit system. For example, the multivalent aptamer can be combined into a biosensor system or kit for use to detect any suitable target analyte, such as, and without being limited thereto, a wide range of small molecule, protein and nucleic acid analytes, including infection-causing pathogens in point-of-care testing for screening, diagnostics and / or health monitoring.

[0112] In some embodiments, the sample is a biological sample, and the presence of the target in the sample is indicative of, or associated, with a disease, disorder or condition. In some embodiments, the target is a pathogen. Accordingly, provided is a method of detecting a pathogen infection in a subject comprising testing a sample from the subject for the presence of a target using the multivalent aptamer combined into a biosensor, biosensor system and / or kit, wherein presence of a target indicates that the subject has an infection.

[0113] In accordance with another aspect, there is provided a kit for detection of a target in a sample comprising the multivalent aptamer combined into a biosensor or biosensor system and instructions for use.

[0114] To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way.EXAMPLESEXAMPLE 1: PREPARATION OF MULTIVALENT TETRAMERIC ANDOCTAMERIC APTAMERS

[0115]

[0111] Self-assembling G-quadruplex-based tetrameric aptamers (GTAs) were designed by introducing a DNA spacer / linker, such as Poly thymidine (T), along with a G-rich sequence at either the 3' or 5 ’ ends of the aptamer sequence, as illustrated in Figure 1 and Table 1. GTAs were prepared through a simple process of annealing and freezing of individual monomeric aptamers in the presence of metal ions (such as K+and Na+). This results in GTAs with significantly improved binding affinity and stability when compared to their monomeric counterparts, as well as a high yield in production, without DNA modifications.

[0116] Exemplary constructs were prepared. Two aptamers were chosen for experimentation: 1) MSA52, targeting the S-protein of SARS-CoV-2, and 2) Apt23 targeting alkaline phosphatase. These aptamers were extended at their 3'-end with a polyT spacer / linker ranging from 6 to 25 nucleotides in length, and a G-rich sequence (G). Through the annealing and freezing process in the presence of metal ions, GTAs were prepared. Their production yield, binding affinity, and characters were determined.

[0117] In addition to forming tetramers, octameric aptamers were prepared with the capability to bind two distinct targets by linking two monomeric aptamers, MSA52 and Apt23, using appropriate linkers and G-rich sequences. This innovative “Janus” octamer is referred to as GTA5 in Table 1.

[0118] The examples presented showcase the incorporation of G-rich sequences at the 3'- ends of two specific aptamers targeting two particular targets. This methodology is versatile and adaptable, allowing for the creation of various types of GTAs with different configurations. This includes the option to add G-rich sequences at the 5'-end of aptamers, use of other G-rich sequences to form quadruplex, utilize other monomeric aptamers, andtarget a wide range of different targets. The flexibility of this approach opens up numerous possibilities for the development of customized GTAs tailored to specific applications and research objectives.Chemical Synthesis of Multivalent Tetrameric and Octameric Aptamers.

[0119] The synthesis of tetrameric and octameric aptamers was carried out using an automated oligonucleotide synthesizer, specifically the Mermade 12 Synthesizer from Biosearch Technologies. The synthesis process followed the manufacturer's instructions and utilized the recommended reagents and default coupling times, unless specified otherwise by Glen Research. The synthesis followed by the addition and protection of each nucleotide from the 3’ to 5’ end. After the synthesis of the nucleotides according to the sequences outlined in Table 1, lum was placed in a 3.3mM sodium cacodylate (pH 7.2) solution and heated to 90°C for 2 min. Approximately 1.0-100mM of KC1 was added and the temperature was increased to 90°C for an additional 3 min. After thorough mixing, the solution was slowly cooled to room temperature for 4h. The mixture was then kept at 4°C for 12 h. The final step was to store the suspension at -20°C from 1 to 7 days. It was observed that both the temperature and the potassium ions gave rise to the self-assemble of the G-quadruplex formation with high yield.EXAMPLE 2: EXEMPLARY TETRAMERIC APTAMERS FOR BINDING SARS- COV-2 S-PROTEINS.

[0120] To evaluate the versatility of tetrameric aptamers, the SARS-CoV-2 spike (S) protein for the BA5 variant was selected. A universal monomer for SARS-CoV-2 called MSA52 was used and formed into both the trimeric and tetrameric form. The DNA sequences used in the preparation of these aptamers are presented in Table 1.Table 1. Sequences of DNA Oligonucleotides

[0121] A tetrameric aptamer was assembled with the monomeric aptamer subunit GTA2, which comprises the MSA52 aptamer, to target the SARS-CoV-2 S-Protein of the BA5 variant. The design of the tetrameric aptamer was based on the size of the S-Protein which has a diameter of 7 - 14 nm with a spacing of 10 nm between each monomer center. Each aptameric branch of the tetrameric aptamer contains 60 nucleotides which is around 20 nm. Due to the flexibility of the independent arms, the tetrameric aptamer should allow for fluctuations between 0 to 34 nm. This range completely covers the distance range between each subunit of the trimeric S-protein of SARS-CoV-2, providing enough flexibility for the tetrameric aptamer GTA2 to bind to the trimeric S-protein.EXAMPLE 3: VERIFICATION OF TETRAMER FORMATION

[0122] Three different tetrameric aptamers were prepared, GTA1, GTA2, and GT A3 having the sequences indicated in Table 1. The formation and yield of GTA formation of these exemplary aptamers was verified by agarose and PAGE gels.

[0123] 10 pL Ctrl, G4, GTA1, GTA2, GT A3 and annealed MSA52 were mixed with 2 pL 6 x loading dye, respectively, and then loaded into 3% agarose in 1 x TBE buffer. The electrophoresis was run at 70 V for 2 h at room temperature. 10 pL G4 was mixed with 2 pL 6 x loading dye, respectively, and then loaded into 10% native-PAGE gel in 1 x TBE buffer. The electrophoresis was run at 250 V for 4 h at room temperature. After electrophoresis, the PAGE gel was stained by l x SYBR Gold for 30 min. All gels were analyzed with a molecular imager (Typhoon). The gel images are shown in Figure 2.

[0124] Ctrl and MSA52 served as negative controls and are unable to form G-quadruplex structures due to the absence of a G-rich sequence.

[0125] G4 (with only G-rich sequence, without linking aptamers) acts as a positive control, demonstrating the ability to form G-quadruplex structures.

[0126] GTAs (GTA1, GTA2, and GTA3) are derived from the MSA52 aptamer. These exemplary GTAs have been extended with varying lengths of polyT linkers and G-rich sequences at their 3 '-ends. As shown in Figure 2(a) on the agarose gel, these GTAs exhibit the formation of G-quadruplex structures.

[0127] The length of the spacer / linker has impact on the yield of GTAs. Specifically, GTA2, which features a 15-nt polyT linker, achieves the highest yield of 93.8%.

[0128] These results showed the successful formation of G-quadruplex -based GTAs.EXAMPLE 4: THE FORMATION OF AN OCTAMER

[0129] An octameric aptamer was prepared using the same method for preparing the tetrameric aptamer. The octameric aptamer features different monomeric aptamers [A] on one side of the G-rich linkage sequence (G) and on the 3' side (B). The exemplary GTA5 sequence is shown in Table 1.

[0130] GTA5 includes the MSA52 aptamer, followed by a 15 nucleotide poly-T linker, followed by the G-rich section, another 15 nucleotide poly-T linker with a second aptamer selected for its binding of Alkaline Phosphatase (ALP). The synthesis method used for GTA5 was the same for GTA2 that is described above in Example 1.EXAMPLE 5: ANALYSIS OF TETRAMERIC STRUCTURE USING CIRCULAR DICHROIC (CD) SPECTROSCOPY

[0131] Circular dichroism (CD) spectroscopy is a powerful analytical technique used in chemistry, biochemistry, and structural biology to study the chiral properties of molecules. It measures the differential absorption of left- and right-handed circularly polarized light by optically active molecules. Chirality refers to the asymmetry in the spatial arrangement of atoms in a molecule, resulting in non-superimposable mirror images, known as enantiomers. In addition to providing chirality, the CD spectrum provides specific information about DNA formation. The most well known B-form, discovered by Watson and Crick, is a right-handed double helix whose base pairs are perpendicular to the helix. A positive maximum at 290 nm, negative maximum around 245 nm, and a broad positive peak between 260-280 nm are typically seen for B-form structures. The A-form is more compact, right-handed double helix structure that is typically observed for RNA and exhibits a positive peak at 260 nm and a negative peak at 210 nm. The Z-form structure is a left-handed double helix that displays a negative band at 290 nm, a positive peak at 260 nm, and negative maximum -200 nm. G- quadruplex (G4) secondary structures are unique in that they fold into four-strands that are rich in guanine. These quadruplexes can form either parallel or anti-parallel structures depending on the stacking interactions of the guanosine residues. Parallel G4 structures havea positive band at 260 nm while the anti-parallel structures exhibit a negative band at 260 nm and a positive peak at 290 nm.CD Spectroscopy results for Tetramers

[0132] A CD spectrometer was used to analyze four separate G-rich tetrameric structures including a control. The control DNA had very limited absorption, while all G-rich tetramers had strong peaks between 266 and 280 nm (Figure 3). Importantly, G4, GTA2, GTA4, GTA5 all had negative peaks at 243 nm which demonstrates that each tetramer is being formed in the same orientation. Since the G4 peak of 266 is close to the parallel peak that is characteristic for parallel G-quadruplexes, this confirms a parallel structure. The peaks of GTA2, GTA4 and GTA5 are slightly shifted towards higher values which is consistent with the linkage of aptamers with the G4 structure according to literature. Based on this data, the formation of the G-rich linked tetrameric structure is in a parallel formation with each arm aligned in the same direction of the G-rich structure.CD Spectroscopy results for Octamers

[0133] The CD spectroscopy of GTA5 identified a minimum at 245 nm with a maximum peak at 277nm which when compared to the parallel G-quadruplex with a minimum of 243 nm and a maximum of 266nm can be interpreted as a parallel structure that confirms that an octamer has been synthesized with different aptamers on each side of the G-rich structure.

[0134] Figure 3 provides insights into the structural characteristics of the formed GTAs, showing that they possessed parallel G-quadruplex structures. This determination was made through the analysis of CD spectra, which indicates that all four monomeric aptamer DNA strands align parallel to one another on the same side, as illustrated in Figure 1.

[0135] In the CD spectra analysis, the following controls and observations were made:

[0136] 1). Ctrl: This control serves as a negative control and cannot form G-quadruplex structures.

[0137] 2). G4: a positive control, G4 forms a parallel G-quadruplex structure, evident from its CD spectrum and literature reports with a characteristic negative peak at 243nm and a positive peak at 266nm.

[0138] 3). GTA2, GTA4, and GTA5 : These GT As, which consist of G4 linked with different aptamers (MSA52, Apt23, or both), exhibit CD spectra patterns similar to the G4 control. The shift observed at approximately 266 nm in their spectra can be attributed to the linkage of aptamers with the G4 structure.

[0139] These CD spectra results provide evidence that the GT As, including those formed by linking G4 with specific aptamers, maintain a parallel G-quadruplex conformation, supporting the gel results in Figure 2.EXAMPLE 6: ASSESSMENT OF BINDING AFFINITY

[0140] The binding affinity of the synthesized multivalent Aptamers (including monomeric, trimeric and tetrameric aptamers) were assessed using the standard dot blot assays, a technique that has been widely used to determine the affinity of protein binding aptamers. Dot blot assays involve the use of two distinct membranes: nitrocellulose and nylon to separate the bound aptamers with proteins from the unbound aptamers. The aptamers were labeled with32P to generate a radioactive signal, allowing the calculation of the fraction of bound aptamers to proteins. By fitting the fraction of bound aptamers, the dissociation constants (Kd values) of the assessed aptamers can be derived. The Kd values were then used to determine and compare the strength of the binding interaction between the aptamers and target proteins. A lower Kd value indicates a higher affinity, meaning that the binding between aptamers and proteins is stronger and less likely to dissociate.

[0141] Dot Blot Binding Assays. Dot blot assays were performed by using a Whatman Minifold- 1 96-well apparatus and a vacuum pump. Before experiments, nitrocellulose membranes and nylon membranes were incubated in lx binding buffer for 1 h. y-[32P] labelled DNA aptamers (10 pM) were dissolved in the binding buffer and heated at 90 °C for 5 min, and then cooled at room temperature for 20 min. Proteins were dissolved anddiluted in the same buffer. 5 pL of the above aptamer solution was mixed with 15 pL of protein with different concentrations. The mixture was incubated at room temperature for 1 h. The dot blot apparatus was assembled with a nitrocellulose membrane on the top, a nylon membrane in the middle and a wetted Whatman paper in the bottom. After washing each well with 100 pL of binding buffer, the binding mixtures were loaded and drained by the vacuum pump (force: 550 mmHg for 8 seconds). The wells were then washed twice with 100 pL binding buffer. The membranes were imaged using a Typhoon 9200 imager (GE Healthcare) and analyzed using Image J software. Each binding assay was performed 3 times. The bound fraction was quantified and plotted against the concentration of the protein. The Kd values were derived via curve fitting using Origin 8.0 using the equation Y= BmaxX / (Kd + X) (Y is the bound fraction of aptamer with protein, Bmax is the maximum bound fraction of aptamer, and X is protein concentration).

[0142] Radiolabelling of DNA Aptamers. DNA aptamers were labeled with y-[32P] ATP at the 5'-end using PNK reactions according to the manufacturer's protocol. Briefly, 2 pL of 1 pM DNA aptamers were mixed with 2 pL of y-[32P] ATP, 1 pL of 10 x PNK reaction buffer A, 10 U (U: unit) of PNK and 4 pL water. The mixture was incubated at 37 °C for 20 min, and then purified by 10% dPAGE.SARS-CoV-2 S Protein Variants

[0143] The affinity of GTA2 for the variant S-proteins of SARS-CoV-2, omicron BA5, XBB and BF.7 was assessed using dot blot assays. The results of the dot blots are provided in Figure 4A, and the derived Kd values are shown in Figure 4B. GTA2 effectively bound to the three variant S-proteins, with similar Kd values ranging from 12.4 to 15.6 pM, confirming its universal ability to bind variant SARS-CoV-2 S-proteins.

[0144] Based on these results, the binding affinity of the MSA52 monomer, a trident formed trimer called TMSA52 and the tetramer GTA2 were assessed for binding affinity using dot blot assays. The results of the three versions of the same monomer can be found in Figure 5A and the derived Kd values are shown in Figure 5B. The monomer MSA52had a binding affinity of 4.4 nM while the trimer TMSA52 and GTA2 had an affinity of 36.9 and 15.6 respectively. These results confirm that even though TMSA52 has a significant increase in binding affinity over the monomer which might be expected due to the trimer symmetry of the spike protein, that the additional fourth arm of the tetramer increases further. In addition, it should be noted that low pM binding affinity is reaching the dot blot lower limits of detection. It is worth noting that the monomeric aptamer MSA52 is a universal aptamer known for its ability to bind to a wide range of variants of the SARS-CoV- 2 S-protein. Consequently, GTA2 also exhibited a universally high affinity toward all three tested variants.EXAMPLE 7: COOPERATIVE BINDING BY TETRAMERIC APTAMERS

[0145] Cooperative cross-binding of target proteins, with all four branches of the tetrameric aptamer was assessed. We introduced the antisense (AS) DNA of each branch of the tetrameric aptamers to block the binding between the branch and proteins. This blockade could result in an increase in the Kd value due to reduced affinity caused by the antisense DNA. We tested GTA2, that was synthesized using the above-described method, to bind its respective target protein. The samples were analyzed by native polyacrylamide gel electrophoresis (nPAGE).

[0146] Figure 6 shows the functional roles of the four arms within GTA2 for binding to the target. The results revealed a gradual decrease in the binding activity of GTA2 as the AS was added. This led to a corresponding increase in Kd values:At a GTA2 : AS ratio of 1 :0, the Kd was 0.015 nM.At a GTA2 : AS ratio of 1 : 1, the Kd increased to 0.042 nM.At a GTA2 : AS ratio of 1 :2, the Kd further increased to 0.95 nM.At a GTA2 : AS ratio of 1 :3, the Kd reached 4.6 nM.At a GTA2 : AS ratio of 1 :4, no binding observed (Kd > 50nM)

[0147] These findings suggest that the four arms of GTA2 function cooperatively in binding to the S-protein. GTA2 may engage in cross-binding with S-proteins.EXAMPLE 8: KINETIC ANALYSIS OF TETRAMERIC APTAMER BINDING

[0148] Optimization through addition of poly-thymine (poly T) linkers was performed to determine the optimal distance from the G-quadruplex to the functional end of the aptamer arms as it affects target binding. Binding affinity was optimized when an intermediate length 15-T linker was installed adjacent to the G-quadruplex - the resulting tetrameric aptamer was named GTA2.

[0149] Following the confirmation of affinity enhancement in these tetrameric aptamers, the factors contributing to the improved affinity was studied compared to the monomeric aptamers. The Bio-layer Interferometry (BLI) method was used to assess and compare the kinetic rates of the tetrameric and monomeric aptamers. The results and derived kinetic values are presented in Figures 7, 8, and 9.

[0150] Bio-layer Interferometry (BLI) tests. The binding of tetrameric aptamers binding with protein was determined using the BLI Octet RED96. Octet® High Precision Streptavidin (SAX) Biosensors (Lot number: 2303010111, Sartorius AG) were first incubated in binding buffer for 10 min. Subsequently, the biosensors were loaded with biotin-labeled monomeric and trident aptamers by immersing them in 100 nM aptamer solutions. For the binding experiment, the BA5 S-protein concentration was set at 200 nM. Basic Kinetics mode in the Octet Data Acquisition software was employed to define sample positions and assay steps, ensuring a significant decrease in signal during the dissociation phase, with a 15-minute duration for specific target affinity. Following the necessary procedures, including sensor ligation and reference signal subtraction, the signals were aligned to the baseline. Finally, a 1 : 1 binding model was fitted to the data. This streamlined approach effectively enabled the use of the Octet 96 instrument for protein binding assays involving biotin-labeled aptamers.Results

[0151] The monomeric, trimeric and tetrameric form of the MSA52 aptamer’s dissociation rates were assessed using BLI. Each form displayed a similar association rate (kon). The monomeric aptamer displayed a kon of 6.31 * 105M’1s'1in a first study and 6.49* 105M’1s’1in a second study, the trimeric aptamer displayed a kon of 3.12>< 106M’1s’1, and the tetrameric aptamer displayed a kon of 6.67* 105M’1s’1. This indicates that the monomeric aptamer was capable of binding to the protein as rapidly as the trident and tetrameric aptamer. The tetrameric aptamer had the slowest association rate which is likely caused by the larger molecular weight. However, the monomeric aptamer exhibited a significantly higher dissociation rate (koff = 4.30 x 10’4s’1and 5.59 10’4s’1), than the trident aptamer (koff = 7.72 x 10’5s’1) and the tetramer (koff = 1.07 x 10’6s’1). Surprisingly, the tetramer has a dissociation rate that is 402 fold smaller than the monomer and 72-fold smaller than the trimer. These results suggest that the presence of four arms in the tetramer increases the concentration of available binding aptamer units to the tetrameric spike protein which results in a much slower dissociation.

[0152] By calculating the Kd values from the BLI results using Kd = koff / kon, we determined that the tetrameric aptamer had a Kd value of 1.6 pM, which corresponds to an approximately 425-fold to 537.5-fold higher affinity than the monomeric aptamer with a Kd of 0.680 nM and 0.860 nM, respectively. The tetramer also has a 15.5-fold higher Kd than the trimer with a Kd of 24.8 pM.

[0153] While GTA2 produced marginally slower association kinetics likely due to its size, a significant decrease in dissociation kinetics was likely the product of multiple simultaneous associations of each GTA2 molecule with targets, resulting in an overall ~540-fold decrease in Kd, indicating unexpectedly strong binding between the tetramer and its target. In conclusion, the significantly enhanced affinity exhibited by the trident aptamers can be attributed to their reduced dissociation rates (Table 2).Table 2. Comparative BLI kinetic analysis of the MSA52, TMASA52, and GTA2 binding with the BA5 variant of the S-protein of SARS-CoV-2.6.49x10s+ 5.59X10'4+MSA52 860 + 407.82X1034.78x10s3.12x10s± 7.72x105±TMSA52 24.8 + 1.23.93X1043.81x10s6.67x10s+ 1.07x10s+GTA2 1.6 + 0.264.59X1041.54x10sMSA52 / TMSA52 34.7 0.21 7.24MSA52 / GTA2 537.5 0.97 522.43 EXAMPLE 9: PREPARATION OF HOMOTRIMERIC APTAMER TMSA52

[0154] The homotrimeric aptamer for the trimeric spike protein of SARS-CoV-2, that was used in the comparative studies discussed above, was prepared as follows. The aptamer used was MSA52, a monomeric DNA aptamer MSA52 (Table 3) discovered through selection with variant S proteins. MSA52 was found to universally recognize variants that were not analyzed in the original selection experiment, demonstrating that the aptamer is insensitive to emerging S protein mutations. Hence, MSA52 is an ideal candidate for COVID-19 recognition, and the trimerization of this MRE should only enhance its complementarity to the S protein.

[0155] With the use of a 15-thymine linker and DNA synthesizer, the branched structural scaffold of a trebler was harnessed to synthesize a DNA molecule containing three identical MSA52 sequences, which is named TMSA52 (Table 3). Analysis with 10% denaturing polyacrylamide gel electrophoresis (dPAGE) of chemically synthesized TMSA52 showed that TMSA52 was synthesized successfully in reference to monomeric and dimeric MSA52 sequences (Table 3).Materials and Reagents

[0156] DNA oligonucleotides listed in Table 3 were obtained from Yale University or Integrated DNA Technologies and purified using 10% denaturing polyacrylamide gel electrophoresis (dPAGE) containing 8 M urea. Sodium borohydride (NaBE , 98%), sodium hexachloroiridate (III) hydrate (NasIrCE • xEEO, M.W. = 473.9), potassium phosphate monobasic (KH2PO4, >99%), sodium phosphate dibasic (Na2HPC>4, >99%), potassium chloride (KC1, >99%), sodium chloride (NaCl, >99.5%), 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES, >99%), magnesium chloride (MgCE, >99%), acetic acid (HO Ac, >99.7%), 3,3 ',5,5' -tetramethylbenzidine (TMB, > 99%), sodium acetate (NaOAc, > 99%), sulfuric acid (H2SO4, 95-98%), hydrogen peroxide solution (30% H2O2), dimethylformamide (DMF), streptavidin (Cat. No. SA101), bovine serum albumin (BSA, Cat. No. A7906), amylase (Cat. No. A1031), human IgG (Cat. No. 14506) and Tween-20 were all obtained from Sigma-Aldrich. The spike proteins of B.l.1.7 (Cat. No. SPN-C52H6), B.1.617.2 (Cat. No. SPN-C52He), B.1.617.1 (Cat. No. SPN-C52Hr), and B.1.1.529 (Cat. No. SPN-C52Hz) SARS-CoV-2 variants expressed in human 293 cells (HEK293) were obtained from Aero Biosystems. The spike proteins of B.1.351 (Cat. No. 510333-1), B.1.429 (Cat. No. 101057) and P.l (Cat. No. 100989-1) SARS-CoV-2 variants expressed in human 293 cells (HEK293) were obtained from BPS Biosciences Inc. The spike proteins for wild-type SARS- CoV-2 and SARS-CoV-1, the spike protein RBD of seasonal coronavirus 229E and OC43, the control lentiviruses, the pseudotyped lentiviruses expressing the spike proteins of wild-type, B.1.351, and P.l SARS-CoV-2 were obtained from Dr. Matthew Miller’s lab at McMaster University. The pseudotyped lentiviruses expressing the spike proteins of B.l.1.7 (Cat. No. 78112-1), B.1.617.1 (Cat. No. 78205-1), B.1.429 (Cat. No. 78172-1), B.l.617.2 (Cat. No. 78216-1), and B.l.1.529 (Cat. No. 78348-1) SARS-CoV-2 were purchased from BPS Bioscience. Nitrocellulose membranes (Cat. No. 10600125) were from GE Healthcare Inc. Nylon membranes (Cat. No. NEF994001PK) were obtained from PerkinElmer Inc. The pooled human saliva (Lot 31887) was from Innovative Research Inc (Novi, Michigan). T4 polynucleotide kinase (PNK) with lOx buffer was acquired from Thermo Scientific (Ottawa, Canada). [y-32P]-ATP was purchased from PerkinElmer. 96-well microtiter plates (clear,polystyrene, flat bottom) were from Celltreat Inc. Ultrapure water (Milli-Q System, Millipore) was used to prepare all aqueous solutions.Table 3. Synthetic DNA oligonucleotides used in this research. All sequences are written in a 5' to 3' direction. Italic T segments act as linkers.EXAMPLE 10: STABILITY ASSESSMENT OF TETRAMERIC APTAMERS

[0157] The prepared GTA2 was evenly divided into four parts. (A) One was kept in the refrigerator at -20°C. (B) One was freeze-dried and stored at room temperature. (C) One was rotary-dried and stored at room temperature. (D) One serving in solution was kept at room temperature.

[0158] The content (%) of GTA2 tetramers were determined by agarose gel on days 1, 4, 7, 14, 28, 35, 42, 49 and 60.

[0159] The results in Figure 10 demonstrates the remarkable stability of GTA2 following long-period storages under various conditions, including drying, storage at room temperature (RT), and -20 ° C. Even after 60 days of storage, GTA2 retained a content of >80%, showcasing its excellent stability.EXAMPLE 11: STABILITY IN SALIVA

[0160] The GTA2 and MSA52 with the concentration of 1.0 pM was incubated in 25% saliva for different times (0, 5, 20, 20 ,30, 60,90 and 120 min). The samples were heated at 90°C for 5 min and then loaded into 10% d-PAGE gel in 1 * TBE buffer. The electrophoresis was run at 35 W for 1 h at room temperature. After electrophoresis, the gels were stained by 1 x SYBR Gold for 30 min and then analyzed with a molecular imager (Typhoon).

[0161] Figure 11 showed the stability comparison between GTA2 and MSA52 monomer under human saliva conditions. The level of degradation of GTA2 after 30 minutes of incubation was similar to level of degradation of MSA52 in 5 minutes. This indicates the protective role of the G-quadruplex structure in preserving the aptamer from degradation.EXAMPLE 12: IN VITRO ASSESSMENT OF APTAMERS IN NEUTRALIZING SARS-COV-2

[0162] Neutralization efficacy of aptamers was measured in vitro on Vero E6 cells at 3 days postinfection to assess the aptamers ability to neutralize SARS-CoV-2.In Vitro Neutralization Assay

[0163] Briefly, serial dilutions of TMSA52 (IpM) and GTA2 (IpM) were incubated with the virus, and the mixture was plated 5 on Vero E6 cells for 3 days prior to measuring the cell viability. Vero E6 cells (ATCC CRL-1586) were seeded at a density of 1.5x104 cells / well in white flat-bottom TC-treated 96-well plates (Coming, 3917) and incubated at 37°C, 5% CO2. On the day of seeding, at the end of the day, the medium was replenishedwith fresh DMEM fortified with 2% FBS, 1% Penicillin-Streptomycin, 1% HEPES (pH=7.3), and 1% Glutamax. The cells were then further 5 incubated for 24 hours.

[0164] Aptamer dilutions were incubated with the ancestral strain and Omicron Variant (Lineage BA.l) of 10 SARS-CoV-2 (330 plaque-forming units (PFU) / well) for 1 hour at 37°C, 5% CO2. Following this incubation, the mixture was transferred onto the Vero E6 cells and re-incubated for 1 hour under the same conditions. The mixture was then replaced with identical dilutions of the aptamers and incubated for a further 72 hours at 37°C, 5% CO2.

[0165] Post-incubation, cell viability was ascertained using the CellTiter-Glo 2.0 Luminescent Cell Viability Assay Kit 15 (Promega), where luminescence intensity was directly proportional to the number of viable cells. Luminescence was quantified using a BioTek Synergy Hl microplate reader, and neutralization titer was determined as the highest aptamer / antibody dilution that achieved a 50% reduction in luminescence compared to the virus control wells.Discussion:

[0166] By utilizing a well-established microneutralization (MNT) assay, we assessed our aptamers in neutralizing SARS-CoV-2. At the same dilution factor (32x), the trimeric aptamer TMSA52 showed -25% neutralization of SARS-CoV-2 in Vero E6 cells, while the tetrameric GTA2 showed -55% neutralization. This amounts to an increase by a factor of 2.23 in neutralization potential for the Omicron BA. l variant of SARS-CoV-2 between trimeric and tetrameric applications of MSA52 (Figure 12). This increased performance may stem from GTA2 making more viral associations per unit, due to its geometry, as well as increased size and valency.

[0167] The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference.

[0168] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVEPROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A multivalent aptamer comprising a G-quadruplex comprised of four monomeric aptamer subunits, each monomeric aptamer subunit having the general formula:A - SA- [G]n- SB - B, wherein:A is an aptamer specific for one or more epitopes of a target molecule or protein, ranging from 5 to 150 nucleotides in length;G is a guanine-rich sequence that forms a guanine tetrad (G-tetrad) with a corresponding G of associating monomeric aptamer subunits to form the G- quadruplex;SA is a spacer molecule that separates A from G at a distance of up to 20.5 nm;B is a functional molecule selected from an aptamer, a reporter molecule, or a crosslinker that is linked to G at an end opposite to A; andSB is a spacer molecule that separates B from G at a distance of up to 20.5 nm.

2. The multivalent aptamer of claim 1, wherein n comprises 2 or more guanine bases.

3. The multivalent aptamer of claim 1, wherein n comprises 2 to 7 guanine bases.

4. The multivalent aptamer of claim 1, wherein the G-tetrad is stabilized by a cation situated in a central channel between the associating monomeric aptamer subunits.

5. The multivalent aptamer of claim 4, wherein the cation is a sodium or potassium cation.

6. The multivalent aptamer of claim 1, wherein the G-quadruplex is configured with the As oriented in parallel alignment to form a tetrameric aptamer.

7. The multivalent aptamer of claim 1, wherein B is an aptamer, and wherein the G- quadruplex is configured with the Bs oriented in parallel alignment opposite from the As to form an octameric aptamer.

8. The multivalent aptamer of claim 1, wherein SA comprises a single stranded nucleic acid sequence of 0 to 30 nucleotides.

9. The multivalent aptamer of claim 8, wherein SA is a single stranded thymine-rich sequence of 12 nucleotides.

10. The multivalent aptamer of claim 1, wherein SA comprises up to 15 linear alkane chains each comprising up to 12 carbon atoms.

11. The multivalent aptamer of claim 1, wherein SA comprises a linear polyethylene glycol chain of up to 55 ethylene glycol units.

12. The multivalent aptamer of claim 1, wherein B is an aptamer identical to A.

13. The multivalent aptamer of claim 1, wherein B is an aptamer different than A14. The multivalent aptamer of claim 1, wherein B is a reporter molecule selected from an antigen, an enzyme, and a fluorescent molecule.

15. The multivalent aptamer of claim 1, wherein B is a crosslinker selected from thiol, amide, biotin, digoxigenein, azide, alkyne, carboxyl, and a click-chemistry-based crosslinker.

16. The multivalent aptamer of claim 1, wherein SB comprises a single stranded nucleic acid sequence of 0 to 30 nucleotides.

17. The multivalent aptamer of claim 16, wherein SB is a single stranded thymine sequence of 5 nucleotides.

18. The multivalent aptamer of claim 1, wherein SB comprises up to 15 linear alkane chains each comprising up to 12 carbon atoms.

19. The multivalent aptamer of claim 1, wherein SB comprises a linear polyethylene glycol chain of up to 55 ethylene glycol units.

20. The multivalent aptamer of claim 1, wherein the aptamer is a homomeric aptamer comprising identical As.

21. The multivalent aptamer of claim 1, wherein the aptamer is a heteromeric aptamer comprising 2 or more As that are each specific to a different epitope of a target molecule or protein.

22. The multivalent aptamer of claim 1, wherein [G]nis linked to the 3’ end of A.

23. The multivalent aptamer of claim 1, wherein [G]nis linked to the 5’ end of A.

24. The multivalent aptamer of claim 1, wherein the As are in the same 5’ to 3’ orientation relative to G.

25. The multivalent aptamer of claim 1, wherein the As are in the same 3’ to 5’ orientation relative to G.

26. The multivalent aptamer of claim 1, wherein A is in a range of 20 to 90 nucleotides in length.

27. The multivalent aptamer of claim 1, wherein the target molecule is SARS-CoV-2 S- protein.

28. The multivalent aptamer of claim 27, wherein A is MSA52.

29. A tetrameric aptamer comprising the multivalent aptamer of claim 1.

30. An octameric aptamer comprising the multivalent aptamer of claim 1.

31. A pharmaceutical composition comprising the multivalent aptamer of claim 1 and one or more pharmaceutically acceptable excipients.

32. Use of the multivalent aptamer of claim 1 in the preparation of a medicament for the neutralization of a disease-causing target in a subject.

33. A biosensor comprising the multivalent aptamer of claim 1 immobilized on and / or in a material.

34. A method for detecting the presence of a target molecule in a sample, the method comprising: a. Contacting the sample with the multivalent aptamer of claim 1, wherein the multivalent aptamer binds the target molecule; and b. Detecting the binding of the multivalent aptamer with the target molecule.

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