Aptamer sequences and uses thereof

Abstract

Provided are vancomycin-sensitive aptamers having defined sequences. The aptamers may be attached to the working electrode of an electrochemical sensor used to detect vancomycin in vivo (for example by contacting the interstitial fluid or blood), or ex vivo (such as in an in vitro diagnostics application).

Description

[0001] APTAMER SEQUENCES AND USES THEREOF

[0002] FIELD

[0003]

[0001] . The present disclosure relates generally to nucleotide sequences in the form of aptamers useful in the detection of analytes. More particularly, the disclosure relates to aptamer sequences for use with electrochemical sensors for the detection of the antibiotic vancomycin. Provided are aptamers per se, electrodes and sensors utilising the aptamers, and also methods for the detection of vancomycin.

[0004] BACKGROUND

[0005]

[0002] . Vancomycin is a potent antibiotic primarily used in medical and veterinary settings to treat bacterial infections. The drug is mainly efficacious against Gram-positive bacteria, and particularly strains that are resistant to other antibiotics.

[0006]

[0003] . Vancomycin is often reserved for treating serious infections caused by Grampositive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). These bacteria are notorious for their resistance to many commonly used antibiotics. Particular applications are to treat infections acquired in healthcare settings, where antibiotic-resistant strains of bacteria may be more prevalent.

[0007]

[0004] . This antibiotic is frequently used in the treatment of infective endocarditis, an infection of the heart valves or lining of the heart chambers. Another use is in combination with other antibiotics to treat bacterial meningitis, an infection of the membranes surrounding the brain and spinal cord.

[0008]

[0005] . In cases of osteomyelitis (bone infection) or septic arthritis (joint infection), vancomycin can be prescribed to target the responsible bacteria.

[0009]

[0006] . Vancomycin is sometimes administered before certain surgical procedures to prevent postoperative infections, particularly in cases where there is a higher risk of encountering antibiotic -resistant bacteria.

[0010]

[0007] . In situations where the specific bacteria causing an infection are unknown, vancomycin may be used empirically until laboratory results can identify the pathogen and guide more targeted therapy.

[0008] . While vancomycin is a powerful antibiotic, its use is not without potential risks for the subject. Of major concern is the potential for nephrotoxicity at higher plasma concentrations. At the same time, plasma concentration must be maintained above the minimum inhibitory concentration for efficacy.

[0011]

[0009] . Monitoring vancomycin concentration in the body and adjusting dosage to maintain the plasma concentrations within a therapeutic window are major challenges for a clinician. A typical approach includes drawing blood from the subject at regular time intervals, and assaying the drug in a laboratory. Upon receipt of the assay results, the clinician adjusts dosage accordingly. This approach is suboptimal because the clinician relies on assay data that is likely at least several hours old.

[0012]

[0010] . Recently, real-time detection of vancomycin has been proposed using electrochemical aptamer-based (EAB) sensors. Such sensors may contact a bodily fluid such as blood or interstitial fluid, with the aptamer component specifically interacting with any vancomycin present.

[0013] [Oi l]. An EAB sensor typically comprises a working electrode being coated in a binding element (such as an aptamer) that undergoes a conformational change upon analyte binding. A redox reporter (such as methylene blue) may be covalently linked to the binding element. The conformational change in the binding element alters the accessibility of the redox reporter to the electrode surface, thereby producing an analyte-induced change in the level of electron transport between the redox reporter and the electrode. In some circumstances, binding of the analyte brings the redox reporter proximal to the electrode surface, thereby increasing the level of electron transport and in turn increasing current through the electrode. In other circumstances, binding moves the redox reporter distal to the electrode surface resulting in the opposite effects. Regardless, binding of the analyte results in a detectable change in electrode current.

[0014]

[0012] . EAB sensors are typically interrogated by the application of an electrical potential across the working electrode and a counter electrode, and then measuring current flow after the potential is removed or changed.

[0015]

[0013] . A challenge in the use of vancomycin-sensitive EAB sensors is the need to provide a sensor with a sufficient dynamic range to cover an expected concentration range. In some instances, it may be necessary to use two or more aptamer species to cover the extended concentration range. This problem exists whether the aptamer is used in an EAB sensor, or in any other sensor format such as localised surface plasmon resonance (LSPR), surface plasmon resonance (SPR), enzyme-linked aptamer-antibody sandwich (ELAAS), optical, or colorimetric-based platforms.

[0016]

[0014] . Aptamers are usually selected from a combinatorial library having a vast number (up to 1015) of different oligonucleotides. Selection of an aptamer that is selective for a given analyte may be facilitated by a process known as SELEX (systematic evolution of ligands by exponential enrichment). While these prior art approaches may be effective, they do not allow for the identification of aptamers having a certain affinity (such as a certain dissociation constant, Kd) required to be operable across a certain vancomycin concentration range. At best, a candidate aptamer is identified, and then further testing is required to determine Kd.

[0017]

[0015] . Attempts to rationally design, screen, or modify known aptamers are hampered by the lack of any information in the prior art as to which bases of an aptamer are important in interacting with the vancomycin molecule.

[0018]

[0016] . In any event, alternatives to prior art vancomycin-sensitive aptamers are sought.

[0019]

[0017] . It is an aspect of the present disclosure to provide an improvement to prior art vancomycin-sensitive aptamers. It is a further aspect of the present disclosure to provide a useful alternative to prior art vancomycin-sensitive aptamers.

[0020]

[0018] . The discussion of documents, acts, materials, devices, articles, and the like is included in this specification solely for the purpose of providing a context for the present disclosure. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0021] SUMMARY

[0022]

[0019] . In a first aspect, but not necessarily the broadest aspect, there is provided an aptamer capable of interacting specifically with a target analyte, the aptamer having a sequence comprising any one or more of: SEQ ID NO: 1 or a variant thereof, or SEQ ID NO:2 or a variant thereof; and / or, SEQ ID NO:3 or a variant thereof, or SEQ ID NO:4 or a variant thereof.

[0020] . In one embodiment of the first aspect, the sequence is not any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.

[0023]

[0021] . In one embodiment of the first aspect, the variant of SEQ ID NO: 1 or SEQ ID NO:2, and / or the variant of SEQ ID NO:3 or SEQ ID NO:4, comprises a variation in 1 or 2 bases of the respective sequence.

[0024]

[0022] . In one embodiment of the first aspect, the variation in 1 or 2 bases is independently selected from: substitution with a natural or a non-natural base, insertion of a natural base or a non-natural base, and deletion of a base.

[0025]

[0023] . In one embodiment of the first aspect, a majority of the bases of SEQ ID NO:1 or variant thereof or SEQ ID NO:2 or variant thereof is not involved in base -pairing with other bases of the aptamer.

[0026]

[0024] . In one embodiment of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof are not involved in base -pairing with other bases of the aptamer.

[0027]

[0025] . In one embodiment of the first aspect, a minority of the bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof is involved in base-pairing with other bases of the aptamer.

[0028]

[0026] . In one embodiment of the first aspect, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof are involved in base-pairing with other bases of the aptamer.

[0029]

[0027] . In one embodiment of the first aspect, no base pairs are formed between any two bases of SEQ ID NO:1 or variant thereof or SEQ ID NO:2 or variant thereof.

[0030]

[0028] . In one embodiment of the first aspect, a majority of the bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof is not involved in base-pairing with other bases of the aptamer.

[0031]

[0029] . In one embodiment of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, or 9 bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof are not involved in base-pairing with other bases of the aptamer.

[0032]

[0030] . In one embodiment of the first aspect, a minority of the bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof is involved in base-pairing with other bases of the aptamer.

[0031] . In one embodiment of the first aspect, no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof are involved in basepairing with other bases of the aptamer.

[0033]

[0032] . In one embodiment of the first aspect, no base pairs are formed between any two bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof.

[0034]

[0033] . In one embodiment of the first aspect, the aptamer comprises a first terminal region which is linked to an electrode surface and a second terminal region which is linked to a redox reporter when the aptamer is incorporated as a sensing element in a electrochemical sensor, and SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof is located closer to the first terminal region than to the second terminal region.

[0035]

[0034] . In one embodiment of the first aspect, the aptamer comprises a first terminal region which is linked to an electrode surface and a second terminal region which is linked to a redox reporter when the aptamer is incorporated as a sensing element in a electrochemical sensor, and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof is located closer to the second terminal region than to the first terminal region.

[0036]

[0035] . In one embodiment of the first aspect, the aptamer has a 5’ terminus and a 3’ terminus, and SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof is located closer to the 5’ terminus than the 3’ terminus.

[0037]

[0036] . In one embodiment of the first aspect, the aptamer has a terminal base, and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases are disposed between the terminal base and SEQ ID NO:1 or variant thereof, or SEQ ID NO:2 or variant thereof, or SEQ ID NO:3 or variant thereof, or SEQ ID NO:4 or variant thereof.

[0038]

[0037] . In one embodiment of the first aspect, the aptamer has a 5’ terminus and a 3’ terminus, and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof is located closer to the 3’ terminus than the 5’ terminus.

[0039]

[0038] . In one embodiment of the first aspect, the aptamer has a terminal base, and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases are disposed between the terminal base and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof.

[0040]

[0039] . In one embodiment of the first aspect, the aptamer comprises SEQ ID NO: 1 or SEQ ID NO:2; and SEQ ID NO:3 or SEQ ID NO:4.

[0040] . In one embodiment of the first aspect, the aptamer comprises a spacer disposed between SEQ ID NO:1 or variant thereof or SEQ ID NO:2 or variant thereof, and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof.

[0041]

[0041] . In one embodiment of the first aspect, the spacer comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0042]

[0042] . In one embodiment of the first aspect, the spacer, alone or in combination with other bases of the aptamer, forms a hairpin structure, a stem structure, a loop structure, or a stem / loop structure.

[0043]

[0043] . In one embodiment of the first aspect, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof, and / or 1, 2, 3, 4, 5, 6, 7, 8, or 9 bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof contribute to the hairpin structure, a stem structure, a loop structure, or a stem / loop structure.

[0044]

[0044] . In one embodiment of the first aspect, the aptamer has base-pairing between bases of SEQ ID NO: 1 or variant thereof, or SEQ ID NO:2 or variant thereof; and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof.

[0045]

[0045] . In one embodiment of the first aspect, the base-pairing occurs exclusively or predominantly between cytosine and guanine bases.

[0046]

[0046] . In one embodiment of the first aspect, the aptamer comprises a redox reporter covalently attached to any point along the length of the aptamer, and a linking element at one terminus configured to facilitate attachment of the aptamer to an electrode.

[0047]

[0047] . In one embodiment of the first aspect, the aptamer comprises at or toward a terminus a redox reporter, and at the other terminus a linking element configured to facilitate attachment of the aptamer to an electrode.

[0048]

[0048] . In one embodiment of the first aspect, the redox reporter is methylene blue or a functional equivalent thereof.

[0049]

[0049] . In one embodiment of the first aspect, the target analyte is an organic molecule.

[0050]

[0050] . In one embodiment of the first aspect, the organic molecule is a glycopeptide antibiotic.

[0051]

[0051] . In one embodiment of the first aspect, the glycopeptide antibiotic is vancomycin.

[0052] . In one embodiment of the first aspect, the aptamer comprises any one of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO:130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141, or a variant thereof.

[0052]

[0053] . In one embodiment of the first aspect, the variant thereof has a sequence identity of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the reference sequence.

[0053]

[0054] . In one embodiment of the first aspect, the aptamer comprises a reporter, a tag, or another modification used to identify the extent to which a target analyte interacts with the aptamer.

[0054]

[0055] . In a second aspect, there is provided a sensor for the detection of vancomycin or another glycopeptide antibiotic comprising an aptamer of any embodiment of the first aspect.

[0055]

[0056] . In one embodiment of the second aspect, the sensor is an electrochemical sensor comprising a working electrode having an aptamer of any embodiment of the first aspect associated therewith, or linked thereto.

[0056]

[0057] . In one embodiment of the second aspect, the working electrode is capable of penetrating the skin of a mammal such that the aptamer contacts a biological fluid of the mammal.

[0057]

[0058] . In one embodiment of the second aspect, the working electrode is a wire, a needle, or a microneedle.

[0058]

[0059] . In one embodiment of the second aspect, the sensor further comprises a counter electrode, and optionally a reference electrode.

[0059]

[0060] . In a third aspect, there is provided a method of producing an aptamer for the detection of vancomycin or another glycopeptide antibiotic, the method comprising the step of incorporating SEQ ID NO: 1 or a variant thereof, or SEQ ID NO:2 or a variant thereof; and / or SEQ ID NO:3 or a variant thereof, or SEQ ID NO:4 or a variant thereof, in a complete aptamer sequence.

[0060]

[0061] . In one embodiment of the third aspect, the method is not for producing an aptamer according to any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, or SEQ ID NO: 10, or a product of the method is not an aptamer according to any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10.

[0061]

[0062] . In a fourth aspect, there is provided a method for identifying an aptamer for the detection of vancomycin or another glycopeptide antibiotic, the method comprising providing a library of candidate aptamers, selecting and / or enriching for aptamers capable of binding to vancomycin or another glycopeptide antibiotic, wherein most or all of the candidate aptamers comprise SEQ ID NO: 1 or a variant thereof, or SEQ ID NO:2 or a variant thereof; and / or SEQ ID NO:3 or a variant thereof, or SEQ ID NO:4 or a variant thereof.

[0062]

[0063] . In one embodiment of the fourth aspect, at least 10% of the candidate aptamers comprise SEQ ID NO: 1 or a variant thereof, or SEQ ID NO:2 or a variant thereof; and / or SEQ ID NO:3 or a variant thereof, or SEQ ID NO:4 or a variant thereof.

[0063]

[0064] . In one embodiment of the fourth aspect, the method comprises multiple rounds of selecting and enriching for aptamers capable of binding to vancomycin or another glycopeptide antibiotic.

[0064]

[0065] . In one embodiment of the fourth aspect, the method is an augmented SELEX (systematic evolution of ligands by exponential enrichment) method.

[0065]

[0066] . In a fifth aspect, there is provided an aptamer identified by the method of any embodiment of the fourth aspect.

[0066]

[0067] . In a sixth aspect, there is provided a method for detecting and / or quantitating vancomycin or another glycopeptide antibiotic, the method comprising the steps of contacting a test fluid or a biological fluid in vivo with an aptamer of any embodiment of the first aspect, or the apparatus of any embodiment of the fifth aspect.

[0067]

[0068] . In one embodiment of the sixth aspect, the aptamer is a component of the sensor of any one embodiment of the second aspect.

[0068] BRIEF DESCRIPTION OF THE FIGURES

[0069]

[0069] . FIG. 1 is a flow diagram illustrating experimental work leading to the identification of vancomycin-binding motifs in DNA aptamers.

[0070]

[0070] . FIG. 2, FIG. 3, FIG. 4, and FIG. 5, each illustrate an aptamer and predicted secondary structure, and the effect of various modifications on the affinity of the aptamer for vancomycin. The secondary structure is that predicted at 35 °C, and at approximately physiological ionic strength. The free energy of the structure was -1.92 kcal / mol. The aptamer is predicted to assume the secondary structure drawn for the temperature range 35°C to 61°C.

[0071]

[0071] . FIG. 6 is a graphical representation of relative kD values for some of the better performing vancomycin sensitive aptamers.

[0072] . FIG. 7A through 7E illustrate various parameters of vancomycin sensitive aptamer SEQ ID NO: 100.

[0072]

[0073] . Unless otherwise indicated herein, features of the drawings labelled with the same numeral are taken to be the same features, or at least functionally similar features, when used across different drawings.

[0073]

[0074] . The drawings are not prepared to any particular scale or dimension and are not presented as being a completely accurate presentation of the various embodiments.

[0074] DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

[0075]

[0075] . After considering this description it will be apparent to one skilled in the art how the disclosure is implemented in various alternative embodiments and alternative applications. However, although various embodiments of the present disclosure will be described herein, it is understood that these embodiments are presented by way of example only, and not limitation. As such, this description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure. Furthermore, statements of advantages or other aspects apply to specific exemplary embodiments, and not necessarily to all embodiments, or indeed any embodiment covered by the claims.

[0076]

[0076] . Throughout the description and the claims of this specification the word “comprise” and variations of the word, such as “comprising” and “comprises” is not intended to exclude other additives, components, integers, or steps.

[0077]

[0077] . Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may.

[0078]

[0078] . As used herein, a “biological fluid” may be any biological fluid of a subject, including but not limited to, interstitial fluid (ISF), blood, saliva, a lacrimal secretion, a lactational secretion, a nasal secretion, a tracheal secretion, a bronchial secretion, an alveolar secretion, a gastric secretion, a gastric content, a glandular secretion, a vaginal secretion, a uterine secretion, a prostate secretion, semen, urine, sweat, cerebrospinal fluid, a glomerular filtrate, an hepatic secretion, bile, or an exudate, any of which are contacted in use with a working electrode.

[0079]

[0079] . The present disclosure is predicated at least in part on the discovery of certain nucleotide sequence motifs, and the disposition of motifs in regard to the overall aptamer structure (both primary and secondary) that may be critical, important, or preferred in a vancomycin-sensitive aptamer. With that information, it will be possible to more rationally design, screen, or modify, aptamers for a given application. For example, where an existing aptamer requires modification to increase binding affinity, insertion of a nucleotide sequence known to interact with vancomycin may be introduced. Alternatively, the bases of an existing motif may be mutated so as to more closely accord with a motif known to be important. Where a decrease in binding affinity is required, modification of an existing motif known to strongly interact with vancomycin may be pursued.

[0080]

[0080] . The motifs identified may be used in an augmented SELEX method to identify further vancomycin-sensitive aptamers. This SELEX method utilises a library that preserves the motifs and having randomised other regions of the aptamer.

[0081]

[0081] . The motifs disclosed herein are described primarily by way of nucleotide base sequences of a DNA molecule. Included within the scope of the present disclosure are variants of those DNA molecules.

[0082]

[0082] . A variant may include a non-natural nucleic acid molecule, being a compound which is analogous (structurally similar) to naturally occurring RNA and DNA, used in medicine and in molecular biology research. Nucleic acids are polymers of nucleotide monomers, each of which is composed of three parts: a phosphate backbone, a pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analogue may have any of these elements altered. Typically, analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases (A, T, G and C), and phosphate-sugar backbone analogues such as PNA, which affect the properties of the chain (PNA can even form a triple helix). Non-natural nucleic acids are also called Xeno Nucleic Acids (XNA).

[0083]

[0083] . A non-natural nucleic acid may derive from a naturally occurring nucleic acid, but having had an alteration to its chemical structure such that the chemical structure is considered non-natural. More typically, the non-natural nucleic acid will be synthesised de novo in an altered form.

[0084]

[0084] . The term “non-natural nucleic acid” is not intended to include a nucleic acid that has been synthesised by, or with the assistance of man, but nevertheless having a natural chemical structure. While such molecules are not the product of nature, they nevertheless have the same chemical structure as a nucleic acid found in nature.

[0085]

[0085] . A non-natural nucleic acid molecule useful in the context of the present disclosure (for example, in a working electrode of an EAB sensor) may be an altered form of an aptamer retaining the ability to detect the analyte, and optionally resist degradation by an agent in a biological fluid.

[0086]

[0086] . Non -limiting examples of non-natural backbone oligomers include, but are not limited to, 2' -fluoroarabinoside nucleic acid (FANA), 2'-O-methyl RNA, locked nucleic acid (LNA), unlocked nucleic acid (UNA), and threose nucleic acid (TNA). Because they are not produced in nature, XNAs are generally highly resistant to enzymatic degradation. In some embodiments, the non-natural nucleic acid may be a peptide nucleic acid (PNA).

[0087]

[0087] . Non-natural nucleic acids include also, morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA), and hexitol nucleic acids (HNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. Aptamers comprising the so-called “fifth letter” 7-(2-thienyl)imidazo[4,5- b]pyridine (Ds) are included, to possibly improve the affinity of any aptamer to a target analyte.

[0088]

[0088] . Mirror-image aptamers made from chirally inverted nucleic acids are also included.

[0089]

[0089] . The DNA aptamer sequences disclosed herein are presented running 5’ to 3’. A variant includes the same sequence, but running 3’ to 5’ . As a simple example, the sequence 5’-CTAG-3’ is considered a variant of the sequence 3’-CTAG-5’.

[0090]

[0090] . An aptamer of the present disclosure capable of interacting with vancomycin may comprise a first motif as follows:

[0091]

[0091] , SEQ ID NO: 1 GAGGGTACC.

[0092]

[0092] . As an alternative to SEQ ID NO: 1, the first motif may be as follows:

[0093]

[0093] , SEQ ID NO:2 CGAGGGTACCGC.

[0094] . An aptamer of the present disclosure capable of interacting with vancomycin may comprise a second motif as follows:

[0094]

[0095] , SEQ ID NOG TGGGTCG.

[0095]

[0096] . As an alternative to SEQ ID NOG, the second motif may be as follows:

[0096]

[0097] , SEQ ID NO:4 GTGGGTCGG.

[0097]

[0098] . Preferably both first and second motifs are present in the aptamer, however in some embodiments only the first or the second motif is present.

[0098]

[0099] . The first and second motifs have been identified by sequence analysis of aptamers known to interact with vancomycin, and the testing various aptamer sequences for the ability to interact with vancomycin as detailed in the Examples herein.

[0099]

[0100] . In some embodiments of the disclosure, the aptamer is not an aptamer defined by any of the following sequences:

[0101] . The present aptamers may assume certain secondary structures, including any one or more of a hairpin, stem, loop, or stem / loop. Any secondary structure assumed may be reliant on the temperature of the aptamer’s environment. Where secondary structures are referred to or drawn herein, those structures are predicted for the range 35 °C to 61 °C. That temperature range encompasses normal mammalian body temperature, and therefore the structures may be applicable to uses of the aptamers when contacting a bodily fluid of an animal in situ, such as will be the case for applications of real-time vancomycin monitoring.

[0100]

[0102] . Certain complete vancomycin-sensitive aptamers have been found, as follows:

[0101]

[0103] . The complete aptamers listed above may, in respect of any motif contained herein (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) comprise any of the variants detailed above in relation to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0102]

[0104] . In relation to the complete aptamer sequences listed above, a variant may have a sequence identity of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0103]

[0105] . The term sequence identity refers to a measure of the identity of nucleotide sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se, has a recognised meaning in the art and can be calculated using published techniques (see, for example, Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide sequences, the term “identity” is known to skilled artisans as defining identical nucleotides at a given position in the sequence (Carillo, H. & Lipton, D., SIAM J Applied Math 48: 1073 (1988)).

[0104]

[0106] . Whether any particular nucleic acid molecule is at least x% identical to, for instance, can be determined conventionally using known computer programs such as DNAsis software (Hitachi Software, San Bruno, Calif.) for initial sequence alignment followed by ESEE DNA / protein sequence software (cabot @trog.mbb.sfu.ca) for multiple sequence alignments.

[0105]

[0107] . When using any sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present disclosure, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleic acid or amino acid sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0108] . Another method for determining the best overall match between a query sequence and another sequence in a global sequence alignment, is using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment, the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U’s to T’s. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomisation Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

[0106]

[0109] . For example, a polynucleotide having 95% “identity” to a reference nucleotide sequence of the present disclosure, is identical to the reference sequence except that the polynucleotide sequence may include on average up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polynucleotide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.

[0107]

[0110] . The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol.

[0108] Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. It can be accessed at the NCBI website, together with a description of how to determine sequence identity and sequence similarities using this program.

[0109]

[0111] . The aptamers of the present disclosure may be used in a sensor (including a biosensor), or as an assay reagent to detect and optionally quantify an analyte. In some cases, the aptamer may be modified or otherwise adapted for use in a certain sensor or assay type including have a tag or a reporter attached, or be bound to a solid surface or a matrix, or a bead or a nanoparticle, for example.

[0110]

[0112] . In some embodiments, the aptamer may be adapted for use in an optical sensor.

[0111] Such sensors may be Anorogenic, colorimetric, or rely on other modes for optical detection of analyte. In an optical sensor, reliance is had on the aptamer undergoing a conformational change upon binding to a specific target analyte. Quencher / fluorophore pairs can be used as dual aptamer labels to generate Anorogenic aptamers. These aptamers exhibit an increase in Auorescence upon binding. A quencher attached to an aptamer can absorb energy from a Auorophore that is directly adjacent when the aptamer is in a certain configuration, preventing Auorescence. When binding of target analyte occurs, the aptamer exhibits a conformational change which results in the Auorophore and quencher being separated from one another physically, generating a Auorescent signal. Fluorophores can be organic dye molecules, quantum dot nanocrystals, or any light-emitting moiety. Quenchers can be any type of molecule, particle or moiety that can absorb that light or associated energy, whether through true Auorescence resonance energy transfer (FRET), plasmonic interactions (e.g., with gold particle-gold particle interactions), bioluminescence resonance energy transfer (BRET), time-resolved Auorescence resonance energy transfer (TR-FRET) or other interactions. The Auorophore or other light-emitting entity becomes unquenched, giving rise to a Auorescent or similar signal. These types of aptamers can be used to develop homogenous solution-based assays for use with Auorescence microplate readers, Auorometers, miniAuorometers, luminometers and other Auorescence or light detection instruments. Another type of plasmonic interaction between nanogold particles can give rise to colorimetric signal changes in which the visible colour of the solution changes when two particles are brought into close proximity.

[0112]

[0113] . Another type of optical sensor utilises an aptamer that contains an analyte binding site as well as a second site for binding to a chromophore. When chromophore binding takes place, the aptamer complex becomes Auorescent. If designed correctly, the chromophore binding site is generated only when the target analyte is also bound.

[0113]

[0114] . Surface-Enhanced Raman Scattering (SERS) may be used with aptamers to detect analyte binding, particularly for small molecule analytes. Raman signal stems from photon scattering caused by a molecule’s electric cloud. The signal is unique for each analyte. While Raman scattering signal was originally too low for sensitive detection, the addition of colloidal nanoparticles offers enhanced signal generation and improved sensitivity. Raman scattering involving nanoparticles, though more sensitive, originally lacked specificity and reproducibility. This has been overcome by the use of highly specific aptamers.

[0114]

[0115] . The aptamers of the present disclosure may be used in the context of an electrochemical sensor. In many cases, one end of the aptamer is covalently attached to an electrically conductive surface, such as a gold-coated electrode. A moiety that can contribute free electrons, such as ferrocene or methylene blue may be attached to any other nucleotide or spacer at any point on the aptamer, including the distal end of the aptamer. When the aptamer binds to the target analyte, the free electron donor comes into close proximity with the surface or can be pushed further away from the surface, either way giving rise to a change in current.

[0115]

[0116] . The present aptamers have been found to bind specifically to vancomycin, thereby allowing for detection and also quantification of that molecule. Accordingly, a sensor incorporating the present aptamers may be used for the monitoring of vancomycin levels in a subject undergoing therapy for an infection, such as MRSA. Such monitoring may be used to address the problem of vancomycin therapy that levels of the drug should be above a minimum so to be at least inhibitory to a target microorganism, but should also be under a maximum level so as to limit the potential for toxicity in the subject.

[0116]

[0117] . An EAB sensor having the working electrode coated with an aptamer of the present disclosure may be used to monitor vancomycin levels in real-time. In an exemplary method, the aptamers may be modified with a redox reporter such as methylene blue and attached to the working electrode of an EAB sensor. The working electrodes (and also the counter electrode and reference electrode) may be wires, needles, or microneedles, that can be inserted into the skin of the subject so as to contact the interstitial fluid and / or blood. Vancomycin will be detectable in either fluid, thereby allowing for continuous real-time monitoring of the drug. Administration of the drug (typically by way of infusion) may be controlled so as to maintain levels within a therapeutic window.

[0117]

[0118] . The present aptamers display varying affinities (typically expressed as Kd) for vancomycin, and accordingly a specific aptamer may be selected according to need. For example, a prior art vancomycin-sensitive aptamer may have a high affinity for the drug, and is therefore well suited to detecting low levels (for example, a trough concentration). That aptamer may be saturable at higher vancomycin concentrations and therefore nor useful in measuring peak levels of the drug. Thus, a second type of aptamer having a lower affinity for vancomycin may be combined in the sensor with the prior art aptamer to increase the dynamic range of the sensor thereby allowing for the quantitation of drug around the clinically important trough and peak concentrations. Where the aptamers are used as sensing elements in an electrochemical sensor, the first and second type of aptamer may be mixed on a single working electrode, or separated over two discrete electrodes.

[0118]

[0119] . The first motif (SEQ ID NO: 1, or SEQ ID NO:2) and the second motif (SEQ ID

[0119] NO:3, or SEQ ID NO:4) may be exploited for identifying aptamers capable of interacting with vancomycin. In one such application, selection of an aptamer that is selective for vancomycin may be facilitated by a process known as SELEX. The process may be considered as two alternating stages. In the first stage, the library oligonucleotides are amplified by a polymerase chain reaction (PCR) to the desired concentration. According to the present disclosure, the library comprises at least 10% (and preferably more) oligonucleotides comprising one or both of the first and second motifs. For the selection of DNA aptamers, a pool of single-stranded oligodeoxyribonucleotides is generated by strand separation of double-stranded PCR products. In the second stage, the products of amplification are incubated with target drug and oligonucleotides which bind the drug are used in the next SELEX round.

[0120]

[0120] . Separation of oligonucleotides with higher affinity for target drug and removal of unbound oligonucleotides are achieved through intense competition for binding sites. The selection pressure rises with every SELEX round. Maximum enrichment of the oligonucleotide pool with aptamers with the strongest affinity for the target molecule is usually achieved after many rounds, e.g., 5 to 15 rounds. A higher than expected proportion of highly binding aptamers would be expected, given that a high proportion of the starting library comprises motif(s) that will assist in vancomycin binding.

[0121]

[0121] . The present disclosure will now be more fully described by reference to the following non-limited Examples.

[0122] EXAMPLE 1: Determination of putative vancomycin interacting motifs in DNA aptamers by sequence analysis

[0123]

[0122] . As outlined in FIG. 1, a SELEX method was implemented to generate a pool of aptamers potentially capable of binding to vancomycin. The sequences of aptamers drawn from the pool were analysed against others from the pool, and also by reference to vancomycin-binding aptamers from other sources. Two sequence motifs were identified: a first toward the 5’ terminus CGAGGGTACCGC and a second toward the 3’ terminus GTGGGTCGG. It was considered that one or both of these consensus sequences may be critical for, or at least contributory to, vancomycin sensitivity and may therefore be considered putative binding motifs. It was further considered that alteration of one or bases in either or both of the putative binding motifs could increase or decrease the affinity of an aptamer for vancomycin, with such altered aptamers being potentially useful for certain applications, such as to broaden the dynamic range of a vancomycin sensor. For example, an aptamer having an affinity intermediate to the two aptamer sequences may be identified.

[0124]

[0123] . Experiments detailed in further Examples herein tested aptamers having preserved the putative binding motifs, but incorporated varying non-binding stem-loop regions. Subsequent testing of the modified aptamers for vancomycin binding by SPR confirmed that all these modified aptamers showed binding capability. Furthermore, whenever the putative binding motifs were altered, in the majority of instances the aptamers lost vancomycin binding capability.

[0125]

[0124] . Reference is made to FIG. 2, FIG. 3, FIG. 4 and FIG. 5 showing modifications made to aptamers obtained from the SELEX process, and the effect thereof on vancomycin binding capability.

[0126]

[0125] . Various aptamers comprising putative binding motifs were tested for binding with vancomycin according to the following SPR method.

[0127] SPR chips:

[0128]

[0126] . The SPR assay method used CM5 chips from Cytiva, Series S, (cat#29149603).

[0129] Before use, the chips were pre-immobilised with ‘capture’ oligos comprised of poly-dT oligonucleotides tethered at one terminus to the SPR chip. When not in use, the SPR chips were stored at 4°C in nuclease -free, sterile water. This is as per Cheng et al. Anal. Chem. 86, 3273-78.

[0130]

[0127] . The SPR assays were typically run on a Biacore S200 SPR Instrument, managed by the Biacore control software, and the resulting data analysed by the Biacore S200 evaluation software.

[0131] Buffer preparation:

[0128] . A solution of lx PBS, pH 7.2 + 0.7 mM MgC12 by diluting an appropriate amount of an aqueous solution of IM MgC12 (Sigma cat#M10280) into Sigma lx PBS (cat#806552) buffer, pH 7.4. This solution was filtered through a Corning bottle top filter (cat#CLS430517) and de -gassed. The degassed lx PBS, pH 7.4 + 0.7 mM MgCh solution was used as the SPR running buffer and vancomycin preparation buffer.

[0132]

[0129] . For aptamer immobilisation onto the SPR chip surface, de -gassed lx PBS, pH 7.4

[0133] + 0.7mM MgCh was used.

[0134] Aptamer sequences:

[0135]

[0130] . All aptamers were synthesised with a 24-poly-dA sequence appended to the 5’ end of the aptamer, as part of the DNA sequence.

[0136] Aptamer sequence preparation:

[0137]

[0131] . Aptamers were received lyophilised from an external DNA synthesis vendor and dissolved to 100 pM in an aqueous solution of 10 mM Tris, pH 8.0, 0.1 mM EDTA buffer. For use in the SPR, aptamers were diluted to 0.125 pM in sterile, degassed, lx PBS, pH 7.4 + 0.7 mM MgCh solution. The aptamers were then foldedby incubating at 95°C for 8 minutes, 4°C for 15 minutes, and 25 °C for 15 minutes.

[0138] Vancomycin preparation:

[0139]

[0132] . A stock solution of 20 mM vancomycin was prepared in nuclease -free, sterile water.

[0140]

[0133] . The 20 mM vancomycin solution in water was used to prepare a working stock of

[0141] 40 pM vancomycin in lx PBS + 0.7 mM MgCh solution and filter sterilised. The filter sterilised stock was then further diluted to the desired vancomycin concentrations for the SPR assay with additional sterile, degassed, IX PBS + 0.7 mM MgCh solution.

[0142] SPR set up:

[0143]

[0134] . The amount of each aptamer immobilised was calculated per standard methods.

[0144]

[0135] . Flow rates for vancomycin were typically 30 pL / min for 60-240 seconds for each vancomycin concentration tested. Typically, 5-7 different concentrations of vancomycin were run, depending on the particular experimental set up. Analysis temperature was set to 37°C. Dissociation time for the vancomycin was typically 120-240 seconds, depending on the expected dissociation time for a particular aptamer.

[0145] Data processing:

[0136] . Data was imported into the Biacore S200 software and was analysed as per standard kinetics modelling to determine the ‘on’ and ‘off rate and dissociation constant (Kd) value of the vancomycin for a particular aptamer.

[0146] EXAMPLE 2: Aptamer performance in electrochemical sensor.

[0147]

[0137] . Stainless steel acupuncture needles were gold plated using electrodeposition.

[0148] Briefly, a strike layer was applied before exposure of the needles to a soft gold plating solution at 50° C. The resultant smooth soft gold layer was cleaned with oxygen plasma.

[0149]

[0138] . An electroactive area was isolated using heat shrunk polyolefin and sensors were functionalized with aptamer (DNA concentration 500 nM; incubation time approx. Ih; TCEP and functionalization buffer IX PBS + 2 mM MgCh pH 7.4 + / - 0.15) followed by a MCH passivation layer (concentration 5 mM; incubation time about 14h; Functionalization Buffer: IX PBS + 2 mM MgCh pH 7.4 + / - 0.15 and rinsed with a solution buffered with HEPES (concentration 50 mM; incubation time 10 min - single rinse -; drying time up to 10 min) prior to testing sensor functionality.

[0150]

[0139] . The working electrode was incorporated into an electrochemical sensor circuit having a counter electrode and a reference electrode (Ag|AgCl). The counter electrode was a smooth soft gold coated acupuncture needle, insulating with polyolefin as for the working electrode.

[0151]

[0140] . A Palmsens™ potentiostat was used to apply an interrogating electrical current to the working electrode, but not to the reference electrode.

[0152]

[0141] . Sensors were subjected to a testing regime to assess performance of the aptamer concerned. The test matrix was 1 x PBS; 0.7 mM MgCh, pH 7.4 + / -0.15.

[0153]

[0142] . Sensors aptamers were initially interrogated by square wave voltammetry over a range of frequencies and over a broad vancomycin concentration range (0 pm to 500pm) to determine optimal frequencies for vancomycin detection.

[0154]

[0143] . Sensors were then interrogated at three different temperatures (30°C, 35°C, 42°C) to determine any temperature dependency.

[0144] . A compilation of the results from all the aptamers screened by initial testing is shown Those which had a high number of desirable characteristics were screened further. Results for the affinity testing by SPR and are shown in the table below.

[0155]

[0145] . It will be noted that each of the aptamers demonstrated some affinity for vancomycin when tested by SPR. In some cases, an aptamer is characterised as a “weak binder”. That characterisation arises because a consistent affinity value is not available by the SPR testing method and analysis algorithm used. However, there is nevertheless a weak level of binding for each aptamer and thus it is classed as a “weak binder”.

[0156]

[0146] . Aptamers that demonstrated a binding affinity about the clinically relevant range for vancomycin and demonstrated a usable signal were subjected to additional testing to determine any lag in response or signal drift over 24 hours of continuous interrogation.

[0147] . Reference is made to the Table below providing further detailed experimental data for aptamers that were subject to sensor testing.

[0157]

[0148] . Aptamers were assessed based on the performance according to the following criteria.

[0158] • affinity for vancomycin within the clinically relevant range, • minimal shift in Kd with variation in temperature,

[0159] • minimal change in signal gain with variation in temperature,

[0160] • large change in the electrochemical response upon exposure to target,

[0161] • little or no increase in response (lag) minutes after target exposure,

[0162] • response which is stable and reproducible over 24 hours of continuous interrogation, and

[0163] • ability be functionalized to the electrode surface such that a large signal to noise is achievable.

[0149] . Particularly preferred aptamers are: SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID

[0164] NO:31, SEQ ID NO: 100, SEQ ID NO: 123, SEQ ID NO: 138, each demonstrating a large response to target (MaxKDM > 1.2), negligible lag at 35 °C, stable response over 24 hours of in vitro interrogation, and reasonable signal gain temperature dependence. The relative Kd values for each of the aforementioned aptamers is shown at FIG. 6.

[0165]

[0150] . Exemplary data for SEQ ID NO: 100 is shown in FIG. 7A through FIG. 7F. The data shows that Kd is temperature dependent, 1-5.3 pM (1.6 pM 10|200 KDM response Kd at 35°C). Good signal gains were demonstrated, with a low temperature dependence.

[0166]

[0151] . Without wishing to be limited by theory in any way, it is considered that the affinity for vancomycin in each case is due to one or both of the first and / or second binding motifs discovered by the inventors.

[0167]

[0152] . It will be further noted that the aptamers display a wide range of affinities.

[0168] Accordingly, these aptamers may form a pool from which one or more aptamers are selected for a certain application. Any of the aptamers above may be combined with an aptamer of the prior art, or an aptamer identified in the future to provide a required dynamic range across which a concentration of vancomycin is expected to fall.

[0169]

[0153] . An aptamer of the present disclosure may not comprise or consist of the following sequence:

[0170]

[0154] . Those skilled in the art will appreciate that the disclosure described herein is susceptible to further variations and modifications other than those specifically described. It is understood that the disclosure comprises all such variations and modifications which fall within the spirit and scope of the present disclosure.

[0171]

[0155] . Accordingly, the spirit and scope of the present disclosure is not to be limited by the foregoing examples, but is to be understood in the broadest sense allowable by law.

Claims

CLAIMS:

1. An aptamer capable of interacting specifically with a target analyte, the aptamer having a sequence comprising any one or more of:SEQ ID NO: 1 or a variant thereof, or SEQ ID NO:2 or a variant thereof, and / orSEQ ID NO:3 or a variant thereof, or SEQ ID NO:4 or a variant thereof.

2. The aptamer of claim 1, wherein the sequence is not any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO: 10.

3. The aptamer of claim 1 or claim 2, wherein the variant of SEQ ID NO: 1 or SEQ ID NO:2, and / or the variant of SEQ ID NO:3 or SEQ ID NO:4, comprises a variation in 1 or 2 bases of the respective sequence.

4. The aptamer of claim 3, wherein the variation in 1 or 2 bases is independently selected from: substitution with a natural or a non-natural base, insertion of a natural base or a nonnatural base, and deletion of a base.

5. The aptamer of any one of claims 1 to 4, wherein a majority of the bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof is not involved in base -pairing with other bases of the aptamer.

6. The aptamer of any one of claims 1 to 5, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof are not involved in basepairing with other bases of the aptamer.

7. The aptamer of any one of claims 1 to 6, wherein a minority of the bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof is involved in base-pairing with other bases of the aptamer.

8. The aptamer of any one of claims 1 to 7, wherein no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof are involved in base-pairing with other bases of the aptamer.

9. The aptamer of any one of claims 1 to 8, wherein no base pairs are formed between any two bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof.

10. The aptamer of any one of claims 1 to 9, wherein a majority of the bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof is not involved in base -pairing with other bases of the aptamer.

11. The aptamer of any one of claims 1 to 10, wherein 1, 2, 3, 4, 5, 6, 7, 8, or 9 bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof are not involved in base -pairing with other bases of the aptamer.

12. The aptamer of any one of claims 1 to 11, wherein a minority of the bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof is involved in base-pairing with other bases of the aptamer.

13. The aptamer of any one of claims 1 to 12, wherein no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof are involved in base -pairing with other bases of the aptamer.

14. The aptamer of any one of claims 1 to 13, wherein no base pairs are formed between any two bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof.

15. The aptamer of any one of claims 1 to 14 comprising a first terminal region which is linked to an electrode surface and a second terminal region which is linked to a redox reporter when the aptamer is incorporated as a sensing element in a electrochemical sensor, and SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof is located closer to the first terminal region than to the second terminal region.

16. The aptamer of any one of claims 1 to 15 comprising a first terminal region which is linked to an electrode surface and a second terminal region which is linked to a redox reporter when the aptamer is incorporated as a sensing element in a electrochemical sensor, and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof is located closer to the second terminal region than to the first terminal region.

17. The aptamer of any one of claims 1 to 16 having a 5’ terminus and a 3’ terminus, and SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof is located closer to the 5’ terminus than the 3’ terminus.

18. The aptamer of any one of claims 1 to 17, wherein the aptamer has a terminal base, and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases are disposed between the terminal base and SEQ ID NO: 1 or variant thereof, or SEQ ID NO:2 or variant thereof, or SEQ ID NO:3 or variant thereof, or SEQ ID NO: 4 or variant thereof.

19. The aptamer of any one of claims 1 to 18 having a 5’ terminus and a 3’ terminus, and SEQ ID N0:3 or variant thereof or SEQ ID N0:4 or variant thereof is located closer to the 3’ terminus than the 5’ terminus.

20. The aptamer of any one of claims 1 to 19, wherein the aptamer has a terminal base, and 0,I, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases are disposed between the terminal base and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof.

21. The aptamer of any one of claims 1 to 20, comprising SEQ ID NO:1 or SEQ ID NO:2, and SEQ ID NO:3 or SEQ ID NO:4.

22. The aptamer of claim 21, comprising a spacer disposed between SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof, and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof.

23. The aptamer of claim 22, wherein the spacer comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,I I, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

24. The aptamer of claim 23, wherein the spacer, alone or in combination with other bases of the aptamer, forms a hairpin structure, a stem structure, a loop structure, or a stem / loop structure.

25. The aptamer of claim 24, wherein 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof, and / or 1, 2, 3, 4, 5, 6, 7, 8, or 9 bases of SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof contribute to the hairpin structure, a stem structure, a loop structure, or a stem / loop structure.

26. The aptamer of any one of claims 21 to 25 having base -pairing between bases of SEQ ID NO: 1 or variant thereof or SEQ ID NO:2 or variant thereof, and SEQ ID NO:3 or variant thereof or SEQ ID NO:4 or variant thereof.

27. The aptamer of claim 26, wherein the base-pairing occurs exclusively or predominantly between cytosine and guanine bases.

28. The aptamer of any one of claims 1 to 27 comprising a redox reporter covalently attached to any point along the length of the aptamer, and a linking element at one terminus configured to facilitate attachment of the aptamer to an electrode.

29. The aptamer of any one of claims 1 to 28 comprising at or toward one terminus a redox reporter, and at the other terminus a linking element configured to facilitate attachment of the aptamer to an electrode.

30. The aptamer of claim 28 or claim 29, wherein the redox reporter is methylene blue or a functional equivalent thereof.

31. The aptamer of any one of claims 1 to 30, wherein the target analyte is an organic molecule.

32. The aptamer of claim 31, wherein the organic molecule is a glycopeptide antibiotic.

33. The aptamer of claim 32, wherein the glycopeptide antibiotic is vancomycin.

34. The aptamer of any one of claims 1 to 33 comprising any one of: SEQ ID NO:1, SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NOTO, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NOTO, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NOTO, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NOTO, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ IDNO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ IDNO: 140, and SEQ ID NO: 141, or a variant thereof.

35. The aptamer of claim 34, wherein the variant thereof has a sequence identity of 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% to the reference sequence.

36. The aptamer of any one of claims 1 to 35 comprising a reporter, a tag, or another modification used to identify the extent to which a target analyte interacts with the aptamer.

37. A sensor for the detection of vancomycin or another glycopeptide antibiotic comprising an aptamer of any one of claims 1 to 36.

38. The sensor of claim 37, being an electrochemical sensor comprising a working electrode having an aptamer of any one of claims 1 to 36 associated therewith, or linked thereto.

39. The sensor of claim 38, wherein the working electrode is a capable of penetrating the skin of a mammal such that the aptamer contacts an interstitial fluid of the mammal.

40. The sensor of any one of claims 37 to 39, wherein the working electrode is a wire, a needle, or a microneedle.

41. The sensor of any one of claims 37 to 40 further comprising a counter electrode, and optionally a reference electrode.

42. A method of producing an aptamer for the detection of vancomycin or another glycopeptide antibiotic, the method comprising the step of incorporating SEQ ID NO:1 or a variant thereof, or SEQ ID NO:2 or a variant thereof; and / or SEQ ID NO:3 or a variant thereof, or SEQ ID NO:4 or a variant thereof, in a complete aptamer sequence.

43. The method of claim 42, that is not for producing an aptamer according to any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NOTO, or a product of the method is not an aptamer according to any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NOT, SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO: 10.

44. A method for identifying an aptamer for the detection of vancomycin or another glycopeptide antibiotic, the method comprising providing a library of candidate aptamers, selecting and / or enriching for aptamers capable of binding to vancomycin or another glycopeptide antibiotic, wherein most or all of the candidate aptamers comprise SEQ ID NOT ora variant thereof, or SEQ ID NO:2 or a variant thereof; and / or SEQ ID NO:3 or a variant thereof, or SEQ ID NO:4 or a variant thereof.

45. The method of claim 44, wherein at least 10% of the candidate aptamers comprise SEQ ID NO: 1 or a variant thereof, or SEQ ID NO:2 or a variant thereof; and / or SEQ ID NO:3 or a variant thereof, or SEQ ID NO:4 or a variant thereof.

46. The method of claim 45 comprising multiple rounds of selecting and enriching for aptamers capable of binding to vancomycin or another glycopeptide antibiotic.

47. The method of claim 46 that is an augmented SELEX (systematic evolution of ligands by exponential enrichment) method.

48. An aptamer identified by the method of any one of claims 44 to 47.

49. A method for detecting and / or quantitating vancomycin or another glycopeptide antibiotic, the method comprising the steps of contacting a test fluid or a biological fluid in vivo with an aptamer of any one claims 1 to 36, or the apparatus of claim 48.

50. The method of claim 49, wherein the aptamer is a component of the sensor of any one of claims 37 to 41.

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