FIELD OF THE INVENTION

The present invention relates to the field of aptamers and their use in detecting the levels of a small molecule in a sample. The invention also relates to devices containing the aptamers.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created March 16, 2023, is named 51638-002WO2_Sequence_Listing_3_16_23 and is 51 ,142 bytes in size.

BACKGROUND

There is a need for improved methods and devices for specific and accurate point-of-care detection of analyte levels in samples using aptamers, specifically for drugs that, e.g., treat neurological and/or neuronal hyperexcitability disorders.

In one example, there are approximately 3.5 million patients with active epilepsy in the US, the vast majority of whom are being treated with one or more anti-epileptic drugs (AEDs). Neurologists and other medical professionals have long sought the ability to rapidly monitor AED blood levels to optimize therapy at the point-of-care. The therapeutic window for many AEDs is very narrow, and epilepsy patients therefore risk breakthrough seizures or significant drug-related side-effects when AED blood levels are too low or high, respectively. Epilepsy patients with poorly-controlled seizures incur significantly higher direct and indirect costs, and experience far more personal suffering, and even death, than those with well- controlled seizures. Optimization of AED therapy is vitally important to minimize morbidity and mortality and maximize quality of life in epilepsy patients. Previous techniques for measuring AED blood levels have relied on traditional immunoassays and HPLC determinations, among other methods. While accurate, such techniques require venous blood draws by trained phlebotomists, with samples sent to CLIA-certified laboratories distal to the point-of-care. The significant delays required by these methods to obtain readings have precluded rapid confirmation of AED blood levels that would enable the physician’s prescribing decisions within the time window of the usual office visit rather than waiting one or more days for the central laboratory reading during which time the patient may have incurred unnecessary suffering.

SUMMARY OF THE INVENTION

In general, the invention provides aptamers useful for detecting the levels of small molecules in a sample, as well as methods of using the aptamers and devices containing the aptamers.

In a first aspect, the invention provides an aptamer having a nucleobase sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-53. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one SEQ ID NOs: 1-14. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 8, and 12. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 1 . In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 4. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 7. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 8. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 12. In some embodiments, the percent sequence identity is at least 75%. In some embodiments, the percent sequence identity is at least 80%. In some embodiments, the percent sequence identity is at least 85%. In some embodiments, the percent sequence identity is at least 90%. In some embodiments, the percent sequence identity is at least 95%. In some embodiments, the percent sequence identity is at least 99%. In some embodiments, the percent sequence identity is 100%.

In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1 - 53, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1 -14, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1 , 4, 7, 8, and 12, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 1 , excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 4, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 7, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 8, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 12, excluding the primer binding regions. In some embodiments, the percent sequence identity is at least 75%. In some embodiments, the percent sequence identity is at least 80%. In some embodiments, the percent sequence identity is at least 85%. In some embodiments, the percent sequence identity is at least 90%. In some embodiments, the percent sequence identity is at least 95%. In some embodiments, the percent sequence identity is at least 99%. In some embodiments, the percent sequence identity is 100%.

In some embodiments, the aptamer is a structure-switching aptamer against a small molecule. In some embodiments, the small molecule is capable of treating a neuronal hyperexcitability disorder. In some embodiments, the small molecule is capable of treating a neurological disorder. In some embodiments, the neurological disorder is a seizure disorder, e.g., epilepsy.

In some embodiments, the small molecule is carbamazepine. In some embodiments, the aptamer is not a structure-switching aptamer against oxcarbazepine or carbamazepine-10,11 -epoxide. In some embodiments, the aptamer is selective for carbamazepine over oxcarbazepine. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as oxcarbazepine as determined by dissociation constant. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as oxcarbazepine as determined by an output from a device designed to measure a conformational change in the aptamer.

In some embodiments, the aptamer is selective for carbamazepine over carbamazepine-10,11- epoxide. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as carbamazepine-10,11 -epoxide as determined by dissociation constant. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as carbamazepine-10,11 -epoxide as determined by an output from a device designed to measure a conformational change in the aptamer.

In some embodiments, the aptamer is selective for carbamazepine over both oxcarbazepine and carbamazepine-10,11 -epoxide.

In some embodiments, the aptamer forms a G-quadruplex structure. In some embodiments, the aptamer includes one or more CO or CA loops.

In another aspect, the invention provides a method of determining the levels of a small molecule in a subject, the method including a) obtaining a sample from the subject, b) contacting the sample with an aptamer to induce a conformational change in the aptamer if the small molecule is present, c) measuring the conformational change in the aptamer, and d) determining the level of the small molecule from the measuring.

In some embodiments, the measuring of a conformational change includes measuring an electrical change. In some embodiments, the measuring of a conformational change includes measuring the normalized current. In some embodiments, the small molecule that is measured is capable of treating a neurological disorder. In some embodiments, the neurological disorder is a seizure disorder, e.g., epilepsy. In some embodiments, the small molecule is carbamazepine. In some embodiments, the small molecule is capable of treating a neuronal hyperexcitability disorder.

In some embodiments of the methods described herein, the method includes detecting the presence or determining the level of a small molecule that is carbamazepine. In some embodiments, the aptamer used in the method is a structure-switching aptamer against carbamazepine. In some embodiments, the aptamer used in the method is not a structure-switching aptamer against oxcarbazepine or carbamazepine-10,11 -epoxide.

In some embodiments, the aptamer is selective for carbamazepine over oxcarbazepine. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as oxcarbazepine as determined by dissociation constant. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as oxcarbazepine as determined by an output from a device designed to measure a conformational change in the aptamer.

In some embodiments, the aptamer is selective for carbamazepine over carbamazepine-10,11- epoxide. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as carbamazepine-10,11 -epoxide as determined by dissociation constant. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as carbamazepine-10,11 -epoxide as determined by an output from a device designed to measure a conformational change in the aptamer.

In some embodiments, the aptamer is selective for carbamazepine over both oxcarbazepine and carbamazepine-10,11 -epoxide.

In some embodiments, the sample is a blood sample. In some embodiments, the blood sample is whole blood. In some embodiments, the whole blood is obtained from a capillary source. In some embodiments, the blood sample is obtained from a fingerstick. In some embodiments, the blood sample is serum or plasma.

In some embodiments, the sample is urine or saliva. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is interstitial fluid. In some embodiments, the sample is lacrimal fluid.

In some embodiments of the methods disclosed herein, the aptamer used in the method is a nucleic acid aptamer. In some embodiments, the aptamer is a DNA aptamer. In some embodiments, the aptamer is an RNA aptamer.

In some embodiments of the methods disclosed herein, the aptamer is any aptamer disclosed herein. In some embodiments of the methods disclosed herein, the aptamer used in the method has a nucleobase sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -53. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one SEQ ID NOs: 1-14. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 8, and 12. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 1 . In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 4. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 7. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 8. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 12. In some embodiments, the percent sequence identity is at least 75%. In some embodiments, the percent sequence identity is at least 80%. In some embodiments, the percent sequence identity is at least 85%. In some embodiments, the percent sequence identity is at least 90%. In some embodiments, the percent sequence identity is at least 95%. In some embodiments, the percent sequence identity is at least 99%. In some embodiments, the percent sequence identity is 100%.

In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1- 53, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1-14, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1 , 4, 7, 8, and 12, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 1 , excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 4, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 7, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 8, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 12, excluding the primer binding regions. In some embodiments, the percent sequence identity is at least 75%. In some embodiments, the percent sequence identity is at least 80%. In some embodiments, the percent sequence identity is at least 85%. In some embodiments, the percent sequence identity is at least 90%. In some embodiments, the percent sequence identity is at least 95%. In some embodiments, the percent sequence identity is at least 99%. In some embodiments, the percent sequence identity is 100%.

In another aspect, the invention provides a device for detecting levels of a small molecule in a sample. The device includes an electrode functionalized with an aptamer, wherein the aptamer undergoes a conformational change when bound to the small molecule.

In some embodiments, the device is capable of measuring the conformational change in the aptamer.

In some embodiments, the measuring of a conformational change includes measuring an electrical change. In some embodiments, the measuring of a conformational change includes measuring the normalized current.

In some embodiments, the small molecule detected by the device is capable of treating a neurological disorder. In some embodiments, the neurological disorder is a seizure disorder, e.g., epilepsy. In some embodiments, the small molecule is carbamazepine. In some embodiments, the small molecule is capable of treating a neuronal hyperexcitability disorder. In some embodiments of the devices disclosed herein, the aptamer used in the device is a nucleic acid aptamer. In some embodiments, the aptamer is a DNA aptamer. In some embodiments, the aptamer is an RNA aptamer.

In some embodiments of the devices disclosed herein, the aptamer used in the device has a nucleobase sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -53. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one SEQ ID NOs: 1-14. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 , 4, 7, 8, and 12. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 1 . In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 4. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 7. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 8. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 12. In some embodiments, the percent sequence identity is at least 75%. In some embodiments, the percent sequence identity is at least 80%. In some embodiments, the percent sequence identity is at least 85%. In some embodiments, the percent sequence identity is at least 90%. In some embodiments, the percent sequence identity is at least 95%. In some embodiments, the percent sequence identity is at least 99%. In some embodiments, the percent sequence identity is 100%.

In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1- 53, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1-14, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NOs: 1 , 4, 7, 8, and 12, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 1 , excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 4, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 7, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 8, excluding the primer binding regions. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to SEQ ID NO: 12, excluding the primer binding regions. In some embodiments, the percent sequence identity is at least 75%. In some embodiments, the percent sequence identity is at least 80%. In some embodiments, the percent sequence identity is at least 85%. In some embodiments, the percent sequence identity is at least 90%. In some embodiments, the percent sequence identity is at least 95%. In some embodiments, the percent sequence identity is at least 99%. In some embodiments, the percent sequence identity is 100%.

In some embodiments, the aptamer used in the device is a structure-switching aptamer against carbamazepine. In some embodiments, the aptamer used in the device is not a structure-switching aptamer against oxcarbazepine or carbamazepine-10,11 -epoxide.

In some embodiments, the aptamer used in the device is selective for carbamazepine over oxcarbazepine. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as oxcarbazepine as determined by dissociation constant. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as oxcarbazepine as determined by an output from a device designed to measure a conformational change in the aptamer.

In some embodiments, the aptamer used in the device is selective for carbamazepine over carbamazepine-10,11 -epoxide. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as carbamazepine-10,11 -epoxide as determined by dissociation constant. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as carbamazepine-10,11 -epoxide as determined by an output from a device designed to measure a conformational change in the aptamer.

In some embodiments, the aptamer used in the device is selective for carbamazepine over both oxcarbazepine and carbamazepine-10,11 -epoxide.

Definitions

Various terms used throughout the present description may be read and understood as follows, unless the context indicates otherwise: “or” as used throughout is inclusive, as though written “and/or”; singular articles and pronouns as used throughout include their plural forms, and vice versa; similarly, gendered pronouns include their counterpart pronouns so that pronouns should not be understood as limiting anything described herein to use, implementation, performance, etc. by a single gender; “exemplary” should be understood as “illustrative” or “exemplifying” and not necessarily as “preferred” over other embodiments. Further definitions for terms may be set out herein; these may apply to prior and subsequent instances of those terms, as will be understood from a reading of the present description.

As used herein, the term “about” represents a value that is in the range of ±10% of the value that follows the term “about.”

As used herein, the term “aptamer” refers to a targeting agent that binds to various molecular targets including small molecules. Aptamers may be nucleic acid aptamers (e.g., DNA aptamers or RNA aptamers) or peptide aptamers. Nucleic acid aptamers refer generally to nucleic acid species that are engineered through in vitro selection or an equivalent method (e.g., systematic evolution of ligands by exponential enrichment (SELEX)) to bind specific molecular targets. Peptide aptamers typically include a variable peptide loop attached at both ends to a protein scaffold that increases the binding affinity of the peptide aptamer to levels comparable to those of an antibody with the same intended molecular target. In certain embodiments, the aptamer is a nucleic acid.

The term “bicyclic sugar moiety,” as used herein, represents a modified sugar moiety including two fused rings. In certain embodiments, the bicyclic sugar moiety includes a furanosyl ring.

The term "complementary," as used herein in reference to a nucleobase sequence, refers to the nucleobase sequence having a pattern of contiguous nucleobases that permits an oligonucleotide having the nucleobase sequence to hybridize to another oligonucleotide or nucleic acid to form a duplex structure under physiological conditions. Complementary sequences include Watson-Crick base pairs formed from natural and/or modified nucleobases. Complementary sequences can also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs.

The term “contiguous,” as used herein in the context of an oligonucleotide, refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.

The terms “detecting,” “detection,” and “determining” when used in reference to a small molecule in a sample are used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels. Detecting or determining may be direct or indirect.

The term “duplex,” as used herein, represents two oligonucleotides that are paired through hybridization of complementary nucleobases.

As used herein, the term “epilepsy” refers to any of a variety of types of epilepsy syndromes, including, but not limited to, frontal lobe epilepsy, occipital lobe epilepsy, medial temporal lobe epilepsy, parietal lobe epilepsy, benign myoclonic epilepsy in infants, juvenile myoclonic epilepsy, childhood absence epilepsy, juvenile absence epilepsy, epilepsy with generalized tonic clonic seizures in childhood, infantile spasms, Lennox-Gastaut syndrome, West syndrome, sleep-related hypermotor epilepsy, progressive myoclonus epilepsies, febrile fits, epilepsy with continuous spike and waves in slow wave sleep, Laudau Kleffner syndrome, Rasmussen's syndrome, epilepsy arising from an inborn error in metabolism, epilepsy of infancy with migrating focal seizures, autosomal dominant nocturnal frontal lobe epilepsy, Ohtahara syndrome, early myoclonic encephalopathy, focal epilepsy, and/or multifocal epilepsy.

As used herein, the term “fingerstick” refers to a means for obtaining a blood sample from a subject by piercing a portion of the finger.

The term “internucleoside linkage,” as used herein, represents a divalent group or covalent bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. An internucleoside linkage is an unmodified internucleoside linkage or a modified internucleoside linkage. An “unmodified internucleoside linkage” is a phosphate (-O-P(O)(OH)-O-) internucleoside linkage (“phosphate phosphodiester”). A “modified internucleoside linkage” is an internucleoside linkage other than a phosphate phosphodiester. The two main classes of modified internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, phosphorodithioate linkages, boranophosphonate linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidate. Non-limiting examples of non-phosphorus internucleoside linkages include methylenemethylimino ( — CH2 — N(CHs) — O — CH2 — ), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ), siloxane ( — O — Si(H)2 — O — ), and N,N'-dimethylhydrazine ( — CH2 — N(CHs) — N(CHs) — ). Phosphorothioate linkages are phosphodiester linkages and phosphotriester linkages in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. In some embodiments, an internucleoside linkage is a group of the following structure: where

Z is O, S, B, or Se;

Y is -X-L-R ¹ ; each X is independently -O-, -S-, -N(-L-R ¹ )-, or L; each L is independently a covalent bond or a linker (e.g., optionally substituted Ci-eo hydrocarbon linker or optionally substituted C2-60 heteroorganic linker); each R ¹ is independently hydrogen, -S-S-R ² , -O-CO-R ² , -S-CO-R ² , optionally substituted C1-9 heterocyclyl, a hydrophobic moiety, or a targeting moiety; and each R ² is independently optionally substituted C1-10 alkyl, optionally substituted C2-10 heteroalkyl, optionally substituted Ce-w aryl, optionally substituted Ce-w aryl C1-6 alkyl, optionally substituted C1-9 heterocyclyl, or optionally substituted C1-9 heterocyclyl C1-6 alkyl.

The term “nucleobase,” as used herein, represents a nitrogen-containing heterocyclic ring found at the 1 ’ position of the ribofuranose/2’-deoxyribofuranose of a nucleoside. Nucleobases are unmodified or modified. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2- thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5- trifluoromethyl uracil, 5-trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e g., 5-substituted pyrimidines; 6- azapyrimidines; N2-, N6-, and/or 06-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-th iouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl ( — CEC — CH3) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5- bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N- benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N- benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1 ,3-diazaphenoxazine-2-one, 1 ,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1 ,3- diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deazaadenine, 7- deazaguanine, 2- aminopyridine, or 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991 , 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163- 166 and 442-443.

The term “nucleoside,” as used herein, represents sugar-nucleobase compounds and groups known in the art (e.g., modified or unmodified ribofuranose-nucleobase and 2’-deoxyribofuranose- nucleobase compounds and groups known in the art). The sugar may be ribofuranose. The sugar may be modified or unmodified. An unmodified sugar nucleoside is ribofuranose or 2’-deoxyribofuranose having an anomeric carbon bonded to a nucleobase. An unmodified nucleoside is ribofuranose or 2’- deoxyribofuranose having an anomeric carbon bonded to an unmodified nucleobase. Non-limiting examples of unmodified nucleosides include adenosine, cytidine, guanosine, uridine, 2’-deoxyadenosine, 2’-deoxycytidine, 2’-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the group consisting of nucleobase modifications and sugar modifications described herein. A nucleobase modification is a replacement of an unmodified nucleobase with a modified nucleobase. A sugar modification may be, e.g., a 2’-substitution, locking, carbocyclization, or unlocking. A 2’-substitution is a replacement of 2’-hydroxyl in ribofuranose with 2’-fluoro, 2’-methoxy, or 2’- (2-methoxy)ethoxy. A locking modification is an incorporation of a bridge between 4’-carbon atom and 2’- carbon atom of ribofuranose. Nucleosides having a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides.

The term “nucleotide,” as used herein, represents a nucleoside bonded to an internucleoside linkage or a monovalent group of the following structure -X ¹ -P(X ² )(R ¹ )2, where X ¹ is O, S, or NH, and X ² is absent, =O, or =S, and each R ¹ is independently -OH, -N(R ² )2, or -0-CH2CH2CN, where each R ² is independently an optionally substituted alkyl, or both R ² groups, together with the nitrogen atom to which they are attached, combine to form an optionally substituted heterocyclyl.

The term “oligonucleotide,” as used herein, represents a structure containing 10 or more (e.g., 10 to 150 or 10 to 100) contiguous nucleosides covalently bound together by internucleoside linkages. The oligonucleotide may be an aptamer. An oligonucleotide includes a 5’ end and a 3’ end. The 5’ end of an oligonucleotide may be, e.g., hydroxyl, a targeting moiety, a hydrophobic moiety, 5’ cap, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, diphosphrodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. The 3’ end of an oligonucleotide may be, e.g., hydroxyl, a targeting moiety, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer (e.g., polyethylene glycol). An oligonucleotide having a 5’-hydroxyl or 5’-phosphate has an unmodified 5’ terminus. An oligonucleotide having a 5’ terminus other than 5’- hydroxyl or 5’-phosphate has a modified 5’ terminus. An oligonucleotide having a 3’-hydroxyl or 3’- phosphate has an unmodified 3’ terminus. An oligonucleotide having a 3’ terminus other than 3’-hydroxyl or 3’-phosphate has a modified 3’ terminus.

The term “point-of-care” refers to anything that is carried out at or near the time and place of treating and/or obtaining a sample from a subject.

The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and/or individual of interest that may or may not contain an entity that is to be characterized, identified, and/or quantified, for example based on physical, biochemical, chemical, electrochemical, and/or physiological characteristics. In some embodiments, the entity is a small molecule. Samples may include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, interstitial fluid, lacrimal fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, stool, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, cellular extracts, and combinations thereof.

The term “selective” as used herein in reference to an aptamer refers to an aptamer that binds to a target small molecule at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, or at least 50 times) as much as a second small molecule, e.g., as determined by dissociation constant or measured by an output of a device described herein.

The term “small molecule,” as used herein, is an organic or inorganic compound having a molecular weight between 10 and 900 daltons. The compound may or may not regulate a biological process. A small molecule may be, for example, a drug or an endogenous molecule.

The term “subject,” as used herein, represents a human or non-human animal (e.g., a mammal) that is in need of the determination of the levels of a small molecule in a sample obtained from the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal.

A “sugar” or “sugar moiety,” includes naturally occurring sugars having a furanose ring or a structure that is capable of replacing the furanose ring of a nucleoside. Sugars included in the nucleosides of the invention may be non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring (e.g., a six-membered ring). Alternative sugars may also include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, e.g., a morpholino or hexitol ring system. Non-limiting examples of sugar moieties that may be included in the oligonucleotides of the invention include p-D-ribose, p-D-2'- deoxyribose, substituted sugars (e.g., 2', 5', and bis substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S- 2'-deoxyribose, and 4'-S-2'-substituted ribose), bicyclic sugar moieties (e.g., the 2'-O — CH2-4' or 2'-0 — (CH ₂ )2-4' bridged ribose derived bicyclic sugars) and sugar surrogates (when the ribose ring has been replaced with a morpholino or a hexitol ring system).

Enumeration of positions within oligonucleotides and nucleic acids, as used herein and unless specified otherwise, starts with the 5’-terminal nucleoside as 1 and proceeds in the 3’-direction

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the melting-off aptamer selection. Aptamers were selected for a target, with counter-targets and matrix used to improve specificity. A typical round of selection began with preincubating a library with the capture oligo at twice the library mole amount for refolding in Refolding Buffer (heat to 90°C for 1 minute, 60°C for 5 minutes, cool to 23°C for 5 minutes). After refolding was completed, the library was captured on streptavidin-coated magnetic beads (MB). The beads were then washed to remove non-specifically bound library members. After the initial wash, the library was incubated with counter-targets, incorporating natural counter-targets in the matrix for 60 minutes at room temperature. The non-binding library was partitioned from the matrix-binding library by magnetic separation, after which the supernatant was discarded. The beads were then washed repeatedly over the course of an hour to remove any remaining non-specific library members. The remaining bead-bound-library was then incubated with positive target at room temperature for 30 minutes. The recovered library was purified on 10% denaturing PAGE for PCR amplification and propagation to the next round. Library response was defined as the ratio of recovered material (determined by spectrophotometric analysis) to the amount of input library. After subjecting the initial library to six rounds of standard selection, the enriched library was divided into three fractions to perform a parallel assessment against the serum-only, counter-target spiked serum, and positive target spiked serum conditions.

FIG. 2 is an illustration of the melting-off parallel assessment and PAGE analysis. The enriched library undergoing parallel assessment was divided into three equal portions. For each portion, the library underwent standard melting-off SELEX as described, with the exception of the final condition: one portion was exposed to the matrix as the final condition (negative), one portion was exposed to the counter-targets in matrix (counter), and the other portion was exposed to the positive target in matrix (positive) as the final condition. Final-condition libraries were then recovered and purified on 10% denaturing PAGE. Bands represent library recovered for sequencing and analysis.

FIG. 3 is an overview of the bioinformatics and aptamer detection. In Step 1 , sequence data from the ultimate (i.e., post-parallel) round of selection are constructed into families with > 93% homology using the FASTAptamer algorithm. In step 2, families that are common to both the negative and positive final generation data sets are removed from the aptamer pool. In steps 3 and 4, the remaining sequence families are compared against the penultimate (i.e., parallel) generation families to determine enrichment rate - families with the highest enrichment rates are the more highly-ranked aptamers for further analysis.

FIG. 4A and FIG. 4B show the 1 X PBS validation of aptamers. After the melting-off validation protocol was complete, each aptamer portion was examined by denaturing PAGE. Successful aptamer validations display stronger bands in the positive (+) lane than in the negative (-) and counter (x) lanes. An image of the gel was taken after staining/destaining with Gel-Star (Lonza; Walkersville, MD). Lane 1 is 20/100 DNA Ladder (IDT; Coralville, IA). Distortions in gel due to saran wrap underlayer. FIG. 4A shows the gel image of the 1X PBS validation for the aptamers having SEQ ID NOs: 1 , 4, and 7. FIG. 4B shows the gel image of the 1X PBS validation for aptamers having SEQ ID NOs: 8 and 12.

FIG. 5A and FIG. 5B show the serum validation of aptamers. After the melting-off validation protocol was complete, each aptamer portion was examined by denaturing PAGE. Successful aptamer validations display stronger bands in the positive (+) lane than in the negative (-) and counter (x) lanes. An image of the gel was taken after staining/destaining with Gel-Star (Lonza; Walkersville, MD). Lane 1 is 20/100 DNA Ladder (IDT; Coralville, IA). Distortions in gel due to saran wrap underlayer. FIG. 5A shows the gel image of the 100% serum validation for aptamers having SEQ ID NOs: 1 , 4, and 7. FIG. 5B shows the gel image of the 100% serum validation for aptamers having SEQ ID NOs: 8 and 12.

FIG. 6A and FIG. 6B show the response and selectivity of the aptamer having SEQ ID NO: 12 at detecting carbamazepine. FIG. 6A shows the mean signal (measured as the normalized current) as a function of the concentration of carbamazepine in PBS. FIG. 6B shows the results of multiple runs with carbamazepine in PBS, as well as control experiments with oxcarbazepine and the gate.

FIG. 7 shows a general scheme for the method used for assessing aptamer-target affinity to characterize aptamers of the disclosure.

FIG. 8 shows the results from a microplate-based aptamer characterization experiment. The results are plotted as the normalized relative fluorescence units as a function of the nanomolar concentration of carbamazepine. The experiment demonstrated a Kd of 7.504 nM.

DETAILED DESCRIPTION

Disclosed herein are aptamers useful in determining the levels of small molecules in a sample, as well as methods of using the aptamers and devices containing the aptamers. The detection of analytes or molecules in samples is an important tool for a variety of applications. Quantitative analysis of analytes in samples often provides critical information for physicians and patients. One approach to measuring analytes involves assays that take advantage of the high specificity of antigen-antibody reactions. More specifically, an antigen or antibody may be detected in a sample based on binding between the antigen and an antibody on the assay, or vice versa. Alternatively, aptamers may be used. Aptamers are singlestranded oligonucleotides that bind with high affinity and high specificity to various targets ranging from various ions and small organic compounds to large proteins and live cells. Aptamers are advantageous to antibodies for several reasons, including (i) they are able to be produced using cell-free chemical synthesis, (ii) they exhibit low variability between batches, (iii) they are minimally immunogenic, and (iv) they are small in size. Point-of-care testing enhances the speed and accuracy of diagnoses, while also providing a reduction in turnaround time, cost, and dependence on sophisticated equipment skilled personnel. Aptamers

Aptamers are useful in determining the levels of a small molecule in a sample obtained from a subject. The aptamers may undergo a conformational change upon contacting the small molecule. This conformational change can be measured and/or detected. An exemplary method of measurement/detection is electrochemical measurement/detection. The aptamers may detect a small molecule that is a drug that has been administered to the subject, or an active moiety thereof, a metabolite thereof, or a biomarker therefor. For example, the small molecule may be a biomarker produced by the subject in response to the drug. Such biomarkers include molecules having positive or negative effects on the subject in response to administration of the drug. In particular, the aptamers may detect a small molecule that is used to treat a neurological disorder or a neuronal hyperexcitability disorder. More particularly, the aptamers may detect a drug that treats a seizure disorder, such as an epilepsy drug (e.g., carbamazepine).

Nucleic acid aptamers may be identified through a process referred to as systematic evolution of ligands by exponential enrichment (SELEX). The process begins with the synthesis of a large oligonucleotide library having randomly generated sequences of fixed length flanked by constant 5' and 3' ends that serve as primers. The sequences in the library are exposed to the target ligand (e.g., a protein or a small molecule). The aptamers that do not bind the target are removed, usually by affinity chromatography or target capture on magnetic beads. The bound sequences are eluted and amplified by PCR. Then, subsequent rounds of selection are carried out in which the stringency of the elution conditions can be increased to identify the tightest-binding sequences. The SELEX process and various modifications thereof have been discussed in, for example, US Patent Nos. 5,475,096; 5,580,737; 5, 683,867;5,696,249; 5,670,637; 5,763,177; 5,843,653; 5,858,660; 5,861 ,254; 6,110,900; 6,291 ,184; 6,331 ,398; 6,376,474; 6,387,620; 6,730,482; 6,855,496; 6,933,116; 8,367,627; 8,409,795; the disclosure of each of which are incorporated herein by reference. FIG. 1 provides a graphical representation of the melting-off SELEX approach used to identify the aptamers of the invention.

The aptamers used herein may be structure-switching aptamers specific against a small molecule. This may allow for the qualitative detection of the small molecule in a sample and/or the quantitative analysis of the levels of the small molecule in the sample, for example by using one of the devices described herein. Nucleic acid aptamers have the advantage of being highly selective and specific against a particular target due to the SELEX procedure used to generate the aptamers.

Quantification of the levels of a small molecule in a sample (e.g., a blood sample) obtained from a subject may be useful for determining a treatment regimen of a drug. Testing of a sample may allow for comparison to known therapeutic levels of circulating drugs. The dosing regimen may be adjusted up or down accordingly based on this determination. The testing may also be useful for determining the half-life of a drug, by detection of the levels of the drug or of a metabolite thereof.

Aptamers may be useful for the detection of a drug that is used to treat a neurological or neuromuscular disorder or a neuronal hyperexcitability disorder. Exemplary neurological or neuromuscular disorders include Alzheimer's Disease, amyotrophic lateral sclerosis (ALS), ataxia, Bell's Palsy, brain tumors, cerebral aneurysm, epilepsy and seizures, Guillain-Barre Syndrome, headache (e.g., cluster headaches, tension headaches, migraine headaches), hydrocephalus, meningitis, multiple sclerosis, muscular dystrophy, neurocutaneous syndromes, Parkinson's Disease, stroke, and encephalitis.

Aptamers may be specific to drugs that treat a seizure disorder (e.g., epilepsy). Subjects being treated with anti-seizure medication may require frequent monitoring ofthe circulating levels of these drugs. If the levels of the drug are too low, the subject is at risk of seizure. If the levels of the drug are too high, the subject is at risk of adverse effects. The aptamers can bind to anti-seizure or anti-epilepsy medications and undergo a conformational change. The high selectivity and ease of detecting the conformational change in the aptamers allow for use in devices used at the point-of-care that produce rapid results.

Aptamers of the invention may be unmodified or may include one or more modified nucleobases. Unmodified nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5- halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5-trifluoromethyl uracil, 5-trifluoromethyl cytosine, 7- methyl guanine, 7-methyl adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3- deazaguanine, 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e g., 5-substituted pyrimidines; 6-azapyrimidines; N2-, N6-, and/or 06-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2- thiothymine and 2-thiocytosine, 5-propynyl ( — CEC — CH3) uracil, 5-propynylcytosine, 6-azouracil, 6- azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5- halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7- deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N- isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N- benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1 ,3- diazaphenoxazine-2-one, 1 ,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1 ,3-diazaphenoxazine-2- one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deazaadenine, 7-deazaguanine, 2-aminopyridine and 2- pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991 , 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T„ Ed., CRC Press, 2008, 163-166 and 442-443. Aptamers of the invention may be unmodified or may include one or more sugar modifications in nucleosides. Nucleosides having an unmodified sugar include a sugar moiety that is a furanose ring as found in ribonucleosides and 2’-deoxyribonucleosides.

Sugars included in the nucleosides of the invention may be non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring (e.g., a six-membered ring). Alternative sugars may also include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, e.g., a morpholino or hexitol ring system. Non-limiting examples of sugar moieties useful that may be included in the oligonucleotides of the invention include p-D-ribose, p-D-2'-deoxyribose, substituted sugars (e.g., 2', 5', and bis substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose, and 4'-S-2'-substituted ribose), bridged sugars (e.g., the 2'-O — CH2-4' or 2'-0 — (CH2)2-4' bridged ribose derived bicyclic sugars) and sugar surrogates (when the ribose ring has been replaced with a morpholino or a hexitol ring system).

Typically, a sugar modification may be, e.g., a 2’-substitution, locking, carbocyclization, or unlocking. A 2’-substitution is a replacement of 2’-hydroxyl in ribofuranose with 2’-fluoro, 2’-methoxy, or 2’- (2-methoxy)ethoxy. A locking modification is an incorporation of a bridge between 4’-carbon atom and 2’- carbon atom of ribofuranose. Nucleosides having a sugar with a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides.

Aptamers of the invention may be unmodified or may include one or more internucleoside linkage modifications. The two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidate. Nonlimiting examples of non-phosphorus internucleoside linkages include methylenemethylimino ( — CH2 — N(CH3) — O — CH2 — ), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ), siloxane ( — O — Si(H)2 — O — ), and N,N'-dimethylhydrazine ( — CH2 — N(CH3) — N(CH3) — ). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are known in the art.

Internucleoside linkages may be stereochemically enriched. For example, phosphorothioate- based internucleoside linkages (e.g., phosphorothioate diester or phosphorothioate triester) may be stereochemically enriched. The stereochemically enriched internucleoside linkages including a stereogenic phosphorus are typically designated Sp or Rp to identify the absolute stereochemistry of the phosphorus atom. Within an oligonucleotide, Sp phosphorothioate indicates the following structure:

Within an oligonucleotide, Rp phosphorothioate indicates the following structure: The oligonucleotides of the invention may include one or more neutral internucleoside linkages. Non-limiting examples of neutral internucleoside linkages include phosphotriesters, phospho roth ioate triesters, methylphosphonates, methylenemethylimino (5'-CH2 — N(CHs) — 0-3’), amide-3 (5'-CH2 — C(=O) — N(H)-3’), amide-4 (5'-CH ₂ — N(H)— C(=O)-3’), formacetal (5'-O— CH ₂ — 0-3’), and thioformacetal (5'-S— CH2 — 0-3’). Further neutral internucleoside linkages include nonionic linkages including siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester, and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65).

Aptamers of the invention may include a terminal modification, e.g., a 5’-terminal modification or a 3’-terminal modification.

The 5’ end of an oligonucleotide may be, e.g., hydroxyl, a thiol moiety, hydrophobic moiety, a targeting moiety, 5’ cap, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, diphosphrodithioate, triphosphorodithioate, phosphonate, phosphoramidate, or a neutral organic polymer. An unmodified 5’-terminus is hydroxyl or phosphate. An oligonucleotide having a 5’ terminus other than 5’-hydroxyl or 5’-phosphate has a modified 5’ terminus.

The 3’ end of an oligonucleotide may be, e.g., hydroxyl, a thiol moiety, a targeting moiety, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, or a neutral organic polymer (e.g., polyethylene glycol). An unmodified 3’-terminus is hydroxyl or phosphate. An oligonucleotide having a 3’ terminus other than 3’-hydroxyl or 3’-phosphate has a modified 3’ terminus.

Oligonucleotides of the disclosure may include thiol moieties, which may be conjugated at the 5’- end or 3’-end of the oligonucleotide or at some point in between (e.g., at an internucleoside linkage). Thiols may be incorporated into the oligonucleotides to allow for binding of the oligonucleotide to a surface. The thiol moiety may be incorporated in the oxidized disulfide form, allowing for subsequent reduction to reveal the free thiol when necessary. These free thiols may be useful for various processes (e.g., adsorption of an oligonucleotide onto an electrode). The thiol moiety may additionally include a linker or a spacer between the oligonucleotide, which may be a substituted or unsubstituted alkyl chain. Nonlimiting examples of thiol moieties include the following:

Aptamers of the invention may also include any combination of a modified sugar, base, internucleoside linkage, terminal modifications, and thiols.

Aptamers of the invention may form secondary structures that are known to bind hydrophobic moieties. Such secondary structures may be beneficial for the binding of small molecules (e.g., antiseizure medications, such as carbamazepine). For example, the aptamers may form a three-junction structure (Yang et al, Journal of the American Chemical Society, 2012, 134:1642-1647). Alternatively, aptamers may form a G-quadruplex secondary structure.

A “G-quadruplex” (also known as a G-tetrad or G4-DNA) is a nucleic acid structure formed from a sequence that is guanine-rich and thus capable of forming a square arrangement of guanines (a tetrad), which is stabilized by Hoogsteen hydrogen bonding and further stabilized by the existence of a monovalent cation (especially potassium) in the center of the tetrads. The SELEX technique for generating aptamers can be used to generate sequence variants that produce variants of the G-quadruplex structure.

The G-quadruplex domain has a sequence capable of forming at least two layers of G-tetrads that will form a G-quadruplex. In various embodiments the G-quadruplex domain can comprise a sequence capable of forming 2, 3, 4, 5, 6 or more G-tetrad layers, preferably two or three G-tetrad layers. The G- quadruplex domain sequence may include about 8 to about 24 guanines as four pairs (gg), triplets (ggg), or quadruplets (gggg), wherein the pairs, triplets or quadruplets are separated by at least one intervening base, which may be any nucleotide base.

G-rich aptamers that form G-quadruplex have several advantages compared with unstructured sequences. They are thermodynamically and chemically stable, show no immunogenicity, are resistant to numerous serum nucleases, and have enhanced cellular uptake. The stability of the G-quadruplex structure improves electrostatic interactions with the positively charged binding ligands because its structure has twice negatively charged density per unit length compared to the duplex DNA.

G-quadruplex domains include loops regions between members of the tetrads. The loops may include CO and/or CA motifs.

Aptamers may also include regions for binding primers for replication, e.g., at the 3’ and 5’ ends. Primer binding regions may be 10-30 nucleotides in length and may differ at each end. Primer binding sites may also be complementary to one another or include a complementary portion thereof, e.g., 6-12 nucleotides in length. Thus, aptamers may include a hairpin stem.

The aptamers may be particularly useful in detecting drugs used to treat seizures due to epilepsy, including brivaracetam, cannabidiol, carbamazepine, cenobamate, clobazam, clonazepam, diazepam, divalproex, eslicarbazepine acetate, ethosuximide, felbamate, fenfluramine, gabapentin, lacosamide, lamotrigine, levetiracetam, lorazepam, midazolam, oxcarbazepine, perampanel, phenobarbital, phenytoin, pregabalin, primidone, rufinamide, stiripentol, tiagabine hydrochloride, topiramate, valproic acid, vigabatrin, and zonisamide.

Even more particularly, the aptamers may be specific to the epilepsy drug carbamazepine. These aptamers may be advantageously selective against carbamazepine without responding to its metabolite carbamazepine-10,11 -epoxide or the closely related drug oxcarbazepine. carbamazepine oxcarbazepine

In some embodiments, the aptamer used in the device is selective for carbamazepine over oxcarbazepine. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times, e.g., between 3 and 50 times, 50 and 100 times, or 100 and 1000 times) as much as oxcarbazepine as determined by dissociation constant. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times, e.g., between 3 and 50 times, 50 and 100 times, or 100 and 1000 times) as much as oxcarbazepine as determined by an output from a device designed to measure a conformational change in the aptamer. In some embodiments, the aptamer used in the device is selective for carbamazepine over carbamazepine-10,11-epoxide. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times, e.g., between 3 and 50 times, 50 and 100 times, or 100 and 1000 times) as much as carbamazepine-10,11-epoxide as determined by dissociation constant. In some embodiments, the aptamer binds to carbamazepine at least 3 times (e.g., at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times, e.g., between 3 and 50 times, 50 and 100 times, or 100 and 1000 times) as much as carbamazepine-10,11-epoxide as determined by an output from a device designed to measure a conformational change in the aptamer. In some embodiments, the aptamer used in the device is selective for carbamazepine over both oxcarbazepine and carbamazepine-10,11-epoxide.

For detecting carbamazepine, the aptamers may have at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the sequences set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 75%. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 80%. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 85%. In some embodiments, the percent sequence identity to a sequence es set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 90%. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 95%. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 99%. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is 100%.

Table 1

The aptamers in Table 1 include an 18 and a 25 nucleotide long primer binding region at the ends. In certain embodiments, an aptamer may include different primer binding regions. The aptamers may have at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) sequence identity to the sequences set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, excluding the primer binding regions. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 75%. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 80%, excluding the primer binding regions. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1 -14 or 1 , 4, 7, 8, or 12, is at least 85%, excluding the primer binding regions. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 90%, excluding the primer binding regions. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 95%, excluding the primer binding regions. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1-14 or 1 , 4, 7, 8, or 12, is at least 99%, excluding the primer binding regions. In some embodiments, the percent sequence identity to a sequence set forth in Table 1 , e.g., SEQ ID Nos: 1 -14 or 1 , 4, 7, 8, or 12, is 100%, excluding the primer binding regions.

Devices

An aptamer may undergo a conformational change upon contacting a small molecule. The present disclosure also includes devices that may be used to measure and/or detect this conformational change resulting from binding a small molecule. The devices may be useful for qualitatively determining the presence or absence of the small molecule. The devices also may be useful for quantitative analysis of the levels of the small molecule in a sample. The devices may be used at the point-of-care or in a laboratory setting. Particularly advantageous devices are used at the point-of-care during the course of a routine office visit. In general, the devices contain an aptamer immobilized on a surface allowing for the detection of a conformational change in the aptamer.

The devices contain a means of detecting a conformational change in the aptamer. For example, the device may be in the form of an optical biosensor. An optical biosensor contains a biorecognition element, e.g., an aptamer. Upon contacting an external stimulus (such as a target small molecule), the aptamer may undergo a conformational change that causes an optical signal to be generated. This signal can be detected by using, e.g., colorimetric, fluorescent, or luminescent methods. Alternatively, the device may be in the form of an electrochemical biosensor. In such a device, an aptamer is immobilized on the surface of an electrode. The aptamer may be immobilized on the electrode at the 5’-end or the 3’-end or at some point in between (e.g., at an internucleoside linkage). The electrode may be, for example, a gold electrode. The attachment can occur through chemical adsorption of a thiol group onto the gold electrode. Upon contacting an external stimulus (such as the presence of a target small molecule), the aptamer may undergo structural or conformational changes leading to an alteration of the electrochemical signal. The device may provide a readout of the normalized current.

Devices may be capable of detecting more than one small molecule target. The device may contain a single means for detection that can be used with different aptamers. The different aptamers may be in the form of a “cartridge” that can be inserted to the device, allowing a single device to be used with multiple aptamers.

A possible method of using the device involves generating a standard curve, wherein known concentrations of the target small molecule are measured (for example, by measuring the normalized current). These values may then be plotted, and a regression analysis conducted. This allows for quantification of resulting samples based on the regression model. These devices may be advantageous as they can be used at the point-of-care and have reduced time to produce a result than traditional laboratory detection methods. For example, in seizure disorders (e.g., epilepsy) the monitoring of the levels of drugs is critical, as levels below the therapeutic range put the subject at risk for seizure, while levels above the therapeutic range put the subject at risk for adverse events. Methods known in the art for monitoring the levels of drugs in patients with seizure disorders require a venous blood draw by a trained phlebotomist, and the samples must be processed in a laboratory setting. This can therefore require days to receive results. The devices disclosed herein are useful for generating data on a small amount of sample (e.g., a drop of blood from a fingerstick) and providing an output within minutes or hours. This can be useful both for routine monitoring and in emergency situations.

Samples

Samples may be any type of composition obtained from a subject that can be contacted with an aptamer described herein. Samples may include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, interstitial fluid, lacrimal fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, stool, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, cellular extracts, and combinations thereof.

In some embodiments, the sample is a blood sample (e.g., serum, plasma, or whole blood). A sample of whole blood may be venous blood or capillary blood. A sample of venous blood may be obtained, for example, by way of a blood draw by a phlebotomist. A sample of capillary blood may be obtained, for example, by way of a fingerstick. Use of a fingerstick is advantageous, as it allows for the detection of a small molecule with a small amount of blood (i.e., one drop).

Methods of the Invention

The invention provides methods of using the aptamers and devices described herein. A method of the invention may be a method of determining the levels of a small molecule in a subject, including the steps of contacting a sample with an aptamer to induce a conformational change in the aptamer if the small molecule is present; measuring the conformational change in the aptamer; and determining the level of the small molecule from the measuring.

The measuring of the conformational change may be through optical methods (e.g., colorimetric, fluorescent, or luminescent methods). Alternatively, the measuring may be through electrochemical methods, such as by measuring the normalized current. The measuring may be by a device described herein.

The measuring of the levels of a small molecule in subject may be useful for therapeutic monitoring of a drug or an active moiety thereof, a metabolite thereof, or a biomarker therefor. The levels of the small molecule can be compared to known therapeutic windows. If the level of the small molecule is below the known therapeutic window, the dose of the drug can be increased, or the drug can be administered more often, or both. If the level of the small molecule is above the known therapeutic window, the dose of the drug can be decreased, or the drug can be administered less often, or both. Alternatively, treatment with the drug can be ceased. The dosage and therapeutic window of small molecules can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the subject to be treated. One of skill in the art can determine the appropriate dosage and modifications to the dosage based on the above factors.

The therapeutic window for many anti-seizure medications is very narrow, and epilepsy patients therefore risk breakthrough seizures or significant drug-related side-effects when AED blood levels are too low or high, respectively. For example, the conventionally accepted therapeutic plasma concentrations in adult humans is 4-12 pg/mL (Greenberg et al., Clin. Neuropharmacol., 2016, 39:232-240). The methods of the invention may be useful in therapeutic monitoring of carbamazepine.

EXAMPLES

The following materials, methods, and examples are illustrative only and are not intended to be limiting.

Materials and Methods

Pooled human serum was purchased from Valley Biomedical (Winchester, VA), divided into 400 pL aliquots, and stored at -40°C. Target carbamazepine and counter-target carbamazepine-10,11 -epoxide were purchased from Sigma-Aldrich, St. Louis, MO. A second counter-target, oxcarbazepine, was originally purchased from Selleck Chemicals, LLC (Houston, TX). PBS and PBS-T, to be used as components of the library re-folding and capture buffer, were purchased from American Bioanalytical (Natrick, MA). DEPC-treated nuclease-free water was purchased from Amresco (Solon, OH). MPC protein precipitation reagent for post-selection isolation of the library was purchased from Lucigen Corp (Middleton, Wl). Pall Nanosep® 10K Omega spin filters were purchased from the Pall Corporation (Port Washington, New York).

The initial aptamer library template and primers were synthesized as single-stranded DNA. The DNA library was designed with a central random region flanked by two constant sites that should form a stable stem upon target binding. An additional biotinylated “capture” oligo complementary to the 5’ stemforming site of the library was synthesized by IDT. The reverse (antisense) primer contains poly(dA) sequence and PEG spacer (hexaethylene glycol) to generate single-stranded library by denaturing PAGE purification after PCR amplification. A fluorescein-tagged primer was also synthesized by IDT to enable enrichment-tracking over the course of selection. All synthesized material underwent desalting purification. Oligonucleotides were reconstituted in nuclease-free water for preliminary analysis and aptamer screening. MyOne Streptavidin T1 Dynabeads™ (10 mg/mL), to be used as a partitioning medium, were purchased from Life Technologies Corporation (typically 1 pmole of biotinylated material is used with every 20 pg of Dynabead™) (Carlsbad, CA).

The hairpin structure of the library provides additional stability in lieu of primer-blocking oligonucleotides. During selection, the library was amplified using Titanium Taq DNA polymerase from Clontech (Mountain View, CA) using 2-step PCR (10 seconds at 95°C, 30 seconds at 60°C, with initial HotStart activation of 60 seconds at 95°C) and purified on denaturing polyacrylamide with 8 M urea (Sequel NE Reagent, Part A and Part B), which was purchased from American Bioanalytical (Natick, MA). Gel elution buffer for 4 °C overnight post-purification library recovery was prepared to 0.5 M NH4OAc, 1 mM EDTA (both purchased from Teknova, Hollister, CA), 0.2% SDS (purchased from Amresco), pH 7.4.

Example 1. Screening of Aptamers

Library screening was conducted using a Melting-Off approach (FIG. 1). The goal was to select sequences that did not interact with the counter-targets or serum matrix, but specifically bound to carbamazepine. At the beginning of a selection round, an aliquot of Dynabeads (amount varied depending on library used and stringency required) was pre-washed three times with 200 pL PBS-T (final concentration of 0.01% Tween 20, pH 7.4) wash buffer, to reduce non-specific binding of the library once it was prepared. In addition, one aliquot each of the target and counter-targets were prepared. Target and counter-targets were prepared in 1X PBS buffer for the first three rounds of selection, and 100% pooled human serum in all subsequent rounds. Next, a given amount of the library was refolded in 1X PBS buffer, pH 7.4, (1 -minute denaturing at 90°C, 5-minute annealing at 60°C, then 5 minutes at 23°C) with twice the library molar amount of the capture oligo. This is done to allow the library to be captured by the Dynabeads through a streptavid in-biotin binding interaction on the capture probe. After refolding, the library was incubated with the Dynabeads on a shaker for 15 minutes at room temperature. The Dynabeads were separated from solution by incubation at 23°C for three minutes on a magnetic stand, and removal of the supernatant by pipette. The Dynabeads were then washed with 200 pL 1X PBS (pH 7.4) to eliminate any remaining PBS-T and non-specifically bound library species, after which the samples were again magnetically separated, and the supernatant was discarded. Next, the Dynabeads underwent a counter selection incubation in 200 pL of the counter-target preparation at 23°C for 60 minutes. This length of time was sufficient to allow library members to bind to the counter-targets and (in later rounds) the serum components, releasing the non-specific library members from the Dynabeads. Non-specific library members were then discarded. After the counter incubation, the Dynabeads were washed six times with 200 pL of 1X PBS buffer over the course of an hour (7 minutes in 1X PBS buffer, 3 minutes to magnetically separate, supernatant was discarded, and the process repeated) to reduce the probability of counter-target and serum carryover. The washes also help to eliminate any non-specific library members that may survive the counter incubation so that they cannot respond during the positive selection step.

Positive selection was conducted after the completion of the wash steps and included incubating the library-bound Dynabeads with 200 pL positive target preparation at 23°C for 30 minutes. After incubation, the Dynabeads were magnetically partitioned and the supernatant — containing library members that responded to target — was recovered. The supernatant then underwent a second magnetic separation in order to ensure that the Dynabeads had been completely removed. The recovered library underwent heat denaturation and protein precipitation with MPC reagent, followed by ethanol precipitation to desalt and concentrate it, and was then purified by 10% denaturing PAGE with 8 M urea.

Library responses were measured using a Nanodrop 1000 spectrophotometer (260 nm) (Thermo Fisher Scientific; Wilmington, DE) after purification to monitor enrichment by determining how much library made it through a round of selection. Recovered library amounts calculated from A260 NanoDrop readings were compared to input library amounts from the beginning of selection rounds, then amplified using PCR with Titanium Taq DNA polymerase. Amplification products were then purified by 10% denaturing polyacrylamide gel electrophoresis (PAGE) with 8 M urea. Gel slices were excised, eluted overnight at 4°C in gel elution buffer, and quantified for the next round of selection using A260 as measured by NanoDrop- 1000.

For parallel assessments (FIG. 2), the library to be assessed was divided into three equal portions for refolding as above. Three aliquots of Dynabeads were also pre-washed as before, and additional aliquots of counter-targets and targets were prepared as appropriate. Each portion was then run through the standard SELEX protocol as described above but with the following change: one portion was incubated in 100% serum in place of a positive incubation, one portion was incubated in counter-target spiked serum, while the last portion was incubated with the positive target spiked serum as normal. Responsive library members from each assessment condition were collected as described above and then analyzed on 10% denaturing PAGE with 8 M urea then recovered and used for the post-parallel round. An additional positive control was optionally added to the parallel assessment. In this positive control, target carbamazepine was spiked into 100% serum to a final concentration of 100 nM and allowed to incubate for 30 minutes at room temperature. The serum was then filtered through a 10 kDa molecular weight cut-off filter by centrifugation according to manufacturer recommendations. The filtered serum should contain small proteins and free target, with minimal target-serum complexes. This control should represent library binding of free carbamazepine in a serum-based solution.

For the post-parallel round, a crossover analysis was conducted. The post-parallel round of selection is designed to maximize the difference between the response of each library to its opposing condition, and thereby reduce the complexity of identifying good aptamers during bioinformatics. The protocol was similar to the parallel assessment, with the following exception: the negative and counter libraries were incubated with the positive target spiked serum during the “counter” incubation step, while the positive library underwent the “counter” incubation step with the counter-targets as normal. Otherwise, each library was then run through the standard parallel assessment protocol as described above: negative library against negative serum, counter library against counter-targets in serum, positive library against positive target in serum. Responsive library members from each assessment condition were collected and then analyzed on 10% denaturing PAGE with 8 M urea, then recovered and used for sequencing and bioinformatics.

The low library recovery seen in the Round 1 selection is typical, a result of collecting sequences that bind to carbamazepine from over 1014 possible species. The following two rounds used decreasing concentrations of target down to 100 nM in order to select for aptamers with high affinity towards carbamazepine. The first three rounds of SELEX used simple 1X PBS buffer as a selection matrix, rather than serum. This is because there are numerous possible interactions between a hydrophilic nucleic acid library and serum components, and many of those interactions heavily favored over the interaction between a library and a small hydrophobic target like carbamazepine. In order to ensure that the library was enriching towards the carbamazepine and not to the serum matrix, the carbamazepine was kept isolated until sufficient library binding was observed. Once it was observed that the library response to carbamazepine remained stable despite a 10-fold drop in the target concentration in round 3, the library was deemed sufficiently enriched to handle the inclusion of serum. From round 4 onwards, serum was used as the matrix during the counter and positive incubations, with 1X PBS buffer used to prepare the library, and to conduct washes. After observing a plateau in the library’s response between rounds 5 and 6, it was decided to conduct a parallel assessment of the library’s performance to determine whether or not it continued to enrich specifically towards carbamazepine. Additional wash steps with 100% serum were conducted between the 1X PBS washes from Round 5 onward to reduce the probability of library response to serum proteins. The parallel assessment produced strong results, with a significantly greater response to 100 nM carbamazepine than to serum alone, or to serum spiked with counter-targets (to a final concentration of 10 pM). As a result, the library was deemed sufficiently enriched to proceed to a crossover fitness test, followed by sequencing and bioinformatics.

Example 2. Sequencing

The Illumina (San Diego, CA) MiniSeq system was implemented to sequence the aptamer libraries after the post-parallel selection using a single-end read technique. Libraries recovered from the cross-over fitness test were prepared by using a PCR protocol to attach library-specific sequencing adaptors and index primers. Constructs were purified using 10% denaturing PAGE. Prepared libraries were then pooled and diluted to a final 1 .5 pM concentration for use with an Illumina MiniSeq Mid Output Kit. Deep sequencing and subsequent data analysis reduces the traditional approach of performing a large number of screening rounds, which may introduce error and bias due to the screening process (Schutze et al., PLoS ONE, 6, e29604, 2011). Hundreds of thousands of sequences were analyzed from the parallel- exposed final libraries. From these sets of data, sequence families were constructed at 93% homology in their variable regions — i.e., the constant primer sites were excluded from homology considerations — and examines sequence similarity with consideration for mutations, deletions, and insertions. Aptamers were identified from these processed sets of data.

The initial library was subjected to 6 rounds of Melting-Off selection followed by 2 rounds of parallel selections. The SELEX process is designed to enrich for sequences over multiple rounds of selection that bind to the given targets of interest, in this case carbamazepine, and remove sequences that bind to oxcarbazepine, carbamazepine-10,11-epoxide, or serum components. As a result, the population to be sequenced is expected to contain multiple copies of potential aptamers.

Example 3. Validation and Bioinformatics

Each aptamer was split into three equal samples then captured onto Dynabeads™ with a biotinylated capture probe according to the exact same protocol described in the initial report. The beads were then washed briefly (three times with 1X PBS, for a total wash time of 15 minutes) and then incubated in negative, counter, and positive conditions for 30 minutes at room temperature. Eluted aptamers were then concentrated using ethanol precipitated and run on a denaturing polyacrylamide gel. In simple buffer systems such as 1X PBS, where an optimal protocol for generating a high signal-to-noise ratio is easy to identify qualitatively, successful aptamers will ideally display bands in the positive lanes and not display bands in the negative and counter lanes. In more complex buffer systems, a larger variety of factors may influence the desorption of the aptamers and their successful recovery. These factors include protein binding to the Dynabeads™ causing displacement of the aptamer, potential variation in pH or salt conditions that may disrupt aptamer structure, etc. Recovery of the aptamer from the matrix also affects the efficiency of the assay, as some aptamer material will be lost during the recovery process. Taken together, these factors require a slightly more stringent assay to address, and so the optimized testing in 100% serum required six washes over the period of 30 minutes to reduce background signals.

The overall bioinformatics procedure is summarized in FIG. 3. Example 4. Secondary Structure of Aptamers.

Aptamers were investigated for secondary structures known to bind to hydrophobic molecules; namely, a G-quadruplex structure. The G-Quadruplex motif is readily observable by eye — a sequence comprised of four guanosine repeats separated by ‘loop’ regions.

53 aptamer sequences were flagged (see Table 2). Aptamers were examined using the QGRS Mapper software (Kikin et al., Nucleic Acid Research, 2006, 34:W676-W682) to confirm the likelihood of G- Quadruplex formation via a G-Score produced by the algorithm.

After confirming that aptamers were likely to form G-Quadruplex structures, the loop regions of the G-Quadruplexes were examined to determine if conserved motifs existed and the aptamer candidates possessed multiple conserved loops. For example, it was observed that particular aptamers possess a CA or CO loop.

Table 2

Example 5. Validation of Aptamers

Aptamer validation was carried out using a low-stringency melting-off approach based on the SELEX experiments used to enrich the library as described above. Five aptamers (SEQ ID NOs: 1 , 4, 7, 8, and 12) were tested first in 1X PBS buffer to ensure they met the requirement of binding to carbamazepine free in solution (FIG. 4). From this validation, it appears that each of the five aptamers displays specific binding to carbamazepine in 1X PBS with varying degrees of signal intensity. In particular, the aptamer having SEQ ID NO: 4 (FIG. 4A) and SEQ ID NO: 12 (FIG. 4B) displayed high signal intensities, while SEQ ID NO: 7 (FIG. 4A) had very low signal intensity. Upon confirming specific carbamazepine binding in 1X PBS, the aptamers were subsequently tested using 100% serum as the matrix, with target and countertargets spiked in. The initial run produced high background signal intensities due to the shortened washing protocol (data not shown), so a repeat was conducted using a higher stringency wash protocol (FIG. 5). While the serum-containing assay did still display a higher background signal in the negative and counter lanes than the 1X PBS assay due to complicating influences of the matrix, the control signals appear notably weaker than the positive signal for each aptamer. Additionally, the lanes containing the counter- responsive sample do not appear stronger than the negative serum lanes, indicating that a more developed assay/system should be able to distinguish free carbamazepine specifically with minimal background.

Example 6. Response and Selectivity

The aptamer having SEQ ID NO: 12 was tested for its response and selectivity against carbamazepine (FIG. 6). The first graph shows the measurements of the normalized current across multiple experiments. The graph demonstrates that the aptamer produces a dose-dependent response to the presence of carbamazepine in PBS as measured by the normalized current. Additionally, the second graph shows the results of multiple runs with carbamazepine. This graph also shows that the aptamer is selective to carbamazepine and does not respond to the presence of the structurally similar drug oxcarbazepine, even at high concentrations. Example 7. Assessment of Aptamer-Target Affinity

FIG. 7 shows a general scheme for assessing aptamer-target affinity used to characterize an aptamer having SEQ ID NO: 12. Assessment followed a similar method to that used in Example 1 . Aptamers were individually prepared by combining aptamer (SEQ ID NO: 12) and a Capture Oligo having the sequence GTCGTCCCGAGAGCCATA (SEQ ID NO: 54), which was attached at the 3’ end to a triethylene glycol linked biotin in 1X SELEX Buffer. Each test sample contained 0.5 pM aptamer and 1 pM Capture Oligo in 50 pL. Control samples were also prepared containing aptamer only, Capture Oligo only, or 1X PBS only. Samples were mixed well before refolding (90°C for 5 minutes, 60°C for 5 minutes, 23°C for 5 minutes). While samples were refolding, Streptavidin T1 Dynabeads™ (10 mg/mL) were washed 3 times using 1X PBS-T.

Refolded material was then incubated for 15 minutes at room temperature with Streptavidin T1 Dynabeads™ such that 1 pmole of Capture Oligo (or 0.5 pmole of aptamer) was combined with 1 pL of Dynabeads. After incubation, each sample of bead-bound material was washed 4 times using 1X PBS. Washed beads were then incubated for 30 minutes at room temperature with various concentrations of target carbamazepine (0 nM, 8 nM, 16 nM, 32 nM, 64 nM, 128 nM, and 256 nM).

Samples were then magnetically separated, and the supernatant (containing eluted aptamer) was transferred to separate wells of a black-walled black bottom Maxisorp 96-well microplate. To ensure accurate and consistent reads, transferred samples were combined with an equal volume (50 pL) of 1X PBS to bring the total read volume to 100 pL. Optimal results were obtained after 60 seconds of shaking, followed by 6 flashes of excitation at 651 nm and a top read of 670 nm. Fluorescence values from triplicate samples were normalized, then analyzed in GraphPad using a One Site -- Total Binding model.

An aptamer having SEQ ID NO: 12 responded to various concentrations of target as described earlier, and signal was read at 670 nm via a fluorescence plate reader. The results are shown in FIG. 8. Collected data were fit to a one-site binding model for affinity determination (N = 3). Surprisingly, the aptamer of SEQ ID NO: 12 demonstrated a Kd of 7.504 nM against target carbamazepine. This is an unexpected and beneficial result, as aptamer affinity for small targets is generally weaker (Kd closer to 1 pM). In conjunction with previous experiments, this aptamer demonstrates particularly high affinity for its target.

OTHER EMBODIMENTS

Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims.