Nucleic acid sequences responsive to vancomycin and other target molecules

Abstract

The present disclosure provides methods for modifying an aptamer to change the affinity of a modified aptamer to a ligand or produce a modified aptamer having a desired half-point of response value. Also disclosed are modified aptamers produced by the methods disclosed herein, and aptamers that bind to specific ligands or target molecules. Additionally provided are electrochemical sensors comprising one or more of the disclosed aptamers.

Description

[0001] TITLE OF INVENTION

[0002] NUCLEIC ACID SEQUENCES RESPONSIVE TO VANCOMYCIN AND OTHER TARGET MOLECULES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims the benefit of US Provisional Patent Application Serial Numbers 63 / 613,496 filed 21 December 2023, 63 / 614,115 filed 22 December 2023, and 63 / 551 ,336 filed 08 February 2024; each of which is incorporated herein by reference in its entirety for all purposes.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under grants GM 138843, GM1 04960, and DK126739 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] MATERIAL INCORPORATED-BY-REFERENCE

[0008] The sequence listing that is contained in the file named Columbia_88800730- 505WO_SequenceListing.xml,” which is 135 kilobytes as measured in Microsoft Windows operating system and was created on 17 December 2024 is filed electronically herewith and incorporated herein by reference.

[0009] BACKGROUND OF THE INVENTION

[0010] Aptamers that recognize small molecules are selected from vast random libraries of oligonucleotides through affinity-based enrichment-amplification cycles. An important advantage of aptamers over antibodies is in the ability to adapt them to a variety of novel biosensor formats while relying on no more than partial knowledge of their presumed secondary structures. For biosensors to be useful, however, the properties of underlying aptamers need to match practical requirements, which raises an issue of adjusting affinities of aptamers to intended applications. Take, for example, a vancomycin aptamer that was previously reported (Dauphin- Ducharme, et al., ACS Sens. 4:2832, 2019) (SEQ ID NO:1 ). It has an affinity of about 110 nM, but in its reported sensor form, it has the half-point of response of about 40 pM, which makes it too high, that is, unsuitable for quantification of a lower range of concentration in blood or in diluted fluids (e.g., effluent fluid in dialysis).

[0011] Therefore a method of tuning the response of aptamer-based sensors, and the aptamers thus produced, would represent a tremendous advance in the field.

[0012] SUMMARY OF THE INVENTION

[0013] Provided herein are methods for tuning the response of aptamer-based sensors using structural engineering, aptamers produced by these methods, aptamers that bind to specific target molecules, and sensors comprising the aptamers. The methods generally comprise engineering at least one of the stem length, loop structure, or body of an aptamer to achieve, through a screening process, a half-point that matches what is needed for both fluorescence and electrochemical sensors. Also provided are methods for therapeutic drug monitoring.

[0014] The present disclosure provides a method for identifying an aptamer having a desired half-point of response value, the aptamer comprising an internal loop, an external loop, a first stem between the internal loop and the external loop, and a second stem connected to the internal loop, the method comprising: a) modifying at least one of the external loop, first stem or second stem of each of a plurality of aptamers to create a plurality of modified aptamers; b) screening at least a portion of the plurality of modified aptamers to determine the half-point of response of the at least a portion of the modified aptamers; and c) selecting a modified aptamer from the plurality of aptamers with the desired half-point of response. In certain embodiments the first end and second end of the second stem of the aptamer are labeled. In some embodiments the aptamer is complexed with a redox label dye. In further embodiments binding of a ligand to the aptamer displaces the redox label dye.

[0015] The present disclosure also provides an aptamer having a desired half-point of response value, the aptamer comprising an internal loop, an external loop, a first stem between the internal loop and the external loop, and a second stem connected to the internal loop, identified by a method comprising: a) modifying at least one of the external loop, first stem or second stem of each of a plurality of aptamers to create a plurality of modified aptamers; b) screening at least a portion of the plurality of modified aptamers to determine the half-point of response of the at least a portion of the modified aptamers; and c) selecting a modified aptamer from the plurality of aptamers with the desired half-point of response.

[0016] The present disclosure additionally provides a method for modifying the affinity of an aptamer to a ligand, the aptamer comprising an internal loop, an external loop, a first stem between the internal loop and the external loop, and a second stem connected to the internal loop, the method comprising: a) modifying at least one of the external loop, first stem or second stem of the aptamer to create a modified aptamer; and b) screening the modified aptamer to determine the affinity of the modified aptamer for the ligand, thereby generating a modified aptamer having a different affinity for the ligand than the aptamer.

[0017] Furthermore, the present disclosure provides an aptamer having a modified affinity for a ligand compared to a starting aptamer, produced by a method comprising: a) modifying at least one of the external loop, first stem or second stem of the aptamer to create a modified aptamer; and b) screening the modified aptamer to determine the affinity of the modified aptamer for the ligand, thereby generating a modified aptamer having a different affinity for the ligand than the starting aptamer.

[0018] Additionally, the present disclosure provides an aptamer having a first loop and a second loop separated by a first stem, and a second stem connected to the first loop, the second stem comprising a first end and a second end, wherein the first loop binds to a ligand, and the second loop is complementary to a portion of the aptamer, wherein at least one of the second loop, the first or the second stem is modified. In certain embodiments the first end and second end of the second stem are labeled. In some embodiments the first end and second end of the second stem are labeled with different labels. In other embodiments the first end of the second stem is labeled with tetramethylrhodamine (TAMRA) and the second end of the second stem is labeled with fluorescein (FAM). In further embodiments the aptamer is complexed with a redox label dye. In yet further embodiments the aptamer and the redox label dye are part of separate moieties. In still further embodiments binding of a ligand to the aptamer displaces the redox label dye, wherein the displaced redox label dye is detected by an electrode. In yet other embodiments at least one of the aptamer or the redox label dye are attached to a linker, wherein the linker is attached to an electrode. In still other embodiments binding of a ligand to the aptamer displaces the redox label dye. In various embodiments the aptamer comprises the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0019] The present disclosure also provides an aptamer for detecting a target molecule in a biological sample, comprising a single-stranded deoxyribonucleic acid (DNA) strand, wherein the aptamer is configured to bind to target molecule and undergoes a change upon binding of the target molecule. In certain embodiments, the target molecule is vancomycin, amikacin, tobramycin, cocaine or serotonin. In some embodiments, the target molecule is vancomycin. In other embodiments, the aptamer comprises the nucleotide sequence of any one of SEQ ID NQs:3-104. In yet other embodiments, the target molecule is cocaine. In particular embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 105 or SEQ ID NO: 113. In further embodiments, the target molecule is serotonin. In yet further embodiments, the aptamer comprises the nucleotide sequence of SEQ ID NO: 106 or SEQ ID NO:114. In still other embodiments, the target molecule is amikacin. In some embodiments, the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs: 135-143. In additional embodiments, the target molecule is tobramycin. In other embodiments, the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs: 144-152.

[0020] In certain embodiments, the biological sample comprises blood, serum, urine, effluent, saliva, sweat, tears, a body fluid, or a combination thereof. In other embodiments, the aptamer is configured to be immobilized to a substrate for sensing the target molecule. In certain embodiments, the aptamer is configured to be incorporated into a sensor device. In various embodiments, the sensor device comprises the aptamer and a field effect transistor, a gold substrate, a fiber-optical cable, or a quartz surface. In further embodiments, the aptamer comprises a fluorophore, a quencher, an enzyme, a redox dye, or a combination thereof.

[0021] The present disclosure additionally provides a method for detecting a target molecule in a biological sample, comprising contacting at least a portion of the biological sample comprising the target molecule with an effective amount of an aptamer for detecting a target molecule in a biological sample, comprising a single-stranded deoxyribonucleic acid (DNA) strand, wherein the aptamer is configured to bind to target molecule and undergoes a change upon binding of the target molecule, and detecting a change after the aptamer contacts the target molecule in the biological sample. In certain embodiments, the change comprises a change of a conductance, a fluorescence, or an electrochemical readout. In some embodiments, the target molecule is vancomycin, amikacin, tobramycin, cocaine or serotonin. In other embodiments, the biological sample comprises blood, serum, urine, effluent, saliva, sweat, tears, a body fluid, or a combination thereof.

[0022] Moreover, the present disclosure provides a method of therapeutic drug monitoring in a patient in need thereof, comprising contacting at least a portion of a biological sample comprising a target molecule from the patient with an effective amount of an aptamer for detecting a target molecule in a biological sample, comprising a single-stranded deoxyribonucleic acid (DNA) strand, wherein the aptamer is configured to bind to target molecule and undergoes a change upon binding of the target molecule, detecting a change after the aptamer contacts the target molecule, wherein the change is indicative of the concentration of the target molecule in the biological sample, performing high-precision control over a plasma level of the target molecule based on the concentration of the target molecule in the sample, and providing the patient with a treatment based on the concentration of the target molecule in the sample. In certain embodiments, the method further comprises detecting a change after the aptamer contacts the target molecule, wherein the change is indicative of the concentration of the target molecule in the biological sample at a predetermined frequency.

[0023] The present disclosure also provides an electrochemical sensor for detecting a molecule, comprising an aptamer comprising a redox reporter that binds to the molecule and undergoes a change in the electron kinetics upon binding of the molecule, wherein the aptamer is attached to a surface.

[0024] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0025] DESCRIPTION OF THE DRAWINGS

[0026] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0027] FIG. 1 illustrates an example of a disclosed aptamer (SEQ ID NO:111) that can bind various ligands through its structural rearrangement without changing the primary order of the sequence.

[0028] FIG. 2 shows an example of a sequence (SEQ ID NO: 111) that binds many small molecule ligands.

[0029] FIG. 3 illustrates a mechanism of how this sequence (SEQ ID NO:111 ) binds many small molecule ligands.

[0030] FIG. 4 shows a mechanism of derivatization of more specific analogs of a sequence (top, SEQ ID NO:112) that binds many small molecule ligands via mutations, insertions, and reselections, such as cocaine (left, SEQ ID NO: 113) and serotonin (right, SEQ ID NO:114).

[0031] FIG. 5A, FIG. 5B, FIG. 5C, FIG.5D and FIG. 5E each is a schematic of the engineering of a vancomycin aptamer, and the results of ThT dye displacement. FIG. 5A is an illustration of a vancomycin aptamer (SEQ ID NO:1 ). FIG. 5B is an illustration of the vancomycin aptamer with modifications to the second loop and first stem (SEQ ID NO:2). FIG. 5C is a general illustration of modification of the vancomycin aptamer, labeled at the 5’ end with tetramethylrhodamine (TAMRA) and the 3’end with fluorescein (FAM) FIG. 5D is the results of ThT dye displacement of the vancomycin aptamer shown in FIG. 5A. FIG. 5E is the results of ThT dye displacement of the vancomycin aptamer shown in FIG. 5B.

[0032] FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D are illustrations of a modified vancomycin aptamer (FIG.6A; SEQ ID NO:3) and the results of ThT dye displacement (FIG. 6B), FRET spectrum (FIG. 6C) and FRET signal (FIG. 6D).

[0033] FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D are illustrations of a modified vancomycin aptamer (FIG.7A; SEQ ID NO:4) and the results of ThT dye displacement (FIG. 7B), FRET spectrum (FIG. 70) and FRET signal (FIG. 7D).

[0034] FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D are illustrations of a modified vancomycin aptamer (FIG. 8A; SEQ ID NO:5) and the results of ThT dye displacement (FIG. 8B), FRET spectrum (FIG. 8C) and FRET signal (FIG. 8D).

[0035] FIG. 9A, FIG. 9B, FIG. 9C and FIG. 9D are illustrations of a modified vancomycin aptamer (FIG. 9A; SEQ ID NO:6) and the results of ThT dye displacement (FIG. 9B), FRET spectrum (FIG. 9C) and FRET signal (FIG. 9D).

[0036] FIG. 10A, FIG. 10B, FIG. 100 and FIG. 10D are illustrations of a modified vancomycin aptamer (FIG. 10A; SEQ ID NO:7) and the results of ThT dye displacement (FIG. 10B), FRET spectrum (FIG. 10C) and FRET signal (FIG. 10D).

[0037] FIG. 11 depicts therapeutic drug monitoring. Panel A depicts current approaches to therapeutic drug monitoring (TDM). Panel B depicts at-home finger-prick measurements of plasma drug levels. Panel C depicts continuous, real-time measurements performed via a wearable device. Panel D depicts improved ability to accurately dose and properly deliver pharmacological therapies.

[0038] FIG. 12 depicts fabrication of a new, vancomycin-detecting electrochemical aptamer-based (E-AB) sensor. Panel A depicts a solution-phase aptamer selection scheme that utilizes a key stem-loop closing step. Panel B depicts adaptation of the aptamer to the E-AB platform, which relies on a binding-induced change in the electron transfer kinetics of a redox reporter-modified (i.e. represented as “R”, which stands for methylene blue), surface-attached aptamer to produce a change in electron transfer rate that can be monitored using square-wave voltammetry. Panel C shows the response of the vancomycin sensor, which is a strong function of square-wave frequency, producing “signal-off,” “signal-on,” or “non-responsive” behaviors. Panel D shows the response of the vancomycin sensor in undiluted, flowing whole blood.

[0039] FIG. 13 shows calibration free measurement of vancomycin in whole blood. Panel A shows the fabrication of sensors using 75 pm diameter gold, silver and platinum wires that can easily be immersed in small volumes. Panel B shows the measurement of vancomycin over its entire clinical concentration range via a rapid (seconds) and single step.

[0040] FIG. 14 shows high-precision measurement of patient-specific pharmacokinetics using the vancomycin E-AB sensor. Panel A shows insertion of a fine-wire sensor into the right jugular vein (via a 22G catheter) of a rat, and insertion of an infusion line in the left jugular vein through vancomycin is administered intravenously. Panel B shows measurement of vancomycin concentration over time in three rat subjects.

[0041] FIG. 15 shows feedback-controlled drug delivery. Panel A shows seconds- resolved E-AB measurements as the input to a proportional integral-derivative (PID) controller that computes the dosing rate required to best reach the desired plasma concentration administered via a PID-controlled drug-delivery infuser. Panel B shows plasma vancomycin concentration over time. Panel C shows maintenance of the vancomycin concentration in the face of ~3-fold variation in the drug’s elimination rate.

[0042] FIG. 16 shows binding of the parent vancomycin-binding aptamer sequence via a quenching / dequenching aptameric assay. Panel A shows partial hybridization of the fluorescein-modified aptamer to a quencher-modified oligonucleotide. Panel B shows that increasing the concentration of the quencher-modified strand results in a decrease in the fluorescence of the fluorescently-labelled aptamer. Panel C shows binding of vancomycin to the hybridized assembly displaces the quencher strand. Panel D shows obtaining a dissociation constant following the resultant increase in fluorescence. Panel E shows molecules presenting similar chemical structures than vancomycin but devoid of its glycan moiety.

[0043] FIG. 17 shows adapting the parent aptamer sequence to the E-AB platform. Panel A shows the rate of electron transfer from the parent aptamer remains unchanged upon addition of saturating (1 mM) vancomycin. Panel B shows destabilization of the parent aptamer via removal of 4 base pairs from its stem (“4-trunc”) leads to a binding- induced conformational change as observed via circular dichroism. Panel C shows the “4-trunc” variant exhibits a binding-induced change in its electron transfer rate thus supporting E-AB signaling.

[0044] FIG. 18 shows addition of 100 pM to the vancomycin E-AB sensor leads to response to its target within the 4 s required to acquire a square-wave voltammogram.

[0045] FIG. 19 shows high precision feedback-drug delivery using the E-AB vancomycin sensor. Panel A shows setting the PID controller to maintain a fixed concentration of 5 pM for more than 4 hours. Panel B shows the control algorithm maintains the set point concentration despite fairly significant changes in elimination rate of the drug over the course of the experiment. Panel C shows releasing the PID controller following establishment of steady-state plasma drug concentrations results in elimination of the drug.

[0046] FIG. 20A, FIG. 20B, FIG. 20C and FIG. 20D. Electrochemical aptamer-based (EAB) sensors. Principles of sensing based on aptamers: Conformational changes upon ligand binding events lead to changes in distances between labels (FIG. 20A). Bold blue-colored font signifies invariant sequence common to all vancomycin aptamers with minimized fully stable aptamer, Vmin shown (SEQ ID NO:114; FIG. 20B). Discovery of a base-flip in Va (SEQ ID NO:115) that leads to Vb (SEQ ID NO:116) and improves the affinity about fourfold (FIG. 20C). This same base-flip was applied to Vc (SEQ ID NO;117), which led to V28 (SEQ ID NO;118), with the same effect on the affinity (FIG. 20D).

[0047] FIG. 21 A and FIG. 21 B. FIG. 21 A. The proposed conformational change (or secondary structure switching; magenta-colored sequences are complementary) in V28 (SEQ ID NO;118) upon binding of vancomycin is used to construct the FRET / Quenching sensor, after conjugation of donor and acceptor dyes (fluorescein and TAMRA). Vancomycin concentration dependent fluorescent emission spectra of the solution of this sensor is shown upon excitation at 480 nm. FIG. 21 B. The proposed conformational change (or secondary structure switch; magenta-colored sequences are complementary) in V28 (SEQ ID NO;118) upon binding is used to construct the EAB sensor by conjugating a redox indicator dye (methylene blue or MB). Sulfide (thiol) is used to attach the aptamer to gold surface. For this EAB vancomycin sensors vancomycin concentration-dependent changes in currents are shown at different square wave voltammetry frequencies.

[0048] FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D, FIG. 22E, FIG. 22F, FIG. 22G, FIG. 22H, FIG. 22I, FIG. 22J, FIG. 22K, FIG. 22L, FIG. 22M, FIG. 22N, FIG. 220, FIG. 22P, FIG. 22Q, FIG. 22R, FIG. 22S, FIG. 22T and FIG. 22U. FIG. 22A. Introduction of a new loop can be used to control affinity of sensors (SEQ ID NO:119; FIG. 22A). Affinities are impacted by changes in this new loop, as well as changes in the length of stems and further mutations. Example of sensor identified through screening (SEQ ID NO: 120; FIG. 22B). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 1 pM; FIG. 22C). Example of sensor identified through screening (SEQ ID NO: 121 ; FIG. 22D). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 2 pM; FIG. 22E). Example of sensor identified through screening (SEQ ID NO: 122; FIG. 22F). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 4.4 pM; FIG. 22G). Example of sensor identified through screening (SEQ ID NO: 123; FIG. 22H). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 8 pM; FIG. 22I). Example of sensor identified through screening (SEQ ID NO: 124; FIG. 22J). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 9.6 pM; FIG. 22K). Example of sensor identified through screening (SEQ ID NO: 125; FIG. 22L). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 11 pM; FIG. 22M). Example of sensor identified through screening (SEQ ID NO: 126; FIG. 22N). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 15 pM; FIG. 220). Example of sensor identified through screening (SEQ ID NO:127; FIG. 22P). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 17 pM; FIG. 22Q). Example of sensor identified through screening (SEQ ID NO: 128; FIG. 22R). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 22 pM; FIG. 22S). Example of sensor identified through screening (SEQ ID NO: 129; FIG. 22T). Graph with fluorescent spectra obtained by adding increasing concentrations of vancomycin to displace the ThT dye from aptamer (50%, 97.5 pM; FIG. 22U). The results indicate relative shifts in the affinity of sensors.

[0049] FIG. 23A, FIG. 23B and FIG. 23C. Example of FRET sensor constructed from the new aptamer analogs selected from examples shown in FIG. 22B (SEQ ID NO: 130; FIG. 23A). Characterization of sensors, concentration dependent emission spectra, upon excitation at 480 nm (FIG. 23B, FIG. 23C).

[0050] FIG. 24A, FIG. 24B and FIG. 24C. Example of FRET sensor constructed from the new aptamer analogs selected from examples shown in FIG. 22B (SEQ ID NO: 131 ; FIG. 24A). Characterization of sensors, concentration dependent emission spectra, upon excitation at 480 nm (FIG. 24B, FIG. 24C).

[0051] FIG. 25A, FIG. 25B and FIG. 25C. Example of FRET sensor constructed from the new aptamer analogs selected from examples shown in FIG. 22B (SEQ ID NO: 132; FIG. 25A). Characterization of sensors, concentration dependent emission spectra, upon excitation at 480 nm (FIG. 25B, FIG. 25C).

[0052] FIG. 26A, FIG. 26B and FIG. 26C. Example of FRET sensor constructed from the new aptamer analogs selected from examples shown in FIG. 22B (SEQ ID NO: 133; FIG. 26A). Characterization of sensors, concentration dependent emission spectra, upon excitation at 480 nm (FIG. 26B, FIG. 26C).

[0053] FIG. 27A, FIG. 27B and FIG. 27C. Example of FRET sensor constructed from the new aptamer analogs selected from examples shown in FIG. 22B (SEQ ID NO: 134; FIG. 27A). Characterization of sensors, concentration dependent emission spectra, upon excitation at 480 nm (FIG. 27B, FIG. 27C).

[0054] FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D, FIG. 28E, FIG. 28F, FIG. 28G, FIG. 28H, FIG. 28I, FIG. 28J, FIG. 28K, FIG. 28L, FIG. 28M, FIG. 28N, FIG. 280, FIG. 28P and FIG. 28Q. Basic structure, tobramycin aptamer, as isolated directly from selection (SEQ ID NO: 135; FIG. 28A). ThT displacement assay, which can be used to approximate the relative binding order of analogs (FIG. 28B). Circularly permuted aptamer, with similar binding affinity (SEQ ID NO: 136; FIG. 28C), and ThT displacement assay (50%, 0.2 pM; FIG. 28D). Introduction of loop and adjustment of stem lengths (SEQ ID NO: 137; FIG. 28E). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 138; FIG. 28F), and ThT displacement assay (50%, 0.45 pM; FIG. 28G). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 139; FIG. 28H), and ThT displacement assay (50%, 1 pM; FIG. 28I). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 140; FIG. 28J), and ThT displacement assay (50%, 2.5 pM; FIG. 28K). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 141 ; FIG. 28L), and ThT displacement assay (50%, 6.5 pM; FIG. 28M). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 142; FIG. 28N), and ThT displacement assay (50%, 12 pM; FIG. 280). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO:143; FIG. 28P), and ThT displacement assay (50%, 17 pM; FIG. 28Q). Blue color indicates matching complementary regions, while red color indicates adjustable spacers.

[0055] FIG. 29A, FIG. 29B, FIG. 29C, FIG. 29D, FIG. 29E, FIG. 29F, FIG. 29G, FIG. 29H, FIG. 29I, FIG. 29J, FIG. 29K, FIG. 29L, FIG. 29M, FIG. 29N, FIG. 290, FIG. 29P and FIG. 29Q. Basic structure, amikacin aptamer, as isolated directly from selection (SEQ ID NO: 144; FIG. 29A). ThT displacement assay, which can be used to approximate the relative binding order of analogs (FIG. 29B). Circularly permuted aptamer, with similar binding affinity (SEQ ID NO: 145; FIG. 29C), and ThT displacement assay (50%, 0.24 pM; FIG. 29D). Introduction of loop and adjustment of stem lengths (SEQ ID NO: 146; FIG. 29E). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 147; FIG. 29F), and ThT displacement assay (50%, 0.7 pM; FIG. 29G). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO:148; FIG. 29H), and ThT displacement assay (50%, 1.9 pM; FIG. 29I). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 149; FIG. 29J), and ThT displacement assay (50%, 4.4 pM; FIG. 29K). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 150; FIG. 29L), and ThT displacement assay (50%, 7 pM; FIG. 29M). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 151 ; FIG. 29N), and ThT displacement assay (50%, 12 pM; FIG. 290). Example of analog with loop introduction and adjustment of stem length (SEQ ID NO: 152; FIG. 29P), and ThT displacement assay (50%, 18 pM; FIG. 29Q). Blue color indicates matching complementary regions, while red color indicates adjustable spacers.

[0056] DETAILED DESCRIPTION OF THE INVENTION

[0057] The present disclosure is based, at least in part, on the discovery of methods of tuning the affinity and signal of aptameric sensors, and aptamers produced by these methods. The methods generally comprise engineering at least one of the stem length, loop structure or body of an aptamer to achieve, through screening process, a half-point that matches what is needed in both fluorescence and electrochemical sensors.

[0058] The maximum sensitivity of most aptameric sensors is achieved at the point of half-response, that is usually connected to the dissociation constant. Thus, if an aptamer has a dissociation constant of 110 nM, it is already saturated at 2 pM, becoming unresponsive.

[0059] When engineering aptamers to reduce their affinity, structures are obtained that do not have the same properties that lead to the transduction of recognition events into signal (e.g., fluorescent, electrochemical or electronic), which leads to unequal signals.

[0060] The present disclosure shows that aptamers that have two stems connected by two single stranded linking regions can be engineered to achieve desired range of responses, while keeping the ability to construct high-quality FRET and electrochemical sensors from these constructs. As shown herein below, in contrast to traditional engineering approaches, the presently disclosed methods can accomplish a near continuous fine-tuning of affinity. Specifically, a known vancomycin aptamer was modified by removing a large external loop, thereby obtaining a minimal sequence that has high affinity (<150 nM). The minimal stem is then substituted with various sequences that are partially complementary to sections of the aptamer, thereby producing new aptamers that have various affinities that are nearly continuous, thus allowing the selection of a modified aptamer that has the specific affinity and half-point of response desired for the particular application to be used.

[0061] Aptamers, or oligonucleotide-based receptors as described herein, provide unique advantages as analytical tools. For example, an aptamer can be incorporated in simple and rapid mix-and-measure assays or readily attached to a surface, e.g., suitable for integration in biosensors.

[0062] For example, a mixture of nucleic acid molecules (e.g., candidate aptamers, such as a library of DNA molecules) are modified to change the affinity to a particular ligand, and / or to achieve a desired half-point of response value. The stem length, loop structure and / or body of an aptamer mixture is modified, and the resulting mixture of modified aptamers is screened to identify one or more modified aptamers having a different affinity for a particular ligand, or one or more modified aptamers having the desired half-point of response value.

[0063] In certain embodiments of the present disclosure the modified aptamer(s) can be complexed with a label, such as a redox label, and in other embodiments either the modified aptamer, the label, or both can be attached to an electrode via a linker. A linker can be, for example, an organic molecule with at least one end having a functional group. Various linker groups are known in the art; except as otherwise specified, compositions described herein can include state of the art linker groups. For example, a state of the art linker molecule can be any such molecule capable of coupling a modified aptamer, a label, such as a redox label, or both to an electrode. A linker group can include one or more of the following exemplary functional groups: carboxylic acid or carboxylate groups (e.g., Fmoc-protected-2,3-diaminopropanoic acid, ascorbic acid), silane linkers (e.g., aminopropyltrimethoxysilane (APTMS)), or dopamine. A linker group, such as carboxylic acid, dopamine, or silane (or another state of the art linker group), can provide sites for binding. Other linkers can include alkane, alkene, or alkyne linkers of various size (e.g., n = 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more). A linker can include chemical motifs such as disulfides, hydrazones, or peptides (e.g., cleavable), or thioethers (e.g., non-cleavable). A linker can include maleimide, or sulfhydryl reactive groups, or succinimidyl esters.

[0064] TARGET MOLECULE

[0065] As disclosed herein, an aptamer can be modified to exhibit a different affinity to a target molecule or ligand, or modified to have a half-point that matches what is needed in both fluorescence and electrochemical sensors. A target molecule can be, for example, a small molecule, a protein, or a nucleic acid, or structures or compositions containing any of these. For example, a target molecule can be a protein, peptide, an amino acid or an analog or derivative thereof, or small molecule displaying one or more nucleophilic groups. As another example, a target molecule can be a small molecule selected from a carbohydrate molecule, a fatty acid molecule, an amino acid, or a derivative or a combination thereof. A target molecule can also be an antibiotic, such as, but not limited to, vancomycin.

[0066] A target molecule can occur in solution or be attached to a substrate. For example, a target molecule can be a sugar molecule on the surface of a cell. A target molecule can be an essential amino acid, such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, or valine, or a non- essential amino acid, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, ornithine, proline, serine, or tyrosine.

[0067] A target molecule can be a proteinogenic amino acid. For example, a target molecule can be an amino acid selected from histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, selenocysteine, or pyrrolysine.

[0068] A target molecule can be a non-proteinogenic amino acid. For example, a target molecule can be an amino acid selected from lanthionine, 2-aminoisobutyric acid, dehydroalanine, N-formylmethionine, gamma-amino-butyric acid (GABA), hydroxyproline, carnitine, ornithine, S-adenosylmethionine, citrulline, beta alanine (3- aminopropanoic acid), canavanine, mimosine, or aspartame.

[0069] A target molecule can be an amino acid derivative, such as 5-hydroxytryptophan, L-dihydroxyphenylalanine, or eflornithine.

[0070] A target molecule can be a sugar. A target molecule can be a carbohydrate. A target molecule can be a saccharide. A target molecule can be a monosaccharide, including but not limited to glucose, dextrose, fructose, or galactose.

[0071] A target molecule can be a disaccharide, including but not limited to sucrose (glucose and fructose), maltose (glucose and glucose), or lactose (galactose and glucose). A target molecule can be a hydrogenated form of carbohydrate, whose carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. A target molecule can be a sugar alcohol, such as a polyol, polyhydric alcohol, polyalcohol, or glycitol. A target molecule can be a sugar alcohol, such as methanol, glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, or polyglycitol, or disaccharide combinations thereof.

[0072] As described herein, target molecule can be a lipid target molecule, such as fatty acids, steroids, sphingollipids, or phospholipids complexed with a derivatization agent.

[0073] A target molecule can be a naturally-occurring fatty acid molecule having at least about 4 up to about 28 carbon atoms. A target molecule can be a fatty acid molecule derived from a monoglyceride, diglyceride, triglyceride, phospholipid, sphingolipid, or ganglioside.

[0074] A target molecule can be a free fatty acid molecule. A target molecule can be a short-chain fatty acid (e.g., fatty acid with aliphatic tails of fewer than six carbons, such as butyric acid); a medium-chain fatty acid (e.g., a fatty acid with aliphatic tails of 6-12 carbons, which can form medium-chain triglycerides); a long-chain fatty acid (e.g., fatty acids with aliphatic tails 13 to 21 carbons); or very long chain fatty acids (e.g., fatty acids with aliphatic tails longer than 22 carbons).

[0075] A target molecule can be a saturated fatty acid molecule. For example, a target molecule can be a saturated fatty acid molecule selected from caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, or cerotic acid, or derivatives thereof.

[0076] A target molecule can be an unsaturated fatty acid molecule. For example, a target molecule can be an unsaturated fatty acid molecule selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, or docosahexaenoic acid, or derivatives thereof. As another example, a target molecule can be an unsaturated fatty acid molecule selected from linolenic acid (LA), a-linolenic acid (ALA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA).

[0077] A target molecule can be a steroid molecule. A steroid is understood as a type of organic compound containing a characteristic arrangement of four cycloalkane rings joined to each other. A target molecule can be, for example, a steroid molecule selected from a cholestane, a cholane, a pregnane, an androstane, a gonane, or an estrane. A target molecule can be, for example, a steroid molecule selected from cholesterol, estradiol, testosterone, progesterone, medrogestone, [3-sitosterol, or dexamethasone.

[0078] A target molecule can be a sphingolipid or a phospholipid. For example, a target molecule can be a sphingosine-phosphate, sphingomyeline, ganglioside, or phosphatidyl-choline.

[0079] A target molecule can be a small molecule. For example, a target molecule can be a catechol (e.g., dopamine and L-DOPA (L-3,4-dihydroxyphenylalanine)).

[0080] A target molecule can be an approved, clinically used drug, drug candidates, or a lead-like small molecule or a drug-like small molecule. For example, a target molecule can be a hydrophobic lead-like or a hydrophobic drug-like molecule. A lead-like small molecule is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see, e.g., Angewante, Chemie Int. Ed. Engl. 24:3943-3948, 1999). In contrast, a drug-like small molecule is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski, J. Pharm. Tox. Methods 44:235-249, 2000).

[0081] APTAMER

[0082] An aptamer, as the term is used herein, is understood as a nucleic acid species engineered through repeated rounds of selection (e.g., in vitro selection) to bind to a target molecule, such as small molecules, proteins, nucleic acids, cells, tissues, or organisms. A modified aptamer is defined as a starting aptamer that has at least one of the external loop, first stem or second stem of the aptamer changed, or modified, in order to change the desired half-point of response value to a target molecule or ligand, or to change the affinity to a target molecule or ligand.

[0083] For those small molecules for which an aptamer has been previously successfully isolated, such as amino acids, various embodiments of the methods described herein can provide modified aptamers having significantly superior affinity, a desired half-point of response value, or reduced aptamer size, or some or all of these attributes.

[0084] Before, during, or after recognition of a target molecule, an aptamer can bind by complementary nucleic acid base pairing, which can create a secondary structure, such as a short helical arm or a single stranded loop. A combination of these secondary structures can result in the formation of a tertiary structures, which can allow an aptamer to bind to a target molecule via van der Waals forces, hydrogen bonding, or electrostatic interaction (similar to an antibody binding to an antigen). When such tertiary structure forms, some, most, or all of the aptamer can fold into a complex (e.g., a stable complex) with the target molecule forming an aptamer-target complex. This three-dimensional structure can allow an aptamer to function like an antibody, which contrasts to conventional thinking which held that polynucleic acids were merely linear, information holding structures.

[0085] An aptamer can be a nucleic acid aptamer. An aptamer can be a DNA aptamer. A DNA aptamer can be relatively more stable, cheaper, and easier to produce than an RNA aptamer. An aptamer can be an RNA aptamer. An RNA aptamer can have a relatively more diverse three-dimensional structure than a DNA aptamer. An aptamer can be an XNA aptamer. An aptamer can be a smart aptamer, selected with a predefined equilibrium constants (Kd), rate constants (koff, kon), and thermodynamic (AH, AS) parameters of aptamer-target interaction.

[0086] A modified aptamer as described herein can be further modified, e.g., to resist degradation in a sample. For example, sugar modifications of nucleoside triphosphates can render a resulting aptamer resistant to nucleases found in serum. As another example, changing a 2'OH group of ribose to a 2'F or 2'NH2 group can yield an aptamer having increased stability or a longer half-life, such as in blood-containing sample or in an in vitro or in vivo environment (see, e.g., Brody and Gold, Rev. Molec. Biol. 74:5-13, 2000). As another example, conjugating an aptamer to a higher molecular weight vehicle can increase stability or half-life in an in vitro or in vivo environment. As another example, an aptamer can be conjugated to a nanomaterial.

[0087] An aptamer or modified aptamer as described herein can be at least about 15 oligonucleotides. An aptamer or modified aptamer as described herein can be up to about 100 oligonucleotides. For example, an aptamer or modified aptamer as described herein can be at least about 15 oligonucleotides up to about 100 oligonucleotides. As another example, an aptamer or modified aptamer as described herein can be at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, or more oligonucleotides. It is understood that recitation of each of these individual values includes ranges there between.

[0088] Nucleic acid sequences for exemplary modified aptamers are provided herein. It is understood that a modified aptamer can have a nucleic acid sequence according to the discrete exemplary sequence provided, or a sequence at least 80% identical thereto (e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) and binding (e.g., selective or non-selective) a target molecule. One of ordinary skill will understand that certain regions of the modified aptamer are more robust with respect to nucleic acid substitution. For example, stem regions of a secondary or tertiary structure of a modified aptamer may have reduced impact on target molecule binding, and so, nucleic acid substitutions in these regions can be more freely made. In contrast, secondary or tertiary structure regions of a modified aptamer associated with binding to target molecules may be more sensitive, and so, may require more conservative substitutions or fewer substitutions. Similarly, regions of a modified aptamer important or critical to certain secondary or tertiary structural features may be more sensitive, and so, may require more conservative substitutions or fewer substitutions. In some embodiments, nucleic acid sequence identity can be lower in stem regions (e.g., at least about 80%, at least about 85%, or at least about 90%) compared to regions associated with binding a target molecule or regions important or critical to secondary or tertiary structural features (e.g., at least about 90%, at least about 95%, or at least about 99%).

[0089] A modified aptamer as described herein can have an equilibrium constant Kd of about 1 pM up to about 100 pM. An aptamer having a Kd as low as about 1 pM to about 100 pM can be with respect to a target molecule, such as a sugar, natively on a surface, such as a cell surface. An aptamer as described herein can have an equilibrium constant Kd of about 1 pM up to about 10.0 pM. As another example, an aptamer as described herein can have an equilibrium constant Kd of about 1 pM up to about 10.0 pM; about 1 pM up to about 1.0 pM; about 1 pM up to about 100 nM; about 100 pM up to about 10.0 pM; about 100 pM up to about 1 .0 pM; about 100 pM up to about 100 nM; or about 1 .0 nM up to about 10.0 pM; about 1 .0 nM up to about 1 .0 pM; about 1 nM up to about 200 nM; about 1.0 nM up to about 100 nM; about 500 nM up to about 10.0 pM; or about 500 nM up to about 1 .0 pM.

[0090] As another example, a modified aptamer as described herein can have an equilibrium constant Kd of about 1 pM, about 50 pM, about 100 pM, about 150 pM, about 200 pM, about 250 pM, about 300 pM, about 350 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM, about 600 pM, about 650 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, about 1 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM. It is understood that ranges between different combinations of Kd listed above are contemplated.

[0091] In certain embodiments, a modified aptamer as described herein can have a nucleic acid sequence with one or more unpaired nucleic acid bases. A modified aptamer with one or more unpaired nucleic acid bases can form a binding pocket providing for binding of the target molecule.

[0092] DIAGNOSTICS

[0093] The modified aptamers described herein can be used in diagnostic applications. Various embodiments of the method can address specific bioanalytical needs, such as mix-and-measure assays of diagnostic increases in small molecules in challenging biological matrices. The simplicity and general applicability of methods described herein or broad availability of synthetic receptors for small molecules provide applications of modified aptamers in clinical chemistry that have not been previously possible.

[0094] Conventional aptamer diagnostic protocols can be adapted by an artisan of ordinary skill for use with modified aptamers disclosed herein. Aptamer usage in diagnostics is well known (see, e.g., Jayasena, Clin. Chem. 45:1628-1650, 1999; Mascini, “Aptamers in Bioanalysis,” Wiley-lnterscience, 1stEd., ISBN-10: 0470148306, 2009). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0095] An aptamer described herein can be immobilized on a surface suitable for diagnostic applications, such as electrodes, gold films, gold particles, silicates, silicon oxides, polymers, metallic substrates, biocoatings including avidin or avidin derivatives, quantum dots, carbon nanotubes, superparamagnetic iron oxide nanoparticles, or carbohydrates (see, e.g., Balamurugan et al., Anal. Bioanal. Chem. 390:1009-1021 , 2008; Famulok et al., Chem. Rev. 107:3715-3743, 2007; Lee et a / ., Adv. Drug Deliv. Sys. 62:592-605, 2010). Chemical protocols for covalent attachment of aptamers to functionalized surfaces is understood in the art and such protocols can be adapted for modified aptamers disclosed herein. For example, an aptamer of the present disclosure can be attached to an electrode or solid surface array.

[0096] A modified aptamer described herein can be used in conjunction with a fluorescent, colorimetric, magnetic resonance imaging, or electrochemical sensor or protocol (see, e.g., Lee et al. Adv. Drug Deliv. Sys. 62:592-605, 2010).

[0097] A modified aptamer described herein can be used to detect a target molecule in a sample. A sample can be a biological sample. A sample can be a biological sample from a subject. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, goats, pigs, mice, rats, monkeys, hamsters, guinea pigs, turkeys, ducks, geese, and chickens, and humans. For example, the subject can be a human subject.

[0098] A biological sample can be a fluid sample or a solid sample. A biological sample can be a urine sample, a saliva sample, a blood sample, a serum sample, a plasma sample, an amniotic fluid sample, a cerebrospinal fluid sample, a sweat sample, an exhaled breath condensate sample, or a solid tissue sample. For example, the sample can be a blood sample, such as a peripheral blood sample. As another example, a sample can be a urine sample.

[0099] For example, a modified aptamer described herein can be used to detect an amino acid. Such amino-acid specific aptamers can be used to detect an amino acid in a biological sample (e.g., a urine sample). For example, a modified aptamer described herein can be used to detect a clinically used drug or a drug candidates. Such amino-acid specific aptamers can be used to detect a drug or drug in a biological sample, such as, e.g., a urine sample, a whole blood or serum sample, a sweat sample, or saliva sample, or cerebrospinal fluid sample, or any other bodily fluid.

[0100] For example, a modified aptamer described herein can be used to detect a clinically used drug or a drug candidates in waste products, effluents and filtrates, of therapy, such as hemodialysis, peritoneal dialysis, or continuous renal replacement therapy (CRRT).

[0101] For example, a modified aptamer described herein can be used repeatedly to monitor time courses an characterize distribution and clearance of a clinically used drug or a drug candidates.

[0102] A modified aptamer described herein can be used to detect a target molecule associated with a disease or condition. Diagnostic methods using a modified aptamer described herein can be performed on a subject having, diagnosed with, suspected of having, or at risk for developing a disease or condition associated with a target molecule. A determination of the need for diagnosis can be assessed by a history and physical exam consistent with the disease or condition at issue. Conventional diagnostic protocols of a disease or disorder associated with a target molecule can be adapted accordingly to use modified aptamers as disclosed herein.

[0103] A target molecule can be a lead-like small molecule or a drug-like small molecule. For example, a target molecule may be an approved, clinically used drug, drug candidate, a lead-like small molecule, or any drug.

[0104] Amino acids that are diagnostic or contribute to diagnosis for specific disorders are known in the art (see, e.g., Blau “Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases,” 2d Ed., Springer, ISBN-10: 354042542X, 2004). Methods described herein for isolating an aptamer specific for an amino acid can be directed towards an amino acid known to be diagnostic or contribute to diagnosis for specific disorders. A modified aptamer isolated according the approach described herein can be used to detect an amino acid in a sample, thereby providing or contributing to a diagnosis for the associated disease or disorder. For example, a modified aptamer described herein can be used as a diagnostic of a congenital disease associated with the target molecule. As another example, a modified tyrosine-specific aptamer can be used to diagnose tyrosinemia in a subject. As another example, a modified aptamer specific for the amino acid citrulline can be used to diagnose or aid in the assessment of small intestinal function (e.g., transplant recipients, including graft-vs-host disease). As another example, a modified aptamer specific for the carbohydrate galactose can be used to diagnose or aid in the diagnosis of several forms of galactosemia.

[0105] Furthermore, a modified aptamer developed as described herein can be used to monitor an amino acid associated with a disease or disorder and to measure compliance. For example, several amino acid disorders are known to be treated by specific diets and modified aptamers described herein can provide a tools allowing a subject or caregiver to monitor the efficacy of a specific diet. As another example, modified aptamers specific for valine, leucine, or isoleucine (branched chain amino acids) can be used for evaluation of nutritional status (e.g., dietary supplement used by athletes).

[0106] Several inborn errors of amino acid metabolism can be treated by special diets that either restrict protein intake (e.g., urea cycle defects, phenylketonuria, tryosinemia, glycine cleavage deficiency and others) or supplement amino acids (e.g., 3- phosphoglycerate dehydrogenase deficiency, MELAS syndrome and others). Conventional treatment involves weekly or monthly determination of amino acid profiles and there are no methods or tools that allow monitoring individual amino acids on a daily basis (cf. glucose profiles in diabetes). Modified aptamers described herein and sensitive to the amino acid associated with such inborn errors of amino acid metabolism can be used to monitoring individual amino acids on an hourly, daily, weekly, monthly, or yearly basis. Exemplary inborn errors of amino acid metabolism are provided in the TABLE 1.

[0107] A method based on modified aptamers developed as described herein can improve current diagnostic approaches in clinically relevant conditions, extend diagnostic capacity to low-prevalence conditions that remain undiagnosed due to economic and technical reasons, uncover yet unrecognized alterations in metabolism, or be used in monitoring the general health of populations. Such methods can be effective when a health issue is characterized through a truly gross shift in patterns of metabolite families, typical for serious metabolic problems, such as metabolic disorders due to genetic polymorphisms (inborn errors). Gross shifts of dominant components in the range of micro-to-millimolar concentrations can be well suited for analysis as described herein, including urinalysis for metabolic errors. Furthermore, analysis described herein (e.g., via arrays) can be useful in other biological fluids, such as serum, saliva, amniotic fluid, and CSF.

[0108] Over 98% of newborns in the US participate in a comprehensive program for mass screening for inborn errors of metabolism on blood spots; this process, made relatively fast and inexpensive by tandem mass spectroscopy coupled with computer analysis, covers 30+ inborn errors of metabolism, treatable if caught at early stages, and 20+ untreatable conditions. While newborn screening is an undeniable success in developed countries, serious problems remain. For example, the rate of false positives can be as high as 1 .3%, with the positive predictive value of the test ranging from 3% (meaning 97% of positives are false) to 50%, depending on individual states (overall leading to estimated 200,000 false positives each year in the US).

[0109] Approaches described herein can diagnose or monitor inborn errors that interfere with metabolic processes involving amino acids. If identified early, the most serious consequences of these errors, such as mental retardation, can be prevented, e.g., by careful changes in diet and by providing supplements / drugs. The National Academy of Clinical Biochemistry stresses in its “Practice Guidelines to Follow-up Testing for Metabolic Diseases Identified in Newborn Screening” that a comprehensive amino-acid analysis provides relevant and timely contribution to the differential diagnosis, with most tests for amino acids performed in serum. The current analytical standard for amino acid analysis in urine is post-derivatization cation exchange chromatography with photometric detection of ninhydrin adducts; iTRAQ®-LC-MS / MS, and post-derivatization GC-MS are being studied as alternatives.

[0110] In some embodiments, diagnostic methods disclosed herein may not fully eliminate the need for chromatography and other diagnostic steps (e.g., genetic). But such diagnostic methods can give a rapid single-step option for sorting out cases identified initially as low-to-moderate risk; thus, as a fast second-tier confirmatory test, it can allow early focus on correct diagnosis, and, if false positive is established, it can provide important relief to a subject. In post-prandial periods in patients with metabolic disorders that interfere with utilization of amino acids, there can be a transient strong elevation above the renal reabsorption threshold of relevant metabolites in plasma. This can result in spillage into urine, useful to confirm the initial diagnosis or result in analysis of acidic components in urine as part of a differential diagnosis. To preserve homeostasis, unnecessary or toxic compounds are rapidly excreted, thus, increases in urine can be more dramatic than in blood.

[0111] In metabolic errors, the shifts in patterns of amino acids can be gross for screened diseases. For example, it is known that in primary aminoacidopathies: (i) in tyrosinemia type 1 , changes in concentrations of tyrosine in urine were >20-fold (~2000 pmol / g creatinine); branched chain amino acids change only minimally; (ii) in homocystinuria (e.g., cystathionine beta synthase deficiency), it is known that homocystine becomes detectable in urine (from ~0 to about 100 pmol / g creatinine); (iii) in urea cycle disorders, e.g., citrullinemia (ASD) and argininosuccinicaciduria (ALD) (of interest for late onset as well), it is known that citrulline in urine increases more than 50- fold from < 200 to >10,000 pmol / g creatinine) and argininosuccinicate from ND to >1000 pmol / g creatinine; (iv) in MSLID at day 3, it is known that leucine (also other branched- chain amino acids) increases >10-fold in serum (e.g., from <200 to ~2000 pM) with expected spillage into urine; any detection of allo-isoleucine in either urine or serum (ND to av. 200 pM) is known to be indicative of the diagnosis. In each of these cases, a modified aptamer can be developed, as described herein, to be sensitive to a metabolite above and thus contribute to diagnosis of the associated disease or disorder.

[0112] Provided below is a list of specific diseases and conditions associated with a disruption of levels of an amino acid. Methods described herein for isolating a modified aptamer specific for an amino acid can be directed towards an amino acid known to be diagnostic or contribute to diagnosis for a specific disease or disorder appearing in the table below. A modified aptamer isolated according the approach described herein can be used to detect an amino acid in a sample, thereby providing or contributing to a diagnosis for the associated disease or disorder appearing in Table 2. TABLE 2: Pathological values / differential diagnosis of inborn errors

[0113]

[0114]

[0115]

[0116] Diagnostic methods discussed above can be useful for urine samples. Urine is presently understood to be a complex matrix for analysis, dependent on kidney filtration and reabsorption efficacy, often requiring collection of 24-hour urines, often under professional supervision in metabolic wards. Aside from standardization against creatinine, many analytes require deconjugation procedures, derivatizations, extraction, or solid state isolation steps. Methods described herein can replace traditionally challenging procedures, typically used for confirmatory second-tier assays, with simple and rapid protocols suitable for routine use “next-to-subject”.

[0117] In the context of newborn screening, urinalysis has been validated based on post-derivatization GC-MS with standard additions for more than 130 different inborn metabolic inflictions (Matsumoto, Mass Spectrom. Rev. 15:43-57, 1996). Methods described herein can avoid the more laborious and complicated GC-MS analysis. Such a breakthrough is provided by aptameric sensors using the presently described modified aptamers, with their ability to transduce adaptive binding into a signal, described herein. In healthy urine, sets of two specific or optimized differentially responsive modified aptameric sensors can have very similar ratios of responses; in urines with gross shifts, these ratios can change dramatically, regardless of renal filtration. For example, aside from the detection of allo-isoleucine, the diagnosis of MSLID is conventionally made based on the ratio of leucine and isoleucine to phenylalanine in chromatographs of derivatives. According to compositions and methods described herein, the same effect can be achieved in a single-step measurement.

[0118] MOLECULAR BIOLOGY

[0119] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X / Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0120] Generally, conservative substitutions can be made at any position so long as the required activity is retained. Deletion is the replacement of a nucleic acid by a direct bond. Positions for deletions include the termini and linkage positions. Insertions are introductions of nucleic acids into the chain, a direct bond formally being replaced by one or more nucleic acids. Nucleic acid sequence can be modulated with the help of art-known computer simulation programs. KITS

[0121] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to target molecule, one or more modified aptamer(s), or materials or reagents for identification or isolation of a modified aptamer. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0122] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0123] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD- ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0124] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel, “Condensed Protocols from Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717, 2006; Ausubel et al., “Short Protocols in Molecular Biology,” 5th ed., Current Protocols, ISBN-10: 0471250929, 2002; Sambrook and Russel, “Molecular Cloning: A Laboratory Manual,” 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773, 2001 ; Elhai, and Wolk, Meth. Enzymol. 167:747-754, 1988; Studier, Protein Ex pr. Purif. 41 :207-234, 2005; Gellissen, ed. “Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,” Wiley-VCH, ISBN-10: 3527310363, 2005; Baneyx, “Protein Expression Technologies,” Taylor & Francis, ISBN-10: 0954523253, 2004).

[0125] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0126] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value, while in other embodiments it is used to indicate a value that is + 10% of the recited value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein serves as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Similarly, it is understood that recitation of ranges of values herein serves as a shorthand method of referring to ranges between each of the recited values.

[0127] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0128] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0129] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0130] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0131] Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0132] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as nonlimiting examples.

[0133] EXAMPLES

[0134] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0135] EXAMPLE 1: VANCOMYCIN, COCAINE AND SEROTONIN APTAMERS

[0136] The following example describes various vancomycin aptamers (SEQ ID NOs:8- 104) that are responsive to clinically relevant ranges of vancomycin in a sample, cocaine (SEQ ID NO: 105) or serotonin (SEQ ID NO: 106).

[0137] The presently disclosed aptamers can form a complex against vancomycin, and the formation of such a complex can be characterized with dissociation constants in the range between about 10-9and about 10-6M or in the range between about 10-6and about 10'3M. The disclosed aptamers can be presented as a single-stranded DNA incorporating an oligonucleotide sequence, such as SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, or SEQ ID NO:11 , and its FRET form (SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14 and SEQ ID NO: 15). These sequences constitute part of a larger structure within the disclosed aptamers, flanked by two partially complementary primers. Additional alternative sequences that can be incorporated into the disclosed aptamers are disclosed herein. More generally, the incorporated sequences within the disclosed aptamer (e.g., SEQ ID NOs:8-15) can be further connected to other sequences W1W2... Wn and Vn...V2Vi on either side, respectively. In non-limiting embodiments, W1 and Vi can form a base pair, W2 and V2 can form a base pair, ... and Wn and Vncan form a base pair. For example, if W1 or W2 is G, Vi or V2 can be either C or T. If W1 or W2 is C, Vi or V2 can be G. If W1 or W2 is A, Vi or V2 can be T. If W1 or W2 is T, Vi or V2 is either G or A.

[0138] The disclosed aptamers can be at least about 60% identical (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) to its incorporated sequences and binds (e.g., selective or non-selective) to vancomycin. The disclosed aptamers can also include certain modifications (e.g., substitution, insertion, deletion, and / or inversion) as long as it is at least about 60% identical (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) to its incorporated sequences and binds (e.g., selective or non-selective) to vancomycin.

[0139] The disclosed aptamers can include a single-stranded DNA strand and / or an oligonucleotide sequence with at least one modified base. For example, the modified base can include RNA, modified RNA, modified DNA, PNA, or combinations thereof. The disclosed aptamers can include a single-stranded DNA strand and / or an oligonucleotide sequence with modifications in the phosphodiester backbone. For example, these modifications can include atom substitutions, neutralization of negative charges, or introduction of positive charges.

[0140] The disclosed aptamers can be immobilized to a substrate (e g., metal substrate) and used for sensing a target molecule / analyte, in this Example vancomycin. In nonlimiting embodiments, upon binding to the target analyte, the disclosed aptamers can reorient (change conformation) and affect the behavior of charge carriers in a substrate (e.g., semiconductor), resulting in a change in the conductance. For example, the disclosed aptamers can be labeled for electrochemistry and attached to an electrode (e.g., gold electrode). Upon binding to vancomycin, the aptamer structure can be modified to approach to the electrode so the conductance can be altered. The disclosed aptamers can be configured to modify its structure to move away from a substrate upon binding to a target molecule / analyte.

[0141] The disclosed aptamers can be synthesized to include functional groups that otherwise do not exist in natural nucleic acids. In non-limiting examples, the groups can include thiols (-SH), products of their reactions (e.g., thioethers), amino (-NH2) and / or carboxyl (-COOH) groups and products of their reactions (amides), aldehydes and / or products of their reactions (imines, hydrozones, hydrazides, or amines), biotin and / or its analogs, and / or any of the functional groups that can be used in click chemistry (e.g., alkynes, azides, strained alkenes, tetrazines) or products of their reactions (e.g., triazoles).

[0142] The disclosed aptamers can constitute a fragment of a larger sequence capable of folding into a double helical stem-loop structure, where the incorporated sequences within the disclosed aptamers are associated with at least one stem within this structure. Such a stem-loop is not necessarily stable in the absence of vancomycin, and its formation can be induced by the presence of vancomycin.

[0143] The disclosed aptamers can be used as a sensor to provide an electronic, electrical or optical device for measuring vancomycin. The sensor can report the presence or absence of analytes through a change in fluorescence or a change in an electrochemical readout. The sensor can include a combination of a field-effect transistor and one or more of the disclosed aptamers, a gold surface and one or more of the disclosed aptamers, a fiber-optical cable and one or more of the disclosed aptamers, and / or a quartz surface and one or more of the disclosed aptamers. The disclosed aptamers can be a part of a larger sequence that can have more than one conformation, with ligand binding impacting the equilibrium between the conformations. For example, the distance between an internal base within the disclosed aptamers and the 5’ end of the aptamers can increase or decrease.

[0144] The interaction of vancomycin and the disclosed aptamers within a larger sequence can change the extent of binding to dye, resulting in a measurable impact on optical or electrochemical properties. For example, the disclosed aptamer can include the truncated sequence SEQ ID NO:8 and its FRET form sequence with donor and acceptor / quencher fluorophores (5’-tetramethylrhodamine, 3’-fluorescein; SEQ ID NO: 12). This FRET form is partially complementary and thus hybridized. Titrating this sequence with an increasing amount of vancomycin produces an increasing fluorescence signal because this sequence can release the quencher portion in response to the vancomycin binding. Specifically, the binding of vancomycin to the partially hybridized assembly displaces the quencher portion, which can be due to a binding-induced conformational change. Alternatively, the disclosed aptamer including the truncated sequence (SEQ ID NO:5) and its FRET form (SEQ ID NO: 12) can detect vancomycin by measuring their electron transfer kinetics, which is monitored using square-wave voltammetry. The electrochemical response of the disclosed aptamer to vancomycin strongly varies with the square-wave frequency, showing “signal-off”, “signal-on”, or “non-responsive” behaviors across clinically relevant concentrations in undiluted whole blood. By using the non-responsive frequency output alongside specific constants, such as a (the ratio of the current at the nonresponsive frequency), KD (the dissociation constant of the aptamer), and y (signal gain), vancomycin’s concentration can be determined without calibration.

[0145] The disclosed aptamers can be attached to larger objects without the ability to freely diffuse through a solution. The disclosed aptamers can form a part of a freely diffusing molecular device with a molecular weight between about 5,000 D and about 1 ,000,000 D. For measuring vancomycin based on interactions involving an oligonucleotide-based receptor, the diffusing molecular device can include the disclosed aptamer. This diffusing molecular device functions by detecting the presence of vancomycin through alterations in the optical or electrical properties of a solution.

[0146] The diffusing molecular device, which can be either freely diffusing or attached to larger objects, can include a fluorophore, a quencher, an enzyme, or a redox dye. A fluorophore, a quencher, a redox dye, and / or a chemical modifier can be connected at 5’ end, 3’ end, or both ends of the disclosed aptamers. The fluorophore can be any available fluorophore, e.g., Fluorescein (FAM), tetramethylrhodamine (TAMN), Alexa Fluor, Cy2, Cy3, Cy5, Cy7, coumarin, TRITC, FITC, Qdot, DAPI, SYTOX™ Green, SYTO™ 9, TO-Pro-3, eFluor, PE-efluor™, PE-cyanine®7, Pacific Blue, Pacific Orange, Texas Red, etc. The redox dye can be any available redox dye. For example, the redox dye can be methylene blue and its analog, malachite green, or its analog, anthracene, Neutral red, Safranin T, Phenosafranin, Indigomono sulfonic acid, Indigo carmine, Indigotrisulfonic acid, Indigotetrasulfonic acid, Thionine, Sodium o-Cresol indophenol, Sodium 2,6-Dibromophenol-indophenol, Viologen, Diphenylamine, Diphenylbenzidine, Sodium diphenylamine sulfonate, o-Dianisidine, 5,6- Dimethylphenanthroline, 2,2'-Bipyridine, N-Ethoxychrysoidine, 1 ,10-Phenanthroline iron(ll) sulfate complex, N-Phenylanthranilic acid, Nitrophenanthroline, 2,2'-bipyridine, or analogs thereof. The quencher can be any available quencher (e.g., dabcyl, Iowa Black®, TAMRA, BHQ, BBQ, Atto, MGB, or Dab). The enzyme can be any enzyme that can be used in electrochemistry (e.g., glucose oxidase, glucose hexokinase, etc.).

[0147] The disclosed aptamers can bind to vancomycin despite physiological variations in concentrations of ions, such as magnesium, potassium, calcium, or sodium, or combinations thereof. For example, Mg(ll) ion can vary from 0.25-1 .75 x1 O'3M, and the extent of the complex formation with the aptamers encompassing the disclosed sequences can minimally change.

[0148] The disclosed aptamers and systems can be unresponsive to Mg(ll), potassium, and sodium concentrations in their physiological ranges, which can make them uniquely suitable for direct measurements in certain samples even without further buffering. The sample can include blood, serum, urine, effluent, any body fluids, or combinations thereof. The disclosed aptamers and systems can also provide sensitivity to vancomycin in the concentration range between about 10’6to about 10’3M, which can make them also suitable for direct use in serum, whole blood or interstitial, lymphatic, dialysis effluent fluids, saliva, sweat or tears.

[0149] The disclosed aptamers and systems can detect vancomycin in diluted or undiluted samples. For example, by mixing the disclosed aptamers with samples and reading fluorescent or electrical or colorimetric signal that is proportional to the concentration of vancomycin, the disclosed systems can detect vancomycin in the diluted samples. The sample can include blood, serum, urine, effluent, any body fluids, or combinations thereof.

[0150] The disclosed aptamers can be part of larger structures for sensing vancomycin at predetermined frequencies (e.g., once every hour, once every ten minutes, once every minute, once every second, or any predetermined period in between).

[0151] The rate of change of vancomycin can be used to assess therapeutic drug monitoring (TDM). For example, the rate of vancomycin change in the blood (e.g., ex vivo in unprocessed, finger-prick-scale volumes of whole blood), plasma, serum, urine, or effluent fluid during any form of dialysis or hemofiltration can be used without calibration to adjust parameters for dialysis or hemofiltration or to adjust the dosing of other drugs or identify circumstances where vancomycin concentrations can be fluctuating.

[0152] The disclosed aptamer can be presented as a single-stranded DNA incorporating an oligonucleotide sequence such as, for example, SEQ ID NO: 103. The disclosed aptamers can bind ligands through its structural rearrangement without changing the primary order of the sequence, as illustrated in FIG. 1. The disclosed aptamers can bind to various small molecule ligands, as illustrated in FIG. 2. These small molecule ligands include, but are not limited to, cocaine, serotonin, benzoylcholine, lidocaine, quinine, (+)-coca, testosterone, pseudoephedrine, naloxone, ephedrine, caffeine, norcocine, ecgonine methyl ester, benzoylecgonine, and tropane. The binding constants of the disclosed aptamers to the aforementioned small molecule ligands range from 10'8M to 10'3M.

[0153] The disclosed aptamers can adapt their structure to bind ideally unlimited ligands (Li to Ln), as illustrated in FIG. 3. The structural adaptation of the aptamers can include engaging canonical and non-canonical base pairings, switching the canonical and non- canonical base pairings, or the repulsion of one or more bases so that extra space can be made to accommodate different spatial arrangements for ligand binding. The structural adaption of the aptamer can include some or all of the aforementioned conditions. The disclosed aptamers can be a starting point for generating any analog aptamer against very challenging structures, as illustrated in FIG. 4. The disclosed aptamers can undergo mutations, insertions, and reselections of the bases in its sequence to bind various small molecule ligands. The disclosed aptamer can be presented as a single-stranded DNA incorporating an oligonucleotide sequence such as, for example, SEQ ID NO: 104. An analog aptamer derived from SEQ ID NO: 104 can be presented as a single-stranded DNA incorporating an oligonucleotide sequence such as SEQ ID NO: 105. This analog aptamer can specifically bind cocaine. An analog aptamer derived from SEQ ID NO: 104 can be presented as a single-stranded DNA incorporating an oligonucleotide sequence such as SEQ ID NO: 106. This analog aptamer can specifically bind serotonin.

[0154] EXAMPLE 2: MODIFIED VANCOMYCIN APTAMERS

[0155] The following example describes the modification of a known vancomycin aptamer, and the effect of the modified vancomycin aptamers on ThT dye displacement and FRET spectrum and signal.

[0156] One embodiment of a method of modifying a known vancomycin aptamer is shown in FIG. 5A, FIG.5B and FIG.5C. In this embodiment, the method involves modifying the known vancomycin aptamer shown in FIG. 5A (SEQ ID NO:1 ) by reducing the size of the external loop structure to produce the modified aptamer shown in FIG. 5B (SEQ ID NO:2). Next, the length of the stem connecting the internal and external loops is adjusted, and the external loop is modified by making various substitutions as shown in FIG. 5C.

[0157] Using this technique, a variety of modified vancomycin aptamers were produced (FIG. 6A (SEQ ID NO:3), FIG. 7A (SEQ ID NO:4), FIG. 8A (SEQ ID NO:5), FIG. 9A (SEQ ID NO:6) and FIG. 10A (SEQ ID NO:7)). The thioflavin T (ThT) dye displacement results, FRET spectrum and FRET signal for the modified vancomycin aptamer shown in FIG. 6A are shown in FIG. 6B, FIG. 6C and FIG. 6D, respectively. The thioflavin T (ThT) dye displacement results, FRET spectrum and FRET signal for the modified vancomycin aptamer shown in FIG. 7A are shown in FIG. 7B, FIG. 7C and FIG. 7D, respectively. The thioflavin T (ThT) dye displacement results, FRET spectrum and FRET signal for the modified vancomycin aptamer shown in FIG. 8A are shown in FIG. 8B, FIG. 80 and FIG. 8D, respectively. The thioflavin T (ThT) dye displacement results, FRET spectrum and FRET signal for the modified vancomycin aptamer shown in FIG. 9A are shown in FIG. 9B, FIG. 9C and FIG. 9D, respectively. The thioflavin T (ThT) dye displacement results, FRET spectrum and FRET signal for the modified vancomycin aptamer shown in FIG. 10A are shown in FIG. 10B, FIG. 10C and FIG. 10D, respectively.

[0158] The results show that the modified vancomycin aptamers have an altered ThT dye displacement, FRET spectrum and FRET signal, thereby providing that the affinity and desired half-point of response value of the original vancomycin aptamer can be modified to produce modified vancomycin aptamers having greater utility in various fluorescence and electrochemical applications.

[0159] EXAMPLE 3: ELECTROCHEMICAL APTAMER-BASED SENSORS FOR IMPROVED THERAPEUTIC DRUG MONITORING AND HIGH-PRECISION, FEEDBACK-CONTROLLED DRUG DELIVERY

[0160] The Electrochemical Aptamer-Based (E-AB) sensing platform is a convenient (rapid, single-step, calibration-free), modular approach to measure concentrations of specific molecules (irrespective of their chemical reactivity) directly in blood and even in situ in the living body. Given these attributes, the platform provides significant opportunities to render therapeutic drug monitoring (the clinical practice in which dosing is adjusted in response to plasma drug measurements) as frequent and convenient as the measurement of blood sugar has become for diabetics. The ability to measure arbitrary molecules in the body in real time enables closed-loop feedback control over plasma drug levels in a manner analogous to the recently commercialized controlled blood sugar systems. This example describes the selection of an aptamer against vancomycin, a narrow therapeutic window antibiotic for which therapeutic monitoring is a critical part of the standard of care, and its adaptation into an electrochemical aptamer-based (E-AB) sensor. Using this sensor: (1 ) rapid (seconds), convenient (single-step, calibration-free) measurement of plasma vancomycin in finger prick-scale samples of whole blood; (2) high-precision measurement of subject-specific vancomycin pharmacokinetics (in a rat animal model); and (3) high precision, closed-loop feedback control over plasma levels of the drug (in a rat animal model) is demonstrated. The ability to not only track (with continuous-glucose-monitor-like measurement frequency and convenience), but also actively control plasma drug levels provides improved therapeutic drug monitoring and, more generally, personalized, high-precision delivery of pharmacological interventions.

[0161] The therapeutic windows (range of plasma concentrations over which a molecule is therapeutically effective without causing significant adverse effects) of most drugs are wide relative to typical metabolic variability and thus indirect predictors of patient metabolism, such as age, body mass, or genotype, are sufficient to ensure effective dosing. The therapeutic windows of many drugs, however, are narrow relative to interpatient metabolic variability and the accuracy of indirect pharmacokinetic predictors is insufficient to ensure safe dose determination and drug delivery. Under these circumstances, therapeutic drug monitoring (TDM), the clinical practice in which plasma drug concentrations are measured to adjust dosing, remains the standard of care. This is particularly true for the most grievously ill patients, as this is simultaneously the population most likely to exhibit altered metabolism and the population for which the margin for therapeutic error is narrowest.

[0162] The need for and problems associated with TDM are compellingly illustrated by the difficulty in properly delivering vancomycin, a “key access antibiotic.” Because plasma levels below the drug’s therapeutic window can lead to dangerously poor efficacy (e.g., death due to sepsis) and above the window are associated with permanent hearing loss and nephrotoxicity, TDM is a recommended element of the standard of care in vancomycin treatment. Current methods of monitoring plasma vancomycin levels, however, rely on venous blood draws that are analyzed using immunoassays or HPLC-MS and are thus slow, cumbersome processes requiring fully- equipped laboratories and returning an answer only hours to days after sample collection (Figure 11 , panel A). Specifically, vancomycin’s standard of care requires a “peaks and troughs” measurement ( / .e., the highest and lowest concentration of the drug) in which often complex, multi-phase pharmacokinetics are fitted. As a result, TDM of vancomycin is performed more rarely than is optimal and, even when performed, provides only a low resolution (few-data point) “snapshot” that may not accurately measure a patient’s metabolism. More generally, the slow, cumbersome nature of current drug measurement approaches remains a major hurdle hindering the wider adoption of TDM. Here, in contrast, the ability of electrochemical aptamer-based (E-AB) sensors to improve on the convenience and accuracy of TDM is shown. This platform renders plasma drug level measurements as easy and convenient as current blood sugar measurements using at-home finger-prick measurements of plasma drug levels (Figure 11 , panel B), or continuous, real-time measurements performed via a wearable device (Figure 11 , panel C), vastly improves the ability to accurately dose and properly deliver this and many other pharmacological therapies (Figure 11 , panel D).

[0163] In response to the need for improved methods of performing TDM, electrochemical aptamer-based (E-AB) sensors were developed, a reagentless, single- step sensing platform for the rapid, and even real-time, measurement of plasma drug levels in whole blood and even directly in the body. E-AB sensors are comprised of an electrode-bound redox-reporter-modified aptamer sequence that undergo a binding- induced change in electron transfer kinetics easily monitored using, for example, square-wave voltammetry. The E-AB platform was studied as a means of performing TDM by selecting the first reported aptamer against vancomycin and adapting it into an E-AB sensor. This new sensor was then applied to: (1 ) the rapid, calibration-free quantification of vancomycin ex vivo in unprocessed, finger-prick-scale volumes of whole blood; (2) achieving seconds-resolved, high-precision vancomycin pharmacokinetic measurements; and (3) performance of high-precision feedback control over plasma levels of this historically difficult to dose drug. Materials and Methods

[0164] Aptamer Selection

[0165] All chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO), unless otherwise noted. Oligonucleotides used in aptamer selection and the optical characterization of the aptamer were obtained from Integrated DNA Technologies (Coralville, IA). The vancomycin stock solution (10 mM) was prepared in nuclease-free water, filtered through a 0.22 pm sterile filter, aliquoted into 10 pl volumes and stored at -20°C. Commercially available phosphate buffered saline (PBS; Corning), pH 7.4 was used with the addition of 2 mM MgCh

[0166] The initial selection library was composed of ~27 pg of DNA sequences in which a 45- base random element is flanked with two fixed primer regions (SEQ ID NO: 107). The elements of this library were immobilized on a streptavidin column via hybridization to a partially complementary capture strand (SEQ ID NO: 108), with a 3’ biotin tag. The column was then exposed to varying concentrations of vancomycin (Round 1 -2, 200 pM; Round 3, 150 pM; Round 4, 100 pM; Round 5, 50 pM; Round 6, 20 pM; Round 7, 10 pM; Round 8, 4 pM; Round 9-10, 2 pM; Round 11 -12, 1 pM; and Round 13-14, 0.5 pM) to elute aptamer candidates. At each step the eluted sequences were amplified using 10 ± 1 PCR cycles where PCR was held for 120 s at 95°C and then cycled [95°C, 15 s ~>60°C, 30 s — >72°C, 45 s] followed by holding at 72°C for 120 s. The oligonucleotide pool that was obtained following the last ( / .e., 14th) round of selection was cloned and sequenced; the protocol was uneventful, required no gel purification steps and all products were within expected errors in synthesis ( / .e., usually N or N-1 ). A single sequence (SEQ ID NO: 109) was then extracted from this pool for further characterization. To test the binding of this “parent” aptamer a fluorescent quenching / dequenching assay was employed (FIG. 16). This consists of conjugating a fluorescein at the 5’ terminus of the parent sequence and hybridizing this to a partially complementary, 3’ -dabcyl- modified quencher strand (SEQ ID NQ:110) in a 1 :10 ratio. Titrating this with an increasing amount of vancomycin produces an increasing fluorescence signal as a result of the parent sequence releasing the quencher strand in response to target binding. Following the resultant increase in fluorescence a dissociation constant (KD) of ~ 0.12 pM was obtained. Parallel studies with vancomycin analogs lacking the glycan moiety (oritavancin and vancomycin aglycon) indicate that the aptamer binding is in part associated with the drug’s glycan group (FIG. 16, panels D and E).

[0167] Sensor Fabrication and Testing

[0168] Sodium phosphate monobasic, sodium chloride, sodium hydroxide, potassium chloride and potassium phosphate dibasic were acquired from Fischer Scientific (Waltham, MA). 6-mercapto-1 -hexanol, phosphate buffered saline (1X PBS, pH = 7.4) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were obtained from Sigma Aldrich (St. Louis, MO). Sulfuric acid was obtained from EMD (Burlington, MA) and vancomycin hydrochloride (USP grade) was purchased from Gold BioTechnology, Inc. (St. Louis, MO).

[0169] Bovine blood with heparin was acquired from Hemostat Laboratories (Dixon, CA) and employed in a circulatory flow system maintained by a gear pump purchased from Cole-Parmer (Vernon Hills, IL). Catheters (22G) and 1 mL syringes were acquired from Becton Dickinson (Franklin Lakes, NJ). Channel connector cables for in vivo probe fabrication were obtained from PlasticsOne (Roanoke, VA). Polytetrafluoroethylene (PTFE) insulated gold, platinum and silver wires (75 pm diameter, 64 pm insulation thickness) were purchased from A-M Systems (Sequim, WA) and were further insulated using a heat-shrink PTFE insulation (PTFE, HS Sub-Lite-Wall, 0.02 in, black-opaque, Lot #17747112-3) from ZEUS (Branchburg Township, CA). Fritted Ag|AgCI reference and platinum counter electrodes were acquired from CH Instruments (Austin, TX) and used in a three-electrode setup (FIG. 13). The in vitro characterization of the sensors was completed using a CHI1000C, and employed a CHI1240C potentiostat for in vivo monitoring (Austin, TX). All of the above materials and reagents were used as received.

[0170] Electrode fabrication and electrochemical cleaning. 7.75 cm segments of gold, platinum, and silver wires were cut and their insulating coatings were stripped at both ends using a surgical blade. These were then bundled together using heat-shrinkable PTFE tubing, leaving 5 mm at the end of each wire exposed. To avoid shorts these were bundled with a physical gap separating each wire (FIG. 13, panel A). Finally, the end of the gold electrode was trimmed by 2 mm to produce an exposed electrode length of 3 mm.

[0171] Following the assembly of the electrodes the gold surface was converted into an E-AB sensor using an established protocol. In brief this starts with electrochemical cleaning as follows: (1 ) cycling the potential 300 times between -1 and -1 .6 V in a solution of 0.5 M NaOH at 1 Vs- 1 to remove any residual thiol / organic contaminants on the electrode surface; (2) pulsing between 0 and 2 V for 16,000 cycles with a pulse length of 0.02 s in 0.5 M H2SO4 to increase the electrodes microscopic surface area (and thus the signaling current); (3) To modify these freshly cleaned electrodes with aptamer, first a DNA stock solution (2 pL of 100 pM) of the desired construct was reduced using a 1000x molar excess of an aqueous solution of TCEP (2 pL of 10 mM) for 1 h at room temperature. The concentration of the reduced DNA was then adjusted to 500 nM based on its adsorption at 260 nm using 1X PBS solution. The freshly cleaned gold electrodes were immersed in this solution for 1 h at room temperature, rinsed the electrodes with 1X PBS, and then incubated in 5 mM 6-mercapto-1- hexanethiol solution in 1X PBS overnight. The silver wire reference was immersed overnight (separately from the gold working electrode) in a concentrated hypochlorite solution (commercial bleach).

[0172] In vitro sensor characterization. The sensors were immersed directly in flowing whole blood (flowing at 1 mL s- 1 ), or in 100 pL drop of undiluted bovine whole blood spiked with the respective concentrations of vancomycin (see FIG. 13, panel B) and recorded square-wave voltammograms by sweeping the potential between -0.1 and -0.5 V (all potentials are reported versus Ag|AgCI) while varying the square-wave frequency (from 2 Hz to 1000 Hz) and fixing the potential increment to 1 mV and the square-wave amplitude to 25 mV. The peak currents of these voltammograms were used to determine the optimal frequencies to sample the current at these sensors while also determining the electron transfer kinetics of the unbound and bound state of the aptamer (FIG. 7). Following the determination of the optimal square-wave frequencies 10, 25 and 300 Hz were selected as the “signal-off,” “non-responsive,” and “signal-on” frequencies from which full binding curves were acquired directly in flowing whole blood. These binding curves serve as calibration curve to estimate the concentration of vancomycin in vivo while determining the constant, a (i.e. , constant to calculate the current in absence of target - 3.6 + 0.2), to use the signal seen at the “non-responsive” frequency to estimate the signal that the sensor would produce at the responsive frequency in the absence of target. These are also used to determine the KD value (45.5 + 2.2 pM) and signal gain, y (125 + 3%).

[0173] In vivo testing and animal procedures. The miniaturized sensors were inserted in a 22G catheter placed in the right jugular vein of Sprague-Dawley rats males (4-5 months old; acquired from Charles River Laboratories of Santa Cruz, CA). The rats weighed between 400-600 g and were pair-housed in a standard light cycle room (12:12 regular light cycle with lights on at 8AM). They were allowed ad libitum access to food and water. Anesthesia was induced under 5% isofluorane in a Plexiglas anesthesia chamber and then maintained anesthesia during the entire length of the experiment using a 2-3% isofluorane / oxygen mixture. A catheter and infusion line was installed into the left jugular vein of the animal where injection of vancomycin can be performed. To do so the area above each vein was shaved and cleaned using betadine and 70% ethanol followed by a small incision done above each vein using spring-loaded microscissors that allows the insertion of a silastic catheter constructed with a bent steel cannula with a screw-type connector (Plastics One, Roanoke, VA) and silastic tubing (11 cm, i.d. 0.64 mm, o.d. 1.19 mm, Dow Corning, Midland, Ml) for infusions. Both the E-AB sensor and the infusion line were tied off using a sterile 6-0 silk suture (Fine Science Tools, Foster City, CA). 30 units of heparin was infused in the rats prior to any recording.

[0174] Gold-coated aptamer sensors, platinum and silver wires were used in a three- electrode setup where a minimal 20 min baseline was recorded prior to drug injection. Real-time plotting of data along with real-time dose infusion control was achieved by employing a custom-made MATLABTM script. Briefly these scripts enable extraction of peak current in real-time and directly derives the kinetic differential measurement required for monitoring vancomycin in vivo to ultimately calculate the necessary dose to infuse in animals.

[0175] Results

[0176] E-AB sensor development requires the availability of an aptamer that achieves clinically relevant affinity and specificity. To identify such an aptamer a solution-phase selection based on a stem-loop closing scheme was performed. This selection starts with a random DNA library of ~1014 unique sequences in which each member is composed of a 45-base random element flanked by two, partially complementary primers (FIG. 12, panel A). The library members are attached to an agarosestreptavidin solid support via hybridization to a biotinylated anchor strand complementary to one of the two primers, which also prevents the primers from hybridizing with one another to form a double-stranded stem. Sequences that undergo a binding-induced conformational change leading to detachment (presumably due to stabilization of the stem) are then collected, amplified, and regenerated. Repeating this cycle 14 times produced an aptamer against vancomycin with a dissociation constant (KD) of ~0.1 pM when measured free in bulk solution using an optical assay (FIG. 16). To support E-AB sensing an aptamer must undergo a binding-induced conformational change that, in turn, produces a significant change in electron transfer kinetics (FIG. 12, panel B). The full-length (“parent”) aptamer does not exhibit this property (FIG. 17, panel A), presumably due to the stability of the folded structure. Circular dichroism studies suggest, however, that the removal of four base pairs from the stem produces the necessary binding-induced conformational change (FIG. 17, panel B). Consistent with this, when adapted into the E-AB platform the truncated aptamer supports good E-AB signaling, exhibiting a relative signal change upon the addition of saturating target of ~120% and a KD of 45 pM when challenged in flowing whole blood (FIG. 12, panel C). These attributes support vancomycin measurements across the entire 6 to 35 pM clinical range of the antibiotic. The sensor is also rapid: when challenged with 10 pM vancomycin it is effectively completely equilibrated in the 9 s it takes to acquire a pair of square-wave voltammograms (FIG. 18).

[0177] By analogy to the impact that the home glucose monitor has had on diabetes care, the E-AB platform significantly improves on current TDM methods by moving it from laboratory-based tests to at-home “self-testing” (FIG. 11 , panel B). To demonstrate this vancomycin-detecting sensors were fabricated using wire electrodes small enough to immerse in sub-100 pL volumes (FIG. 13, panel A) and then deployed them in a calibration-free manner. Briefly, this relies on the strong square-wave frequency dependence of E-AB signaling (FIG. 12, panel C) to produce a “non- responsive” current that is independent of target concentration used to correct for sensor-to-sensor variation. Using this approach to measure vancomycin spiked into 100 pL samples of whole bovine blood returns concentration estimates within ± 20% of the known concentration of the drug over the entire clinically relevant range (FIG. 13, panel B), showing that E-AB sensors support convenient, calibration-free, finger-prick- style self-testing closely analogous to the home-glucose-meter testing employed by many diabetics.

[0178] Although self-testing of plasma vancomycin levels using finger-prick samples could improve the convenience of TDM, such measurements still only provide poorly time-resolved “snapshots” of metabolism. In contrast, the ability of indwelling E-AB sensors to achieve sub-second resolved measurements directly in the living body provides an unprecedented route towards not only measuring plasma drug levels, but also measuring patient-specific pharmacokinetics. To demonstrate this E-AB sensors were placed in the circulatory systems of live Sprague-Dawley rats via a 22-gauge catheter (FIG. 14, panel A). To remove the drift seen in in vivo deployments a drift correction approach was used: kinetic differential measurements (KDM). This takes the difference between the sensor’s response measured at two square-wave frequencies - one in which the sensor is “signal-on” and the other at which the sensor is “signal-off” - but that drift in concert. Using KDM corrected sensors can measure 9-s-resolved plasma vancomycin levels in real time after, for example, the intravenous injection of human-equivalent, 30 mg kg-1doses (FIG. 14, panel B). The resultant pharmacokinetic curves obtained from this study produces peak concentrations and plasma half-lives consistent with values reported in the literature. The typically ~15 min time resolution of these prior measurements, however, is poorly matched to the metabolic timescale of the drug and thus the precision with which they define its distribution (a) and elimination (P) time constants is limited. In contrast, high-frequency E-AB measurements return parameter estimates with precision (defined as 95% confidence intervals) of better than 20%, which is more than sufficient to identify statistically significant pharmacokinetic differences between individual animals (FIG. 14, panel B).

[0179] The goal of TDM is to increase the time during which plasma drug levels remain within a drug’s therapeutic window. The ultimate realization of this would be to measure these levels in real-time and then use this information to optimize dosing on a timescale faster than the metabolic timescale of the drug. The seconds-resolved, real time measurements provided by indwelling E-AB sensors support just such feedback- controlled delivery. To illustrate this an indwelling vancomycin sensor was placed into the right jugular vein of a rat and a drug-delivery catheter into the left jugular vein that was attached to a syringe pump (FIG. 15, panel A). The sensor’s output was then employed to drive a proportional-integral-derivative (PID) controller that, in turn, adjusted the flow rate of the syringe pump every 9 s. Using this system rapid achievement (~30 min) and accurate maintenance (± 2 pM) of plasma vancomycin concentrations was obtained over the course of hours (FIG. 15, panel B and FIG. 8) even in the face of several-fold variations in the rate with which the drug is eliminated from the body during this same period (FIG. 15, panel C). Note: the limited duration of these experiments is due to animal welfare concerns (which preclude keeping rats under anesthesia for more than 6 h) and not by loss of sensor performance.

[0180] A rat animal model has been used to demonstrate the application of E-AB sensors to the problem of performing TDM and, as the ultimate goal in TDM, feedback controlled, high-precision drug delivery. To do so a vancomycin-binding aptamer was selected via a solution-phase selection scheme followed by its reengineering and adaptation into an E-AB sensor. Using this sensor: (1 ) the rapid (seconds), convenient (single step, calibration-free), accurate (± 20%) determination of vancomycin levels in finger-prick-scale volumes of unprocessed whole blood; (2) the high precision (± 20%) measurement of subject-specific vancomycin pharmacokinetics; and (3) the high precision (± 20%) feedback control of plasma vancomycin levels in the face of significant, hour-to-hour changes in drug metabolism was performed.

[0181] TDM was introduced into clinical practice to counter the relatively narrow therapeutic windows of some drugs and, in doing so, to improve the safety and efficacy of treatments. TDM has been shown, for example, to significantly improve patient outcomes upon treatment by vancomycin and the aminoglycoside antibiotics. TDM can likewise improve patient compliance (less of a critical concern for vancomycin considering that it is administered / monitored in a hospital setting, but a serious health issue for many other drugs) and the cost-effectiveness of treatments. Unfortunately, however, studies have repeatedly shown that clinicians often fail to achieve these goals, suggesting that improvements in the ability to perform TDM are a critical component of the move towards high-precision, personalized medicine. Specifically, the present development of a platform technology for convenient measurements of plasma drug levels represents a paradigm shift in how TDM is performed, and significantly improves outcomes, reduces complications, and reduces health costs.

[0182] EXAMPLE 4: METHOD FOR TUNING AFFINITY OF APTAMERS

[0183] A therapeutic drug is a candidate for therapeutic drug monitoring (TDM) if (1 ) it shows a clear plasma concentration-effect relationships; (2) it has a wide interpatient pharmacokinetic variability, (3) there is a narrow therapeutic window that prevents us simply arbitrarily increasing its concentration to achieve the desired effect; and (4) its effects cannot be simply titrated through a direct clinical observation; vancomycin, an antibiotic with substantial toxicity for kidneys, is the prime example of such drugs. A particularly challenging example which requires therapeutic drug monitoring of vancomycin (and other drugs) is if a patient is in intensive care unit undergoing continuous renal replacement therapy (CRRT, a form of dialysis) due to acute kidney injury (ICU).

[0184] The leading method to monitor drugs continuously are electrochemical aptamerbased (EAB) sensors as described herein (FIG. 20A); here, the word aptamers is used to describe oligonucleotide-based receptors for small molecules), that can provide a real-time monitoring of drugs irrespective of their intrinsic spectroscopic properties, while operating well in complex media (e.g., in the bloodstreams of live animal subjects). An alternative approach, previously used mostly in imaging, is based on aptamer-based fluorescent sensors, including FRET.

[0185] There are three main considerations that need to be taken into account simultaneously while designing aptamer-based sensors for monitoring: First, the sensitivity of an aptameric sensor needs to be adjusted to be at its most sensitive in the range of clinical interest; for vancomycin that is typically between 5-40 pM, if that drug is being monitored in whole blood. Second, an aptamer that has otherwise a stable secondary structure, needs to be engineered to signal ligand binding through a gross change in that structure, which can conflict with the first requirements. Third, the change in signal needs to be maximized upon ligand binding. On top of these essential considerations, any introduction of hydrophobic moieties, such as redox-active labels, or fluorescent dyes, could impact affinity of the resulting sensor, in the manner that is not fully predictable.

[0186] These conflicting requirements were recognized early, with various proposed solutions, including those relying on secondary structure prediction programs, random screening or various combinations thereof. We describe here, in this example, a vancomycin sensor, and a systematic approach to fine tune its affinity properties, while keeping conformational changes that lead to transduction of binding events into a readable signal almost constant.

[0187] Minimal vancomycin binding sequence The set of vancomycin aptamers that were isolated from a SELEX, led to the common region (blue color) identified through comparative studies combined with an extensive minimization, which included deletions and mutations (FIG. 20B). This common region overlapped with a domain identified to interact with vancomycin through docking studies, and was the basis for all subsequent designs.

[0188] From minimal original aptamer to V28

[0189] If a stem in an aptamer is shortened and the affinity of that aptamer remains approximately the same, the simplest explanation is that the resulting shorter stem is still stable. And, vice versa, if a shortening of a stem leads to a substantial decrease in affinity, the first instinct is to propose that the stem became unstable. This process, shortening of the stem until there is a drop in affinity, was the basis of original folding sensors (also known as molecular beacon aptamers) and early electrochemical sensors.

[0190] An intriguing observation was made during the minimization and mutagenesis studies of V28 (FIG. 20C): flipping of one base pair, C-G into G-C, led to a substantial (severalfold) improvement in affinity, as judged by the half-point displacement of ThT dye (FIG. 20C). This effect was extended to the analog with only two base pairs (FIG. 20D), with a very similar result. This process yielded V28, the shortest aptamer with the unstable lower stem, significant conformational change, but still displaying a submicromolar half-point displacement of the dye. The difference from the full length aptamer in affinity was approximately three-fold, that is, below 1 kcal / mol. This particular structure-switching variant was thus selected for further optimization.

[0191] These results are consistent with two effects of the single base flip described above: First, 5’ CCCGA could form a stable stem with TCGGG near the 3’ end, thus providing an alternative, non-binding secondary structure. In this model, flipping of the closing C-G pair, eliminates one base pair in this non-binding structure, while leaving the bonding one with the same number of base-pairs, therefore relatively favoring the latter. Second, focusing on the nearest neighbor effects, the two consecutive CC\GG pairs confer less stability that CG\GC pairs. Here, then, direct additional stabilization of a binding conformation is provided by a terminal pair. Together, these effects account for the most of the difference seen between V28 and original aptamer. Sensors based on V28

[0192] V28 has two main conformations, inactive ( / .e., non-binding) and active ( / .e., binding), and the balance between the two could signal concentration of vancomycin. To demonstrate this labels for both FRET (fluorescence energy resonance transfer, 5’- fluorescein and 3’-TAMRA) and EAB (electrochemical-aptamer based sensing, with 5’- SH for positioning on electrode and 3’-methylene blue as the redox dye of choice) sensing formats were introduced into V28. Both analogs showed functional sensor behaviors (FIG. 21A).

[0193] Specifically, the FRET sensors signaled the presence of vancomycin consistent with the KD of about 160 nM. The sensor, despite labelling, did not act as the pure FRET sensor ( / .e., with expected isosbestic point); namely, the fluorescence spectrum revealed that FRET pair was very close even without the ligand present, while in its bound state, there was a substantial quenching (FIG. 21A).

[0194] Similarly, the electrochemical sensor showed both free and bound forms of sensors positioning redox dye (MB) near gold electrode ( / .e., rapid electron transfer; FIG. 21 B). All results were, thus, consistent with the two-state model as shown in FIG. 21 A and FIG. 21 B.

[0195] Analogs of V28with different affinity

[0196] One potential issue with using V28 directly as the sensor is that its high, nanomolar, affinity could prevent it from being suitable for continuous monitoring of a wide range of micromolar concentrations (covering applications in blood, serum, interstitial fluid, cerebrospinal fluid, and saliva) without further work-up. Thus, a general approach was developed to fine-tune the responsiveness range of this two state-switch, which could be subsequently expanded to other aptamers with two or more stems.

[0197] The third well-defined state to this equilibrium was introduced, in order to fine tune it by using another loop with complementarity to the 5’ end of the aptamer (FIG. 22A). This can adjust affinity by adjusting the length and position of the loop-based complementary region. Indeed, and as expected, by varying sizes of loops, positions and lengths of complementary regions, identification, through screening, of sensors of varying affinities, with nearly arbitrary resolution. Ten examples are shown in FIG. 22B through FIG. 22LI). Further adjustments of each sensor is possible, for example, by modifying individual stems through mutations, extensions, and introduction of mismatches and other modifications.

[0198] On the mechanism of reduction of V28 affinity

[0199] While results were satisfactory from the practical point of view and were validating the initial designs, some of the observations with sensors conflicted with the proposed mechanism. Specifically, resulting FRET sensors (FIG. 23A through FIG. 27C), when compared with those built on V28, were not consistent with the increase in distances between the donor-acceptor pair, in the absence of ligand binding, as expected due to the presence of additional conformation.

[0200] The explanation for these observations was that in a majority of these examples there had been no impact on gross conformational equilibrium through formation of any additional secondary structures. Instead, simple steric interactions were observed between the newly introduced loop and the aptamer core. In support of this mechanism, completely mutating complementarity out of the loop led to very little impact on affinity. This mechanism would then be analogous to early work on allostrically modulated ribozymes in which the activity of nucleic acid enzyme was controlled through the attachment of aptamers to the non-conserved loops, with ligand binding inhibiting enzymatic activity through through-space (steric) interactions.

[0201] The ability to fine-tune vancomycin aptamers to near-arbitrary affinity was achieved through a mixture of engineering and screening. This ability is based on the impact of structural changes introduced in analogs of the initial sensor to impact relative populations of two states, active and inactive, which leads to efficient transduction of binding events into fluorescent or electrochemical signals.

[0202] The ability is general, that is, it goes beyond just vancomycin. Similar results were seen, in lesser detail, starting from tobramycin (FIG. 28A through FIG. 28Q) and amikacin (FIG. 29A through FIG. 29Q) aptamers.

Claims

CLAIMS1. A method, comprising: identifying an aptamer having a desired half-point of response value, the aptamer comprising an internal loop, an external loop, a first stem between the internal loop and the external loop, and a second stem connected to the internal loop, comprising a) modifying at least one of the external loop, the first stem, or the second stem of each of a plurality of aptamers to create a plurality of modified aptamers; b) screening at least a portion of the plurality of modified aptamers to determine the half-point of response of the at least the portion of the plurality of modified aptamers; and c) selecting a modified aptamer from the at least the portion of the plurality of modified aptamers with the desired half-point of response.

2. The method of claim 1 , wherein modifying comprises engineering at least one of the stem length, the loop structure, or the body of each of the plurality of aptamers.

3. The method of at least one of claims 1 -2, wherein the screening determines a half-point of response that matches what is needed for at least one of fluorescence or electrochemical sensors.

4. The method of any one of claims 1-3, wherein the second stem of the modified aptamer includes a first end and a second end, and further comprising, labeling each of the first end and the second end of the second stem of the modified aptamer.

5. The method of any one of claims 1-4, further comprising, complexing the modified aptamer with a redox label dye.

6. The method of claim 5, further comprising displacing the redox label dye via binding of a ligand to the modified aptamer.

7. An aptamer identified by the method of claim 1 .

8. A method, comprising: modifying an affinity of an aptamer to a ligand, the aptamer comprising an internal loop, an external loop, a first stem between the internal loop and the external loop, and a second stem connected to the internal loop, comprising a) modifying at least one of the external loop, first stem, or the second stem of the aptamer to generate a modified aptamer having a different affinity for the ligand than the aptamer; and b) screening the modified aptamer to determine the affinity of the modified aptamer for the ligand.

9. An aptamer produced by the method of claim 8.

10. An aptamer comprising: a first loop; a second loop; a first stem coupled to and separating the first loop and the second loop; and a second stem comprising a first end and a second end, wherein the first end is coupled to the first loop, wherein the first loop binds to a ligand, wherein the second loop is complementary to a portion of the aptamer, and wherein at least one of the second loop, the first stem, or the second stem is modified from its original form.11 . The aptamer of claim 10, wherein the at least a first portion of the second loop is complementary to at least a second portion of the aptamer in the first loop.

12. The aptamer of any one of claims 10-11 , wherein the first end and second end of the second stem are labeled.

13. The aptamer of any one of claims 10-12, wherein the first end and second end of the second stem are labeled with different labels.

14. The aptamer of claim 13, wherein the first end of the second stem is labeled with tetramethylrhodamine (TAMRA) and the second end of the second stem is labeled with fluorescein (FAM).

15. The aptamer of any one of claims 10-13, complexed with a redox label dye.

16. The aptamer of claim 15, wherein the aptamer and the redox label dye are part of separate moieties.

17. The aptamer of claim 15, wherein binding of a ligand to the aptamer displaces the redox label dye, wherein the displacement of the redox label dye is detected by an electrode.

18. The aptamer of claim 15, wherein at least one of the aptamer or the redox label dye is attached to a linker, wherein the linker is attached to an electrode.

19. The aptamer of claim 18, wherein binding of a ligand to the aptamer displaces the redox label dye.

20. The aptamer of claim 10, wherein the aptamer comprises the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.21 . An aptamer for detecting a target molecule in a biological sample, comprising a single-stranded deoxyribonucleic acid (DNA) strand, wherein the aptamer is configured to bind to the target molecule and undergoes a change upon binding of the target molecule.

22. The aptamer of claim 21 , wherein the target molecule is vancomycin, amikacin, tobramycin, cocaine or serotonin.

23. The aptamer of claim 22, wherein the target molecule is vancomycin.

24. The aptamer of claim 23, wherein the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs:3-104.

25. The aptamer of claim 22, wherein the target molecule is cocaine.

26. The aptamer of claim 25, wherein the aptamer comprises the nucleotide sequence of SEQ ID NO:105.

27. The aptamer of claim 22, wherein the target molecule is serotonin.

28. The aptamer of claim 27, wherein the aptamer comprises the nucleotide sequence of SEQ ID NO: 106.

29. The aptamer of claim 22, wherein the target molecule is amikacin.

30. The aptamer of claim 29, wherein the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs: 135-1 3.31 . The aptamer of claim 22, wherein the target molecule is tobramycin.

32. The aptamer of claim 31 , wherein the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs: 144-152.

33. The aptamer of any one of claims 21-32, wherein the biological sample comprises blood, serum, urine, effluent, saliva, sweat, tears, a body fluid, or a combination thereof.

34. The aptamer of any one of claims 21-33, wherein the aptamer is configured to be immobilized to a substrate for sensing the target molecule.

35. The aptamer of any one of claims 21-34, wherein the aptamer is configured to be incorporated into a sensor device.

36. The aptamer of claim 35, wherein the sensor device comprises the aptamer and: a) a field-effect transistor; b) a gold substrate; c) a fiber-optical cable; or d) a quartz surface.

37. The aptamer of any one of claims 21-36, wherein the aptamer comprises a fluorophore, a quencher, an enzyme, a redox dye, or a combination thereof.

38. an aptamer having a modified affinity for a ligand compared to a starting aptamer, produced by a method comprising: a) modifying at least one of an external loop, a first stem, or a second stem of the aptamer to create a modified aptamer; and b) screening the modified aptamer to determine the affinity of the modified aptamer for the ligand, thereby generating a modified aptamer having a different affinity for the ligand than the starting aptamer.

39. A method for detecting a target molecule in a biological sample, comprising: a) contacting at least a portion of the biological sample comprising the target molecule with an effective amount of the aptamer of claim 21 ; and b) detecting a change in the aptamer after the aptamer contacts the target molecule in the biological sample.

40. The method of claim 39, wherein the change in the aptamer comprises a change of a conductance, a fluorescence, or an electrochemical readout.41 . The method of any one of claims 39-40, wherein the target molecule is vancomycin, amikacin, tobramycin, cocaine or serotonin.

42. The method of any one of claims 39-41 , wherein the biological sample comprises blood, serum, urine, effluent, saliva, sweat, tears, a body fluid, or a combination thereof.

43. A method for detecting a target molecule in a biological sample, comprising: contacting at least a portion of the biological sample comprising the target molecule with an effective amount of an aptamer for detecting the target molecule in the biological sample, wherein the aptamer comprises a single-stranded deoxyribonucleic acid (DNA) strand, wherein the aptamer is configured to bind to the target molecule and wherein the aptamer undergoes a change upon binding to the target molecule, and detecting a change in the aptamer after the aptamer contacts the target molecule in the biological sample.

44. The method of claim 43, where the change in the aptamer comprises a change of a conductance, a fluorescence, or an electrochemical makeup of the aptamer.

45. The method of any one of claims 43-44, wherein the target molecule is vancomycin, amikacin, tobramycin, cocaine or serotonin.

46. The method of any one of claims 43-45, wherein the biological sample comprises blood, serum, urine, effluent, saliva, sweat, tears, a body fluid, or a combination thereof.

47. A method of therapeutic drug monitoring in a patient in need thereof, comprising a) contacting at least a portion of a biological sample comprising a target molecule from the patient with an effective amount of the aptamer of claim 21 ; b) detecting a change in the aptamer after the aptamer contacts the target molecule, wherein the change is indicative of the concentration of the target molecule in the biological sample; c) performing high-precision control over a plasma level of the target molecule based on the concentration of the target molecule in the sample; and d) providing the patient with a treatment based on the concentration of the target molecule in the sample.

48. The method of claim 47, further comprising performing the detecting step of step b) at a predetermined frequency.

49. An electrochemical sensor for detecting a molecule, comprising an aptamer comprising a redox reporter that binds to the molecule and undergoes a change in the electron kinetics upon binding of the molecule, wherein the aptamer is attached to a surface.

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