WO2020069318A1

WO2020069318A1 - Cortisol binding aptamer

Info

Publication number: WO2020069318A1
Application number: PCT/US2019/053460
Authority: WO
Inventors: Chongli YUAN; Alexander Wei
Original assignee: Purdue Research Foundation Limited
Priority date: 2018-09-28
Filing date: 2019-09-27
Publication date: 2020-04-02

Abstract

The present invention relates to a novel cortisol binding aptamer, the use thereof for cortisol determination as well as sensors and assays comprising the novel aptamer.

Description

CORTISOL BINDING APTAMER

RELATED APPLICATIONS

This Application claims priority under 35 USC § 119(e) to U.S. Provisional Application Serial No. 62/738,630, filed September 28, 2018, entitled

"CORTISOL BINDING APTAMER", which is incorporated by reference in its entirety.

LIELD OL THE INVENTION

BACKGROUND

Cortisol is a biomarker for stress. It is a glucocorticoid hormone playing a role in various physiological processes. While there exist natural fluctuations of cortisol levels in humans, and mammals, in general, it nevertheless has been confirmed that a correlation can be made between increased levels of cortisol and (emotional) stress. Increased stress levels may have negative consequences for the respective individual and elevated cortisol is implicated in a number of stress-related conditions including chronic fatigue syndrome, irritable bowel syndrome, depression, bipolar disorder and post-traumatic stress disorder. In addition, elevated cortisol levels are a feature of both kidney dysfunction and Cushing’s syndrome (hypercortisolism) which has the symptoms of obesity, hypertension, muscle weakness and osteoporosis. Reduced cortisol levels, in mammals, are a feature of Addison’s disease (hypocortisolism) which has the symptoms of weight loss, fatigue and darkening of skin folds. Increased cortisol levels may also be an indicator of stress in non-verbal subjects such as animals. It is well known that dogs, cats and other domesticated animals can show increased levels of cortisol in response to stress just as people do. Therefore, a reliable and effective method for determining cortisol levels is desirable. As cortisol levels may be determined in various bodily fluids, it is the desire in the art to enable a simple yet effective in vitro determination of cortisol levels which do not require laborious sample preparations and which, preferably also enable Point of Care Diagnostics, so that undesired delays between sample generation, measurement and diagnosis can be avoided.

Aptamers are synthetic receptors that can adopt local or global conformations that enable them to bind molecular analytes with high affinity. Aptamers are generally considered as biopolymers and can be made from natural or unnatural oligonucleotides, amino acids, or hybrid structures.

The use of aptamers for cortisol detection offers advantages over the known approaches employing antibodies since these lead to issues such as permanent denaturation and batch to batch variations. Aptamers have a much longer shelf life than antibodies and are stable over a wide range of conditions. Aptamers are less massive (ca. 20 kDa vs. 160 kDa) than antibodies and can be produced on demand by well-established synthetic methods. Antibodies must be produced through mammalian cultures which require a high overhead and a continuous investment in animal maintenance. WO 2017/035666 Al and WO 2016/056028 A2 disclose aptamer identification using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodologies.

Sanghavi, BJ et ah, disclose in Biosens Bioelectron, 2016, 78, pp 244-252 an approach using aptamers for electrochemical detection of cortisol in a microfluidic device. Other examples of using aptamers for cortisol detection are given in J.A. Martin et ah, Anal Bioanal Chem (2014), 406, pp 4637-4647, as well as in A.S.Z. Abidin et ah, Sensors 2017, 17, 1180.

However, the aptamers reported in the prior art have not shown a desired binding affinity to cortisol, so further advances are required in order to fulfill the general promises aptamer use has in relation with cortisol aptasensing. BRIEF DESCRIPTION

The present invention provides the aptamer as defined in claim 1. Preferred

embodiments are described in claims 2 to 5 as well as in the present specification. In addition, the present invention provides the method, use and device as defined in claims 6 to 8 and as described in the present specification.

DESCRIPTION OF THE FIGURES Figure 1 shows a table of the Cort-Apt sequence, the complementary sequence (CBA- comp) and fragment numbers 1-7 (F1-F7). From top to bottom, sequences correspond to SEQ ID NOs: 1-9.

Figure 2 shows a graph of uncorrected fall-off DNA fragment concentration (ng/pF) against fragment number.

Figure 3 shows a graph of AG fall-off DNA fragment concentration (ng/pL) against fragment number.

PET AIDED DESCRIPTION

The present invention provides the aptamer having the sequence TAGGGAAGAG AAGGACATAT GATGGGCCAG GGGCGTGTTA TATTCCGTAG GGCTTGACTA GTACATGACC ACTTGA (SEQ ID NO: 1) (Cort-Apt), as well as variants thereof, wherein in the variants, 20% or less, preferably 10% or less, more preferably 5% or less, more preferably 2% or less, more preferably 1% or less, of the deoxyribonucleotides of SEQ ID NO: 1 are changed. In this regard the present invention considers variations by using the nucleotides A, C, G and T only as well as variations using other bases, such as FT. Preferably the variation, however, is as small as possible, such as only 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. Most preferred in accordance with the present invention is SEQ ID NO: 1. The aptamers in accordance with the present invention have been identified by the SELEX procedure and may be synthesized using well known synthetic concepts for the preparation of aptamers. The SELEX procedure involves exposing a sequence library to a specific target, in this case cortisol, and amplifying the bound molecules which are then subjected to additional rounds of selection. Methodologies for producing aptamers include those disclosed in WO 2004/042083.

The aptamers in accordance with the present invention have shown the ability to selectively bind to cortisol, so that a selective aptasensing of cortisol is made possible. The dissociation constant of the aptamers reported herein are much lower, compared with the dissociation constants reported in the prior art, such as for the aptamers disclosed in J.A. Martin et al., Anal Bioanal Chem (2014), DOI 10.1007/S00216-014- 7883-8. Accordingly, the present invention provides molecules having a better suitability for the preparation of sensors or other devices employing aptamers for cortisol detection.

In some embodiments, the aptamers as herein described are provided for detecting the presence or absence of cortisol in a sample. Optionally, the aptamer is provided for the quantification of cortisol levels in a sample. Such detection or quantification may be used diagnostically for example for the diagnosis of a disease, disorder or condition in which cortisol is implicated.

In some embodiments, a method comprising contacting the aptamer as described herein with a sample and detecting the presence or absence of binding of the aptamer to cortisol is provided.

The aptamers disclosed herein enable selective cortisol detection, with a high binding affinity for cortisol, so that effective aptasensing of cortisol is made possible. As regards the methodology of cortisol detection, the present invention is not bound to a particular method. The aptamers in accordance with the present invention enable cortisol detection using known strategies, including the use of gold nanoparticles (enabling visual color based confirmation of detection results) as well as using surface immobilization techniques, such as described in A.S.Z. Abidin et ah, Sensors 2017, 17, 1180. The aptamers in accordance with the present invention may provide cortisol detection using existing assays and techniques including but not limited to serum control assays, immunoassays, liquid chromatography - tandem mass spectrometry and synacthen and dexamethasone suppressions tests. The present invention, however, also considers sensors as well as assays enabling a confirmation of cortisol detection and quantification by means of generating a fluorescent signal, which may be easily detected and quantified using known technologies.

In this manner easy to handle devices and methods for cortisol level evaluation can be provided, which enable an effective yet easy to handle measurement. Accordingly the present invention enables point of care diagnosis, which may be based on the cortisol level determinations provided using the aptamer of the present invention, the result of which is typically displayed within a couple of minutes.

The sample to be analyzed is one that is suspected of containing cortisol. The sample may be a biological sample or non-biological sample. Biological samples include bodily fluids. As cortisol is present in various bodily fluids, the method according to the present invention as well as the devices described and claimed herein may carry out aptamer cortisol sensing using bodily fluids, including but not limited to blood, serum, saliva, cerebrospinal fluid, interstitial fluid and urine. It has been found that the aptamer disclosed herein is in particularly suitable to enable cortisol detection using small volumes of urine, so that in particular also micro(fluidic) devices are made possible.

In some embodiments, the aptamers may also comprise at least one chemical modification. The modification may include without limitation a chemical substitution at a sugar position, at either the 5' or 3' end, at a phosphate position, or at a base position of the nucleic acid. In some embodiments the aptamers are modified to incorporate a detection label to serve as a detector agent. In some embodiments, the modification is selected from biotin, quantum dots, nanoparticles, linkers, quenchers, dyes and fluorescent labels. In some embodiments, the modification is a sequence of base units complementary or substantially complementary to a sequence of base units in the aptamer.

In some embodiments, the aptamers described herein may be immobilised on a surface. Surfaces may include, but are not limited to gold, dextran, streptavidin, or other functionalized group surfaces. Optionally the aptamers may be immobilised on magnetic beads coated with streptavidin.

In some embodiments, the aptamers have a dissociation constant (K_d) to cortisol of less than 9 mM as measured by Bio-layer Interferometry (BLI). Optionally less than 8, 7, 6, 5, 4, 3 or 2 mM as measured by Bio-layer Interferometry.

One embodiment provides a kit comprising the aptamers. The present invention extends to a kit which may be used to detect cortisol. Kits, e.g., for multiplexed detection of cortisol and a plurality of different target molecules from a sample, are also provided.

One embodiment provides a pharmaceutical composition comprising a therapeutically effective amount of the aptamers described herein, and a pharmaceutically acceptable carrier or diluent.

One embodiment provides the aptamers described herein for use in medicine.

Optionally for use in the treatment or prevention of a disease, disorder or condition in which cortisol is implicated. One embodiment provides a method of treatment or prevention of a disease, disorder or condition, the method comprising the step of administering an effective amount of the aptamers described herein to thereby treat or prevent the disease, disorder or condition. Optionally the disease, disorder or condition is a disease, disorder or condition in which cortisol is implicated. Typically the administration is to a subject in need thereof.

For the avoidance of doubt, insofar as is practicable any embodiment of the present invention may occur in combination with any other embodiment of the present invention. In addition, insofar as is practicable it is to be understood that optional or preferred feature of any embodiment of the present invention should also be considered as an optional or preferred feature of any other embodiment of the present invention. EXAMPLES

Cort-Apt identification

Starting aptamer libraries and biotinylated oligos for tethering to the surface were synthesized commercially (IDT DNA). Magnetic beads coated with streptavidin (Dynabeads M270, Invitrogen) were used as the surface for tethering oligos.

Biotinylated oligos were tethered to Dynabead surface via streptavidin-biotin interactions. The starting aptamer library was hybridized with the coated Dynabeads via annealing (95 °C for 5 minutes followed by slow cooling to room temperature). The annealed beads then undergo selection by incubating with cortisol of defined concentrations (100, 75, 50, 25, or 0 mM). Aptamers falling off the surface were further amplified using PCR via Taq DNA polymerase (NEB) to generate a new enriched double- stranded library. The library was further purified into a single- stranded form via NaOH dissociation followed by ethanol precipitation. The single- stranded library was then annealed to new Dynabead surface followed by the same procedure as described previously. The selection was repeated 12 times at different cortisol concentrations.

Two negative selection rounds using progesterone were included in round 5 and round 10. The final library was annealed to T vectors (T-easy, Promega), transformed into E coli (XL Blue Ultra II, Stratagene) and selected for Sanger sequencing to identify the sequence of the aptamers.

Binding affinity measurements

Binding affinity measurements were carried out using BLI (Bio-layer Interferometry). Specifically, the identified aptamer was loaded onto High Precision Streptavidin Biosensor (SAX) via streptavidin-biotin interaction. The loaded sensor was then dipped into a series of cortisol solutions with varying concentrations to determine the association and dissociation curves under those respective conditions. The collected dataset was then fitted globally to determine the binding affinity of the aptamer. The detailed experiment procedure is as follows:

1) Remove excess DNA

( step not needed if starting with fresh Dynabeads)

1. Wash all the Dynabeads 3x with an original volume of SELEX buffer (150 mmol L-l NaCl, 3 mmol L-l KC1, 25 mmol L-l Tris, 0.05% Tween 20); and resuspend in SELEX buffer after final wash.

2. Combine all the Dynabeads together in a single tube.

3. Wash Dynabeads 2x with 0.125 M NaOH (original volume) and measure final wash in UV-Vis to ensure all excess DNA has been removed.

4. Wash Dynabeads 3x with BW Buffer, (17.5 g/L NaCl; 50 mM phosphate buffer NaH2P04/Na2HP04; 0.1 % (v/v) Tween 20; pH 7.5), and measure the final wash in the UV-Vis to ensure all excess DNA has been removed.

2) Immobilization

( step not needed if starting with Dynabeads that are already bound to the biotinylated aptamer)

1. Resuspend beads in IX BW Buffer to twice the original volume.

2. To immobilize, add 250 pmol of biotinylated cortisol binding aptamer (BCBA) per 100 pL (original volume) of Dynabeads.

3. Incubate for 15 min at room temperature using gentle rotation. 4. Separate the biotinylated DNA coated beads with a magnet for 2 min.

5. Wash the left over Dynabeads 2-3 times with a lx BW Buffer.

6. Measure the concentration of the final wash using the UV-Vis to make sure no excess aptamer remains

3) Partition Dynabeads

1. Wash the Dynabeads 3x with SELEX buffer.

2. Add 100 pL of BCBA bound Dynabeads to fresh 1.5 mL tubes, one tube per test sample

4) Prepare oligomer samples

1. Make a 5 pM solution in SELEX buffer of the oligomer to be tested.

2. Measure the concentration of the prepared solution in the UV-VIS. 5) Anneal oligomer

1. Remove the SELEX buffer and add the 5 pM oligomer solution to the respective Dynabeads.

2. Incubate for 95°C for 5 min.

3. Cool on ice for 15 min.

4. Incubate for 30 min at room temperature with gentle rotation.

5. Wash the beads 3x with SELEX buffer measuring the oligomer concentration in the supernatant with each wash.

6) Prepare cortisol solution

1. Prepare a solution of 20 pM cortisol in SELEX buffer.

2. Measure the cortisol solution in the UV-Vis.

7) Cortisol binding

1. Resuspend the Dyna beads with the 20 pM cortisol solution.

2. Incubate for 30 min at room temperature with gentle rotation

3. Remove the supernatant and measure Cortisol/oligomer concentration in the UV-Vis. 8) Calculate the oligomer concentration in the supernatant

3. Equations 1-2 can combined to give

9) From the UV-Vis measurements, you can calculate the amount of oligomer annealed and the amount of oligomer dissociated by the addition of cortisol. Results:

Binding affinity measurements were carried out using BLI (Bio-layer Interferometry) for Cort-Apt and for the cortisol binding aptamer described in J.A. Martin et ah, Anal Bioanal Chem (2014), 406, pp4637-4647 (GGAATGGATC CACATCCATG

GATGGGCAAT GCGGGGTGGA GAATGGTTGC CGCACTTCGG CTTCACTGCA GACTTGACGA AGCTT, SEQ ID NO: 10).

The results demonstrate that the Cort-Apt has a lower dissociation constant for cortisol than the aptamer described in the prior art and hence Cort-Apt has an improved binding affinity compared to the prior art.

Hypersensitive Binding Site Identification

A tiling assay was performed to identify hypersensitive binding sites of the Cort-Apt. A tiling assay is a way of identifying the segments of the aptamer sequence that are most important to cortisol binding. Oligonucleotides are hybridised to different segments of the aptamer in different experiments. After purification the analyte is added and the quantity of displaced oligonucleotide is measured (by UV-Vis of the supernatant or by attaching a fluorescent probe to the oligonucleotide). The oligomer which shows the highest amount of displacement is bound to the aptamer section with the highest affinity for cortisol (i.e. the cortisol displaces the hybridised oligonucleotide from the aptamer). The fragments are listed in Figure

1. Each fragment (F1-F7) has a 5 base pair overlap with the previous fragment.

Procedure for the tiling assay:

1. Biotinylated Cort-Apt were bound to magnetic beads at room temperature for 2 hrs, (tiling fragments were used in 5x excess).

2. Fragments were annealed to Cort-Apt at 95 °C for 5 min and the samples were cooled to RT in ~ 2 hrs.

3. The annealed construct were washed twice with SELEX buffer to remove unbound ssDNA.

4. Each fragment was measured.

5. Steps 1-4 were repeated twice for each fragment.

Results:

1. Uncorrected results shown in Figure 2 show the hypersensitive binding site of Cort-Apt is located on the 5’ end of the aptamer (nucleotide positions 1- 15) where the SELEX adapters were attached.

2. To correct for the base pair composition of each DNA, the Gibbs free energy of each fragment was calculated. The results were corrected accounting for difference in binding energies. The corrected results shown in Figure 2 show the same hypersensitive site toward the 5’ (nucleotide positions 1- 15) of the aptamer.

Claims

CLAIMS What is claimed is:

1. An aptamer comprising a sequence TAGGGAAGAG AAGGACATAT

GATGGGCCAG GGGCGTGTTA TATTCCGTAG GGCTTGACTA GTACATGACC ACTTGA (SEQ ID NO: 1), as well as variants thereof.

2. The aptamer according to claim 1, wherein in the variants 20% or less of the deoxyribonucleotides of the sequence are changed.

3. The aptamer according to claim 1 or 2, wherein in the variants 10% or less of the deoxyribonucleotides of the sequence are changed

4. The aptamer according to any one of claims 1 to 3, wherein in the variants 5% or less of the deoxyribonucleotides of the sequence are changed

5. The aptamer according to any one of claims 1 to 4, wherein the variations are generated by using the building blocks A, C, G and T only.

6. The aptamer according to any one of claims 1 to 4 wherein the variations are generated using building blocks also including other bases, such as U.

7. A method for cortisol detection comprising employing the aptamer according to any one of claims 1 to 5 as a cortisol sensing molecule.

8. A method comprising using the aptamer according to any one of claims 1 to 5 as a sensing molecule.

9. A device for cortisol detection, comprising the aptamer according to any one of claims 1 to 5.