Patent Application
20140342918
The present disclosure is in the field of chemical processes, namely affinity purification of target ligands capable of binding to switchable aptamers. The disclosure further relates to the isolation of switchable aptamers for targeting cells, viruses and antibodies.
The Systematic Evolution of Ligands by EXponential enrichment method, or SELEX, is a combinatorial chemistry technique for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to one or more target ligands. The method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve a desired level of binding affinity and selectivity. SELEX has been used to evolve nucleic acid aptamers of extremely high binding affinity to a variety of targets. Some of these targets include, for example, lysozyme (Potty et al.), thrombin (Long et al.), human immunodeficiency virus trans-acting responsive element (HIV TAR) (Darfeuille et al.), hemin (Liu et al.), interferon gamma (Min et al.), vascular endothelial growth factor (VEGF) (Ng et al.), prostate specific antigen (PSA) (Savory et al.; Jeong et al.).
Aptamers have found applications in many areas, such as biotechnology, medicine, pharmacology, microbiology, and analytical chemistry, including chromatographic separation and biosensors.
Interestingly, structure-switching aptamers or SwAps have also found multiple applications. A review of SwAps as biosensors was published in 2009 by Sefah et al. Changes in fluorescence intensities between free and bound aptmamer complexes have been described to detect cocaine by aptamer-based capillary zone electrophoresis (Deng et al.). Multiple small molecule analytes have been detected by a similar method (Zhu et al.) High surface area, solid phase sol-gel-derived macroporous silica films have also been shown to be suitable platforms for high-density affinity-based immobilization of functional single stranded-aptamer molecules, allowing for binding of both large and small target ligands through SwAps with robust signal development (Carrasquilla et al.)
Aptamers have further been used for protein and small molecule purification using affinity chromatography. Indeed, aptamer affinity chromatography has been applied to protein purification (Romig et al.) and in the separation of mature dendritic cells from immature dendritic cells (Berezovski et al.). However, aptamer affinity chromatography has not to the inventors' knowledge been shown in the prior art to apply to the purification of cells, viruses or antibodies.
A major problem encountered when dealing with aptamer-based affinity chromatography to purify target ligands such as viruses, cells and certain other biological materials is the need for elevated temperatures or the addition of detergents to alter the conformation of the SwAp and to subsequently allow the release of the captured biomolecular target ligand from the solid medium or chromatography column. These harsh regeneration techniques decrease significantly the viability of cells and viruses, denature proteins and irreversibly change the structure of biomolecules. Furthermore, the lack of an efficient regeneration technique that can be generalized to other target-specific aptamers has been a challenge to the widespread use of aptamers for purification. Concerns have also been raised with regard to the possible cross-reaction between aptamers and other contaminants that might exist in the mixture containing the biomolecule to be purified. As such, until now, these problems have made the utilization of aptamers for the purification and recovery of purified targets such as viruses and cells very difficult to achieve.
The methods currently available for purification of viruses include: differential centrifugation, size exclusion chromatography (SEC) and heparin affinity column chromatography. These techniques are not without challenges. Sucrose differential gradient centrifugation is conventional for virus isolation in small quantities, but it is difficult to scale-up, is labour-intensive and requires long processing times, which may decrease the infectivity of viruses (Diallo et al.) SEC does not separate well from cell debris or large molecular aggregates with similar sized viruses, and is followed with additional concentration steps such as ultrafiltration or polyethylene glycol-6000 precipitation. The heparin column purification utilizes sepharose beads conjugated to linear anionic heparin molecules. This technique is used to purify proteins containing a heparin-binding domain as well as retroviruses. Although, this heparin method yields a purer product than the density gradient method, it still requires additional SEC purification from cationic proteins and salt.
The present disclosure is directed to the purification of a target ligand of interest using aptamer molecules which exhibit a switchable affinity for the target in the presence or absence of a binding ion. According to various embodiments, the target can be a virus, a eukaryotic cell which may be receptor-positive for a selected receptor, a prokaryotic cell or an antibody.
According to one aspect, the disclosure relates to a method of isolating a switchable aptamer having affinity for a selected target ligand from a pool comprising a mixture of aptamers. The mixture may consist of a randomized pool of aptamers. According to this aspect, the method comprises the steps of:
At least steps a through c may be performed at room temperature, for example a maximum temperature of 25° C.
The method may comprise the further step of amplifying the switchable aptamer isolated in step d.
The method may comprise the further step of measuring the affinity of said switchable aptamer for the target in the presence and absence of the binding ion.
Another aspect relates to an iterative process wherein two or more switchable aptamers are isolated and steps a through d are repeated using the two or more switchable aptamers in place of the mixture of aptamers in said pool wherein a switchable aptamer is isolated which has an increased affinity for the target relative to others of said switchable aptamers. From about 5 to about 20 such iterations or rounds (or between 5 and 20) may be performed for sequentially achieving higher purification levels, or from about 7 to about 15 (or between 7 and 15) rounds, or about 10 rounds.
Suitable targets for the method include a virus such as Vesicular Stomatis Virus (VSV) or a cell. Cellular targets include a receptor positive cell for a selected receptor such as a Neuropilin 1 (NRP) receptor, a Leukemia inhibitory factor (LIF) receptor, a Patched 1 (PTCH1) receptor, a Delta-Like Ligand 4 (DLL4) receptor or a plasminogen activator/urokinase receptor (PLAUR). A prokaryotic cell such as a bacteria may also comprise a target.
The binding ion may be a monovalent or divalent ion. Suitable divalent ions include calcium or magnesium or a combination thereof.
Suitable chelating agents include ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA) or a combination thereof.
The target-aptamer complexes and the unbound aptamer molecules may be separated by centrifugation or by immobilizing the target-aptamer complexes and washing away unbound aptamer.
Suitable aptamers comprise nucleotides. The aptamers in said pool may comprise a random region of between 20 and 60 (or from about 20 to about 60) nucleotides, for example about 40 nucleotides.
According to a further aspect, the disclosure relates to a switchable aptamer having a high affinity for a selected target in the presence of a binding ion and a low affinity for said target in the absence of said binding ion. The switchable aptamer may be obtained through the method described herein. The switchable aptamer may comprise the nucleotide sequence represented in any one of SEQ ID NOS: 3 through 17 or 21 through 89, or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NOS: 3 through 17 or 21 through 89. The aptamer may have a target as described above.
According to a further aspect, the disclosure relates to a method for purifying a selected target from a complex mixture. According to this aspect, the method comprises the steps of:
Suitable conditions and targets for said method may be the same as described herein for the method of isolating the switchable aptamer. The method may be performed at room temperature (maximum of 25° C.).
The method may comprise the further step of re-using the separated switchable aptamer to repeat said steps a through e on the same or a different complex mixture.
The switchable aptamer used in said method may comprise the switchable aptamer isolated according to the method described herein.
In the present specification, the following definitions apply, unless the context clearly requires otherwise:
“SwAps” or “switchable aptamers” means an aptamer with switchable affinity for one or more target ligands, wherein the affinity may be selectively increased or decreased by changing the environment of the aptamer such as contacting the aptamer with one or more ions.
“Target ligand” or “target” means a virus, a prokaryotic cell, a eukaryotic cell an antibody or a biomolecule whether synthetic or naturally produced which is capable of being bound to an aptamer.
“Biomolecule” means any molecule that is produced by a living organism or which is a synthetic analogue of such a molecule, including large macromolecules such as proteins, protein antibodies, peptides polysaccharides, lipids, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and natural products.
“Aptamer” means a molecule that has an affinity for and binds to a specific target molecule via an interaction between the nucleic acid sequence of the aptamer and the target. This base pairing creates secondary structures such as short helical arms and single stranded loops. Combinations of these secondary structures results in the formation of tertiary structures that allow aptamers to bind to targets via van der Waals forces, hydrogen bonding and electrostatic interaction—aligning with the same ways antibodies bind to antigens. When this tertiary structure forms, the entire aptamer folds into a stable complex with the target ligand.
An aptamer may constitute a nucleic acid, which may be RNA or DNA or a peptide for a peptide aptamer.
“Chelating agent” is a molecule which binds to a metal ion. Chelation involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central atom. Usually these ligands are organic compounds, and are called chelants, chelators, chelating agents, or sequestering agents.
“Binding ion”—an ion participating in formation aptamer-target complex
“Complex mixture”—a mixture of a target and non-target molecules, viruses, cells, and particles.
The objects and advantages of the present disclosure will become more apparent upon reading the following non-restrictive description of the preferred embodiments thereof, given for the purpose of exemplification only, with reference to the accompanying drawings.
SwAps.
While the disclosure will be described in conjunction with example embodiments, it will be understood that it is not intended to limit the scope of the disclosure to such embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included and defined by the appended claims.
The disclosure provides for the selection and purification of SwAps with selectively variable binding to a target and the use of such SwAps for the purification of the target from a complex mixture. In general, this can be achieved by the steps of:
In one aspect, the present method for the purification of the target using
SwAps assumes that the target needs to be purified from a complex solution. The complex solution may comprise a mixture of biological molecules and may also contain non-biological molecules. For example, the target may be in a solution containing debris and impurities.
In one aspect, the target is a virus, such as VSV. It will be understood that the virus can also be a virus other than VSV.
Step a) involves providing a library of randomized nucleic acid sequences and isolating an aptamer with a relatively high degree of switchable affinity to a specific target from this pool, in which the pool has variable degrees of affinity for the target. In some embodiments, the randomized library comprises a mixture of different nucleic acids, each of which has a region of about 20-60, preferably about 40 randomized nucleotides as set out in SEQ ID NO: 18. Other lengths of the randomized region are from 15 to 100 nucleotides. The desired aptamers to be isolated are those with switchable affinity to the target in the presence or absence of a binding ion. This step a) may be performed separately from the purification, or as the first step in purifying the target of interest.
Isolation of the switchable aptamer is performed through a modified cell-SELEX process. More specifically, a pool of randomized aptamers is incubated with the target for a length of time, such as 30 minutes, in the presence of binding ions (such as Ca2+, Mg2+, or a combination of Ca2+ and Mg2+) to produce an aptamer-target mixture. This mixture is washed to remove any unbound aptamers. In some embodiments, the unbound aptamers are removed by centrifugation, leaving only the aptamers, which are bound to the target in the mixture. In other embodiments, the unbound aptamers are washed away from immobilized aptamer-target complexes using a washing agent.
The mixture is then subjected to treatment with a chelating agent, for example ethylenediaminetetraacetic acid (EDTA) and/or ethylene glycol tetraacetic acid (EGTA). The chelating agent will be chosen in accordance with the binding ion. For example, EDTA may be used to chelate Ca2+ ions, whereas EGTA may be used to chelate Mg2+ ions. The addition of the chelating agent and the corresponding reduction in free binding ions induces a conformational change in some aptamers in the randomized pool, which allows them to become unbound from the target. Such aptamers with variable target affinity to the target are examples SwAps.
The unbound SwAps are then separated from the target and target-aptamer complexes. In some embodiments, the chelated mixture is centrifuged to remove the unbound target and the non-switchable aptamers still bound to the target. In other embodiments, the unbound SwAps are separated by washing or elution from target and target-aptamer complexes immobilized on a surface. The SwAps are then amplified, for example by polymerase chain reaction (PCR), thereby enriching the SwAps within the aptamer pool. These amplified SwAps can then be re-isolated any number of times to further enrich the aptamer pool for SwAps with switchable affinity to the target.
In some embodiments, the steps of incubating the pool, separating the complexes, chelating the complexes, and separating the released SwAps is conducted at room temperature. This is especially preferred where the target is temperature sensitive, such as where the target is a virus or a cell. Amplification can be conducted at higher temperatures, as dictated by the polymerase chain reaction (PCR) protocol used.
Optionally, the selection method may also include an assay of the binding affinity of the SwAp to the target in the presence or absence of the binding ion. Such assessments may be useful for the identification of SwAps of particular interest for purification of the target, particularly those which exhibit a large change in affinity in the presence or absence of the binding ion. Affinity measurements also permit the selection to be conducted in an iterative fashion until a SwAp having a desired affinity is obtained. In one embodiment, the affinity assay is performed using flow cytometry. In other embodiments, target affinity may be measured using electrochemical means, such as by impedimetric assays. Each of these affinity assays are described in further detail below. Affinity assays may also be carried out in a variety of other ways known to the person of skill in the art. Examples include use of a gel-shift assay, filter-binding assay, surface plasmon resonance, stopped-flow assay or isothermal calorimetry.
For example, as discussed further below, an aptamer isolated for the purification of VSV may comprise any one of the nucleotide sequences set out in SEQ ID NOs: 3 to 17, each of which were derived from the selection method according to the present disclosure. In another embodiment, the aptamer has a sequence which has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NOs: 3 through 17.
In one embodiment, SwAps that have been isolated in Step a) are labelled with a tag. This tag is, for example, biotin. Other tags such as fluorescent dyes and markers are also contemplated.
In further embodiments of the disclosure, SwAps isolated in Step a) are immobilized on a surface, such as the stationary phase of a chromatography column, on a magnetic bead, on a membrane or in an agar medium prior to being contacted with the target. For example, as discussed further below, one or more biotin labelled aptamers may be immobilized on streptavidin-coated magnetic beads. Various other means of immobilizing SwAps would be apparent to those of skill in the art. For example, SwAps may be immobilized on a glass substrate modified with organosilanes or other fixing agents. Gold surfaces may also be used in conjunction with thiol-modifications to immobilize SwAps. A variety of other physical adsorption, covalent bonding, affinity binding, and matrix entrapment techniques are known in the art.
Immobilization of the SwAps aids in the separation and washing steps during purification, particularly in respect of the separation of the aptamer-target complexes from the complex solution and the recovery and re-use of SwAps for further rounds of purification. For example, a SwAp immobilized on a magnetic bead may form aptamer-target complexes which can be separated from the complex solution as shown schematically in
In Step b), the SwAps obtained through the isolation in Step a) are incubated with a binding ion. The binding ion can be, for example, a divalent ion like calcium or magnesium. Both calcium and magnesium together can also act as the binding ion. It will be understood that a monovalent or divalent cation can be used as the binding ion. Submillimolar levels of the binding ion (0.01-1 mM) induces conformational changes in the aptamer DNA and stabilize secondary and tertiary structures of the switchable aptamers. In the early folding stages, aptamers form secondary structures stabilized through the binding of monovalent cations or divalent cations in order to neutralize the polyanionic backbone. The later stages of this process involve the formation of DNA tertiary structure, which is stabilized almost largely through the binding of divalent ions such as magnesium and calcium with contributions from potassium binding. As such, the SwAps bind to their targets in the presence of Mg2+ and Ca2+ ions and release their targets once the ions are removed by the addition of the binding ion chelator in Step d).
Step c) calls for the separation of the target-aptamer complexes of Step b) from the complex mixture. Where the SwAps are immobilized, this typically involves washing the target-aptamer complexes with a washing agent to remove debris or impurities. For example, the washing agent can be Dulbecco's phosphate-buffered saline (DPBS), which is particularly useful where the binding ion is Mg2+ or Ca2+. If the aptamers are not immobilized, other means of separation known in the art may be employed, such as centrifugation or electrophoresis.
The above steps result in a highly purified aptamer/target complex, such that the subsequent separation of these components produces essentially a two-component mixture comprising an isolated target and an isolated switchable aptamer that is specific to this target.
Step d) describes the addition of a chelating agent to the mixture of target-aptamer complexes to chelate the binding ion. The chelating agent will be selected in accordance with the binding ion. For example, if the binding ion is Ca2+, the binding ion chelator will be ethylenediaminetetraacetic acid (EDTA). Similarly, if the binding ion is Mg2+, the binding ion chelator will be ethylene glycol tetraacetic acid (EGTA). It follows that if Ca2+ and Mg2+ are both used together as binding ions, both EDTA and EGTA will be used a chelators. The chelators remove the binding ion, and consequently allow for the release of the target by the SwAps.
Step e) describes the collection of the purified target released by chelation of the SwAps. In some embodiments, such as the embodiment show in
In some embodiments, the purification process is performed in an iterative manner to achieve higher levels of purity, such that the purified target from the first round of purification acts as the complex solution in subsequent rounds of purification.
The following examples detail the use of SwAps with controlled affinity for the purification of Vesicular Stomatis Virus (VSV). As shown below, the virus captured with such SwAps can be recovered upon treatment with a simple eluent, containing a mixture of EDTA/EGTA at room temperature and neutral pH. Using the method described herein, a total of 15 sequences were obtained as a first pool of aptamers with varying affinity to VSV. Each sequence was assessed for affinity and switchability by both flow cytometry and impedimetric analysis. SwAps clones 5, 6, 7 and 9 were selected for further study. SwAps clone 6 was the candidate that demonstrated the best affinity and switchability for VSV. The aptamers switchability is a function of the divalent cations and the affinity of the SwAps to VSV can be terminated upon chelation of these cations. The resulting system provides an efficient and efficiency and simplicity of the SwAps-based purification technique described here above.
The protocol of VSV production and harvesting was previously described elsewhere (Diallo et al.). Briefly, 6 plates of Vero cells were grown until confluent and then were infected with VSV (approx. 106 PFU/plate). After 24 hours, the supernatant was collected into 50-mL tubes and centrifuged to removed the cell debris. Subsequently, the supernatant was passed through a 0.2 μm filter (Pall Inc., USA) and the pellet containing the virus was then re-suspended in DPBS. Finally, it was aliquoted and stored at −80° C.
N40 ssDNA library, with the sequence (5′CTCCTCTGACTGTAACCACG-(N40)-GCATAGGTAGTCCAGAAGCC3′) (SEQ ID NO:90) was used for all experiments. It consists of total of 80 nucleotides and contains two flanking primer regions of 20 nucleotides each, whereas the central region contains 40 random nucleotides. Fluorescently-labeled 5′-primer (6-FAM/5′CTCCTCTGACTGTAACCACG3′) (SEQ ID NO:1) and the non labeled 3′-primer (5′GGCTTCTGGACTACCTATGC3′) (SEQ ID NO:20) and the library were all obtained from Integrated DNA Technologies, U.S.
After the selection process described herein, some aptamers can be slightly longer or shorter due to mutations happened in PCR steps or original synthesis of the DNA library.
The larger the size of this region, the more combinations of nucleotides will result and the greater the chance of an exact “hit” with a target. In some embodiments, practical limits for are about 20-60 nucleotides for the central region.
Submillimolar levels of calcium and magnesium (0.01-1 mM) induce conformational changes of DNA and stabilize secondary and tertiary structures. In the early folding stages, aptamers form secondary structures stabilized through the binding of monovalent cations or divalent cations in order to neutralize the polyanionic backbone. The later stages of this process involve the formation of DNA tertiary structure, which is stabilized almost largely through the binding of divalent ions such as magnesium and calcium with contributions from potassium binding. Here the use of these cations through a modified cell-SELEX technique to create aptamers with switchable affinity has been exploited. These aptamers have a switchable functionality allowing them to bind to their targets in the presence of Mg2+ and Ca2+ ions and to release their targets once the ions are removed. To allow for this functionality, an additional step in the cell-SELEX process was added using two strong chelating agents, namely 2.5 mM EDTA and EGTA in PBS. The SwAps selection scheme through modified cell-SELEX is presented in
The process for the selection of SwAps involves 5 steps: 1) Incubation of aptamers with 2.5×109 pfu mL−1 of VSV in DPBS for 30 minutes; 2) Washing VSV to remove unbound aptamers by centrifugation; 3) Treatment with EGTA & EDTA to remove Mg2+ and Ca2+ and release VSV; 4) Collection of unbound switchable aptamers through centrifugation; and 5) PCR amplification (symmetric+asymmetric).
In more ample details, the selection of the switchable aptamers begins with adding VSV to a pool of aptamers in DPBS, along with Ca2+ and Mg2+, followed by incubation for 30 min at room temperature. This allowed for binding between aptamers and VSV to reach equilibrium.
Centrifugation allowed for the removal of unbound DNA aptamers, where the aptamers bound to VSV became a part of the pellet after the centrifugation step, whereas the non-bound DNA remained in the supernatant and was discarded. Addition of EGTA & EDTA to remove Ca2+ and Mg2+ allowed for the collection of aptamers exhibiting switchable affinity. These aptamers were collected and amplified by symmetric and asymmetric PCR.
The selection scheme involved 5 steps and was repeated for 10 rounds. The scheme involved (1) incubation of aptamer pool with VSV, (2) separation of bound aptamers, (3) addition of EDTA and EGTA, (4) collection of unbound aptamers, and (5) PCR to amplify the desired aptamer pool. Each aptamer pool was denatured by heating at 95° C. for 5 min in Dulbecco's phosphate buffered saline (DPBS), containing 0.901 mM CaCl2, 0.493 mM MgCl2, 2.67 mM KCl, 137.93 mM NaCl, 1.47 mM KH2PO4, and 8.06 mM Na2HPO4 (D8662, Sigma-Aldrich, U.S.) and was allowed to re-fold on ice for 10 min. Prior to each round of selection, 2.5×109 PFU mL−1 of VSV was incubated with 100 nM of FAM-labeled aptamer pool in a total volume of 50 μL (DPBS) for 30 min on a shaking incubator at 25° C. and 400 r.p.m. The mixture was then centrifuged at 17 200 r.c.f. for 15 min. Next, the supernatant was discarded and 50 μL DPBS was added and the mixture was centrifuged again. This washing step was repeated 3 times for rounds 1-5 and increased to 5 times for rounds 6-10. Upon completion of the last washing step, the pellet was re-suspended in 50 μL of an equimolar mixture of 2.5 mM EDTA (EMD Chemicals, U.S.)/EGTA (Bio Basic Inc., Canada) in PBS (2.67 mM KCl, 1.47 mM KH2PO4, 8.06 mM Na2HPO4 and 137.93 mM NaCl) for 30 min. Afterward, the mixture was centrifuged for 15 min at 17 200 r.c.f. and the supernatant was transferred to a separate tube for storage at −20° C. Finally, aptamers were amplified by PCR and the cycle was repeated.
Aptamer pools were amplified using bundled symmetric and asymmetric PCR after each subsequent round of selection. Symmetric PCR amplifies and produces dsDNA, where 5 μL of the supernatant collected during selection and containing the bound aptamers were mixed with 45 μL of symmetric PCR master mix. The master mix contained the following reagents in final concentrations: 1×PCR buffer (Promega Corporation, U.S.), 2.5 mM MgCl2, 0.028 U μL−1 GoTaq Hot Start Polymerase (Promega Corporation, U.S.), 220 μM dNTPs, 500 nM forward primer (5′CTCCTCTGACTGTAACCACG3′) (SEQ ID NO:1), and 500 nM reverse primer (5′GGCTTCTGGACTACCTATGC3′) (SEQ ID NO:20) (Integrated DNA Technology, U.S.). Upon completion, 5 μL of the symmetric master mix were added to the asymmetric PCR master mix containing the same reagents as the symmetric master mix but with 1 μM forward FAM-labeled primer (FAM-5′CTCCTCTGACTGTAACCACG3′) (SEQ ID NO:1) and 50 nM reverse primer. Asymmetric PCR has low amplification power but it produces ssDNA. Both symmetric and asymmetric PCR used the following program: preheating for 2 min at 95° C., 15 cycles for symmetric PCR or 10-15 cycles for asymmetric PCR of 30 sec at 95° C., 15 s at 56.3° C., 15 s at 72° C., and hold at 4° C.
A total of 10 rounds of SwAps selection were performed, and the selected aptamers were analyzed by flow cytometry.
For affinity testing, pools were purified by loading the mixture onto 30 kDa cut-off filter (Nanosep, U.S.). This was followed by centrifugation at 3 800 r.c.f. for 13 min at 16° C. Subsequently, an equal volume DPBS was added for two additional washing steps for 10 min each. The purity was tested by running the raw and purified samples on 3% agar gel (Sigma-Aldrich, U.S.) at 150V. Finally, concentration of sample was measured using NanoDrop-2000 UV-Vis spectrophotometer, U.S.
Aptamer pool/clone affinity to VSV and switchability were measured using a FC-500 Flow Cytometer (Beckman Coulter Inc., U.S.). All samples, contained 100 nM of purified FAM-labeled aptamer, were incubated with 2.5×107 PFU mL−1 at room temperature for 30 min in DPBS. The samples were then divided into two portions; the first portion had DPBS added to it the second had 10 mM EDTA/EGTA 30 min at room temperature. All samples were made to 250 μL prior to flow analysis. Control experiments were performed using the aptamer pool 8 and a sample of VSV was stained using TOTO-3 dye (Invitrogen, U.S.) to allow for identification on flow cytometry.
Ten selected pools of aptamers were examined for two criteria; the affinity of the aptamer pool to VSV and the ability of this pool to release VSV upon treatment with the EDTA/EGTA mixture which is denoted here by the Coefficient of Switching (CoS). Rather than using the N40 DNA library as a standard, it was decided to compare the aptamer pools to the initial pool which was used to start selection. This was decided as a better representation because unlike with typical SELEX protocols where the DNA library would represent “round 0”, here our starting pool was pre-selected to bind to VSV. Thus, comparing to the native library would not have provided us with information as to whether the selection scheme was truly creating switchable aptamers. All pools were FAM-labeled, purified and made to a total volume of 100 μL in DPBS with 50 nM aptamer pool and 107 PFU mL−1 VSV. Flow cytometry results were analyzed by Kaluza software; one can see two trends in
How effectively the aptamers can switch from their bound and unbound form can be compared. Round 0 is the lowest, followed by rounds 2, and 8, all showing a CoS of <0.20. The CoS was small, which indicates that the binding of the pool to VSV was largely unaffected by the presence or absence of Ca2+ and Mg2+. Large CoS values was exhibited by pools 3, 7 and 10. Since rounds 1 and 2 represent the beginning of selection, it was expected that they would not show a good switching functionality. One would think that as the number of rounds of selection increases, the binding and switchability characters would also increase linearly. This is seldom the case in aptamer selection as after each round, mutations were introduced during PCR, which may be beneficial or detrimental to binding. Pool 10 showing the highest affinity and switchability was thus selected for cloning. More washing steps were employed during the later rounds which resulted in more specificity. Flow cytometry histograms of round 10 are demonstrated in
Aptamer pool 10 was cloned and a total of 15 SwAps sequences were obtained. All clones were tested for their respective affinities to VSV and the switchability as was described above.
Aptamer pool 10 (SEQ ID NO: 18) which showed both high affinity and switchability was selected for cloning to obtain individual aptamer sequences. The resulting sequences are provided in SEQ ID NOs: 3 through 17, also reproduced below at Table 3.
Briefly, the pool was amplified using symmetric PCR to obtain dsDNA and then was purified using a DNA gel extraction kit (AxyGen Biosciences, U.S.). Cloning was performed according to the protocol obtained with the M13mp18 perfectly blunt cloning kit (Novagen, U.S.). White colonies identifying the presence of the insert were then grown in LB+ Ampicillin overnight in a shaking incubator. PCR was then performed to ensure the presence of the insert with the following master mix. For each PCR reaction, 2 μL of the cell suspension was mixed with 18 μL of the PCR Master Mix containing: 1×PCR GC buffer, 2.5 mM MgCl2 (Mallinckrodt Baker, Inc., U.S.), 0.01 U μL−1 KAPA 2G Robust Hot Start DNA polymerase (Kapa Biosystems, U.S.), 220 μM dNTPs, 0.5 μM forward FAM-labeled primer, and 0.5 μM reverse primer. Amplification was performed using a PCR program (preheating for 5 min at 95° C., 30 cycles of 30 s each at 95° C., 15 s at 56.3° C., 15 s at 72° C., and hold at 4° C.). Upon identifying the clones with the plasmid containing the insert, amplification was performed. This was carried out using the same aforementioned master mix but with the M13 forward primer (5′GTAAAACGACGGCCAGT3′) (SEQ ID NO:91) and M13 reverse primer (5′AGCGGATAACAATTTCACACAGG3′) (SEQ ID NO:92). The unpurified PCR product obtained was sent to McGill University and Genome Québec Innovation Centre, Canada for sequencing.
Electrochemical studies, including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were carried out with an electrochemical analyzer (CH Instruments 660D, TX, U.S.) connected to a personal computer. All measurements were performed at room temperature in an enclosed and grounded Faraday cage. A conventional three-electrode configuration printed on a ceramic substrate; including a GNPs-SPCE electrode as the working electrode, carbon counter electrode, and a silver pseudo-reference electrode. A three-electric contacts edge connector was used to connect the screen-printed electrode with the potentiostat (Dropsens, Spain). The open-circuit or rest-potential of the system was measured prior to all electrochemical experiments to prevent sudden potential-related changes in the self-assembled monolayer (SAM). CV experiments were performed at a scan rate of 100 mV s−1 in the potential range from −600 to 800 mV. EIS measurements were conducted in the frequency range of 100 kHz to 0.1 Hz, at a formal potential of 250 mV and AC amplitude of 5 mV. The measured EIS spectra were analyzed with the help of equivalent circuit using ZSimpWin 3.22 (Princeton Applied Research, U.S.) and the data were presented in Nyquist plots. Electrochemical measurements were performed in 25 mM sodium phosphate buffer (pH 7), containing 2.5 mM K4Fe(CN)6 and 2.5 mM K3Fe(CN)6.
Prior to experiments, the gold nanoparticles modified screen-printed carbon electrode (GNPs-SPCE) (L33×W10×H0.5, Dropsens, Spain) was washed thoroughly with deionized water then dried with pure N2. Subsequently, the electrode was incubated with equimolar amounts (1 μM) of the selected aptamer and an HPLC purified, Thiol-spacer-5′GGCTTCTGGACTACCTATGC3′ (SEQ ID NO:20) modified at the 5′ position with a 6-hydroxyhexyl disulfide group, detection probe (Integrated DNA Technologies, U.S.) in 25 mM sodium phosphate buffer, pH 7, for 5 days at 4° C. Finally, the electrode was incubated with 1 mM 2-mercaptoethanol in ethanol for 5 min to back-fill the empty spots of the electrode surface, thus reducing the non-specific adsorption onto the surface.
The electrochemical characteristics of the developed impedimetric sensor is provided in
The rationale behind this electrochemical approach is that the binding between the virus and the respective aptamer, will further block the charge transfer from the solution-based redox probe to the electrode surface. Consequently, RCT will become increasingly high and can be used to monitor the binding event (Ayyar et al.; Wei et al.). A schematic representation of the sensor preparation and performance is provided in
More specifically, incubation of 1×106 PFU of VSV in Dulbecco's phosphate buffered saline (DPBS) with the respective SwAp immobilized onto the electrode surface for 1 hour at room temperature, caused an increase in the interfacial resistance and consequently in the value of RCT. Fifteen aptamer sequences were tested and the affinity between the virus and each sequence was expressed as the change in RCT value, which is calculated from the difference between RCT after incubation with the virus (RCTV) and the baseline resistance obtained after the preparation of the aptasensor, RCTB. Afterward, each aptasensor was incubated with an equimolar mixture of EDTA and EGTA (50 mM) for 30 min at room temperature. As can be seen in
The switching ability of each SwAp can be expressed as the Coefficient of Switching (CoS), which can be calculated from the formula 2:
Where RCTS represents the resistance to charge transfer after aptamer switching due to EDTA/EGTA treatment. A control experiment was performed, under the same conditions, using the original aptamer pool employed in selection. The circuit elements calculated for each SwAp are provided in Table 2. In
As can be seen in Table 3, the values of RCTV-RCTB and CoS were determined for each aptamer sequence and both parameters were employed to assess the efficiency of each SwAp for VSV purification. In other words, SwAps exhibiting high virus affinity and switching ability, including SwAps clones 9, 5, 7 and 3, have the potential to be further integrated into affinity chromatography units involved in VSV purification. A slight variation of the results obtained using the impedimetric sensor was observed when compared to the flow cytometry data. This could be ascribed to the different forms of aptamers used in each method where free aptamers were used in flow cytometry, whereas immobilized aptamers were used to develop the sensors. Thus, they may adopt different tertiary structures and alter their binding capabilities.
5′GCATAGGTAGTCCAGAAGCC3′ (SEQ ID NO: 2)
The electrochemical characteristics of the developed aptasensor were investigated by both CV and EIS. As shown in
The switching ability of aptamer pools was hypothesized to stem from the removal of Ca2+ and Mg2+ which can bind to phosphate backbone of ssDNA. To confirm that this was indeed the case and not the result of a change in buffer ionic strength, a flow cytometric analysis was performed as shown in
Briefly,
In a low DNA binding tube, 200 nM of 5,6,7 and 9 clones were mixed together with either biotin-labeled forward (5′CGTGGTTACAGTCAGAGGAG3′/3Biotin) (SEQ ID NO: 19) or reverse (5Biosg/5′GGCTTCTGGACTACCTATGC3′) (SEQ ID NO: 20) complementary primers. An annealing protocol was used to hybridize the probes as follows; heat to 95° C. for 2 min and then gradually decrease the temperature to 20° C. by 1° C. every 3 sec. Streptavidin-coated magnetic beads (Cat#Z5482, Promega, U.S.) were used for the purification procedure. Briefly, 1 mL of beads was added to low DNA binding tube and the buffer was exchanged with penicillin-streptomycin (P4333, Sigma-Aldrich, U.S.) and incubated for 2 hrs at 37° C. The beads were then washed with sterile DPBS, followed by the addition of 200 nM aptamer clone mixture and incubation at 25° C. for 2 hrs (shaking every 10 min to resuspend beads). A magnetic stand (Z5331, Promega, U.S.) was used to separate the beads from the aptamer mixture and the beads were then washed 3 times with DPBS. A mixture of VSV and cell debris collected from the VSV harvesting procedure was mixed with aptamer-bead complex for 1 hr. Subsequently, the beads were washed 3 times with DPBS followed by the addition of 25 mM EDTA/EGTA and incubation for 30 min at 25° C. This allowed for the liberation of VSV which was examined using flow cytometry and viral plaque assays.
For the selection of SwAps with switchable affinity to VSV, the mixture of SwAps clones contained equal amount of 5, 6, 7, and 9 with either forward or reverse biotin primers. These clones were chosen because 5, 7 and 9 showed promising results when assessed using the developed aptasensors. The conjugation of SwAps to magnetic beads mimics the approach used for aptasensor analysis and thereby would suggest that the SwAps selected via electrochemical impedance spectroscopy would function well for purification. Although SwAps6 showed modest results in the aptasensor analysis, it was selected to be included among the best SwAps due to its superior performance in free form as indicated by the flow cytometry data.
Biotinylated aptamers (SwAps6) are conjugated to streptavidin coated magnetic beads and mixed with VSV and cell debris solution obtained from the virus harvesting protocol. SwAps bind to VSV. Debris is removed by washing the beads with DPBS. EDTA and EGTA are added to chelate the magnesium and calcium ions causing a conformational change which releases VSV. VSV is then collected.
As shown in
The analysis was carried out using a plaque forming assay as seen in
An aptamer which showed both high affinity and switchability was selected for cloning to obtain individual aptamer sequences to the NRP receptor Pool. This pool was designed to be specific to NRP positive cells. The resulting sequences are provided in SEQ ID NOs: 21 through 30, also reproduced below at Table 4. Briefly, the pool was amplified, purified and cloned using the same method as described in Example 5 above.
Flow cytometry data indicated the aptamers that demonstrated the best switching capability. These are NRP-SMG-E1; NRP-SMG-E2 and NRP-SMG-E3. This data is reproduced at
An aptamer pool selected specifically to LIF positive cells and which showed both high affinity and switchability was selected for cloning to obtain individual aptamer sequences to the NRP receptor. The resulting sequences are provided in SEQ ID NOs: 31 through 47, also reproduced below at Table 5. Briefly, the pool was amplified, purified and cloned using the same method as described in Example 5 above.
Flow cytomtry data showed that the aptamers with the best switching capability are LIF-SMG-E46, E8, E16, E6, E45, E13, E7 and E55. This data is reproduced at
An aptamer pool selected specifically to PTCH1 positive cells and which showed both high affinity and switchability was selected for cloning to obtain individual aptamer sequences to the NRP receptor. The resulting sequences are provided in SEQ ID NOs: 48 through 59, also reproduced below at Table 6. Briefly, the pool was amplified, purified and cloned using the same method as described in Example 5 above.
Flow cytometry data shows that aptamers PTCH1-SMG-E1, E2 and E3 have the best switching capability. This data is reproduced at
An aptamer pool selected specifically to DLL4 positive cells and which showed both high affinity and switchability was selected for cloning to obtain individual aptamer sequences to the NRP receptor. The resulting sequences are provided in SEQ ID NOs 60 through 74, also reproduced below at Table 7. Briefly, the pool was amplified, purified and cloned using the same method as described in Example 5 above.
Flow cytometry data showed that DLL4-SMG-E25, E43, E69, E24, E76, E31,E9, E7 and E1 had the best switching capability. This data is reproduced at
An aptamer pool selected specifically to PLUR/PLAUR) positive cells and which showed both high affinity and switchability was selected for cloning to obtain individual aptamer sequences to the NRP receptor. The resulting sequences are provided in SEQ ID NOs: 75 through 89, also reproduced below at Table 8. Briefly, the pool was amplified, purified and cloned using the same method as described in Example 5 above.
Flow cytometry data indicated that PLAUR-SMG-E50, E13, E76, E35, E20 and E31 had the best switching capability. This data is reproduced at
The scope of the disclosure should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole. The claims are not to be limited to the preferred or exemplified embodiments of the disclosure.
To the extent that external references may be incorporated by reference into the present specification, all references identified herein are incorporated into this specification by reference in their entirety.
Ayyar, B. V.; Arora, S.; Murphy, C.; O'Kennedy, R. Methods 2012, 56, 116.
Berezovski M V, Lechmann M, Musheev M U, Mak T W, Krylov S N. “Aptamer-facilitated biomarker discovery (AptaBiD)” J Am Chem Soc. 2008 Jul. 16;130(28):9137-43.
Carrasquilla C, Li Y, Brennan J D. Surface immobilization of structure-switching DNA aptamers on macroporous sol-gel-derived films for solid-phase biosensing applications. Anal Chem. 2011 Feb. 1;83(3):957-65)
Darfeuille, F.; S. Reigadas, J. Hansen, H. Orum, C. Di Primo, J. Toulme (2006). “Aptamers targeted to an RNA hairpin show improved specificity compared to that of complementary oligonucleotides” Biochemistry 45: 12076-12082.
Deng Q P, Tie C, Zhou Y L, Zhang X X, Cocaine detection by structure-switch aptamer-based capillary zone electrophoresis, Electrophoresis. 2012 May;33(9-10):1465-70.
Diallo, J.; Vähä-Koskela, M.; Le Boeuf, F.; Bell, J. Methods in Molecular Biology 2012, 127.
Fitzgerald, J.; Leonard, P.; Darcy, E.; O'Kennedy, R. Methods Mol Biol 2011, 681, 35. Jiang, Y.; Fang, X.; Bai, a. C. Analytical Chemistry 2004, 5230.
Jeong, S.; S. R. Han, Y. J. Lee, S. W. Lee (2010). “Selection of RNA aptamers specific to active prostate-specific antigen.” Biotechnology Letters 32: 379-85.
Liu, M.; T. Kagahara, H. Abe, Y. Ito (2009). “Direct In Vitro Selection of Hemin-Binding DNA Aptamer with Peroxidase Activity”. Bulletin of the Chemical Society of Japan 82: 99-104.
Long, S.; M. Long, R. White, B. Sullenger (2008). “Crystal structure of an RNA aptamer bound to thrombin”. RNA 14 (2): 2504-2512.
Min, K.; M. Cho, S. Han, Y. Shim, J. Ku, C. Ban (2008). “A simple and direct electrochemical detection of interferon-gamma using its RNA and DNA aptamers.” Biosensors & Bioelectronics 23: 1819-1824.
Ng E. W. M; D. T. Shima, P. Calias, E. T. Cunningham, D. R. Guyer, A. P. Adamis (2006). “Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease.” Nature Reviews Drug Discovery 5 (2): 123-132.
Potty, A.; K. Kourentzi, H. Fang, G. Jackson, X. Zhang, G. Legge, R. Willson (2009). “Biophysical Characterization of DNA Aptamer Interactions with Vascular Endothelial Growth Factor”, Biopolymers 91: 145-156.
Romig T S, Bell C, Drolet D W. J Chromatogr B Biomed Sci Appl. 1999 Aug. 20;731(2):275-84.
Savory, N.; K. Abe, K. Sode, K. Ikebukuro (2010). “Selection of DNA aptamer against prostate specific antigen using a genetic algorithm and application to sensing.” Biosensors & Bioelectronics 15: 1386-91.
Sefah et al. (Sefah K, Phillips J A, Xiong X, Meng L, Van Simaeys D, Chen H, Martin J, Tan W. Nucleic acid aptamers for biosensors and bio-analytical applications. Analyst. 2009 September;134(9):1765-75.
Wei, S.; Mizaikoff, B. J Sep Sci 2007, 30, 1794.
Yang, H.; Gurgel, P. V.; Carbonell, R. G. J Chromatogr A 2009, 1216, 910.
Zhu Z, Ravelet C, Perrier S, Guieu V, Roy B, Perigaud C, Peyrin E. Multiplexed detection of small analytes by structure-switching aptamer-based capillary electrophoresis. Anal Chem. 2010 Jun. 1;82(11):4613-20.
| Number | Date | Country | |
|---|---|---|---|
| 61823638 | May 2013 | US |
