Patent Application
20120135540
1. Field of the Invention
The present invention pertains to the field of aptamer- and nucleic acid ligand (DNA and RNA ligand)-based diagnostics. More specifically, the application relates to single-stranded deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) ligand sequences, whether individual or linked together to form longer multiple binding site “receptors,” that specifically target and bind to clinically relevant analytes on one or more binding sites or “epitopes” from humans such a cardiovascular biomarkers, bone metabolism markers, glucose, natural or recombinant human growth hormone (“hGH” or somatotropin) and vitamins. The invention includes general DNA ligand or aptamer-based detection and quantitation of these analytes in body fluids such as blood plasma, serum, sputum or saliva, interstitial, synovial, or cerebrospinal fluid aspirates, mucus, and urine or solid biopsy samples.
2. Background Information
These individual or linked DNA ligand (aptamer) sequences represent valuable target analyte-responsive components of diagnostic devices or biosensors. A “biosensor” is defined as any device that employs a biologically-derived molecule as the sensing component and transduces a target analyte binding event into a detectable physical signal (including, but not limited to, changes in light intensity, absorbance, transmittance, refraction (Surface Plasmon Resonance or “SPR”), wavelength, color, agglutination of cells or particles, fluorescence intensity, fluorescence lifetime, fluorescence polarization or anisotropy, fluorescence correlation spectroscopy (“FCS”), fluorescence or Förster resonance energy transfer (“FRET”; nonradiative dipole-dipole coupling of fluorophores or fluorophores and quenchers), upconverting phosphor, two-photon interaction phenomena, Raman spectroscopy or surface-enhanced Raman spectroscopy (“SERS”), electrical conduction, electrical resistance or other electrical properties, mass, photon or radioactive particle emissions, etc).
Once bonded with the target, these DNA ligand sequences can be used to qualitatively determine the presence of analyte, as well as to quantify or semi-quantify the target analyte amount in a sample using a broad variety of assay types and diagnostic or sensor platforms including, but not limited to, affinity-based lateral flow test strips, membrane blotting, SPR, surface acoustic waveguides (“SAW” devices), magnetic bead (“MB”)-based capture, plastic-adherent sandwich assays (“PASA”), chemiluminescence (“CL”), electrochemiluminescence (“ECL”), radioisotopic, fluorescence intensity, including quantum dot (“QD”) or other fluorescent nanoparticle (“FNP”) of dye-based, fluorescence lifetime, and fluorescence polarization (“FP”) assays or enzyme-linked (“ELISA-like”) microplate assays. ELISA-like assays refer to microwell or microplate assays similar to traditional Enzyme-Linked Immunosorbent Assays (“ELISA”) in which an aptamer or nucleic acid ligand is substituted for the antibody or receptor component or components, but the other components such as peroxidase or alkaline phosphatase enzymes and color-producing substrates remain the same.
In addition, these DNA ligand sequences are valuable in competitive displacement assays which are not solely dependent on high affinity (strong attractive forces between a receptor and its ligand) or high avidity (high tensile or physical strength of receptor-ligand bonds) to produce sensitive detection (sub-nanoMolar or sub-nanogram levels), because the equilibrium constant (generally Ka=106 to 108 to enable competition) must allow reasonable displacement of previously bound target materials to detect a change at or below nanogram or nanoMolar levels.
In a competitive displacement assay, labeled DNA ligand plus labeled analyte complexes compete with unlabeled analyte to bind with the labeled DNA. After allowing the labeled and unlabeled analytes to come to equilibrium with the labeled DNA, the unlabeled target analyte may be quantitatively assayed by fluorescence intensity or other methods. Such assays would include competitive displacement FRET assays or DNA ligand “beacon” FRET assays. In a competitive displacement FRET assay the fluorophore (“F”) and quencher (“Q”) are placed in a putative binding loop or pocket so as to reside within the Förster distance of 60-85 Angstroms to enable quenching.
In an aptamer beacon assay, the F and Q labels are placed on the 5′ and 3′ ends and binding of the target analyte to the beacon opens the beacon beyond the Förster quenching distance so that F is no longer quenched and emits light generally in proportion to the amount of target analyte introduced into the liquid system. Each of these types of aptamer assays and detection platforms has different applications in either central medical laboratories or in point-of-care (“POC”) sensor devices for use in emergency rooms, intensive care units, cardiac care units, or physician's offices and clinics.
The DNA ligand sequences listed herein (Table 9) were derived by iterative cycles of affinity-based selection, washing, heated elution, and polymerase chain reaction (“PCR”) amplification of bound DNA ligands from a randomized library using immobilized target analytes for affinity selection and PCR amplification followed by cloning and Sanger dideoxynucleotide DNA sequencing.
Sanger dideoxynucleotide sequencing refers to DNA chain termination due to a lack of a 3′ hydroxyl (—OH) group to link incoming bases to during DNA synthesis followed by automated fluorescence reading of the DNA sequence from an electrophoresis gel containing all of the terminated DNA fragments. DNA sequencing may be accomplished by PCR doped with dideoxynucleotides lacking hydroxyl groups at the 2′ and 3′ sugar ring positions and thereby disallowing chain formation. PCR refers to the enzymatic amplification or copying of DNA molecules with a thermo-stable DNA polymerase such as Thermus aquaticus polymerase (Taq) with known “primer” regions or short oligonucleotides of known sequence that can hybridize to a longer target DNA sequence to enable priming of the chain reaction (exponential doubling of the DNA target copy number with each round of amplification).
A randomized library can be chemically synthesized by linking together the four deoxynucleotide triphosphate bases (adenine; A, cytosine; C, guanine; G, and thymine; T) in equal amounts (25% each), so that a combinatorial oligonucleotide arises with sequence diversity equal to 4 raised to the nth power (4n) where n is the desired length of the randomized region in bases. In other words, if position 1 in an oligonucleotide is allowed to consist of A, C, G, or T (diversity=4) by equal availability of all 4 bases and these 4 possibilities are multiplied by each base linking to 4 more possible bases at position 2, then this process yields 16 possible 2-base oligonucleotides (i.e., AA, AC, AG, AT, CA, CC, CG, CT, GA, GC, GG, GT, TA, TC, TG, TT) and so on for the entire chosen length (n) of the randomized region. This combinatorial progression displays immense diversity as a function of oligonucleotide chain length. For 1,048,576 unique DNA sequences from which to chose or select one or more DNA sequences that bind a given immobilized target analyte with the strongest affinities.
The randomized oligonucleotide or DNA is designed to be flanked on either side by short primer regions of known and fixed sequences to enable PCR amplification (exponential copying) of the rare sequences that are selected from the random library by binding to the target after the non-binding members of the random library are washed away (not selected).
Additional assays, such as ELISA-like plate assays or fluorescence (e.g., intensity and FRET) assays, may be used to screen or verify the value of particular DNA and RNA ligands or aptamer sequences for detection of a given target analyte in a given assay format or type of biosensor. Some of the sequences operate (bind and transduce the binding signal) more effectively in affinity-based (ELISA-like or fluorescence intensity) assays, while other DNA ligand sequences against the same targets function better in lower affinity competitive or other assays, thereby leading to more sensitive detection with lower limits of detection (sub-nanoMolar or sub-nanogram) and less cross-reactivity or more specificity for the target analyte. “Specificity” or “selectivity” means the ability to selectively exclude molecules similar in structure to the true target analyte that may interfere with the assay and give false indications of detection. All of the listed DNA ligand nucleotide sequences have potential applications in some type of assay format, because they have survived at least 5 rounds of affinity-based selection and enrichment (by PCR amplification), although some of the sequences will undoubtedly perform better in certain assay formats or configurations (in tubes, square cuvettes, membranes, or on biochips) than others.
Combinations of the DNA ligands, whether in whole or in part (i.e., their binding sites of approximately 5-10 or more nucleotides or bases), could be linked together in a linear or 2-dimensional (“2-D”) or 3-dimensional (“3-D”) fashion similar to dendrimers as shown in
The linked aptamer complex will be likely to gain specificity as well since the probability of binding to multiple epitopes with any degree of success is multiplicative. Thus, the ability to bind to epitopes A, B and C equals the product of the probability of binding to A with high affinity times the probability of binding to B with high affinity times the probability of binding to C with high affinity. The product of those three fractional probabilities is clearly much less than the probability of binding to only A, B, or C independently in the absence of binding the other two or any combination of the two epitopes therein in the absence or binding the third. Hence by linking two or more aptamers or their binding regions with or without DNA or other “spacer” regions, the selectivity or specificity of aptamers or DNA ligands can be increased.
This approach to binding site linkage emulates the nature of antibodies which demonstrate linkage of their “hypervariable” (“HV”) regions on the antigen combining sites of the immunoglobulin (“Ig”) light and heavy chains. In the HV regions, the variability of the 20 amino acid types is quite high and essentially represents a selection of one combination from a large combinatorial library in the protein realm (similar to down selection of a few candidate aptamers from a large diverse starting library). The trait of HV region linkage contributes to Ig affinity, avidity and specificity. Similarly, linking aptamers or aptamer binding sites for various epitopes in one, two or three dimensions will enhance larger aptamer or DNA ligand construct affinity, avidity, and selectivity or specificity as illustrated in
The present invention provides specific DNA sequence information for nucleic acid ligands (aptamers) or their linked constructs selected from randomized pools to bind clinically important proteins, peptides, hormones, sugars, vitamins, post-translational N-acetylglucosamine (NAG or O-GlcNAc) modifications of key proteins, etc. in a variety of assay formats and sensor or diagnostic platforms for assessment of human health status. While all of the candidate sequences have been shown to bind their cognate targets, some are shown to function more effectively in affinity-based assays versus fluorescence resonance energy transfer (FRET) or other assay formats that rely more on physical parameters other than affinity such as fluorophore-quencher proximity (i.e., the Förster distance). Therefore, all of the sequences are potentially valuable for simple qualitative detection or quantitative assays, but some may function better in terms of sensitive and specific detection than others in particular assay formats.
There is no single preferred embodiment for use of the DNA aptamer ligand sequences or linked aptamer constructs identified herein. Rather, the sequences are useful to varying extents in a variety of assay formats and sensors or diagnostic devices chosen from at least the following list: lateral flow test strips, Surface Enhanced Raman (SERS), Surface Plasmon Resonance (SPR), Surface Acoustic or Transverse Wave (SAW or STW) detection, electrical, electrochemical, colorimetric absorbance, agglutination, ELISA-like or enzyme-linked microplate assays, magnetic bead-based capture assays, ECL or other chemiluminescence assays, radioisotopic assays and a variety of fluorescence assays including, but not limited to, fluorescence intensity, fluorescence lifetime, fluorescence polarization (FP) and Fluorescence or Förster Resonance Energy Transfer (FRET) assays (both beacon and competitive FRET (Bruno et al., 2010, 2011) in round tubes, square or flat cuvettes, or immobilized on magnetic beads, other types of microbeads, or flat surfaces such as nitrocellulose, nylon, or other membranes or on glass or plastic DNA microarrays or “biochips.”
While there may appear to be considerable variability among sequences that bind the same clinical analyte targets, “epitopes” and their cognate aptamer binding sites are usually quite small (e.g., 5-10 bases) and a single target may contain numerous individual binding sites or epitopes for multiple aptamer binding. In addition, however, there is often a common or consensus sequence (designated herein by slashes between clone numbers in Table 9, e.g. —aptamer clones CTx 2, 13, 19, 20, 25, 32F or R series are identical and only listed once as SEQ ID NO 3 and 4) or common segments of 5-10 or more nucleotides in a row within otherwise different aptamer sequences that can bind a specific target epitope that may dominate the other binding sites by being more physically accessible or having stronger electrostatic, hydrogen bonding, or other attractive forces (summation of van der Waals or other weak forces). Variations in nucleotide sequences around these consensus segments or common binding sequence segments may serve to modulate the binding segment's affinity or specificity or may have no effect at all. These properties must be determined by empirical comparisons.
General methods for developing DNA ligands or aptamers to the immobilized proteins, peptides, or small molecules (defined as less than 1,000 daltons) are as follows. The protein, peptide or an amino-derivative of the small molecule (such as glucosamine in the case of D-glucose or dextrose) is then added to 2×109 tosyl-coated magnetic beads (MBs; e.g., Dynal brand from Invitrogen Corp. Carlsbad, Calif., 2. 8 micron size) for 2 hours at 37° C. The tosyl group is a “leaving” group that allows the formation of a very stable covalent bond between primary amine groups in the target protein, peptide or amino-derivatized small molecule and therefore immobilizes the target on the surfaces of the MBs so that they can be used to probe the randomized DNA library for DNA ligands. Target molecule-conjugated MBs (or target-MBs) are collected for 2 minutes in a magnetic collection device using an external magnet and the supernate is carefully withdrawn with a pipette tip. Target-MBs are then resuspended by vortexing briefly in 1× Binding Buffer (1×BB; 0.5M NaCl, 10 mM Tris-HCl, and 1 mM MgCl2, pH 7.5-7.6,) and washed by agitation for 5 minutes. MBs are collected and washed three times in this manner and then resuspended in 1 ml of 1×BB.
MB-based DNA ligand or aptamer development is then performed using a template library sequence such as: 5′-ATCCGTCACACCTGCTCT-N36-TGGTGTTGGCTCCCGTAT-3′, where N36 represents the randomized 36-base region of the DNA library (maximal sequence diversity=436 in theory). Primer sequences are: 5′-ATACGGGAGCCAACACCA-3′ (designated forward) and 5′-ATCCGTCACACCTGCTCT-3′ (designated reverse) to prime the template and nascent strands for PCR, respectively. The random library is reconstituted in 500 μl of sterile nuclease-free water and heated to 95° C. for 5 minutes to ensure that the DNA library is completely single-stranded and linear. The hot DNA library solution is added to 100 μl of target-MBs (2×108 beads) with 600 μl of sterile 2× Binding Buffer (2×BB). The DNA library and target-MB suspension (1.2 ml) is mixed at room temperature (RT, approximately 25° C.) for 1 hour. Target-MBs with any bound DNA (round 1 aptamers) are magnetically collected. The DNA-target-MB complexes are washed three times in 400 μl of sterile 1×BB. Following the third wash, the DNA-target-MB pellet (about 75 μl) is used in a PCR reaction to amplify the bound DNA as follows. The MB pellet is split into 15 μl aliquots and added to five pre-made PCR tubes which contain most of the nonperishable ingredients of a PCR reaction beneath a wax seal. A total of 3 μl of 1:10 primer mix (10% forward primer plus 10% reverse primer) in nuclease-free deionized water or ˜20 nanomoles of each primer per ml plus 1 μl (5 U) of Taq DNA polymerase and 5 μl of 2 mM MgCl2 are added to each of the five tubes. PCR reactions are supplemented with 0.5 μl of E. coli single-strand binding protein (SSBP, Stratagene Inc., La Jolla, Calif.) to inhibit high molecular weight concatamer (end to end aggregates of the DNA ligands) formation. PCR is carried out as follows: an initial 95° C. phase for 5 minutes, followed by 20 cycles of 1 minute at 95° C., 1 minute at 53° C., and 1 minute at 72° C. followed by a 72° C. completion stage for 7 minute, and refrigeration at 4° C. This constitutes the first of multiple rounds of MB-aptamer development. Iterations of the MB-aptamer development process are repeated until the desired affinity or assay sensitivity and specificity are achieved. Typically, 5-10 rounds of the MB-aptamer development process are required to achieve low ng/ml detection of target analytes. To begin the second round and all subsequent rounds, 4 complete tubes of the original PCR tubes are heated to 95° C. for 5 minutes to release bound DNA from the target-MBs. The fifth tube is always retained and refrigerated as a back-up for that round of the aptamer generation process. All available DNA (25 μl per tube) is siphoned out of the hot tubes without removing the target-MBs before the tubes cool significantly and the DNA is pooled. The 100 μl of hot DNA is added to 100 μl of fresh target-MBs in 200 μl of 2×BB and allowed to mix for 1 hr at RT. Thereafter, the selection and amplification process are repeated for 3-8 more rounds with checking for 72 bp aptamer PCR products by ethidium bromide-stained 2% agarose electrophoresis after each round. Following the last round of aptamer development, aptamers are cloned into chemically competent E. coli using a cloning kit from Lucigen Corp. (Middleton, Wis.) and clones are sent to Sequetech, Inc. (Mountain View, Calif.) for DNA sequencing.
To evaluate, screen, and rank aptamers based on affinity against clinically relevant targets, an enzyme-linked plate assay is conducted by first immobilizing 100 μl of 1:10 diluted target (about 0.1 mg of total protein, peptide or small molecule) in 0.1M NaHCO3 (pH 8.5) overnight at 4° C. in a covered polystyrene 96-well plate. The plate is decanted and washed three times in 250 μl of 1×BB. Each of the different 5′-biotinylated aptamers raised against the target is dissolved in 1×BB at 1.00 nmoles to 4.50 nmoles per 100 microliters and applied to their corresponding plate wells for 1 hour at room temperature (RT; ˜25° C.) with gentle mixing on an orbital shaker. The plate is decanted and washed three times in 250 μl of 1×BB for at least 5 minutes per wash with gentle mixing. One hundred μl of a 1:2,000 dilution of streptavidin-peroxidase from a 5 mg/ml stock solution in 1×BB is added per well for 30 minutes at RT with gentle mixing. The plate is decanted and washed three times with 250 μl of 1×BB per well as before. One hundred μl of ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) substrate with stabilized hydrogen peroxide (Kirkegaard Perry Laboratories, Inc., Gaithersburg, Md.) is added per well for 10 minute at RT. Finally absorbance is quantified using a microplate reader with 405 nm optical filter.
As Tables 1-8 illustrate for several cardiovascular biomarker targets (Brain Natriuretic Peptide; BNP, D-Dimer; DD, Creatine Kinase-MB types I and II; MBI and MB2, Interleukin-18; IL 18, and Troponin-T; Tpn all at (1 μg/ml) the initial ELASA screening is useful for ranking the relative affinity of aptamers for their respective targets by simple ranking of absorbance values at 405 nm from highest to lowest. Each of the Tables (1-8) illustrates general consistency between ELASA trials as well (i.e., the highest affinity aptamers consistently rank among the highest absorbance values between ELASA trials or plates 1-4 maximally).
P - 5/11/15b/19/25
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Once key aptamers have been identified by the commonality of their sequences or their secondary stem-loop structures, the assay developer decides upon secondary structure loops (potential binding pockets) to label with a fluorophore (F) or quencher (Q) as illustrated by dotted lines that define the borders or limits of potential binding loops in
Alternatively, one may label the suspected binding loops internally and place an F or a Q somewhere in the mid-section of the suspected loop other than the 3′ or 5′ end (i.e., intrachain FRET). Attachment of F or Q is usually accomplished via succinimide linkage of F- or Q-succinimides added to amino-modified aptamers at specifically chosen locations in the binding pockets. Primary amine linker moieties such as the “UniLink™” can be added internally at the time of chemical synthesis of aptamers. Typically 1 mg or more of an aptamer sequence is synthesized with a primary amine linker moiety (UniLink™) located at the approximate center of each loop structure (suspected binding pockets). Each of these internally amine-labeled aptamers is then labeled with 100 μl (0.1 mg) of F-succinimide (or alternatively Q-succinimide) for 2 hours in a 37° C. incubator, followed by purification through a 1×BB-equilibrated PD-10 (Sephadex G-25; GE Healthcare) column. In the meantime, an equal molar amount of primary amine-modified target molecule is labeled with 0.1 mg of spectrally matched Q-succinimide (to accept photons from F) at 37° C. for 2 hours and then washed three times by centrifugation at 14,000 rpm for 10 minutes per wash and resuspension in 1 ml of 1×BB. “Spectrally matched” means that most of the wavelengths of light emitted by F can be effectively absorbed by Q because its absorbance spectrum largely overlaps the emission spectrum of F. Naturally, if the aptamer is labeled with a Q-succinimide in the alternate form of the assay, the amine-modified target must be labeled with an appropriately matched F-succinimide to be quenched when bound to the Q-labeled aptamer. Pooled one ml fractions of purified F-labeled DNA aptamers are mixed with an equimolar amount of Q-labeled-amino-target analyte (or vice versa in the alternate embodiment) for 30 minutes at RT with mixing in 1×BB or phosphate buffered saline (PBS, 0.1M phosphates in 8.5 g/L sodium chloride at pH 7.2 to 7.4) and then purified through an appropriate size-exclusion chromatography column (according to molecular weight of the combined F-aptamer plus Q-target complex) to produce a purified “competitive FRET complex” consisting of F-aptamer conjugate bound to Q-labeled target. This competitive FRET complex can later be competed against unlabeled cognate analyte concentrations to increase the fluorescent light output of the liquid assay system and quantify the unlabeled analyte concentration.
Generally, the aptamer beacons or competitive FRET-aptamer complexes are then diluted to a final concentration of 1-5 μg/ml in 1×BB and equally dispensed to polystyrene or methacrylate cuvettes in which 1 ml of unlabeled target at various concentrations in 1×BB, PBS or diluted blood, plasma, serum, saliva, aspirate or urine has been added already. Cuvettes are gently mixed for 15 to 20 minutes at RT prior to reading their fluorescence in the homogeneous beacon or competitive-displacement FRET assay formats using a spectrofluorometer having gratings to vary the excitation wavelength and emission scanning ability or a stationary, handheld or otherwise portable fluorometer having a more restricted or fixed excitation and emission optical filter set with a range of wavelengths for excitation and emission.
The linkage of binding sites is beneficial in terms of enhancing receptor affinity, avidity (tensile binding strength), and selectivity versus complex targets with two or more distinct epitopes. This linkage can be sequential and linear (one-dimensional as in antibody heavy and light chain linkage of HV regions,
For 2-D or 3-D linked aptamer structures a variety of linker chemistries are available, but the preferred embodiment is probably addition of a UniLink™ primary amine group somewhere in the mid-section of a larger multi-aptamer construct followed by covalent linkage of two or more such multi-aptamer constructs by means of bifunctional linkers such as low levels (<1%) of glutaraldehyde, carbodiimides, sulfo-EGS, sulfo-SMCC or other such bifunctional linkers familiar to those skilled in conjugate chemistry. This strategy would result in a larger flower-like 2-D or 3-D structure consisting of two or more lengthy multi-aptamer structures.
Referring to the figures,
Although the invention and DNA ligand (aptamer) sequences have been described with reference to specific embodiments, these descriptions are not meant to be construed in a limited sense. Various modifications of the disclosed embodiments, as well as alternative embodiments of the inventions will become apparent to persons skilled in the art upon reference to the description of the invention. It is, therefore, contemplated that the appended claims will cover such modifications that fall within the scope of the invention.
This application is based upon and claims priority from U.S. Provisional application Ser. No. 61/402,491 filed on Aug. 31, 2010, and Ser. No. 61/463,020 filed on Feb. 10, 2011, which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61402491 | Nov 2010 | US | |
| 61463020 | Feb 2011 | US |
