This invention relates to target-binding bimolecular constructs useful in detecting and quantifying substances in samples and subjects for applications in proteomics, diagnostics, drug discovery, medical devices, systems biology and, more generally, life sciences research and development. Compositions and methods of the invention can be used, for example and without limitation, in instrumented and noninstrumented sensors, transducers, signal processing devices and solid-phase, solution-phase, homogeneous and heterogeneous assay systems.
As background, monomolecular nucleic acid-based detection constructs, such as molecular beacons, are dissimilar from the instant bimolecular constructs and disadvantageous for reasons discussed below. In the typical monomolecular beacon construct, a fluorophore and a quencher are placed on opposite ends of the same nucleic acid strand. In the nonbinding hairpin conformation, the fluorophore and quencher are in close proximity, and fluorescence emission in response to illumination is quenched. In the target-bound conformation, the fluorophore and quencher are separated by sufficient distance to circumvent quenching. In this case, illumination by light of suitable spectral qualities results in target-dependent fluorescence.
An immobilized bimolecular construct of the present invention comprises a solid support, a first oligonucleotide and a second oligonucleotide. The first oligonucleotide is labeled at one end with a fluorophore or quencher and attached at the other end to a solid support. The second oligonucleotide is labeled at one end with a fluorophore or quencher and hybridized at the other end to the first oligonucleotide. Hybridization of the second oligonucleotide with the first oligonucleotide brings the labeled end of the second oligonucleotide in close proximity or physical contact with the labeled end of the first oligonucleotide. In one embodiment the second oligonucleotide is also attached to the solid support in proximity to the first oligonucleotide. In this embodiment, the second oligonucleotide may be first attached to the solid support and then hybridized to the first oligonucleotide or, conversely, first hybridized to the first oligonucleotide and then attached to the solid support.
A bimolecular construct of the present invention is described for the attachment of nucleic acid-based molecular devices to surfaces as illustrated, e.g., by immobilized molecular beacons, aptamers and tunable affinity ligands (TALs). “Bimolecular construct,” as used herein, refers to a molecular complex or its substituent components comprising at least two hybridizably linked or linkable nucleic acid-based molecules, at least one of which is capable of generating a detectable signal or attaching to a surface. The second of the at least two hybridizably linked or linkable molecules enables or facilitates surface attachment or signaling by its hybridizable partner or enhances function compared to a corresponding monomolecular construct as measured, e.g., by attachment effectiveness, efficiency, reliability, stability, sensitivity, specificity, signal-to-noise ratio, versatility, convenience, ease-of-use and/or cost effectiveness. Bimolecular constructs of the invention are advantageously used for signaling molecular interactions and/or detecting the presence or amount of a substance in a sample or subject. As such, in one embodiment of the invention, bimolecular constructs are capable of generating a signal, advantageously a signal corresponding to a specific binding event between a probe or ligand moiety of the construct and a target substance, molecule, sequence and/or cell. In another embodiment, bimolecular constructs are capable of detecting the presence and/or amount of a target substance in a sample or subject, advantageously recognizing the target with a high or controlled degree of selectivity through specific binding interactions well known in the art, e.g., nucleic acid hybridization, ligand-receptor binding, and capitalizing on the signal-generating properties of detectably labeled bimolecular constructs. Bimolecular constructs with signaling and detection functionalities have broad utility in molecular and cellular analysis; clinical, agricultural, veterinary and environmental diagnostics; military, space and forensic uses; and, more broadly, life science and industrial applications.
As used in this disclosure, “nucleic acid” and “nucleic acid-based” refers to constructs comprising a plurality of nucleotides, advantageously a sufficient number of nucleotides to participate in base-pairing, and optionally nonnucleotide monomers, polymers, spacers, linkers and the like. The term “nucleotide” includes any compound containing a heterocyclic compound bound to a phosphorylated sugar by an N-glycosyl link, any monomer capable of complementary base pairing and any analog, mimetic, congener or conjugate thereof, including modified purines and pyrimidines, minor bases, convertible nucleosides, structural analogs of purines and pyrimidines and labeled, derivatized, modified and conjugated nucleosides and nucleotides. Nonnucleotide constituents of nucleic acid-based constructs include, for example and without limitation, sequence modifiers, terminus modifiers, spacer modifiers, backbone modifications, amide linkages, achiral and neutral internucleotidic linkages and nonnucleotide bridges such as polyethylene glycol, aromatic polyamides, lipids and the like. The term “oligonucleotide” means a molecule comprising a sequence of nucleotides, typically at least three and less than about a thousand nucleotides, although the term as used herein is not intended to convey any particular limit on nucleotide sequence length. The term “nonnucleic acid” refers to a molecule or group of molecules other than a nucleic acid or oligonucleotide molecule. The term “nonoligonucleotide” refers to a molecule or group of molecules other than an oligonucleotide or nucleic acid molecule. Nonnucleic acid and nonoligonucleotide molecules are those lacking a sequence of purines, pyrimidines and/or purine or pyrimidine analogs and include, for example, peptides, proteins, sugars, carbohydrates, lipids, inorganic molecules, purine and pyrimidine monomers and naturally occurring and synthetic monomers, dimers, trimers, oligomers, polymers and analogs, mimetics, conjugates and complexes thereof. The term “proximity” with regard to fluorophore/quencher interaction refers to a distance sufficiently small (the “energy transfer distance”) to allow detectable fluorophore-quencher energy transfer, advantageously a distance in the range of the Forster energy transfer distance (“Forster distance”) or a small multiple thereof that allows for energy transfer efficiency of at least about 10%. The Forster distance is based on the principle of fluorescence resonance energy transfer or FRET. Fluorescence resonance energy transfer (FRET) is the distance-dependent transfer of excited state energy from a donor fluorophore to an acceptor fluorophore. The Förster distance is a characteristic distance for energy transfer and provides a spectroscopic ruler. The Förster distance is defined as the distance at which FRET is 50% efficient.
For the purposes of this application, molecular beacons are defined as hairpin-forming nucleic acid-based ligands that, upon binding to target, switch from a quenched conformation to one that fluoresces. Typically, the targets of molecular beacons as described in the art are nucleic acid sequences complementary to the loop region of the molecular beacon hairpin. The hairpin loop is designed to contain a probe sequence optimal for specific hybridization to the target of interest. Bimolecular constructs of the present invention comprehend and incorporate molecular beacon-like hairpin probe regions for specific detection of nucleic acid targets as well as other nucleic acid-based ligands that recognize and detect a diverse assortment of nucleotide and nonnucleotide molecules through hybridization and nonhybridization-based interactions with target molecules. The term “beacon,” as used herein, is occasionally used to refer to a beacon-like structure, component, region or functional element of fluorophore- and quencher-labeled hairpin probes known in the art as molecular beacons. For convenience, terms like “beacon” and “beacon moiety,” e.g., are sometimes used in reference to a hairpin oligonucleotide or fluorophore- or quencher-labeled hairpin oligonucleotide that lacks the full complement of features required for target-dependent signal generation. For example, “beacon,” and “beacon moiety” may be used as generic terms in reference to, e.g., a hairpin-forming oligonucleotide comprising a bimolecular construct or a hairpin-containing precursor of a bimolecular construct.
Bimolecular constructs can be designed to detect and quantify substances over a wide range of sizes, shapes and compositions, including, e.g., cells, cell surface markers, subcellular structures, liposomes, vesicles, microorganisms, nanoparticles, macromolecules, multimers, natural and synthetic polymers, oligomers, monomers and small molecules. Targets may include, for example and without limitation, nucleic acids, proteins, peptides, antibodies, antigens, haptens, carbohydrates, drugs, pharmacophores (including biological, bioderived, bioinspired and synthetic drug candidates, leads, prospects, analogs, congeners, mimetics, agonists, antagonists, competitors and the like), hormones, growth factors, autocoids, transmitters, vitamins, metabolites, cofactors, food pathogens, toxins, environmental pollutants, industrial contaminants, infectious agents, biomolecular complexes (e.g. ribonucleoprotein complexes, multimeric proteins and protein complexes, lipid and lipoprotein particles and protein-carbohydrate complexes), cell surfaces, viruses, and other complex biological targets. Small molecules, as distinct from macromolecules, are intended to comprehend molecules having a number-average/weight-average molecular weight of under about 5,000 Daltons and more typically under about 2,000 Daltons, though the term can also be applied to low molecular weight polymers such as oligonucleotides, oligopeptides, oligosaccharides and the like, for which it is difficult to justify a specific molecular weight cutoff between, say, 5,000 Daltons and 10,000 Daltons. For purposes of this disclosure, “small molecules” shall mean those having a molecular weight less than about 5,000 Daltons with discretion as needed in the case of selected oligomeric species. Biomolecular complexes are intended to comprehend noncovalent associations of biologically occurring molecules, including proteins, nucleic acids, carbohydrates, small molecules and associated ions. Examples include ribosomes and other ribonucleoprotein complexes, biologically functional protein complexes in muscles, the cytoskeleton, secretory processes and nonfunctional biomolecular aggregates (e.g., prion protein precipitates and Alzheimer plaques.) Other complex biological targets include the extracellular biological matrix, biofilms, and other complex associations of living cells, colonies of cells, and associated biopolymer matrices. Proteins are intended to comprehend glycoproteins and lipoproteins.
Molecular beacon target binding sequences can be naturally occurring, rationally designed, or discovered by a combinatorial process such as SELEX. When used in reference to an immobilized detection reagent or solid phase binding assay, the term “surface” refers to a support, advantageously a solid, semi-solid or insoluble substance, material, or matrix, to which molecules can be attached, e.g., for the purpose of distinguishing surface-bound molecules and complexes from solution-phase molecules and complexes. The term “support” refers to the surface/structure to which molecules can be attached or otherwise immobilized, associated, localized and/or insolubilized.
As stated above, in the typical monomolecular construct, a fluorophore and a quencher are placed on opposite ends of the same nucleic acid strand. In the “unbound” (target-free) hairpin conformation that prevails in the absence of target, fluorophore and quencher are in close proximity, and excitation-induced fluorescence is quenched. In the target-bound conformation, the fluorophore and quencher are spatially separated by the intervening probe-target complex, and fluorescence occurs upon illumination by light of suitable wavelength.
In the bimolecular construct, the fluorophore and the quencher are placed on separate strands, thereby providing key advantages over attachment methods using monomolecular beacons. Because use of the preferred bimolecular construct results in the projection of a duplex structure from the attachment surface, a more rigid spacer separates the fluorophore and the quencher from the modified surface. Interaction between the fluorophore and the quencher is therefore favored over interaction between the fluorophore or the quencher and the surface. As a consequence of the bimolecular design, which limits the interaction of fluorophore and/or quencher with the surface, reduced background fluorescence is obtained for bimolecular compared to unimolecular beacons. Another advantage of the bimolecular construct is that surface attachment can occur through both of the duplex stands. The bimolecular construct can thus be attached to the surface by (at least) two covalent bonds, rather than just one. A major advantage of attaching both strands is that rigorous washing procedures can be performed following immobilization to remove nonspecifically bound fluorescent moieties from the surface, without risking removal of the hairpin-forming oligonucleotide from the bimolecular construct. Finally, in the case where the target is a protein or other nonnucleic acid molecule, the bimolecular construct allows greater control over the designed placement and target-dependent separation of the fluor and quencher moieties.
1. Principle of Operation of Molecular Beacons
Molecular beacons are nucleic acid probes that undergo a conformational change and fluoresce brightly when they bind to their target (See, for example, Tyagi and Kramer, 1996; Tyagi, Bratu et al., 1998). These probes are single-stranded nucleic acids that form a stem-and-loop structure (
Since molecular beacons are dark (nonfluorescent) when not hybridized and brightly fluorescent when hybridized to their targets, the course of hybridization can be followed in real time with a spectrofluorimeter.
Just as in any other nucleic acid hybridization reaction, the binding of a molecular beacon to its target follows second order kinetics, and the rate of the reaction depends on the concentration of the probe, the concentration of the target, the temperature, and the salt concentration. Under in vitro and in vivo assay conditions, in which the molecular beacon concentration is chosen so that they will always be more abundant than the target, hybridization is spontaneous and rapid, reaching completion in only a few seconds, and the intensity of the resulting fluorescence is linearly proportional to the amount of target present.
Since the introduction of molecular beacons, they have been used in a number of studies that would have been far more difficult to perform with conventional hybridization probes. Molecular beacons are able to monitor the progress of any amplification reaction where either single-stranded or double-stranded nucleic acids are formed. Real-time monitoring of the synthesis of DNA or RNA sequences have been developed for PCR, NASBA, rolling circle amplification and the isothermal ramification amplification method (See, for example, Marras, 2003b). In addition, molecular beacons have been used to detect the movement of specific RNAs in living cells (See, for example, Bratu, Cha et al., 2003). Other studies use molecular beacons to measure enzymatic activities, duplex and triplex formation in nucleic acids, and interactions between proteins and nucleic acids (See, for example, Marras, Kramer et al., 2003a)).
2. Molecular Beacons with Nonnucleic Acid Targets.
Aptamers are nucleic acid ligands that have been discovered by the combinatorial process known as SELEX (See, for example, Brody and Gold, 2000; Famulok and Mayer, 1999; Wilson and Szostak, 1999). Aptamer beacons are molecular beacons that are constructed using known aptamers and are designed to fluoresce in the presence of target (e.g. a protein) and to be quenched in the absence of target. Ellington and coworkers have designed monomolecular aptamer beacons based on the well-studied thrombin aptamer that fluoresce in protein-binding G-quadruplex form and that are quenched when in the competing hairpin form (See, for example, Hamaguchi, Ellington et al., 2001). Beacons can also be derived using naturally occurring protein-binding nucleic acid sequences, for example in gene-regulatory regions of the chromosome. We will discuss below particular examples of both naturally occurring sequences and aptamer sequences that can be integrated into protein-binding molecular beacon design.
3. Tunable Affinity Ligands (TALs).
TALs are ligands defined by the following properties:
4. Quadruplex-Hairpin Tunable Affinity Ligands (TALs).
As a specific example of TALs, we have focused on nucleic-acid based ligands that can partition between quadruplex and hairpin forms. The partitioning between quadruplex and hairpin depends strongly on the presence of ions such as K+, which coordinate specifically with, and thereby stabilize, quadruplex structures. Bulky ions such as Li+ are unable to coordinate specifically, and will therefore shift the equilibrium toward the hairpin. The binding conformation of a given Tunable Affinity Ligand (TAL) can be biologically derived, e.g. from quadruplex-forming sequences such as genomic G-rich regions, including telomeres, the c-MYC promoter region, and fragile X expansion regions. The binding conformation can also be derived from aptamers, arrived at by the SELEX methodology, e.g. the thrombin aptamer, or the aptamer for the receptor activator of NF-κB (RANK). Finally, the binding conformation can be derived by any combination of procedures involving rational design followed by screening, followed by optimization.
5. Tunable Affinity Ligand (TAL) Beacons.
Tunable Affinity Ligand (TAL) beacons are TALs that exist in either a quenched conformation or an unquenched conformation. We define standard TAL beacons as molecules for which the unquenched conformation shows specific target binding affinity, while the quenched conformation binds the same target with reduced affinity. Molecules for which the quenched conformation binds target specifically and the unquenched conformation binds target with reduced affinity we define as reverse TAL beacons. One example of a TAL beacon design is a monomolecular construct where quencher and fluorophore are on opposite ends of the same molecule, and where one set of conditions favors a stem-loop hairpin conformation, and contact-quenching of fluorescence (See, for example, Hamaguchi, Ellington et al., 2001). Under other conditions, the TAL shifts to a quadruplex conformation that favors target binding, with a separation of fluorophore and quencher:
6. Enhanced Specificity of Molecular Beacons.
Hybridization-based molecular beacons recognize their target nucleic acids with greater specificity than linear oligonucleotide probes (See, for example, Tyagi, Bratu et al., 1998; Marras, Kramer et al., 1999; Bonnet, Tyagi et al., 1999). In a similar manner, protein-binding molecular beacons recognize their target proteins with greater specificity than nonswitchable aptamers, as a consequence of balancing the conformational equilibrium of an active form with a hairpin structure (See, for example, Bonnet, Tyagi et al., 1999). When a molecular beacon binds to its target sequence, the probe-target hybrid occurs at the expense of the hairpin. When a protein-binding molecular beacon binds to its target protein, the equilibrium shifts from an inactive hairpin conformation to an active conformation.
Molecular beacons are designed so that over a wide range of temperatures, only perfectly complementary probe-target hybrids are sufficiently stable to open the stem structure. Mismatched probe-target hybrids do not form except at substantially lower temperatures (See, for example, Marras, Kramer et al., 1999; Bonnet, Tyagi et al., 1999). Therefore a relatively wide range of temperatures exist in which perfectly complementary probe-target hybrids elicit a fluorescent signal while mismatched molecular beacons remain dark. Consequently, assays using molecular beacons robustly discriminate targets that differ from one another by as little as a single nucleotide substitution. This high specificity allows detection of a small proportion of mutant DNA in the presence of an abundant wild-type DNA (See, for example, Szuhai, Ouweland et al., 2001).
Similarly, protein-binding molecular beacons can be optimized so that only specific target complexes are favored, and related protein targets will only form at lower temperatures. This enhanced specificity can be used to discriminate protein binding partners even if the inherent free energy of binding is very similar. In summary, an analog can be made between the balancing of hairpin vs. linear duplex equilibria in nucleic acid target detection, and the balancing of hairpin vs. protein binding equilibria in protein target discrimination with molecular beacons. In the former case, hairpin probes allow enhanced discrimination between fully complementary targets vs. targets with a single mismatch. In the latter case, hairpin probes allow enhanced discrimination among proteins with similar, but not identical binding sites. In both cases, the enhanced discrimination comes at the cost of decreased overall binding.
In solution, conventional molecular beacons show exquisite sensitivity for single base-pair mismatches, and do not require the labeling of target. Fluorescence enhancements are generally around 25×, and enhancements of up to 200× have been reported (See, for example, Yao and Tan, 2004). Over the past few years, several groups have tested molecular beacon arrays for multiplexed SNP detection (See, for example, Yao and Tan, 2004; Culha, Stokes et al., 2004; Steemers, Ferguson et al., 2000; Wang, Li et al., 2002). These studies have demonstrated varying degrees of success. As outlined by Beaucage, the requirements for the successful application of arrayed oligonucleotides include the following: 1) chemically stable attachment chemistry, 2) a sufficiently long linker to minimize steric interferences, 3) hydrophilic linker to ensure solubility in aqueous solution, and 4) minimal nonspecific binding to the glass surface (See, for example, Beaucage, 2001). The requirements for molecular beacon arrays are even more stringent. First, nonspecific interactions of hydrophobic dyes with both surfaces and linkers need to be minimized. Such interactions could result in a partial destabilization of the quenched hairpin state, which could in turn give a high background fluorescence. An additional concern for surface-attached molecular beacons would be maintaining the high discrimination ratio for single nucleotide mismatches that is obtained in solution.
In fact, and in contrast to the solution situation, immobilized molecular beacons in array studies do tend to suffer from a high fluorescence background, with fluorescence enhancements in the single digit range. As we have determined that titrating linkers into solution-phase molecular beacon assays has little effect on assay performance, the high fluorescent background noted in array-based assays appears to result from surface interactions rather than linker interference.
A variety of immobilization methods and surface modifications have been used for the attachment of oligonucleotides in general and molecular beacons in particular to glass slides (See, for example, Beaucage, 2001). These methods include a) robotic deposition of oligonucleotides on polylysine or aminosilane-coated surfaces, b) covalent attachment of oligonucleotides through aminoalkane linkers to aldehyde or epoxide modified glass surfaces, c) physical adsorption of avidin on glass-slides followed by noncovalent attachment of DNA via a biotin linker, d) reductive coupling of amino-linked oligonucleotides to polyacrylamide or agarose gels, e) attachment of oligonucleotides to gold surfaces either directly using thiol-linkers or indirectly to self-assembled monolayers (SAMS) on gold surfaces using biotin-streptavidin cross-links, f) attachment to a polyelectrolyte multilayer surface via biotin-streptavidin linkage (See, for example, Kartalov, Unger et al., 2003).
Hybrid surfaces comprising hydrogels layered on top of gold surfaces provide an additional level of control over surface properties. The standard surface for surface plasmon resonance (SPR) studies is a gold surface that is derivatized with a matrix of carboxymethylated dextran (See, for example, Lofas and Johhsson, 1990). This surface has shown excellent compatibility with a variety of biopolymers, including oligonucleotides, and represents an attractive surface for bimolecular construct immobilization.
1. Design Features of Bimolecular Detection Constructs for Hybridization Analysis.
One design the bimolecular construct of the present invention is illustrated in
2. Design Features of Bimolecular TAL Beacons for Protein Targeting.
In the present invention we have introduced an analogous TAL beacon design (
3. Advantages of Bimolecular Constructs Compared to Monomolecular Beacons.
By placing the fluorophore and the quencher on separate strands, with complementary bases holding them together, a key advantage is obtained over attachment methods using monomolecular beacons: Since a duplex structure projects from the surface, a more rigid spacer separates the fluorophore and the quencher from the modified surface. As a consequence, interaction between the fluorophore and the quencher is favored compared to interaction between the fluorophore or quencher and the surface. Because of the bimolecular design, which limits the interaction of fluorophore and/or quencher with the surface, reduced background fluorescence can provide enhanced signal-to-noise ratios compared to unimolecular beacons. Another advantage is that the ratio of anchor strand and beacon strand can be optimized in order to maximize signal compared to background. A final advantage is that it is simpler, more efficient, and more economical to synthesize the quencher and fluorophore on opposite strands. For a monomolecular beacon, it is necessary to synthesize molecules that have a) a linker group for surface attachment, b) an internal quencher or fluorophore and c) a terminal quencher or fluorophore. For bimolecular constructs, each oligonucleotide need only have one terminal linker for surface attachment, and one terminal fluorophore or quencher.
A fluorescein labeled hairpin DNA Oligonucleotide, HP2, with a ten base-pair linker sequence was machine synthesized and HPLC purified. The sequence of HP2 was:
The underlined stretches in this sequence represent arm sequences that form the stem structure of the hairpin in the absence of complementary nucleic acid target. An anchor-oligo sequence representing the linear complement to the ten base-pair linker sequence of HP2 was also synthesized and HPLC purified. The sequence of this anchor-oligo was:
Finally, the target oligonucleotide complementary to the loop region of HP2 was synthesized and purified. The target oligo sequence was:
The fluorescence background of 150 μl of a 1 mM MgCl2, 20 mM Tris-HCl, pH 8.0 solution was determined, using 491 nm as the excitation wavelength and 515 as the emission wavelength. 10 μl of 1 μM HP2 was added to this solution and the new level of fluorescence was recorded. A two-fold molar excess of anchor oligo was added and the decrease in fluorescence was monitored until it reached a stable level. Finally, a five-fold molar excess of target oligo was added and the increase in fluorescence was monitored.
As shown in
A Dabcyl labeled 2′O-methyl hairpin oligonucleotide, HP3, with a ten base-pair linker sequence was machine synthesized and HPLC purified. The sequence of HP3 was:
CGAG -DABCYL 3′
The underlined stretches in this sequence represent arm sequences that form the stem structure of the hairpin in the absence of complementary nucleic acid target. A 2′O-methyl anchor-oligo sequence representing the linear complement to the ten base-pair linker sequence of HP3 was also synthesized and HPLC purified. The sequence of this anchor-oligo was:
Finally, a target RNA sequence corresponding to the let7b miRNA was synthesized. The let7b sequence was fully complementary to the loop sequence in HP3.
The interaction of FAM-labeled anchor oligo with the Dabcyl-labeled let7b probe gives a decrease in fluorescence as hairpin formation brings the FAM and Dabcyl groups into near proximity. As let7b target is added, quenching is reduced and fluorescence increases as binding of the let7b target opens the hairpin and separates the FAM and Dabcyl groups. See,
A Dabcyl labeled 2′O-methyl hairpin oligonucleotide, HP3, with a ten base-pair linker sequence was machine synthesized and HPLC purified. The sequence of HP3 was:
CGAG -DABCYL 3′
The underlined stretches in this sequence represent arm sequences that form the stem structure of the hairpin in the absence of complementary nucleic acid target. A 2′O-methyl anchor-oligo sequence representing the linear complement to the ten base-pair linker sequence of HP3 was also synthesized and HPLC purified. The sequence of this anchor-oligo was:
Finally, RNA sequences corresponding to the miRNAs let7a, let7b, let7c and let7f were synthesized. The let7b sequence was fully complementary to the loop sequence in HP3. The target oligo sequences were:
The mismatches with respect to the probe let7b sequence are underlined. The bimolecular let7b construct of FAM-labeled anchor oligo and Dabcyl-labeled let7b probe easily discriminates between targets that differ by a single base pair. Notably, under the conditions of these experiments, targets with more than one mismatch have no measurable effect on the fluorescence of the bimolecular construct. See,
Recent data that we have obtained for TAL beacon constructs demonstrate the utility of TALs for protein profiling applications. The thrombin aptamer beacon constructs that we have examined are designed according to the features shown in
α-thrombin was obtained from Haematologic Technologies, Inc. and used without further purification. Oligonucleotides were machine synthesized and HPLC purified. A solution containing 277 nM TAL2 and 1.5 fold molar excess of Dabcyl anchor was prepared in buffer containing 10 mM KCl, 5 mM MgCl2, and 12.5 mM Tris Acetate, pH 6.5, and titrated with a 10-fold excess of α-thrombin. Even though the quenched hairpin dominated in the absence of complementary DNA or protein target, titration with α-thrombin induced a dramatic increase in fluorescence, suggesting protein-induced stabilization of the quadruplex form. See,
α-thrombin was obtained from Haematologic Technologies, Inc. and used without further purification. Oligonucleotides were machine synthesized and HPLC purified. A solution containing 277 nM TAL2 and 1.5 fold molar excess of Dabcyl anchor was prepared in buffer containing 10 mM KCl, 5 mM MgCl2, and 12.5 mM Tris Acetate, pH 6.5, and titrated with increasing concentrations of α-thrombin. When the TAL2 beacon was titrated with α-thrombin, both the limiting fluorescence and the kinetics of fluorescence increased strongly with increasing total concentration of protein. See,
α-thrombin, β-thrombin and γ-thrombin were obtained from Haematologic Technologies, Inc. and used without further purification. Oligonucleotides were machine synthesized and HPLC purified. A solution containing 277 nM TAL2 and 1.5 fold molar excess of Dabcyl anchor was prepared in buffer containing 10 mM KCl, 5 mM MgCl2, and 12.5 mM Tris Acetate, pH 6.5, and titrated with each of the thrombin variants. When a constant concentration of TAL2 beacon was titrated to a constant ratio of 100:1 protein to beacon, clear differences were apparent among the closely related variants, α-thrombin, β-thrombin and γ-thrombin. See,
Specific ion effects were examined using the anchor sequence
The hairpin-forming TAL construct (TAL1) was
The underlined sequences represent arm sequences that form the stem structure of the hairpin in the absence of target. HEG is a hexaethylene glycol spacer.
TAL1 was designed to base pair with the anchor sequence under the appropriate ionic conditions. The molecule is quenched when in the hairpin form and unquenched (i.e., fluorescent) when in the quadruplex form. The effect of a 10 fold molar excess of α-thrombin on the solution fluorescence of a 277 nM solution of TAL1 was compared in KCl buffer and in LiCl buffer. The KCl buffer contained 12.5 mM Tris, pH 8.0, 10 mM KCl and 5 mM MgCl2. The LiCl buffer contained 12.5 mM Tris, pH 8.0, 10 mM LiCl and 5 mM MgCl2. The results demonstrated that the TAL in LiCl solution reached equilibrium much more rapidly than the TAL in KCl. See,
The Dabcyl anchor 5′NH2-(CH2)6-CACGTAGCAG-Dabcyl3′ was compared to the Black Hole Quencher 2 anchor 5′NH2-(CH2)6-CACGTAGCAG-BHQ2-3′ in terms of their effect on the fluorescence reporting behavior of TAL1. 277 nM of TAL1 was titrated with 1.5 fold molar excesses of the Dabcyl anchor and the BHQ2 anchor, and then with a 10:1 excess of α-thrombin. The results shown in
The TAL1 beacon, with an internal hexaethylene glycol linker was compared to the TAL2 beacon, which did not have an internal linker, as components of bimolecular constructs formed using the anchor 5′NH2-(CH2)6-CACGTAGCAG-Dabcyl3′. 277 nM of TAL1 and TAL2 were titrated with a 1.5 fold molar excesses of the Dabcyl anchor, and then with a 10:1 excess of α-thrombin. The results shown in
Experiments were performed using bimolecular constructs on CodeLink™ slides from GE Healthcare. These glass slides are coated with Sepharose®, and derivatized to allow covalent attachment of amino groups via Schiff's base chemistry. The probe DNA oligonucleotide in these experiments was:
CGCGTG TC TGC TAC GTG - 3′
The 5′ amino anchor sequence was: 5′-6 amino-CAC GTA GCA G Dabcyl-3′, and the target DNA oligonucleotide was T GAG GTA GTA GGT TGT ATA GTT. In the first experiment, the probe oligomer and anchor oligomer were pre-hybridized at a ratio of 1:1. This pre-hybridized bimolecular construct was then spotted at a concentration of 10 μM using a GeneMachines Omnigrid microarraying robot and conjugated to the gel surface using the manufacturer's protocol. The slide was then washed extensively with SSC buffer (150 mM NaCl, 25 mM MgCl2, 15 mM sodium citrate, pH 7). In
Experiments were performed using bimolecular constructs on CodeLink slides from GE. The 5′ amino anchor oligo 5′-6 amino-CAC GTA GCA G Dabcyl-3′ was spotted and conjugated onto Code-link slides at a concentration of 10 μM. Using a 16-well gasket to allow multiple conditions on the same slide individual wells on the slide were incubated with variable concentration probeacon oligo for 15 minutes at 50° C. and then cooled to room temperature for 30 minutes. The slide was then rinsed with SSC buffer and the fluorescence monitored with the Gene-Pix scanner. The slide was then incubated with 1 nM target oligo for 15 min. Fluorescence data before and after target addition are shown for 100 fM pro-beacon spots in
We show the ratio of fluorescence before and after target addition in
Bimolecular constructs can also be attached by both strands. This embodiment is preferred since it allows extensive washing to remove nonspecifically associated, fluorescently labeled oligonucleotides. For example, the following strands may be attached in this manner:
CGCGTG TC TGC TAC GTG - C6 amino -3′
anchor-oligo: (5′ amino, DABCYL labeled): 5′-C6 amino-CAC GTA GCA G Dabcyl-3′ following coupling, and extensive washing, these strands are washed extensively to interact to bind
In this embodiment, the probe oligo is pre-hybridized with the anchor oligonucleotide, and then attached via Schiff's base chemistry or other chemistry well-known to those skilled in the art to surfaces with closely spaced reactive groups. Periodate treated agarose is a preferred surface substrate because periodate treatment results in two closely spaced hydroxyl groups. Hydroxyl or epoxide coated glass slides also have a very high density of reactive groups and can be used to attach both strands simultaneously while maintaining hybridization.
A. The anchor sequence, with 3′ quencher is attached via a 5′ amino functionality to an amine-reactive surface.
B. The Tunable Affinity Ligand (TAL) functionality is hybridized to the anchor under conditions favoring hairpin formation (e.g. LiCl solution) and attached via a 3′ amino linker.
C. The Tunable Affinity Ligand (TAL) is switched to a protein-binding conformation (here, a quadruplex) under other conditions (e.g. KCl solution).
D. Protein binding to the active Tunable Affinity Ligand (TAL) conformation shifts the equilibrium toward that conformation.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2006/047523 | 12/13/2006 | WO | 00 | 6/13/2008 |
Number | Date | Country | |
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60750410 | Dec 2005 | US |