Patent Application
20100159455
The present invention relates generally to assay technologies and their use in biodetection and diagnostics. More particularly, the invention relates to compositions and methods of nucleic acid templated chemistry in biodetection of receptors (and their families) and proteins (and their families), and the use of same in diagnostics.
DNA-Programmed Chemistry (DPC) technology is a method that uses oligonucleotides attached to reactive chemical groups or building blocks whereby the oligonucleotides specifically direct chemical reactions via oligonucleotide hybridization. The use of DNA in bringing together reactants controls reaction stoichiometry, lowers energies of activation, and segregates reactants, to ensure highly specific reactions under physiologic conditions. One application of DPC is protein detection. See, e.g., Gartner, et al., 2004, Science, vol. 305, pp. 1601-1605; Li, et al., 2004, Angew. Chem. Int. Ed. Vol. 43, pp. 4848-4870; U.S. Pat. No. 7,070,928; U.S. Pat. No. 7,223,545; WO06128138A2 by Coull et al. As DPC provides the potential for low-noise detection and/or specific detection of biomarkers, methods and compositions useful for efficient and accurate detection and analysis of biomarker profiles are desirable and can improve diagnostic and potential treatment options.
For example, ErbB family of receptor tyrosine kinases are involved in an array of combinatorial interactions involving homo- and heterodimers. ErbB overexpression is associated with cancers of the breast, colon, prostate gland, lung, ovaries and brain. Although ErbB-directed therapeutics are effective against such ErbB-overexpress sing cancers, their efficacy is limited and variable for reasons that are not well understood. For example, although Trastuzumab (Herceptin™) is effective against ErbB2-overexpressing metastatic breast cancers, which can be diagnosed using an immunohistochemical (IHC) test (HercepTest™) for ErbB2, Trastuzumab is nevertheless effective in only a minority of such cases. A principle reason for the efficacy limit is that the current diagnostic tests used to select appropriate patients are unable to distinguish active receptor dimers. This remains a significant technical challenge with current technologies.
The present invention is based, in part, upon the discovery that biomarker profiling, e.g., ErbB receptor family dimerization profiles, can be achieved by DPC-based detection methods. More particularly, immunohistochemical (IHC)-based ErbB dimer assays for all known ErbB family dimers, can be developed using DNA-Programmed Chemistry for highly specific detection of protein complexes. These assays hold the promise of enabling drug response and cancer prognosis predictions in individual patients based on complete ErbB family profiling of cancer tissue samples. These assays, using formalin-fixed, paraffin-embedded tissue samples, will improve diagnosis, treatment, and prognosis of all cancers in which ErbB proteins are dysregulated.
In one aspect, the present invention relates to a method for measuring the dimerization profile of a family of receptors. The method includes: (a) providing an assay comprising a pair of probes, (i) the first probe comprising a first binding moiety having specific binding affinity to a first member of the receptor dimers to be profiled, wherein the first binding moiety is conjugated, optionally via a first linker, to a first oligonucleotide that is associated with a first reactive group; (ii) the second probe comprising a second binding moiety having specific binding affinity to a second receptor of the receptor dimers to be profiled, wherein the second binding moiety is conjugated, optionally via a second linker, to a second oligonucleotide that is associated with a second reactive group; wherein the second oligonucleotide is capable of hybridizing to the first oligonucleotide sequence and the second reactive group is reactive to the first reactive group when brought into reactive proximity of one another; (b) combining the first probe and the second probe with a sample to be measured for the dimerization of the first and second receptor members under conditions where the first and the second binding moieties bind to the first and second receptor members, respectively; (d) allowing the second oligonucleotide to hybridize to the first oligonucleotide to bring into reactive proximity the first and the second reactive groups; and (e) detecting a reaction between the first and the second reactive groups thereby determining the dimerization profile of the first and second receptor members.
In another aspect, the invention relates to a method for measuring the dimerization profile of a family of receptors. The method includes: (a) providing an assay comprising a pair of probes, (i) the first probe comprising (1) a first binding moiety having specific binding affinity to a first member of the receptor dimers to be profiled, and (2) a first oligonucleotide zip code sequence; (ii) the second probe comprising (1) a second binding moiety having specific binding affinity to a second member of the receptor dimers to be profiled, and (2) a second oligonucleotide zip code sequence; wherein the first probe is hybridized to a first reporter probe comprising (1) an anti-zip code sequence of oligonucleotides complementary to the first oligonucleotide zip code sequence, (2) a first reporter oligonucleotide, and (3) a first reactive group; and wherein the second probe is hybridized to a second reporter probe comprising (1) an anti-zip code sequence of oligonucleotides complementary to the second oligonucleotide zip code sequence, (2) a second reporter oligonucleotide, and (3) a second reactive group; wherein the second reporter oligonucleotide is capable of hybridizing to the first reporter oligonucleotide sequence and the second reactive group is reactive to the first reactive group when brought into reactive proximity of one another; (b) combining the first and second probes with a sample to be measured for the dimerization of the first and second receptor members under conditions where the first and the second binding moieties bind to the first and second receptor members, respectively; (c) allowing the second reporter oligonucleotide to hybridize to the first reporter oligonucleotide to bring into reactive proximity the first and the second reactive groups; and (d) detecting a reaction between the first and the second reactive groups thereby determining the dimerization profile of the first and second receptor members.
In yet another aspect, the invention relates to an assay for measuring the dimerization profile of a family of receptors. The assay includes: (a) a first probe comprising a first binding moiety having specific binding affinity to the first member of the receptor dimers to be profiled, wherein the first binding moiety is conjugated, optionally via a first linker, to a first oligonucleotide that is associated with a first reactive group; (b) a second probe comprising a second binding moiety having specific binding affinity to the second receptor of the receptor dimers to be profiled, wherein the second binding moiety is conjugated, optionally via a second linker, to a second oligonucleotide that is associated with a second reactive group; wherein the second oligonucleotide is capable of hybridizing to the first oligonucleotide sequence and the second reactive group is reactive to the first reactive group when brought into reactive proximity of one another.
In yet another aspect, the invention relates to an assay for measuring the dimerization profile of a family of receptors. The assay includes: (a) a first probe comprising (1) a first binding moiety having specific binding affinity to a first member of the receptor dimers to be profiled, and (2) a first oligonucleotide zip code sequence; (b) a second probe comprising (1) a second binding moiety having specific binding affinity to a second member of the receptor dimers to be profiled, and (2) a second oligonucleotide zip code sequence; wherein the first probe is hybridized to a first reporter probe comprising (1) an anti-zip code sequence of oligonucleotides complementary to the first oligonucleotide zip code sequence, (2) a first reporter oligonucleotide, and (3) a first reactive group; and wherein the second probe is hybridized to a second reporter probe comprising (1) an anti-zip code sequence of oligonucleotides complementary to the second oligonucleotide zip code sequence, (2) a second reporter oligonucleotide, and (3) a second reactive group; wherein the second reporter oligonucleotide is capable of hybridizing to the first reporter oligonucleotide sequence and the second reactive group is reactive to the first reactive group when brought into reactive proximity of one another.
In yet another aspect, the invention relates to a method for detecting a biological target. The method includes: (a) providing a first probe, the first probe comprising (1) a first binding moiety having binding affinity to the biological target, (2) a first oligonucleotide sequence, and (3) a first reactive group associated with the first oligonucleotide sequence; (b) providing a second probe, the second probe comprises (1) a second binding moiety having binding affinity to the biological target, (2) a second oligonucleotide sequence, and (3) a second reactive group associated with the second oligonucleotide sequence, wherein the second oligonucleotide is capable of hybridizing to the first oligonucleotide sequence and the second reactive group is reactive to the first reactive group when brought into reactive proximity of one another; (c) combining the first probe and the second probe with a sample to be tested for the presence of the biological target under conditions where the first and the second binding moieties bind to the biological target; (d) allowing the second oligonucleotide to hybridize to the first oligonucleotide to bring into reactive proximity the first and the second reactive groups; and (e) detecting a reaction between the first and the second reactive groups thereby determining the presence of the biological target, wherein the reaction between the first and second reactive groups generates a rhodamine.
In certain embodiments, the family of receptors is the ErbB receptor family and, optionally, the ErbB receptor dimers are selected from the group consisting essentially of (e.g. greater than 80%, greater than 90%, greater than 95%, greater than 97% greater than 99.5%, and/or greater than 99.9%) of homodimers of ErbB1, ErbB2, ErbB3, and ErbB4. In certain embodiments, the ErbB receptor dimers are selected from the group consisting essentially of (e.g. greater than 80%, greater than 90%, greater than 95%, greater than 97% greater than 99.5%, and/or greater than 99.9%) of hetero-dimers of ErbB1, ErbB2, ErbB3, and ErbB4.
In another aspect the present invention relates to a method for analyzing receptor family profits comprising detecting a signal generated via DNA-programmed chemistry. The receptor family in this or any aspect of the invention can be the ErbB receptor family (and, optionally, the signal is generated to analyze ErbB dimerization), the BCL2 family, the IAP family, or the Gβγ subunits of trimeric G proteins.
The term, “DNA programmed chemistry” or “DPC”, as used herein, refers to nucleic acid-templated chemistry, for example, nucleic acid sequence specific control of chemical reactants to yield specific products accomplished by (1) providing one or more templates, which have associated reactive group(s); (2) contacting one or more transfer groups (reagents) having an anti-codon (e.g., complementary sequence with one or more templates) and reactive group(s) under conditions to allow for hybridization to the templates and (3) reaction of the reactive groups to yield products. For example, in a one-step nucleic acid-templated reaction, hybridization of a “template” and a “complementary” oligonucleotide bring together reactive groups followed by a chemical reaction that results in the desired product. Structures of the reactants and products need not be related to those of the nucleic acids comprising the template and transfer group oligonucleotides. See, e.g., U.S. Pat. Nos. 7,070,928 B1 and 7,223,545 and European Patent No. 1,423,400 B1 by Liu et al.; U.S. Patent Publication No. 2004/0180412 (U.S. Ser. No. 10/643,752; Aug. 19, 2003) by Liu et al., by Liu et al.; Gartner, et al., 2004, Science, vol. 305, pp. 1601-1605; Doyon, et al., 2003, JACS, vol. 125, pp. 12372-12373, all of which are expressly incorporated herein by reference in their entireties. See, also, “Turn Over Probes and Use Thereof” by Coull et al., PCT WO07/008,276A2, filed on May 3, 2006.
The terms, “nucleic acid”, “oligonucleotide” (sometimes simply referred to as “oligo”) or “polynucleotide,” as used herein, refer to a polymer of nucleotides. The polymer may include, without limitation, natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadeno sine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). Nucleic acids and oligonucleotides may also include other polymers of bases having a modified backbone, such as a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a threose nucleic acid (TNA), among others.
Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present invention also consist essentially of, or consist of, the recited components, and that the processes of the present invention also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions are immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.
The invention may be further understood from the following figures in which:
In its simplest sense, the invention provides methods and compositions for biomarker family measurement and profiling using DNA-programmed probe chemistry.
For example, the invention provides methods and compositions for the measurement and analysis of the dimerization profiles of the ErbB family of receptor tyrosine kinases.
The family of ErbB receptors includes ErbB1 (also known as human epidermal growth factor (EGF) receptor 1 (HER1) or EGF receptor (EGFR)), ErbB2 (also known as HER2 or Neu), ErbB3, and ErbB4. They consist of an extracellular ligand-binding domain, a single transmembrane domain, an uninterrupted tyrosine kinase domain, and a cytoplasmic tail, and are involved in an array of combinatorial interactions involving homo- and heterodimers. See, e.g., Citri, et al., Nat Rev Mol Cell Biol, 2006. 7(7), 505-16; Muthuswamy, et al. Mol Cell Biol, 1999. 19(10), 6845-57; Hynes, et al. Nat Rev Cancer, 2005, 5(5) 341-54.
They have been implicated in the development of many human cancers and have been intensely pursued as therapeutic targets. See, Hynes, et al. Nat Rev Cancer, 2005, 5(5) 341-54; Baselga, J. Science, 2006 312(5777), 1175-8. ErbB-directed therapeutics have demonstrated clinical efficacy; however, anti-tumor effects are often not as strong as predicted from pre-clinical studies. For example, only 35% of patients with ErbB2 overexpressing metastatic breast cancer respond effectively to Trastuzamab (anti-ErbB2, Herceptin™) therapy. Vogel, et al., J Clin Oncol, 2002, 20(3) 719-26. Moreover, trastuzamab is somewhat, but not always, effective in treating breast cancers that show elevated levels of both ErbB1 and ErbB2. Indeed, ErbB homo- and heterodimers differ in their signaling, response to ligands, and tumorigenic properties. Citri, et al., Nat Rev Mol Cell Biol, 2006. 7(7), 505-16; Muthuswamy, et al. Mol Cell Biol, 1999. 19(10), 6845-57; Hynes, et al. Nat Rev Cancer, 2005, 5(5) 341-54. It is therefore highly likely that variation in the levels as well as dimerization status of ErbB family members in a tumor cell contributes to variations in drug efficacy between patients. For example, co-expression of ErbB2/ErbB1 and ErbB2/ErbB3 may predict responsiveness to Herceptin™ treatment. The efficacy of Herceptin therapies is influenced by the expression of other ErbB receptors, their ligands and the activation of downstream signalling proteins. Smith, et al., Br J Cancer, 2004, 91(6), 1190-4; Wehrman, et al., Proc Natl Acad Sci USA, 2006, 103(50), 19063-8.
Furthermore, anti-cancer therapy includes antibodies that disrupt ErbB1/ErbB2 dimers (e.g. Omintarg™), but treatment responsiveness cannot be predicted, because no suitable assay is available to detect dimer status from tissue samples. In addition, the presence of ErbB2 extracellular domain (ECD)-truncated forms, as a result of alternative initiation of translation within the ErbB2 mRNA, may promote resistance to Trastuzumab. Anido, et al., Embo J, 2006, 25(13), 3234-44. Hence detection of full-length vs. truncated receptor may help to predict responsiveness to Trastuzumab.
Homo- and heterodimerization of ErbB receptors are measured using probe sets. Each set is specifically directed at a particularly dimer. As illustrated in
Illustrated in
Polymethine dye is characterized by a chain of methine (—CH═) groups with an electron donor and an electron acceptor at opposite ends of their polyene chain (
Advantages of DPC-based dimer profiling Include: (1) High sensitivity. Since DNA hybridization increases effective molarity of the reactants (from nM concentration to mM effective concentration, 106 fold), assays can be performed at very low probe concentration. (2) Low non-specific background. Many conventional assays rely on an increase or decrease in fluorescence of a fluorophore-labeled probe upon binding of the probe to a target, or upon liberation (cleavage) of a fluorophore, a quencher, or a protecting group from the target-bound probe by a chemical or enzymatic event. The associated fluorescence backgrounds are usually high. In DPC-based assays, non-specific background can be kept very low (“zero”) when non-fluorescent precursors are used. For example, in the hemicyanine based DPC system, the probes become fluorescent only after binding to the probes' target protein hetero- or homodimer and upon reaction to create a fluorescent signal molecule. (3) Improved specificity and affinity. The assay format of the present invention requires two probes and dual recognition of the target. The analyte recognition involves two independent binding events (protein recognition and nucleic acid hybridization). Thus, the dimerization induced nucleic acid hybridization and DPC greatly increases its detection specificity. For example, only a probe pair with completely matched oligonucleotide sequences can produce a fluorescent hemicyanine product. (4) Detection in functional context. DPC-based assay format allows detection of receptors and dimerization in their functional context. It is minimally invasive to the biological system. It is also very simple and amenable to point-of-care or point-of-sale format. (5) Can be customized to a multiplex platform as described herein.
Methods and compositions of biodetection using nucleic acid-templated chemistry based probes are described in WO06128138A2 by Coull et al., which is incorporated herein by reference in its entirety.
More particularly illustrated in
In
The prefluorophores may reside in an “end of helix” configuration (
The proximity effect afforded by tethering the pair of oligonucleotides may affect the kinetics of annealing of two complementary oligonucleotide sequences compared to the two oligonucleotides free in solution. More importantly, a localized high concentration shifts the melting curve upwards compared to the free complex, i.e. increase the Tm of the complex. In a bulk solution, it is known that Tm has dependence upon total oligonucleotide concentration as illustrated in the equation below. Wetmur, Criti. Rev. in Biochem. And Mol. Biol., 1991, 26, 227-259.
T
m=(1000*ΔH)/(A+ΔS+R ln(Ct/4)−273.15+16.6 log Na+)
where ΔH and ΔS are the enthalpy and entropy for helix formation, R is the molar gas constant, Ct is the total concentration of oligomers, and Na+ is the molar concentration of sodium ion in the solution.
Reaction products of R1 and R2 may be released from the hybridization complex as a result of the chemical transformation. Thus, the fluorophore or chromophore may be separated from the hybridization complex and analyzed independently, or the fluorophore or chromophore and the annealed oligonucleotides may be removed once detected so that additional rounds of interrogation of the sample can be conducted. The reaction between R1 and R2 may or may not covalently link the two probes once the product(s) is formed.
This zip-coded architecture supports creating a single reporter-oligonucleotide conjugate which would assemble with different downstream reporter oligonucleotides through an anti-zip code sequence. Libraries of different reporters linked to a unique anti-zip code may be tested simply by mixing each one with stoichiometric amounts of the binder-zip code oligonucleotide conjugate with its complementary zip code.
One advantage of the “zip coded” approach is the ability to create the reporter oligonucleotides separately, and have them assemble together with binders under conditions retaining the activities of both the binders and of the nucleic acid template-activated chemistry.
The zip-coded system is based upon two pairs of oligonucleotides, with each pair being held together by the base-pairing of a unique zip code and an anti-zip code pair. “Zip codes” are oligonucleotide sequences which bind specifically to their complementary sequences, and preferably are designed such they are not complementary to known genomic sequences (relevant if the sample may contain genomic DNA), have similar Tm values, lack significant secondary structure, and do not anneal to other zip code or anti-zip code sequences in the detection system.
It is worth pointing out the methods of the invention do not require enzymatic or chemical ligation of the first and/or the second oligonucleotide sequences.
Factors that may be considered in optimizing a design of a zip-coded architecture include, for example, (1) spacer groups (e.g., oligonucleotides and/or non-base groups) between the aptamer/antibody and zip codes (spacer 1), e.g., to allow hybridization partners to reach each other, to prevent any steric hindrance; (2) Length of a zip code sequence in order to form a sufficiently stable annealing to the anti-zip code sequence to form the complex; and (3) Spacer groups (spacer 2) between the anti-zip code and the reporter sequence, e.g., to prevent any steric hindrance.
The binders (target binding moieties) attached to the oligonucleotides may be any chemical moieties that specifically bind to a target molecule and allow the design of the invention to work. Examples include a wide range of functionalities, such as (1) antibodies: e.g., IgG, IgM, IgA, IgE, Fab's, Fab′, F(ab)2, Dab, Fv or ScFv fragments; (2) small molecule binders, such as inhibitors, drugs, cofactors; (3) receptors for protein detection, and vice versa; (4) DNA, RNA, PNA aptamers; (5) DNA sequences for DNA-binding and regulatory proteins; (6) peptides representing protein binding motifs; (7) peptides discovered through phage display, random synthesis, mutagenesis; (8) naturally binding protein pairs and complexes; (9) antigens (for antibody detection); and (10) a single polyclonal antibody separately attached to two oligonucleotides may serve as two separate binders of different specificity.
The target binding moieties attached to the oligonucleotides may be of heterogeneous types directed against different sites within the same target. For example, the two binders may be two different antibodies, an antibody and a receptor, an antibody and a small molecule binder, a receptor and a peptide, an aptamer and a cofactor, or any other combination.
The target analytes can be of any type, provided the target supports two (or more) binding sites. Molecules which exist in equilibrium with a monomeric form and a homodimeric or higher polymerization phase may be detected by a pair of probes containing the same binder but different complementary DNA sequences. Suitable targets include proteins, cell surfaces, antibodies, antigens, viruses, bacteria, organic surfaces, membranes, organelles, in situ analysis of fixed cells, protein complexes. The invention may be particularly suited for the detection of fusion proteins (e.g., BCR-ABL in the presence of BCR and ABL).
In the design of the probes, one consideration is the Tm of the two reporter sequences carrying the reactive groups. Since the Tm of the duplex should be below room temperature in the absence of a target, this sequence normally should be short, for example 6-15 bases and/or A-T rich. A typical reporter length of 10 base pairs might have a Tm of around 30° C. at a low salt concentration. Therefore, it is often necessary even with a short sequence to add 10% to 40% volume/volume formamide to further lower the temperature below assay temperature, or to elevate the assay temperature. Very short reporter oligonucleotides may suffer from a lack of specificity and exhibit some binding to zip code sequences (when these are employed) which is undesirable.
Another factor in the design of the probes is the length of oligonucleotide in between the binding moiety and the reporter sequence, including any zip code sequences. These must be long enough for the reporter oligonucleotides to reach each other and anneal. The sequences may be interspersed with polyethylene glycol (PEG) linkers that are flexible and may afford additional protection against any steric hindrance. For example, total lengths of oligonucleotides may be around 35 bases long. Oligonucleotides containing 0, 1, or 2 C18 PEG spacers, or homopolymer tracts may also be utilized (i.e. C10).
A third consideration is the length of zip and anti-zip sequences when these are employed (i.e.
Regarding signal generation, nucleic acid-templated chemistry may be used to create or destroy a label that effects an optical signal, e.g., creating or destroying a fluorescent, chemiluminescent, or colorimetric molecule. Additionally, a detection reaction may be designed to create or destroy a product that directly or indirectly creates a detectable label, for example, a product that catalyzes a reaction that creates an optical label; inhibits a reaction that creates an optical label; is a fluorescence quencher; is a fluorescent energy transfer molecule; creates a Ramen label; creates an electrochemiluminescent label (i.e. ruthenium bipyridyl); produces an electron spin label molecule.
The following examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof. Practice of the invention will be more fully understood from these following examples, which are presented herein for illustrative purpose only, and should not be construed as limiting in anyway.
Oligonucleotides were prepared using standard phosphoramidite chemistry (Glen Research, Sterling Va., USA) and purified by reversed-phase C18 chromatography. Oligonucleotides bearing 5′-amino groups were prepared using either 5′-Amino-Modifier 5 (antizip reporter oligonucleotides) or 5′-Amino-Modifier C6 (zip oligonucleotides). Oligonucleotides bearing 3′-amino groups were prepared using 3′-Amino-Modifier C7 CPG (Glen Research, Sterling Va., USA).
Scheme 1 illustrates the reduction of DAZR (diazidorhodamine, non-fluorescent) to rhodamine (fluorescent) via DNA programmed chemistry.
Both DAZR and phosphine are linked to oligonucleotide (DNA) through amide bond formation. First, an acid precursor was synthesized. The acid precursor was then either converted to the active N-hydroxysuccinimide ester (NHS ester) that reacted with a DNA bearing amine functionality in solution (DNA_DAZR, Scheme 2) or was directly coupled to the DNA bearing amine functionality on the Controlled Pore Glass (CPG) (DNA_bisdiphenylphosphine, Scheme 3).
Scheme 2 illustrates various synthetic routes to DAZR and DNA_DAZR conjugate. The acid derivatives of rhodamine (isomers) were initially synthesized following the literature procedure by condensing 3-aminophenol with 1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxylic acid under very harsh conditions (e.g., high temperature, strong acid and prolonged reaction time) (Scheme 2a). Under those conditions, significant amount of fluorescein and rhodol compounds were generated which were difficult to separate from rhodamine acid. The yield for this reaction was also quite low. The compound isomers (1) are Zwitterion and cannot be purified by Silica gel column. Subsequent reaction of 1 with sodium nitrile and sodium azide afforded only 11% of DAZR (Novikova, et al., Russian Journal of Organic Chemistry, 1998, 34, 1762-1767).
An alternative approach was thus developed to provide more effective access to DAZR acid (Scheme 2b). In this approach, diiodo and dibromo substituted rhodamines (3 & 4) were synthesized and then converted to DAZR acid. Compounds 3 and 4 are not Zwitterions and can be purified easily, and the yields for generating these compounds are good (40 to 60% yield). Around 80% of DAZR acid isomers were generated from dibromo-substituted rhodamines (4).
Synthesis of compound 2 (3′,6′-diazido-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-[and -6]-carboxylic acid mixed isomers, Scheme 2a): To a solution of 1 (3′,6′-diamino-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-[and -6]-carboxylic acid mixed isomers, 600 mg, 0.16 mmol) in 4 mL of 2N HCl was added sodium nitrite (276 mg, 4.01 mmol). After stirring at room temperature (RT) for 30 minutes, ice was added followed by sodium azide (625 mg, 9.62 mmol) dissolved in a minimum amount of water. Solid was precipitated out and was separated out by filtration. The crude product was purified by 4 g of RediSep silica-gel column on a CombiFlash Companion chromatography system (1% triethylamine in DCM/MeOH) to afford 40 mg of mixed isomers 2 (11%).
Synthesis of compound 3 (3′,6′-diiodo-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-[and -6]-carboxylic acid mixed isomers): To a 100 ml flask was added 2,4-benzenetricarboxylic acid anhydride (4.50 g, 23.4 mmoles), 3-iodophenol (10.3 g, 26.8 mmoles) and methanesulfonic acid (40 mL). The mixture was stirred at 125° C. under atmosphere of argon for 16 hrs. The cooled mixture was poured into 100 g of ice. The precipitate was dissolved in acetone/DCM and washed with water. The crude product was purified by 80 g of RediSep silica-gel column on a CombiFlash Companion chromatography system (DCM/acetone) to afford 6.0 g of the product (white powder, 43.0%). Calculated exact mass for C21H10I2O5: 595.86
Observed: 594.8348 (M−H).
Synthesis of compound 4 (3′,6′-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-[and -6]-carboxylic acid mixed isomers): To a 100 ml flask was added 1,2,4-benzenetricarboxylic acid anhydride (2 g, 10.41 mmoles), 3-bromophenol (2.341 g, 13.53 mmoles) and methanesulfonic acid (45 g, 468 mmoles). The mixture was stirred at 135° C. for 14 hours under atmosphere of Argon. The cooled mixture was poured into ice water (100 g). The product was extracted with 75 mL of DCM and then washed with 100 mL of water. The solvent was removed by rotary evaporation. The solid residue was washed with DCM to remove unreacted bromophenol and other nonpolar impurities. The crude product was further purified by silica gel chromatography (0-5% MeOH/DCM) to obtain 3.0 g of product (white powder, 57.4%). Calculated exact mass for C21H10Br2O5: 499.89; Observed: 500.9679 (M+H).
Synthesis of compound 2 (3′,6′-diazido-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-[and -6]-carboxylic acid mixed isomers, Scheme 2b): To a 100 ml flask was added iodofluoran (3) (0.30 g, 0.50 mmoles), CuI (0.01 g), sodium ascorbate (0.02 g), 1,2-dimethaminoethane (0.02 g), sodium azide (0.10 g, 1.50 moles), 15 mL of ethanol and 5 ml of water. The resulting mixture was heated under Argon at 100° C. for 30 minute. The cooled mixture was diluted with water, neutralized with HCl, and extracted with DCM. The crude product was purified by silica gel chromatography (0-5% MeOH/DCM) to obtain 0.13 g of product (white powder, 60.6%).
Synthesis of compound 5 (NHS ester of DAZR 5-[and 6-] isomers): To a 100 ml flask was added compound 2 (0.18 g, 0.422 mmol) and DCM (5 ml), followed by 1-hydroxypyrrolidine-2,5-dione (0.15 g, 1.303 mmol), and N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine (0.15 g, 0.966 mmol). The mixture was stirred at room temperature for 5 minutes. The TLC showed completion of the reaction (DCM/EtOAc, 9:1). The mixture was loaded directly onto a 12 g of RediSep silica gel column and eluted out with gradient 0-15% of DCM/EtOAc solvent system. Obtained was 0.19 g of product (white powder, 86%). Calculated exact mass for C25H13N7O7: 523.09; Observed: 524.1212 (M+H).
Synthesis of DAZR_DNA conjugate: To a 1.5 mL of centrifugation vial containing 55 nmole of DNA in 50 μL of water was added 30 μL of N,N-diisopropylethylamine (DIPEA) and 3 mg of 5 (0.005 mmol) dissolved in 70 μL of N-methyl-2-pyrrolidone (NMP). After reacted for 16 h at RT, the reaction mixture was desalted by gel filtration using Sephadex G-25 and then purified by reversed-phase C18 column. Antizip2 reporter 1_DAZR (DNA: SEQ ID NO: 69): LC-MS: Calcd. for monoisotopic [M 7]-7: 1359.9580; Found 1359.9852. Antizip2 reporter 2_DAZR (DNA: SEQ ID NO: 73): LC-MS: Calcd. for monoisotopic [M-7]−7 1483.8350; Found 1483.8777.
Synthesis of diphenylphosphine_DNA conjugate: Oligonucleotides containing 5′-Amino-Modifier 5 (1 umole scale) were prepared using standard phosphoramidite chemistry (Glen Research, Sterling Va., USA). After trityl group of 5′-amino was removed, oligo-CPG was transferred to a 1.5 mL of centrifuge tube. A solution of 9 (81 mg, 0.15 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl) (28 mg, 0.15 mmol) and DIPEA (33 μL) in NMP (300 μL) was added. After reacting at 37° C. for 2 h, the solvent was removed by centrifugation and oligo-CPG was treated with ammonium methylamine (AMA) at 55° C. for 45 min. The crude oligos released from AMA cleavage was purified by reverse phase C18 HPLC. Antizip5_bisdiphenylphosphine (DNA: SEQ ID NO: 72): MS: Calcd. for monoisotopic [M-4]−4 1931.357; found 1931.465. Antizip5m_disdiphenylphosphine (DNA: SEQ ID NO: 71): MS: Calcd. for monoisotopic [M-4]−4 1925.1583; found 1925.0873.
Scheme 4 illustrates the condensation of an indolinium and an aldehyde to form hemicyanine accomplished using diamine catalyzed DNA programmed chemistry. Both the aldehyde and indolinium precursors were conjugated to DNA through amide bond formation.
Scheme 5 gives one example of synthesizing a DNA-conjugated indolinium compound. The acid functionality is introduced to indoline ring through N-quaternization. Scheme 6 provides an example of synthesizing DNA conjugated aldehyde. The acid functionality in aldehyde precursor is introduced through hydrolysis of a cyano group by hydrogen peroxide (Brady, et al. J. Bio. Chem. 2001, 276, 18812-18818). Scheme 7 provides another example of DNA labeled α,β-unsaturated aldehyde 1. Wittig reagent was used for the two-carbon homologation of aldehydes into the corresponding α,β-enals (Eitel, et al. Synthesis (1989) 364-367). The acid functionality in aldehyde precursor is introduced through hydrolysis of a cyano group by concentrated HCl (Bratenko, et al. Chemistry of Heterocyclic Compounds (2004) 40, 1279-1282).
Additional examples of catalysts are shown in
Synthesis of compound 10: To 5-bromovaleric acid (2.435 g, 13.45 mmole) was added 2,3,3-trimethylindolenine (2.141 g, 13.45 mmole). The reaction mixture was heated with rigorous stirring at 110° C. overnight. The dark red sticky oil obtained was transferred to a Gregar extractor and extracted with EtOAc overnight. A light red solid was obtained. The solid was redissolved in 30 mL of MeOH. MeOH was removed under reduced pressure and the remaining residue was treated with 10 mL of EtOAc. Brownish solid was precipitated out and filtrated. The solid was washed with 2×50 mL of acetone and 2×100 mL of EtOAc. Total 1.590 g of light brownish solid was obtained (35% yield). 1H NMR (DMSO) δppm: 7.98 (m, 1H), 7.84 (m, 1H), 7.61 (m, 2H), 4.49 (t, 2H), 2.84 (s, 3H), 2.30 (t, 2H), 1.84 (m, 2H), 1.63 (m, 2H), 1.53 (s, 6H). MALDI-MS (positive mode): 260.2419.
Synthesis of compound 11: Compound 11 (0.1 g, 0.294 mmole), N-hydroxy succimide (0.068 g, 0.588 mmole) and N,N′-dicyclohexylcarbodiimide (DCC) (0.085 g, 0.411 mmole) were dissolved in 1.5 mL of dimethyl formamide (DMF). The reaction mixture was stirred at 37° C. for 1 hr. The precipitated dicyclohexylurea (DCU) was removed by filtration, and the filtrate was treated with 15 mL of ether. Light orange solid was washed three times with 10 mL of ether and dried under vacuum for several hours. The solid obtained was used directly for the next reaction. MALDI-MS (positive mode): 357.1590.
Labeling DNA with indolinium compound: To a 1.5 mL of centrifugation vial containing 20 nmole of DNA was added 41.6 μL of 0.1 M sodium phosphate buffer (NaPi), pH 7.8, 41.6 μL of compound 10 in NMP (96 mM) and 41.6 μL of NMP. The vial was placed in a shaker and shook for 4 hr at 37° C. The reaction mixture was desalted by gel filtration using Sephadex G-25 and then purified by reversed-phase C8 column. Antizip5_indolinium (DNA: SEQ ID NO: 72): 10% yield. LC-MS (negative mode): Calcd for C307H401N108O180P29 (average): 1340.2614 [M-8H]7−; Found: 1340.2705 [M-8H]7−. Antizip5m_indolinium (DNA: SEQ ID NO 71): 10% yield. LC-MS (negative mode): Calcd for C308H404N109O177P29 (monoisotopic): 1336.4969 [M-8H]7-; Found: 1336.673 [M-8H]7− Antizip3_indolinium (DNA: SEQ ID NO: 70): 5% yield. LC-MS (negative mode): Calcd for C307H404N107O178P29 (monoisotopic): 1332.4034 [M-8H]7-; Found: 1332.6293 [M-8H]7-.
Synthesis of compound 12: In a 50 mL of round-shaped flask containing N-methyl-N-cyanoethyl-4-aminobenzaldehyde (1.024 g, 5.44 mmole) was added 27.2 mL of 5 N NaOH solution and 6.8 mL of 30% H2O2. The reaction mixture was refluxed for 2 hr. After cooling, the reaction mixture was neutralized by the addition of concentrated HCl (37% w.t.) and extracted with 2×100 mL of EtOAc and 1×100 mL of CH2Cl2. The organic layers were combined and washed once with 50 mL of brine and concentrated to dryness. The crude product was purified by a 40 g RediSep silica-gel column on a CombiFlash Companion chromatography system (EtOAc/MeOH). Total 0.702 g of light pinkish solid was obtained (62%). Electrospray MS: M+H 208.0735. (Brady, et al., J. Biol. Chem. 2001, 276, 18812-18818).
Labeling DNA with aldehyde: The NHS ester of 12 was synthesized following the same procedure of compound 11. After removing the DCU by filtration, the filtrate was used directly for DNA conjugation (calculated as 0.2 M product in DMF). To a 1.5 mL of centrifugation vial containing 50 nmole of DNA was added 104 μL of 0.1 M NaPi, pH 8.6, 125 μL of the above filtrate and 83 μL of NMP. The vial was placed in a shaker and shaken overnight at 37° C. The reaction mixture was desalted by gel filtration using Sephadex G-25 and then purified by reversed-phase C8 column. Antizip2 reporter1_A0 (DNA: SEQ ID NO: 69) (30% yield). LC-MS: Calcd for C303H396N110O177P29 (monoisotopic): 1328.2458 [M-7H]7−; Found: 1328.3051 [M-7H]7−.
Synthesis of compound 13: In a 100 mL of round-shaped flask containing N-methyl-N-cyanoethyl-4-aminobenzaldehyde (1.116 g, 5.9 mmole) and ylide (2.71 g, 8.9 mmole) was added 57 mL of dry toluene. The reaction mixture was heated under reflux for overnight, allowed to cool, and filtered through filter paper. After removing the solvent from the filtrate, the residue was first purified by a 40 g RediSep silica-gel column on a CombiFlash Companion chromatography system (Toluene/Ether) and then preparative HPLC C18 column (Agilent Prep-C18, 30×100 mm, 10 um) to afford 0.27 g of pure product (21%). MALDI-MS (positive mode): 215.226.
Synthesis of compound 14: In a 50 mL of round-shaped flask containing compound 13 (0.1 g, 0.47 mmole) was added 30 mL of concentrated HCl. The reaction mixture was heating to boiling and left at room temperature for 1 hr. HPLC analysis indicated that only one product was formed and no starting material remained in the reaction mixture. After removing most of the HCl, the compound was dissolved in water and lyophilized to dryness to afford the product.
Labeling DNA with α,β-unsaturated aldehyde: The NHS ester of compound 14 was synthesized following the same procedure as compound 11, however was purified by silica-gel chromatography (EtOAc/Hexanes) instead. After drying under vacuum for several hours, the NHS ester of compound 14 was dissolved in NMP (96 mM) and was used for labeling DNA following the same procedure as labeling DNA_A0 Antizip2 reporter1_A1 (DNA: SEQ ID NO: 69): yield 40%. Calcd for C305H398N110O177P29 (monoisotopic): 1331.96239 [M-7H]−7; Found: 1332.0778 [M-7H]−7.
Two hemicyanine products were formed by mixing antizip3_indolinium with antizip2 reporter1_A0 and antizip2 reporter1_A1 respectively (DNA: SEQ ID NO: 69). The product (P1) formed between antizip3_indolinium (DNA: SEQ ID NO: 70) and antizip2 reporter1_A0 (DNA: SEQ ID NO: 69) has excitation maximum at 540 nm and emission maximum at 600 nm, while the product (P3) formed between antizip3_indolinium (DNA: SEQ ID NO: 70) and antizip2 reporter1_A1 (DNA: SEQ ID NO: 69) has excitation maximum at 600 nm and emission maximum at 670 nm (
DPC reaction: Reactions were performed with 200 nM each of reagent in 15 mM N,N-dimethyl ethylenediamine (DMEDA), 50 mM sodium phosphate buffer, pH 8.0, 2.5 mM MgCl2 at 30° C. Total reaction volume was 50 μL. Catalyst DMEDA was added after mixing both reagents together in reaction buffer. Fluorescence was recorded immediately after the addition of catalyst DMEDA.
DNAs are covalently attached to antibody or affibody via cross-linkers. Varieties of heterobifunctional cross-linkers can be used to synthesize DNA-antibody conjugate. The most commonly used are: 1) amine-reactive and sulfhydryl-reactive cross-linkers such as SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate); 2) aldehyde-reactive and sulfhydryl-reactive cross-linkers such as hydrazine/hydroxylamine and maleimide/iodoacetate functional group containing compounds; 3) aldehyde-reactive and amine-reactive cross-linkers such as hydrazine/hydroxylamine and succinimidyl functional group containing compounds. See, e.g., Hermanson, G. T. Bioconjugate Techniques, Academic Press 1996. For a precursor to be able to attach to a DNA, it is usually functionalized with a carboxylic acid group and then reacts with an amine-containing DNA. Other functional groups such as aldehyde and sulfhydryl can also be incorporated into DNA. The precursor is functionalized with hydrazone/hydroxylamine and maleimide group accordingly. Scheme 8 provides an example of synthesizing affibody_oligonucleotide conjugate through BMPS. Zip2_ErbB2 (DNA: SEQ ID: NO 66) affibody has been synthesized following this scheme.
Suspensions of cells (A431, BT474, or N87) expressing various levels of EGFR were analyzed for the presence of EGFR homodimers by an assay utilizing the DPC-based hemicyanine synthesis. Cells were incubated with EGF (epidermal growth factor, 200 ng/mL) in phosphate buffered saline (PBS) at 4° C. for up to 1 hr. Control cells (untreated) were incubated under the same conditions without EGF. After treatment, cells were fixed by incubation in 3% formaldehyde in PBS for 30 min. at 4° C. Cells were then washed with PBS and blocked by incubation with PBS containing 2% bovine serum albumin (BSA), 100 μg/ml goat IgG, and 10 μM yeast tRNA at 4° C. for 45 min. The blocked cells were then incubated with anti-EGFR antibody-zip oligonucleotide conjugates (egfr1-zip2 (DNA: SEQ ID NO: 66) and egfr1-zip5 (DNA: SEQ ID NO: 68), 30 nM each) and antizip oligonucleotides (antizip5_A0, (DNA: SEQ ID NO: 72), and antizip2 reporter1_indolinium, DNA: (SEQ ID NO: 69), 60 nM each) for 1 hr. at room temperature. Reactants were removed and the cells washed with PBS containing 1 μM yeast tRNA. Cells were suspended in PBS containing 5 mM N,N-dimethyl ethylenediamine (DMEDA) and incubated for 4 hrs. at room temperature. Cells were then washed in PBS containing 2% BSA, suspended in PBS and analyzed by flow cytometry for fluorescence generated by the DPC reaction. The results of the DPC assay for EGFR homodimers expressed as the product of the percent positive cells times the mean fluorescence intensity (MFI) are shown in
For cells untreated with EGF, a positive DPC signal (i.e., the fluorescent signal resulting from the DPC reaction) was detected indicating the presence of EGFR homodimers. The magnitude of the DPC signal was proportional to the expression level of EGFR (A431 cells express significantly higher levels of EGFR than BT474 or N87 cells). Treatment of cells with EGF resulted in an approximately two fold increase in the magnitude of the DPC signal. These results are consistent with a model of EGFR mechanism of action in which in the absence of EGF, unactivated EGFR homodimers are reversibly formed and the equilibrium level of these dimers is increased upon ligation of EGF, also resulting in the activation of these homodimers as evidenced by autophosphorylation of specific tyrosines in the cytoplasmic domain of the receptor (Citri, et al. Nature Rev. Mol. Biol. 2006, 7, 505-516).
Both homodimers of ErbB2 and EGFR-ErbB2 heterodimers on N87 cells were detected by a DPC assay employing the DAZR reduction reaction. Suspensions of N87 cell were fixed by incubation with 2% formaldehyde in PBS at 4° C. for 30 min. Fixed cells were washed with PBS and then blocked by incubation with PBS containing 2% BSA, 10 μM yeast tRNA, 100 μg/mL goat IgG, and 5% dextran sulfate at 4° C. for 45 min. The blocked cells were then incubated with antibody-zipcodes (anti-erbB2 9G6-zip2 (DNA: SEQ ID NO: 66) and 9G6-zip5 (DNA: SEQ ID NO: 68) for the ErbB2 homodimer assay or anti-EGFR egfr1-zip5 (DNA: SEQ ID NO: 68) for the heterodimer assay, 30 nM concentration for antibody conjugates) and 60 nM antizip2 reporter 2_DAZR (DNA: SEQ ID NO: 73), for 1 hr at 30° C. Cells were centrifuged, reactants removed, and washed twice prior to incubation with 80 nM antizip5_bisdiphenylphosphine (DNA: SEQ ID NO: 72) at 37° C. for 1 hr. Cells were then washed twice and suspended in PBS prior to analysis by flow cytometry for fluorescent cells. The results of the DPC assays for ErbB2 dimers are shown in
The background signal in the assay was assessed by omitting the 9G6-zip5 conjugate, described above. The total amount of ErbB2 (monomer, homodimer, and heterodimer) was assessed by performing the DPC under conditions in which the DPC reaction was obtained independent of the state of EerbB2. The DPC signal obtained for ErbB2-ErbB2 homodimer was significantly greater than that obtained for the EGFR-ErbB2 heterodimer and both dimer DPC signals were significantly lower than that of the total ErbB2. N87 cells express high levels of ErbB2 and significantly lower levels of EGFR, thus the relative magnitudes of the DPC signals are consistent with these expression levels. As in the case of the EGFR homodimers on A431 cells, these results indicate the presence of homo and heterodimers on untreated cells.
A DPC assay was performed on suspensions of A431 cells as described in Example 5 except that instead of EGFR homodimers, ErbB2 homodimers and EGFR-ErbB2 heterodimers were detected. In this assay, cells were either untreated, treated with 200 ng/ml EGF for 15 min at 4° C., or pretreated with the EGFR specific tyrosine kinase inhibitor AG1478 (1 μM, 15 min at 37° C.) prior to treatment with EGF. After the various treatments, cells were fixed in 2% formaldehyde in PBS for 30 min at 4° C. Cells were then washed with PBS and blocked by incubation with PBS containing 2% BSA, 5% dextran sulfate, 100 μg/ml goat IgG, and 10 μM yeast tRNA at 4° C. for 45 min. The blocked cells were then incubated with antibody-zip conjugates, antizip2 reporter1_A0, and antizip5_indolinium for 1 hr at 30° C. For ErbB2 homodimer detection, anti-ErbB2 9G6-zip5 and an anti-ErbB2 affibody-zip2 conjugates were used, while for EGFR-ErbB2 heterodimer detection, anti-EGFR egfr1-zip5 and anti-erBB2 affibody-zip2 conjugates were used. Reactants were removed and the cells washed with PBS containing 1 μM yeast tRNA. Cells were suspended in PBS containing 5 mM DMEDA and incubated for 3 hrs at room temperature. Cells were then washed in PBS containing 2% BSA, suspended in PBS, and analyzed by flow cytometry for fluorescence generated by the DPC reaction. The results of the DPC assay for ErbB2 dimers expressed as the product of the fraction of positively stained cells times the mean fluorescence intensity are shown in
The background signal in the assay was assessed by omitting the one of the zip conjugates. The DPC signals for the ErbB2 homodimers were quite low yet above the background signal (signal/background of 3-4). Such a low DPC signal is consistent with the low level of expression of ErbB2 in A431 cells. The magnitude of the DPC signal obtained in the assay for EGFR-ErbB2 heterodimer was larger and varied significantly in response to treatment. As in the case of EGFR homodimers, there was a signal in the assay of untreated cells indicating a basal level of heterodimer. The DPC signal increased nearly two fold in cells treated with EGF and approximately three fold when the cells were pretreated with AG1478 prior to EGF treatment.
The results of these DPC assays are consistent with many studies (e.g. immunoprecipitation followed by Western blotting) that have shown the existence of EGFR-ErbB2 heterodimers in untreated cells and the induction of additional heterodimers in response to EGF. Treatment with AG1478 alone has been shown to induce formation of EGFR-ErbB2 heterodimers (Arteaga, et al., J. Biol. Chem. (1997) 272, 23247-23254). Furthermore, pre-treatment with the tyrosine kinase inhibitor prevents the autophosphorylation of the cytoplasmic domain of EGFR and prevents the down regulation of the receptor by endocytosis allowing its accumulation (Gannett, et al. J. Biol. Chem. (1997) 272, 12052-12056.)
Five oligonucleotides were prepared using standard phosphoramidite chemistry (Glen Research, Sterling Va., USA). Oligonucleotides bearing 5′-amino groups (Oligo2 and Oligo6) were prepared using 5′-Amino-Modifier 5 and Oligonucleotides bearing 3′-aminogroups (Oligo4 and Oligo5) were prepared using 3′-Amino-Modifier C7 CPG (Glen Research, Sterling Va., USA)
Oligo1, Oligo4 and Oligo5 were removed from the synthesis support and purified by reversed-phase HPLC. The amino groups of Oligo2 and Oligo6 were converted while resin-bound to their triphenyl phosphine derivatives and these were purified and isolated (Sakurai et al., J. Amer. Chem. Soc., 2005, 127, pp 1660-1667) to give Oligo2-TPP and Oligo-6TPP, respectively.
Amino group bearing Oligo4 and Oligo5 were converted to their azidocoumarin derivatives (Oligo4-AzC and Oligo5-AzC, respectively) by reaction of each oligo with the N-hydroxysuccinimide ester of 7-azido-4-methylcoumarin-3-acetic acid (Thevenin et al., Eur. J. Biochem (1992) Vol. 206, pp-471-477). The reaction was performed by adding 1 μL of trifluoroacetic acid to 5 μL of N-methylmorpholine to prepare a buffer to which was added 10 μL of water containing 6.6 nmol of Oligo 4 or Oligo 5, followed by addition of 30 μL of a 0.16 M solution of the coumarin NHS-ester in dimethylformamide. Each reaction was allowed to proceed for 2 hours at room temperature, whereupon 50 μL of 0.1 M aqueous triethylammonium acetate was added. The mixtures were applied to a NAP-5 desalting columns (Amersham Biosciences, Piscataway N.J. USA) and eluted according to the manufacturers instructions the eluate was purified by RP-HPLC to provide Oligo4-AzC and Oligo5-AzC, in yields of 77% and 70%, respectively. Product identity was confirmed by Maldi-ToF mass spectrometry.
To demonstrate the hybridization-specific creation of fluorescence, various combinations of complementary and non-complementary oligonucleotides bearing azido-coumarin and triphenyl phosphine moieties were allowed to react at room temperature in a buffer comprised of 30% aqueous formamide, 50 mM NaCl, and 10 mM sodium phosphate, pH 7.2. The reaction progress was monitored over time using a Victor Multilabel fluorimeter (EG&G Wallach, Turku Finlnad) set to excite the sample at 360 nm and monitor light emission at 455 nm
Results of additional experiments involving ternary complexes are shown in
A model system was prepared which included two twenty-mer oligonucleotides with a ten-base complementary region and ten-base single stranded spacer arms, further linked to a six carbon spacer arm. These oligos were synthesized both with and without a 5′-biotin (with a 6-carbon spacer arm). As shown below, the complementary region is underlined. A third oligo was identical to the (−) strand oligo but with 4 base mismatches (italicized) to the (+) strand.
Melting curves of the 10-base pair oligonucleotide pair (oligo 26+oligo 27) were examined by measuring fluorescence of SYBR dye binding to double stranded DNA in a BioRad iCycler (Lipsky, et al., Clinical Chemistry 2001, 47[4], 635-44). The binding curves are presented as the first derivative of the slope of the melting curve, such that a maximum value represents a point of inflection in the curve (a Tm, or in a mixed population of double stranded sites, a “local” Tm). Binding curves can be obtained up to at least 70° C. as avidin retains biotin binding activity up to this temperature and beyond.
To check the dependence of this particular pair of oligonucleotides upon concentration, melting curves were generated for the oligonucleotide pair varied over the range from 500 to 20 nM (
To test whether binding the (+) and (−) strands to a protein target would cause an increase in Tm, the biotinylated version of these oligonucleotides were incubated in the presence of avidin. Avidin contains 4 equivalent binding sites, which are spaced relatively close together and bind to biotin very tightly (Ka˜<10−15 M) and non-cooperatively.
Presented with equal molar concentrations of oligonucleotides #26 and #27 in biotinylated form, it would be expected that about half of the biotin binding sites are occupied by complementary pairs of oligonucleotides, and about half with the same oligonucleotide (non-complementary pairs). The prediction is that one would observe two melting curve peaks in the presence of avidin. One peak would be the result of any pairs of oligonucleotides which were either not bound to avidin (free in solution) or which had only one partner of the two bound to avidin, which should not exhibit a proximity effect upon Tm. A second peak of significantly higher Tm would represent a pair of biotinylated oligos both bound to avidin, which should exhibit a proximity effect.
Such an experiment was conducted as shown in
Results were essentially identical if the experiment was conducted by adding equimolar amounts of both the oligonucleotides at room temperature, ramping to 60° C., and then obtaining the melting curves. In this method (as well as the hot start method) suitable melting curves can be generated by adding an excess molar of each oligo relative to avidin if desired. (Large excesses of pairs of oligos increases the size of the low Tm peak, however, as predicted.) This was not detrimental in forming high Tm hybrid DNA since the pairs of oligos competed equally for biotin binding sites as long as they were added together in equal molar amounts. If oligos were added one at a time, it was important to add about a 2:1 molar ratio of the first oligo to avidin followed by a 2:1 ratio of the second oligo. With sequential addition, adding an excess molar amount of either oligo relative to avidin occupies all the binding sites of the avidin with the first oligo and prevents occupying adjacent sites with the second, complementary oligo and exhibiting the elevated Tm effect. These observations are consistent with the mechanism being binding of adjacent pairs of complementary oligos to two adjacent biotin binding sites to obtain hybrids exhibiting the elevated Tm peaks.
Experiments were also conducted with a 10-base self-complementary oligonucleotide which was composed entirely of A and T. (Oligo 31: 5′-biotin-spacer arm-TTTTTTTTTTTTTAATTAAA) (SEQ. ID. NO. 27). Because this oligonucleotide was homogeneous in base composition and composed entirely of AT, it melted at a lower Tm than the above-described model system and produced a fairly sharp melting curve. In the presence of avidin, its Tm was increased from 30.5° C. to 61.5° C. (
These experiments were repeated using anti-biotin antibody as a target rather than avidin. Anti-biotin antibody contains two biotin binding sites located near the ends of the Fab portion of the antibody, but the binding sites are much further apart than the biotin binding sites on avidin.
Here, an exemplary system was designed to utilize nucleic acid-templated azidocoumarin (AzC)-triphenylphosphine (TPP) chemistry to detect a protein target upon aptamer binding and annealing of the two complementary DNA probes.
Human PDGF-BB and PDGF-AA was obtained from R&D Systems (220-BB and 220-AA, respectively). Anti-human PDGF-B Subunit monoclonal antibody was obtained from R&D Systems (MAB2201). Buffers included Tris/Mg buffer, at 50 mM Tris/HCl, pH 8.0-10 mM MgCl2. Oligonucleotides used were as follows:
Oligonucleotide Sequences Used in this Example
CAGGCTACGGCACGTAGAGCATCACC
ATGATCCTGCCCCCCCCCCATATTTAA
ACGGCACGTAGAGCATCACCATGATC
CTG
CTACGGCACGTAGAGCATCACCATGA
TCCTG
DPC Reaction conditions. Except as noted, each 100 microliter reaction contained, in a total volume of 100 μl, 1×Tris/Mg buffer, 40 picomoles of TPP and AzC reaction probes, 40 picomoles of target oligonucleotide or of target protein, and typically 25-30% v/v of formamide. Samples were incubated at 25° C. in a Wallac Victor 1420 spectrophotometer and the increase in fluorescence monitored with excitation at 355 nm and emission at 460 nm.
As illustrated in
As shown in
The DNA-dependence of the reaction was critically dependent upon the melting temperature of the DNA relative to the assay temperature. In the presence of 0% formamide (with the calculated and observed Tm>Tassay, the reaction took place in the presence or absence of the target protein PDGF-BB (
DNA melting experiments with the complementary sequences, monitored with SYBR Green had indicated a Tm of the sequence of about 30° C. in the Tris/Mg buffer in the absence of formamide, and about 7° C. lower for every 10% increase in formamide. Tm in the optimal formamide concentration for the detection assay, 30%, was 10° C.
In 0% formamide, the oligonucleotides can form at least a partial duplex even in the absence of PDGF-BB (Tm slightly higher than Tassay). The DNA target-dependence of the reactions in 20% and 30% formamide is explained by the assay being conducted at a temperature greater than the Tm in the absence of protein target. No reaction occurs unless the Tm of the complex is increased by the binding of the two probes to the PDGF-BB target. At 40% formamide, the reaction doesn't occur with any set of reactions. The likely explanation is that either the Tm had been reduced so low that binding to PDGF-BB could not raise it above Tassay, or that formamide had inhibited PDGF-BB binding to the aptamers. A more complex situation is the observed inhibition of reaction rate upon addition of PDGF-BB in the absence of formamide. Since half of the duplexes formed by PDGF-BB are non-productive (50% will be homoduplexes) the reduction in rate is likely due to PDGF-BB binding preventing these homoduplexes from disassociating and then reassociating in solution with complementary pairs to form heteroduplexes. This situation should not occur using pairs of probes specifically directed against different binding sites in a heterodimeric target.
The sensitivity of the assay (
The assay sensitivity was also determined using PDGF-AA as a target. The aptamer monomer is expected to have an affinity for PDGF-AA about ten times weaker than for PDGF-BB. However, since the assay involves forming a complex of two aptamer-dimers to either type of PDGF, the avidity of binding of the dimer is expected to be tighter than the affinity of the monomer, and its affinity should be substantially tighter (lower Ki) than the concentrations tested of the target PDGFs (down to about 1 nanomolar). As shown in
Ratios of TPP to AzC Probes. To confirm the model of the reaction mechanism (
Thus, in this model system fluorescence was not generated unless the aptamers bound and the complementary sequences in the two probes annealed to each other.
The reaction, in 35% formamide at 22° C., was dependent upon the presence of both of the reporter oligonucleotides, both of the aptamer oligonucleotides, and the target, PDGF-BB (
Confirmation of the correctness of the model was obtained with experiments varying the ratio of the TPP and AzC aptamer oligos (
These experiments indicate that the complex can self-assemble in solution, such that each zip code and its anti-zip code anneal to each other with minimal interference with the aptamer sequence or the reporter sequences.
Experiments were also conducted to determine if the order of addition, and thus assembly of the aptamer and reporter probes, was of any importance. Slightly slower reaction rates were obtained if the aptamer oligonucleotides were first incubated with PDGF before adding the reporter oligonucleotides, compared with adding all probes together as a mixture. Somewhat greater reaction rates were obtained if each pair of aptamer oligonucleotides and reporter oligonucleotides was first incubated and allowed to assemble with each other before the two sets were mixed together and incubated with PDGF. The reason for this may be that there is some steric hindrance to zip code-anti zip code annealing to aptamer probe if the aptamer probe is already bound to target.
As a control, a set of one-piece TPP and AzC probes was compared which contained only the zip code sequences and no zip code-anti zip code sequences (
The sequence of the aptamer-containing TPP and AzC probes was also systematically varied to determine any constraints on the design. The aptamer-containing TPP and AzC oligos were synthesized, both having the same sequences as described in
Oligonucleotides used in this example included:
None of these changes resulted in a significant difference in the performance of the system. Experiments 4) and 5) also resulted in a 3 and 6-base single stranded (not annealed to zip code) structure immediately upstream of the C18 spacer in the reporter oligonucleotides.
The results of these experiments indicate that the aptamer-based PDGF detection system can be assembled separating the binding and DPC functions into two separate oligonucleotides. Through the selection of appropriate zip code sequences, the detection format described in
These results indicate that a zip-coded reporting approach can be effectively designed, for example, using aptamer-containing oligonucleotides.
While the results with the aptamer system indicate that a stable complex between binding and reporter sequences can be formed simply by annealing the zip code and anti-zip code regions, it should be noted that there are technologies to covalently and irreversibly link the two oligonucleotides together, with a high likelihood of retaining activity of the reporter reactive groups. For example, the oligonucleotides may be incubated in pairs (a binder oligonucleotide and a reactive oligonucleotide for nucleic acid-template chemistry) at a temperature at which the zip codes and anti-zip codes are mostly double stranded, but the rest of the sequences are single-stranded. Adding an intercalating, photoactivatable cross-linker such as Trioxalen, followed by UV irradiation, may irreversibly crosslink the two strands. Similarly, UV irradiation may introduce thymidine dimers between separate strands of annealed sequences. Alternately, a sequence may be introduced complementary to a short target (splice) DNA, abutting 3′ and 5′, which may then be ligated with DNA ligase. The splice oligonucleotide may alternately be composed of RNA, and removed after ligation with RNase H, which hydrolyzes RNA annealed to DNA. This can result in converting the two oligonucleotides into a single piece of single-stranded DNA. These methods can lead to cost-effective production of oligonucleotide reagents in detection kits against specific targets.
Relevant references for this example include Capaldi, et al., Nucleic Acid Res. 2000, 28[7], e21; Castiglioni, et al., Appl. and Exper. Microbio. 2004, 7161-72; Fang, et al., Chem. BioChem. 2003, 4, 829-34; Gerry, et al., J. Mol. Biol. 1999, 292, 251-62.
In another embodiment, the aptamer sequences are replaced with non-DNA binders such as antibodies. For PDGF and other protein targets, the aptamer sequences are replaced with chemically active groups, such as aldehydes, and reacted with non-DNA binder sequences such as antibodies or receptors to the protein targets (
Referring to
The SoluLink technology for conjugation of the antibody and oligonucleotides first requires modification of the primary amino groups of the antibody with succinimidyl 2-hydrazinonicotinate acetone hydrazone) to incorporate an acetone hydrazone onto the antibody. The primary amines of the oligonucleotides are separately activated with succinimidyl 4-formylbenzoate. The two activated molecules are mixed in the desired ratio (typically 6:1) and reacted at a mildly acidic pH to form a stable hydrazone linkage. The details of this chemistry are available at www.SoluLink.com. Two conjugates were prepared: one containing the zip code to anneal to the AzC-containing reporter oligonucleotide, and the other containing the zip code to anneal to the TPP-containing reporter oligonucleotide.
The antibody-oligonucleotide conjugates received from SoluLink were further purified by gel chromatography on a 1.6×60 cm column of Superdex S-200 (Amersham Biosciences) in PBS buffer (0.01 M potassium phosphate, pH 7.4-0.138 M sodium chloride). The main antibody peak, eluting at about 0.6 times the column volume, was collected and a later eluting peak of contaminating non-conjugated oligonucleotide was discarded. The antibody conjugate was concentrated by reversed dialysis with a Pierce (Rockford, Ill.) 30 K molecular weight cut-off Slide-A-Lyzer using Pierce Concentrating Solution. The protein content was determined using the Bio-Rad Micro BCA Reagent Kit and the oligonucleotide content determined using SYBR Gold DNA binding dye (Molecular Probes (Eugene, Oreg.). The conjugates were both determined to contain an average of approximately 3 oligonucleotides per protein molecule.
Recombinant human PDGF-BB (220-BB) and mouse monoclonal anti-PDGF-BB (MAB220) were obtained from R&D Systems (Minneapolis Minn.).
Sequences used in this study included (where AzC indicates azidocoumarin and TPP indicates triphenylphosphine):
In addition, the 5′ amino modifier C6 was obtained from Glen Research (from Glen Research phosphoramidite 110-1906). The 3′-amino modifier C7 was obtained from Glen Research (from Glen Research CPG 20-2957). The C18 PEG was obtained from Glen Research (from Glen Research phosphoramidite 10-1918).
Assembly of Antibody-Oligo Conjugates with Reporter Oligonucleotides.
The two antibody-oligo conjugates with their reporter were first assembled separately in a volume of 10 μl. Each assembly contained 0.5 μM (5 picomoles) of antibody-oligonucleotide conjugate and 0.15 μM of (15 pmoles) of complementary reporter oligonucleotide in 0.05 M Tris/HCl pH 8-0.01 M magnesium chloride. Each was incubated for at least 15 minutes at 4° C. before use in the detection reaction mixture.
Detection Reaction of Anti-PDGF-BB DPC Conjugates/Reporters with PDGF-BB
To conduct detection reaction, each reaction may contain in a volume of 50 μl:10 μl of each conjugate assembly, prepared as described above, and variable amounts of PDGF-BB, in a buffer of 0.05 M Tris/HCl pH 8-0.01 M magnesium chloride-40% volume/volume formamide. The conjugates are present in this reaction mixture at 0.2 μM. Samples are incubated in the wells of a black 96-well microplate in a Wallac Victor Luminometer at 25° C. Fluorescence can be followed vs. time with excitation at 355 nm and emission at 460 nm.
Reactions typically may be carried out at 25° C., monitoring fluorescence generation at the wavelength optimums of the reaction product, 7-amino coumarin.
The entire disclosure of each of the publications and patent documents referred to herein is incorporated by reference in its entirety for all purposes to the same extent as if each individual publication or patent document were so individually denoted.
The invention may be embodied in other specific forms without departing form the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
This application claims the benefit of and priority to U.S. Patent Application Ser. Nos. 60/845,330, filed Sep. 18, 2006; 60/847,859, filed Sep. 28, 2006, 60/905,364, filed Mar. 7, 2007; and 60/918,023, filed Mar. 14, 2007, the entire disclosure of each of which is incorporated by reference herein for all purposes.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/20223 | 9/18/2007 | WO | 00 | 3/8/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60845330 | Sep 2006 | US | |
| 60847859 | Sep 2006 | US | |
| 60905364 | Mar 2007 | US | |
| 60918023 | Mar 2007 | US |
