WO2013095137A2 - Methods, means and kits for detecting scarce analytes - Google Patents

Abstract

The invention relates methods, means and kits for detecting and quantitation of chemical and biological analytes using catalytic signal amplification. Provided are methods, kits, and reagents for detecting a target molecule or interacting target molecules in a sample based on catalytic signal amplification, comprising the steps of (i) providing at least a first non-fluorescent and a second non-fluorescent probe capable of binding to distinct binding sites on the target molecule(s), the first probe being provided with a first ligand and the second probe being provided with a second ligand, wherein the first and second ligand can form a catalytic complex in the presence of a catalytic compound and wherein the catalytic complex can catalyze a chromogenic reaction; (ii) contacting a sample known or suspected to contain the target molecule(s) with said at least a first and second non-fluorescent probe under conditions that allow for binding of the probes to the target molecule(s) such that, upon binding of the probes to the target molecule(s), the first and second ligand are brought in close proximity of each other and together with the catalytic compound form a catalytic complex immobilized on the target molecule(s); and (iii) detecting the formation of the catalytic complex based on a non-enzymatic conversion of a water- soluble precursor dye into a chromophoric reporter dye.

Description

Title: Methods, means and kits for detecting scarce analytes. The invention relates the field of analytical techniques. In particular, it relates to methods, means and kits for detecting and quantitation of chemical and biological analytes using catalytic signal amplification.

Catalytic signal amplification is a powerful tool for the detection of various types of analytes. In chemistry it has been employed in sensing various toxic metal ions (e.g. Pd2+, Pb2+, Cu2+ and Hg2+)[1] as well as small molecules such as carbon monoxide[2] and thiols[3] . In a biological context, catalytic reactions have enabled highly sensitive detection of scarce analytes. They have been widely utilized, for instance, in the detection and assay of proteins [4], antibodies[5] and nucleic acids. [6]

In the case of nucleic acids, DNA-templated catalytic processes in particular have been successful. Fluorogenic transformations of this type have been exploited for the amplified detection of deoxyribonucleotides (ODNs) both in homogeneous solutions [7] and in living cells [8]. In this approach, DNA probes are labeled with poorly fluorescent precursors and assembled with target nucleic acids into catalytic hybrids. These then chemically convert the precursors into fluorescent reporters (e.g. through the Staudinger reaction and aminolysis).[7e, 9] The turnover rate and detection signal can be further improved by repeated thermal cycling. While such systems have resulted in moderate amplified detection and assays of DNA, RNA and peptide nucleic acids (PNAs), the approach remains limited by several hurdles, e.g. the covalent attachment of profluorescent molecules to probe ODNs and the need for an external stimulus to achieve probe replacement for multiple turnovers.

The present inventors aimed to overcome at least one of these drawbacks. Especially, they set out to provide a highly sensitive detection method which does not require any complex (probe) design nor the use of external stimuli. Preferably, the method allows for detecting and quantitating molecules in the subnanomolar range. A further aim was to design a concept which is applicable not only to the detection and quantitation of single analytes, but also to the detection of interacting molecules.

These goals were met the development of an entirely novel strategy for catalytic signal amplification wherein ligand-labeled probes form a catalytic complex upon binding/hybridizing to a specific target molecule(s). The catalytic center formed on the target(s) when ligands become into close proximity of each other, then efficiently converts a water-soluble profluorescent dye, present in excess, into a highly emissive reporter. As such, each hybridization event can catalyze the chromogenic conversion of many precursor dyes, producing hundreds of fluorophores even in sub- picomolar target concentration. For example, an amplified DNA-detection concept was developed using iodo-BODIPYs and a catalytic DNA-Pd complex allowing multiple signal generation via Pd-catalyzed dehalogenation from a single hybridization event. According to the present invention, ligands for the catalytic complex, rather than dye precursors, are conjugated directly to the probes, yielding an active catalytic complex upon hybridization to the target(s).

Accordingly, in one embodiment the invention provides a method for detecting a target molecule or interacting target molecules in a sample based on catalytic signal amplification, the method comprising the steps of:

providing at least a first non-fluorescent and a second non-fluorescent probe capable of binding to distinct binding sites on the target molecule(s), the first probe being provided with a first ligand and the second probe being provided with a second ligand, wherein the first and second ligand can form a catalytic complex in the presence of a catalytic compound and wherein the catalytic complex can catalyze a chromogenic reaction

(n) contacting a sample known or suspected to contain the target molecule (s) with said at least a first and second non-fluorescent probe under conditions that allow for binding of the probes to the target molecule(s) such that, upon binding of the probes to the target molecule(s), the first and second ligand are brought in close proximity of each other and together with the catalytic compound form a catalytic complex immobilized on the target molecule(s); and

(in) detecting the formation of the catalytic complex based on a non-enzymatic conversion of a water-soluble precursor dye into a chromophoric reporter dye. Thus, based on the juxtapositioning of the target-dependent formation of a catalytic complex, a detectable signal is generated. The present concept provides an

improvement over the standard method of conjugating reporter dye precursors to the probes, which is limited to one fluorophore per target hybridization. Our novel strategy results in an amplified ultrasensitive detection of a target molecule down to a limit of 10 fJVI, to the best of our knowledge 2 orders of magnitude better than that reported for any other target-templated fluorogenic reaction [18a]. Additionally, since neither the precursor nor the reporter is covalently attached to the target, no external trigger is required to amplify the fluorescence signal; e.g. deiodination of iodinated- BODIPY upon diffusion to the catalytic site is sufficient. This ingenious yet technically simple target-directed catalyst assembly opens the possibility of generating multiple detectable e.g. fluorescent signals per hybridization event.

The chromogenic reaction comprising a non-enzymatic conversion of a water- soluble precursor dye into a chromophoric reporter dye can be fluorogenic or colorimetric. As used herein, the term fluorogenic means that the quantum yield of the reporter dye increases during the catalytic reaction. The reaction may result in a shift of the absorption spectrum of a reporter dye or a shift in emission wavelength. Although a fluorogenic reaction is more sensitive, it needs relatively expensive equipment. Chromogenic reactions can be detected with the naked eye, but are typically less sensitive. It may comprise the transformation of a non-colored compound (i.e. no absorption in the visible) to a colored compound (absorption in the visible spectrum). The conversion may also involve a color change i.e. changing the absorption wavelengths within the visible spectrum.

Typically, the method further comprises the step of quantitating the target molecule(s) in the sample by correlating the amount of chromophoric reporter dye detected with the chromophoric signal intensities obtained using a standard curve.

The invention also provides a probe set comprising at least a first non-fluorescent and a second non-fluorescent probe, each probe capable of binding to distinct binding sites of a target molecule of interest, the first probe being provided with a first ligand and the second probe being provided with a second ligand, wherein the first and second ligand can form a catalytic complex in the presence of a catalytic compound and wherein the catalytic complex can catalyze a chromogenic reaction, preferably transition-metal catalyzed dehalogenation.

Sample

It will be understood that the present invention can be applied to the detection of any type of molecule in any type of sample. Because of its high sensitivity, the sample may be known or suspected to contain the target molecule(s) in amount of up to 1 nM, preferably up to 100 pM, more preferably up to 1 pM. Exemplary samples are biological samples, for instance a sample obtained from a human or animal subject. The sample can be a bodily fluid, tissue or tissue extract. As used herein, a "bodily fluid" may be amniotic fluid, aqueous humour, bile, bladder lavage, blood, breast exudate, bronchioalveolor lavage, cerebrospinal fluid, chyle, chyme, cytosol, feces (in semi-fluid or fluid form), interstitial fluid, lymph, menses, mucus, plasma, pleural fluid, pus, saliva, sebum, semen, serum, sputum, sweat, synovial fluid, tears, urine and/or vitreous humour. In an exemplary embodiment of the present invention, the bodily fluid may be blood. In another embodiment, the bodily fluid (sample) may be obtained from a mammal such as a human being.

For example, the method may be advantageously used for (clinical) diagnostic purposes, including high throughput screening for diseases, disorders and/or therapy effectiveness. Exemplary clinical applications include endocrinology, infectious disease (e.g. HIV) testing, oncology, allergy testing and sensing of cardiac markers. In another embodiment, the method is performed in a research setting or in a drug development process. Samples may be obtained from a test animal, in vitro cultured (e.g. mammalian or microbial) cells, cell culturing medium, and the like.

Target molecule(s)

The invention is applicable to any target molecule(s) of interest. In one embodiment, the detection method employs a set of probes to detect a single molecule of interest. The presence of the target in the sample is the determining factor whether or not the ligand-conjugated probes are brought within a short distance from each other, allowing for the formation of a catalytic complex. In another embodiment, the detection method of the invention is designed to detect an interaction between a first target molecule and a second target molecule using a first probe being reactive with the first target and a second probe being reactive with a second, distinct, target. In the absence of an interaction, the ligands on the probes are too far apart to form a catalytic complex. On the other hand, when the first and second probes are in proximity (typically 0.1-10 nm ) due to the interaction of the two molecules, the probe-bound ligands can form a catalytic complex together with the catalytic compound. Thus, the method allows for characterizing distance-dependent interactions on a molecular scale. Similar to Forster resonance energy transfer (FRET), it is one of the few tools available to measure intermolecular and

intramolecular distance interactions both in-vivo and in-vitro.

The target molecule can be of natural, synthetic or semi-synthetic origin. In case of interacting molecules, the nature of the targets may be the same (e.g. both natural compounds) or they may be different (e.g. synthetic compound and biological molecule.

In one aspect, at least one of the targets is a biological molecule. Exemplary biological targets include nucleic acid molecules (DNA, RNA), proteinaceous substances and lipids. The target can also be a small molecule e.g. a molecule of less than 2 kDa, such as ATP.

In a preferred embodiment, the invention provides a method for detecting a nucleic acid molecule. "Nucleic acid" shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof. For example, the target is a stretch of about 10- 1000 nucleotides, preferably 20-500 nucleotides. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art. The target nucleic acid can be single or double stranded. In case of a double stranded DNA target, a PNA probe is suitably used to replace a DNA strand and allow for probe binding.

As is shown herein below, the specificity and sensitivity of a method disclosed herein allows discriminating between distinct oligonucleotide targets which differ in only a single base. This opens up a wide range of diagnostic applications relating to genetic variations, such as gene mutational analysis and single -nucleotide polymorphism (SNP). Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents. For example, a single base difference in the Apolipoprotein E is associated with a higher risk for Alzheimer's disease. SNPs are also thought to be key enablers in realizing the concept of personalized medicine. However, their greatest importance in biomedical research is for comparing regions of the genome between cohorts (such as with matched cohorts with and without a disease) in genome -wide association studies. The study of SNPs is also important in crop and livestock breeding programs (genotyping).

In another preferred embodiment, the target is a disease marker or metabolite thereof. A disease marker (also known as biomarker) is anything that can be used as an indicator of a particular disease state or some other physiological state of an organism. In medicine, is a term often used to refer to a protein measured in blood whose concentration reflects the severity or presence of some disease state.

Biomarkers are characteristic biological properties that can be detected and measured in parts of the body like the blood or tissue. They may indicate either normal or diseased processes in the body. A biomarker is a parameter that can be used to measure the progress of disease or the effects of treatment. The parameter can be chemical, physical or biological. In molecular terms biomarker is "the subset of markers that might be discovered using genomics, proteomics technologies or imaging technologies. Biomarkers play major roles in medicinal biology. Biomarkers help in early diagnosis, disease prevention, drug target identification, drug response etc. Several biomarkers have been identified for many diseases such as serum LDL for cholesterol, the P53 gene and MMPs for cancer etc. Gene based biomarker is found to be an effective and acceptable marker in the present scientific world.

In one embodiment, the invention provides a highly sensitive method for detecting a tumor marker. Tumor markers are typically endogenous proteins or metabolites whose amounts or modifications are indicative of tumor state, progression characteristics, and response to therapies. They are present in tumor tissues or body fluids and encompass a wide variety of molecules, including transcription factors, cell surface receptors (e.g. EGFR), and secreted proteins. In one embodiment, the target molecule is a tumor marker for prostate, colorectal, liver, ovarian, breast, stomach, pancreatic or lung cancer. Cancer biomarkers in clinical practice include alpha-

Fetoprotein/AFP, ErbB2/Her2, CA125/MUC16, Kallikrein 3/PSA, ER alpha/NR3Al, Progesterone R/NR3C3, ER beta/NR3A2 and Progesterone R B/NR3C3. The target or targets may be immobilized on a solid surface, like an array, chip, paper strip or bead.

Probes

The skilled person will appreciate that the concept underlying the invention is not restricted to any type of probe, as long as the probe can be labeled with a ligand and is capable of specific binding to the target. In one embodiment, at least one of the probes is a nucleic acid. For nucleic acid targets, the probe is preferably a nucleic acid with a sequence that is complementary to at least part of the target sequence. For example, the probe is a nucleic acid molecule between 9 and 50 nucleotides in length. Typically, a nucleic acid target is detected using a set of at least two oligonucleotide probes, each probe capable of hybridizing to adjacent sequences on the target. Of course, the probes should not be reactive with each other. The conjugation of ligand to the 3' end of the first probe and to the 5' end of the second probe brings the ligands in close proximity of each other, resulting in target-induced catalytic complex formation and the generation of a detectable signal. See Figure 2A. Thus, also provided is a method for determining whether a sample contains a target polynucleotide, comprising the steps of : placing said sample in contact with the ligand-labeled probes, under conditions which permit said target polynucleotide to hybridize to said probes; adding a catalytic compound and a precursor dye; and determining whether a signal indicative of the presence of said target polynucleotide in said sample has been generated, wherein said signal is generated by the catalytic conversion of the precursor dye which can produce a plurality of signals without being exhausted.

In another embodiment, at least one probe is an oligonucleotide aptamer or oligopeptide aptamers having a selective affinity for the at least one target molecule. The term aptamer is derived from the Latin 'aptus' meaning "to fit" and is based on the strong binding of oligopeptides or oligonucleotides to specific targets based on structural conformation. Oligonucleotide aptamers are single-stranded RNA or DNA oligonucleotides, typically about 10 to 60 bases in length, that bind with high affinity to specific molecular targets; most aptamers to proteins bind with Kds (equilibrium constant) in the range of 1 pM to 1 nM similar to monoclonal antibodies. Aptamers are usually created by selecting them from a large random sequence pool. Preferably, an oligonucleotide or oligopeptide aptamer is obtained by screening a library of candidate aptamers and selecting at least one oligonucleotide or oligopeptide displaying the desired characteristics.

In one embodiment, at least one or each of the probes is an oligonucleotide aptamer, like an RNA or DNA aptamer. The oligonucleotide typically consists of between 5 and 60 nucleic acids, preferably 15-40, nucleotide residues or non-natural nucleotide derivatives. As used herein, the term "nucleotide" encompasses both naturally occurring and (semi) -synthetic nucleotide analogs. Non-natural nucleobase- modified nucleotides, representing the latter class, can be also incorporated by polymerases (S. Jager, M. Famulok, Angew. Chem. Int. Ed. 2004, 43, 3337-3340). A specific ATP-binding RNA motif (the ATP aptamer) was previously isolated by in vitro selection from a pool of random RNA sequences. In a specific aspect of the invention, the first and second probes are aptamer fragments capable of self- assembling in the presence of a target molecule. One such example is a split aptamer that recognizes ATP. But also split aptamer sequences are known for drug molecules like cocaine. See for example M.N. Stojanovic, P. de Prada, D.W. Landry, J. Am. Chem. Soc. 2000, 11547-11548).

Theoretically, it is possible to select aptamers virtually against any molecular target; aptamers have been selected for small molecules, peptides, proteins as well as viruses and bacteria. The aptamers are advantageously selected by incubating the target molecule in a large pool of oligonucleotide, the pool size of the oligonucleotide is from 10¹⁰ to 10²⁰. The large pool size of the oligonucleotide ensures the selection and isolation of the specific aptamer. The structural and informational complexity of the oligonucleotide pool and its functional activity is an interesting and active area to develop an algorithm based development of nucleic acid ligands. Aptamers can distinguish between closely related but non-identical members of a target compound family, or between different functional or conformational states of the same compound. The protocol called systematic evolution of ligands by exponential enrichment (SELEX) is generally used with modification and variations for the selection of specific aptamers. Using this process, it is possible to develop new oligonucleotide aptamers in as little as two weeks. Accordingly, in one embodiment a method of the invention involves the use of an oligonucleotide aptamer as probe, the oligonucleotide aptamer being obtained by a screening process. Starting point of each in vitro selection process is typically a synthetic random DNA oligonucleotide library consisting of a multitude of ssDNA fragments with different sequences. This pool of DNA is used directly for the selection of DNA aptamers. For the selection of RNA aptamers, the library has to be transcribed into an RNA library. The SELEX procedure is characterized by the repetition of successive steps consisting

of (I) selection (binding, partition, and elution), (II) amplification and (III)

conditioning (in vitro transcription or purification of ss DNA). In the first SELEX round the sequence pool and the target molecules are incubated for binding. Non- bound oligonucleotides are removed by several washing steps of the binding complexes. The oligonucleotides that are bound to the target molecule are eluted and subsequently amplified by PCR or RT-PCR. A new enriched pool of selected oligonucleotides is generated by preparation of the relevant ssDNA from the PCR products (DNA SELEX) or by in vitro transcription (RNA SELEX). This selected oligonucleotide pool is then used for the next selection round. In general, 6 to 20 SELEX rounds are needed for the selection of high affinity, target-specific aptamers. The last SELEX round is finished after the amplification step. The enriched aptamer pool is cloned, sequenced and several individual aptamers can be characterized.

In another embodiment, at least one or each of the probes is an oligopeptide aptamer. As used herein, the term oligopeptide refers to any proteinaceous substance consisting of between about 5 and 120 amino acids, either in the L- or D- configuration. As used herein, the term "amino acid" encompasses both naturally occurring and (semisynthetic amino acid analogs. For example, one or more non-natural amino acid analogues can be incorporated into proteins by genetic engineering (C.C.Liu, P:G: Schultz, Ann. Rev. Biochem., 79, 413-44). Typically, a certain minimum length is needed to achieve a satisfactory binding constant and selectivity. In one embodiment, the oligopeptide aptamer consists of from 8- 18 amino acids, preferably 10-13 amino acids.

Methods for selecting an oligopeptide aptamer are also known in the art. For example, it involves expressing a library of candidate oligopeptide aptamers in a recombinant host cell, and selecting at least one host cell expressing a desired aptamer and identifying the oligopeptide aptamer. In another embodiment, it comprises the screening of candidate peptides expressed on the cell surface of the host cell. See for example "Decorating microbes: surface display of proteins on Escherichia coli", Bloois E, Winter RT, Kolmar H, Fraaije MW, Trends in Biotechnology, Volume 29, Issue 2, 79-86, 10 December 2010.

Another suitable method is phage display. Thereby, a library of random peptides is expressed in M13 phages followed by the selection of those phages displaying a peptide that can access and bind to an immobilized target compound.

In another embodiment, the probe is a proteinaceous molecule like an oligopeptide, antibody or antibody fragment. For example, the invention also encompasses a sandwich type ELISA wherein two distinct antibodies bind a target molecule and wherein during the binding process two sites of the ligand-conjugated antibodies come spatially close, which can be exploited for a catalytic reaction (see Figure 7A). Peptide aptamers are short peptides, usually 12 to 20 amino acids in length that can be selected from a random peptide library. They specifically bind to a given target protein under intracellular conditions. Each oligopeptide of the library is displayed in a unique conformation and the library is large enough to contain particular peptides able to recognize and bind a large variety of target structures. Selection occurs through screening of a high complexity peptide library. This can be done intracellularly using yeast or mammalian cells. Alternatively, screening can be performed extracellularly using the phage display technology. The interference of peptide aptamers with the function of crucial viral or cellular regulatory proteins has been shown and validated the concept that aptamers can be employed as inhibitors of crucial biological processes. To optimize the binding properties to their target structures, peptide aptamers are usually presented in constrained configurations. Ligand/ Catalytic compound

The present invention relies on the concept of target(s)-induced catalytic complex formation if (a) probe-conjugated ligands come in close proximity of each other and (b) a catalytic compound is present. The catalytic compound is preferably water-soluble. The ligands may be distinct or they may be the same. In one embodiment, each of the probes carries the same type of ligand. Preferably, the ligand is readily coupled to the probe and does not interfere with binding of the probe to the target molecule.

Typically, the ligand is covalently attached to the probe via a site which is not or only minimally involved in target binding. For example, a nucleotide probe is labeled at the 5' or 3' end with ligand. As another example, a peptide probe is labeled at its N- or C-terminus with ligand. Another possibility is coupling the ligand to a cysteine residue within a peptide sequence.

According to the invention, in the presence of a catalytic compound the ligands form a catalytic complex which catalyzes a chromogenic reaction. In a preferred embodiment, the ligands contribute to the formation of a catalytic complex which catalyzes a transition metal-catalyzed dehalogenation. For example, it catalyzes Pd- catalyzed deiodination. Other suitable metal ions for dehalogenation include Rh³⁺, Ir³⁺, Ru³⁺, Pt²⁺, Cu²⁺. A catalytic compound is preferably a transition metal salt. Exemplary catalytic compounds include PdC , (NH^PdCU, PdBr2, Palladium(II) acetate, Tris(dibenzylideneacetone)dipalladium(0), RI1CI3, IrCle, RuCk, K2PtCl4, and CuSCk

In one aspect, the ligand is a phosphine. Phosphines are among the most widely used ligands used in homogeneous catalysis. These trivalent phosphorus compounds stand out from other metal ligands inter alia because they are, in principle, easily modified by a well-established chemistry. In this regard, the stereo electronic tuning of the ligands is key in order to optimize a given catalyzed transformation. It is known that arylphosphines are more stable against oxidation than alkylphosphines. In a specific aspect of the present invention, the ligand comprises or consists of a phosphine moiety and/or the catalytic compound is a transition metal catalyst. Triphenylphosphine (IUPAC name: triphenylphosphane) is a common organophosphorus compound with the formula P(C6Hs)3 - often abbreviated to PPI13 or PI13P. It is widely used in the synthesis of organic and organometallic compounds. Triphenylphosphine is a relatively inexpensive substance. It can be prepared in the laboratory by treatment of phosphorus trichloride with

phenylmagnesium bromide or phenyllithium. Triphenylphosphine binds well to most transition metals, especially those in the middle and late transition metals of groups 7-10. Illustrative PPI13 complexes include tetrakis(triphenylphosphine)palladium(0) which is widely used to catalyse C-C coupling reactions in organic synthesis (Heck reaction); Wilkinson's catalyst, RhCl(PPh3)3 is a square planar Rh(I) complex of historical significance used to catalyze the hydrogenation of alkenes; Vaska's complex, trans-IrCl(CO)(PPh3)2; Stryker's reagent, [(PPli3)CuH]6, ligand stabilized transition metal hydride used as a catalyst for conjugate reductions. Other suitable phosphine- based ligands include l, l'-Bis(diphenylphosphino)ferrocene, tricyclohexylphosphine and (1, r-Biphenyl-2-yl)dicyclohexylphosphine.

In a further embodiment, the ligand is selected from the group consisting of 2,6-bis[l- (phenyl)iminoethyl] pyridine, for example

R=H, Me, Et, i-Pr, i-Bu

In still a further embodiment, the ligand is a mono-Schiff base ligand, such as 3-[[3- [(E)-[[2,6-bis(l-methylethyl)phenyl]imino]methyl]-4-hydroxyphenyl]methyl]-l-methyl- imidazolium chloride. Macrocyclic ligands like 3,7, 11, 17-tetraazabicyclo[l 1.3.1] heptadeca- l(17), 13, 15-triene or tetrasulfophthalocyanine may also be used. As yet another example, the ligand can be pentamethylcyclopentadiene. In a preferred embodiment, the probes are labeled with triphenylphosphine.

Phosphine-containing probes are advantageously used in combination with a transition metal catalyst, preferably wherein the catalytic compound comprises palladium (Pd), more preferably wherein the catalytic compound is a palladium salt such as Na2PdCl4 , PdC , (NH^PdCU, PdB , Palladium(II) acetate, and

Tris(dibenzylideneacetone)dipalladium(0).

Reporter dyes

In step (iii) of a method of the invention, the formation of the catalytic complex is detected based on the conversion of a water-soluble precursor dye into a chromophoric reporter dye. The conversion is non-enzymatic i.e. the method is based on non- enzymatic signal amplification. The non-fluorescent reporter dye(s) for use in the present invention will be selected based on the reaction that is catalyzed by the target-induced, ligand-dependent catalytic complex. Many types of (chemical) reactions may be used for catalytic signal amplification by conversion of a non- chromogenic substrate into a chromogenic product. These include dehalogenation and other transition-metal catalyzed reactions where dehalogenation takes place like the Heck reaction. In a specific aspect, the reporter dye is converted into a highly emissive fluorophore via a transition metal-catalyzed Heck reaction. For example, if the complex catalyzes a dehalogenation reaction, the precursor dye can be a halogenated molecule which becomes fluorescent upon dehalogenation. In one embodiment, it is an iodinated or brominated molecule, preferably a monoiodinated, bisiodinated, monobrominated or bisbrominated molecule.

Reporter dyes of particular interest for practicing the present invention include boron dipyrromethane (BODIPY) derivatives. BODIPY dyes are notable for their uniquely small Stokes shift, high, environment-independent fluorescence quantum yields, often approaching 100% even in water, and sharp excitation and emission peaks contributing to overall brightness. The combination of these qualities makes BODIPY fluorophore an important tool in a variety of imaging applications. The position of the absorption and emission bands remain almost unchanged in solvents of different polarity as the dipole moment and transition dipole are orthogonal to each other.

In one embodiment, the precursor dye is a halogenated, e.g. iodinated,

BODIPY derivative. Bisiodinated and monoiodinated BODIPY derivatives have been shown to exhibit the heavy atom quenching effect and are non- fluorescent. Removal of one or both of the halogen atoms converts the precursor dye into a highly fluorescent molecule. In a specific aspect, a method of the invention employs a

BODIPY derivative that is mono- or bisiodinated, preferably wherein an iodine atom is incorporated at the C2 and/or C6 positions of the BODIPY core.

Step (iii) of a method provided herein involves detecting the formation of the catalytic complex based on the conversion of a water-soluble non-chromophoric reporter dye into a chromophoric reporter dye. The skilled person will be able to determine the reaction conditions that are suitable for the conversion. This may depend on the reaction type, catalyst, ligands and/or reporter dye employed. For example, in case of Pd-catalyzed dehalogenation of an iodinated BODIPY derivative, the conditions may be acidic e.g. pH 4.5-5.5. Suitable buffers are known in the art and include sodium acetate buffers. The reaction mixture may contain further additives such as salts, e.g. 10- 100 mM NaCl, to optimize binding specificity.

The inventors investigated the scope and utility of the DNA-mediated Pd- catalyzed dehalogenation reaction. Since an inert gas atmosphere complicates the applicability of an analytical assay, they performed the catalysis in the presence of air. It was observed that whereas the presence of oxygen affects the catalysis, the initial rates for the two reactions are very similar (data not shown). This indicates that the transformation still proceeds at a fast rate even in oxygenated environments. In the next step, the catalytic dehalogenation assay was performed in more complex environments. The performance of the catalyst was studied in the presence of crude cell extract and proteins (bovine serum albumine (BSA) and DNA polymerase). The results indicate that the catalyst remains active in crude cell extract and in the presence of the polymerase. These measurements showed that the DNA mediated Pd catalyst system of the invention is robust enough for practical sensing

applications.

Suitable concentrations of reactants, target(s), catalytic compound, probes and dyes are readily determined by routine optimization. The ratio between each of the probes and the catalytic compound is preferably about equimolar. Typically, the probes and catalytic compound are used in the range of 100 nM to 10 μΜ. Reporter dye is typically used at 100 pM to 1 μΜ. It is preferred that the reporter dye is present in at least a 100-fold excess of the expected concentration target molecule(s).

To allow for efficient conversion of the precursor dye by the catalytic complex, it is important that the dye is sufficiently soluble in an aqueous medium. Preferably, its solubility in water is at least 10 mg/ml. To that end, the precursor dye may be modified with one or more hydrophilic moieties. For example, the non-fluorescent reporter dye is an oligoethylene glycol-modified molecule. Preferably, the precursor is an oligoethylene glycol-modified BODIPY derivative, like a BODIPY core modified with at least two, preferably at least three, more preferably at least four polyethylene glycol chains. Water solubility can also be induced or improved by the addition of ionic groups, like carboxylate, sulfonate and quaternized amines.

In one embodiment, the invention provides a BODIPY derivative having the formula

Formula I Formula II

These are not known or suggested in the art. Whereas Zhu et al. (Organic Letters 13, 2011, 438-441) discloses bisiodinated BODIPY derivatives, a mono-iodinated derivative of the invention has the advantage that in the assay just one halogen atom needs to be removed in the catalytic process to render the molecule highly fluorescent. As a consequence, a BODIPY derivative according to Formula I or II has a lower detection limit as compared to the bisiodocompounds known in the art. Also provided is a method for the manufacture of a water-soluble (monoidinated) BODIPY

derivative, for example according to the reaction scheme as set out in Scheme SI herein below.

Kits

A further aspect of the invention relates to a kit of parts for detecting target molecule(s) of interest. The kit comprises a set of probes as described herein above, together with an appropriate catalytic compound. An "appropriate catalytic compound" is meant to refer to a catalytic compound which, together with the particular probe-bound ligands, can form a catalytic complex capable of catalyzing a chromogenic reaction. Any suitable combination of suitable and preferred ligands and catalytic compounds as described herein above may be used. In a preferred

embodiment, the kit comprises at least two phosphine-conjugated probes and at least one transition metal catalyst. Preferred probes include oligonucleotide probes. The ligands are preferably identical. Preferred ligands are phosphine-ligands e.g.

triphenylphosphine. In one specific aspect, the kit comprises a first oligonucleotide probe which is labeled at the 3' end with ligand and a second probe which is labeled at the 5'end with ligand. The kit may further comprise a water-soluble precursor dye, preferably wherein the dye is a halogenated molecule, more preferably wherein the non-fluorescent reporter dye is a BODIPY derivative according to formula I or II disclosed herein above. Other useful kit-components include instructions for use, ready-to-use, concentrated or lyophilized buffers, internal standards, and the like.

Applications

In one embodiment, the invention provides methods, means and kits for (clinical) diagnostic purposes, including high throughput screening for diseases, disorders and/or therapy effectiveness. Exemplary clinical applications include endocrinology, infectious disease (e.g. HIV) testing, oncology, allergy testing and sensing of cardiac markers. For example, the kit comprises a set of probes designed for detecting a clinically relevant biomarker, e.g. a tumor marker or cardiac marker. The tumor marker can be a molecule which is targeted by a therapeutic antibody, such as

Herceptin, Avastin or Rituximab. Exemplary cardiac markers include N-terminal prohormone brain natriuretic peptide (NT-proBNP), troponin I (Tnl), CK-MB and myoglobin. The concept of the invention can be employed in various types of homogenous and heterogeneous systems, ranging from aqueous reaction mixtures to microfluidic chips, or more simple assay format like on paper strips.

In another embodiment, the invention finds its use in the detection of chemical compounds, like food or feed ingredients, pollutants, reactants and the like.

In still a further embodiment, the invention is employed for research purposes. A sensitive detection system, either for individual targets or for interacting targets, is needed in various research fields like biophysics, biomedical imaging, and cell biology. For imaging purposes, ligand-conjugated probes can be designed to study protein interactions and conformational changes of molecules like protease sensors. A method of the invention allows analysis of molecular interactions both in vitro and in vivo. Molecular events can be detected by a microscope, for instance using single molecule spectroscopy experiments.

The invention is exemplified by the experimental section herein below, describing various aspects of an amplified colorimetric and fluorometric target detection system through transition metal-catalyzed dehalogenation. LEGEND TO THE FIGURES

Figure 1 : Chemical structures of the water-soluble BODIPY chromophores and their fluorescence spectra, (a) Pd-Catalyzed dehalogenation of profluorescent mono-/bisiodinated precursor reporter dyes (1 and 2) into fluorescent deiodinated reporters (3 and 4). (b) Fluorescence spectra of the mono- (solid) and bisiodo (dashed) precursors (left panel) and the corresponding reporters (right panel). Numbers in brackets indicate the FQY (Fluorescence quantum yield) (ΦΑ) of each compound, as determined against a cresyl violet reference in methanol.

Figure 2: DNA-directed assembly of catalyst and successive fluorogenic conversion, (a) Pd-Catalyzed detection of nucleotide target sequence (T, 30 mer) mediated by the complexation (Cat.) of phosphine-modified ODN probes L and R (15 and 14 mer, respectively) with catalytic compound Pd. (b and c) Photographs of reaction mixtures (precusor 2, L, R, and Na2PdCl4 in NaOAc buffer) with (left) and without (right) target nucleotide (1 nM) taken under ambient light (b) and UV (365 nm) exposure (c). In the presence of the template, conversion of 2 to 4 resulted in a clear color and fluorescence emission.

Figure 3: Fluorimetric determination of reaction kinetics, limit of detection and catalytic conversion, (a) Evolution of fluorescence intensity over time at target concentrations (CT) of 1 pM to 1 nM. (b) Fluorescence intensities of 1 to 3 conversion for a range of CT (square) and without template (circle). Magnified sub-picomolar range of the graph (inset), (c) Equivalent of bis- / monoiodo (black / grey) dye molecules converted per target. Each threshold was determined by the complete conversion of the dye added in varied equivalents (from 30 to lOOOx) at fixed CT = 10, 100, 500 and 1000 fM. At CT = 10 fM, complete conversion of bisiodo BODIPY 2 could not be detected at any dye concentration (N/D). All fluorogenic dehalogenations were monitored at 510 nm (A_ex = 500 nm).

Figure 4 : Evolution of fluorescence intensity over time. Pd-catalyzed dehalogenation assays were performed in the presence of bacterial cell extracts (curve 1), DNA polymerase (curve 2), and BSA (curve 3). Figure 5: Evolution of fluorescence intensity over time. cDNAs with catalyst (curve 1), single-base-mismatched DNAs with catalyst (curve 2), cDNAs without catalyst (curve 3), and catalyst without template (curve 4) .

Figure 6: Small molecule detection by catalytically active DNA aptamers. The figure shows how a catalytic signal amplification method of the invention can be used for the detection of small molecules, such as ATP. Panel A. schematic representation showing ATP-induced juxtapositioning of probe -conjugated ligands such that a catalytic complex with Pd-catalyst is formed. Complex formation is detected by dehalogenation of reporter dye to yield a fluorescent signal. Panel B. Graph showing that complex formation is fully dependent on presence of target and probes, and that the fluorescence signal depends on ATP concentration.

Figure 7: Schematic representation scheme of several different strategies for detection of small molecules and antigens using ligand modified probes, a) Sandwich type ELISA approach for the detection of small molecules and proteins employing triphenylphosphine modified antibodies or antibody fragments, b) Scheme for the detection of small molecules or proteins using an immobilized short peptide binding probe modified with a phosphine ligand in combination with a ligand modified oligonucleotide binding sequence. The figure shows one possible mode of detection with one peptide binder (immobilized) and a nucleic acid binder that are in proximity and therefore can be used for detection. The second nucleic acid binder can also be a peptide binder. Figure 8. Optical properties of compounds 1, 2, 3 and 4. a) Fluorescence spectra of profluorescent mono (1, solid) and bisiodinated (2, dashed) precursor dyes and b) their corresponding fluorescent reporters (4, solid and 3, dashed), c) Normalized absorption spectra of 1 ( _max = 533 nm) and 3 ( _max = 500 nm). d) Relative fluorescence emission spectra of 1 ( _em = 577 nm) and 3 ( _em = 510 nm). e) Normalized absorption spectra of 2 max = 533 nm) and 4 ( _max = 500 nm). f) Relative fluorescence emission spectra of 1 i em = 552 nm) and 2 ( _em = 510 nm). All spectra have been measured in water at 24°C. EXPERIMENTAL SECTION

Materials and Methods

All chemicals and reagents were purchased from commercial suppliers and used without further purification, unless otherwise noted. The 3,5-dihydroxybenzaldehyde (98%), 2,4-dimethylpyrrole (95%), 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ, 98%) trifluoroacetic acid (99%), iodic acid (99.5%), iodine (99.99%),

tetrabutylammonium iodide (n-Bu4NI, 99%), Cul (99.5%), N-hydroxy-succinimide (NHS, 98%), tri-teri-butylphosphine (P(i-Bu)₃, 98%),

tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3), sodium-tetrachloro-palladate(II) (Na₂PdCl4, 99.99%), 1,4-dioxane (99%), triphenylphosphine carboxylic acid (98%),

N,N '-cUcy clone xylmethylamine (CY2NMe, 97%), and dimethylformamide (99%) were purchased from Sigma-Aldrich and used as received. Other special chemicals obtained from different chemical sources were tris(3-sulfonatophenyl)phosphine hydrate sodium salt (P(p-S03CeH4Na)3, Strem Chemicals), 4-ethynylbenzoic acid (96%, ChemBridge Corporation) and N,N '-dicyclohexyl-carbodiimide (99%, Merck). Both modified and unmodified oligonucleotides (ODNs) were synthesized using standard automated solid-phase phosphoramidite coupling methods on an AKTA oligopilot plus (GE Healthcare) DNA synthesizer. All solvents and reagents for oligonucleotide synthesis were purchased from Novabiochem (Merck, UK) and SAFC (Sigma-Aldrich, Netherlands). Solid supports (Primer SupportTM, 200 pmol/g) from GE Healthcare were used for the synthesis of DNA. Oligonucleotides were purified by reverse-phase High Pressure Liquid Chromatography (HPLC) using a C15 RESOURCE RPC™ 1 mL reverse phase column (GE Healthcare) through custom gradients using elution buffers (A: 100 mM triethylammonium acetate (TEAAc) and 2.5% acetonitrile and B: 100 mM TEAAc and 65% acetonitrile). Fractions were further desalted by either desalting column (HiTrapTM desalting, GE Healthcare) or dialysis membrane (MWCO 2000, Spectrum® Laboratories). Labeled oligonucleotides were purified by HPLC and characterized by MALDI-TOF mass spectrometry using a 3- hydroxypicolinic acid matrix. The spectra were recorded on an ABI Voyager DE-PRO MALDI TOF (delayed extraction reflector) Biospectrometry Workstation mass spectrometer. Ή-NMR and ¹³C-NMR spectra were recorded on a Varian Mercury (400 MHz) NMR spectrometer at 25 °C. High-resolution mass spectra (HRMS) were recorded on an AEI MS-902 (EI+) instrument. Absorption and fluorescence spectra of both the non-templated and templated products of the fluorogenic reactions and the concentration of the DNA were measured on a SpectraMax M2 spectrophotometer (Molecular Devices, USA) using 1 cm light-path quartz cuvette. Column

chromatography was performed using silica gel 60 A (200-400 Mesh).

Example 1: Synthesis and characterization of Water-Soluble iodinated BODIPY Substrates Boron dipyrromethane (BODIPY) derivatives are useful chromophores in view of their (1) high fluorescence quantum yield (FQY), (2) high extinction coefficient and (3) photo-stability. I¹⁷l In this Example, mono- and bisiodinated BODIPY precursor chromophores (1 and 2, respectively, see Figure la) were investigated. To ensure aqueous solubility, which is essential for applications in biological systems, a precursor was modified with four triethylene glycol chains. Both profluorescent compounds 1 and 2 proved highly soluble in aqueous media (> 10 mg / ml) and were synthesized in 10 and 30 % overall yield, respectively (see Schemes S1-S3 for synthetic details and structural characterization). In both compounds, iodine atoms were incorporated at the C2 and / or C6 positions of the BODIPY core, to favor intersystem crossing to the triplet manifold. ^[18lThe photophysical properties of the precursors and their deiodinated products (3 and 4) were initially investigated to confirm their suitability as substrates for fluorogenic reactions in water.

Scheme SI. Synthetic route to the oligoethylene-modified bisiodinated BODIPY precursor.

1.1 3, S-dif l-( l',3 '-bis-(3',6',9'-trioxadecylglyceryl]benzaldehyde ( 8)

The starting material 3,5-dihydroxybezaldehyde (7) was obtained from a commercial source and l,3-bis(3,6,9-trioxadecanyl) glycerol-2-toluenesulfonic ester (6) was synthesized as reported by Lauter et al. (Macromol. Chem. Phys. 1998, 199, 2129- 2140). Compound 6 (16.4 g, 30.4 mmol) and K2C03 (5.6 g, 40.6 mmol) were added to a solution of compound 7 (2.0 g, 14.5 mmol) in dry DMF (10 mL). The mixture was stirred at 65 °C under continuous nitrogen atmosphere for 48 h. The progress of the reaction was monitored by TLC. The reaction mixture was cooled down to RT and a mixture of water (5 mL) and brine (5 mL) was added. The resulting solution was extracted with CH3CI (3 x 20 mL) and the combined organic layers were further washed with brine (3 x 10 mL). The resulting crude mixture was purified by silica gel column chromatography using EtOAc/CLbC /MeOH (5: 10:2 v/v) to yield compound 8 as a colorless oil (1.1 g, 75%). ⁱH NMR (400 MHz, CDCls) δ (ppm): 3.48 (s, 12H), 3.49-3.66 (m, 56H), 4.43-4.46 (m, 2H), 6.82 (s, 1H), 7.12 (s, 2H), 9.83 (s, 1H).

¹³C NMR (100 MHz, CDCls) 8(ppm): 59.21, 70.55, 70.65, 70.68, 70.78, 70.79, 111.0, 111.9, 130.1, 154.7, 191.2.

AEI MS-902 (EI+): Calculated exact mass for C41H74O19 [M+H]⁺: 870.48; found:

870.52.

Elemental analysis: Anal, calculated for C41H74O19: C, 56.54; H, 8.56; found: C, 56.49; H, 8.58.

1.2 4,4-Difluoro-8-{3,5-di[l-(l',3^,-bis-(3^,,6',9^,-trioxadecylglyceryl]}

benzaldehyde-l,3,S, 7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (4)

Compound 8 (2.61 g, 3.0 mmol) and 2,4-dimethylpyrrole (0.571 g, 6.0 mmol) were dissolved in dry CH2CI2 (50 mL) under nitrogen atmosphere. Three drops of trifluoro acetic acid (TFA) were added and the resulting reaction mixture was stirred at room temperature in the dark for 5 h. A solution of 2,3-dichloro-5,6-dicyano- l,4- benzoquinone (1.36 g, 6.0 mmol) in dry CH2CI2 (5 mL) was added dropwise to the reaction mixture. This reaction mixture was stirred for an additional 1 h at room temperature. Subsequently, freshly distilled borontrifluoride diethyl etherate

(BF3 OEt2, 15 mL) was added at 5 °C, followed by triethylamine (15 mL). The reaction mixture was stirred at room temperature overnight and then concentrated under reduced pressure, dissolved in CH2CI2, and washed with water (3 x 20 mL). The organic layer was further washed with brine (20 mL), followed by drying over Na2S04 and concentration in vacuo. The crude product was purified twice by silica gel column chromatography using CHCI3/ Me OH (100:2) and CHCI3/ THF (10:3) as mobile phase, yielding compound 4 as an orange oil with bright green fluorescence (1.48 g, 45%). ⁱH NMR (400 MHz, CDCls) 8(ppm): 1.51 (s, 6H), 2.51 (s, 6H), 3.34 (s, 12H), 3.48-3.71 (m, 56H), 4.43-4.46 (m, 2H), 5.94 (s, 2H), 6.52 (s, 2H), 6.64 (s, IH). ⁱ³C NMR (100 MHz, CDCls) 8(ppm): 14.55, 14.77, 59.21, 70.55, 70.65, 70.68, 70.78, 70.79, 106.21, 108.93, 121.31, 131.25, 136.69, 141.48, 143.17, 155.66, 160.52.

AEI MS-902 (ESI+): Calculated exact mass for C53H87N2O18BF2 [M+Na]⁺: 1112.07; found: 1111.59.

Elemental analysis: Anal, calculated for C53H87N2O18BF2: C, 58.45; H, 8.05; N, 2.57; found: C, 58.44; H, 8.12; N, 2.49.

1.3 4, 4-Difluoro-8-{3, 5-di[l-(l ', 3 '-bis-(3 ',6', 9 '-trioxadecylglyceryl]}

benzaldehyde-l,3,S, 7-tetramethyl-2,6-diiodo-4-bora-3a,4a-diaza-s-indacene (2)

Iodic acid (ΗΊΟβ, 1.2 g, 6.8 mmol) in water (7 mL) was added dropwise to a solution of compound 4 (3.37 g, 3.1 mmol) and iodine (1.0 g, 7.8 mmol) in ethanol (30 mL) over 10 min. After the addition was complete, the mixture was stirred for an additional 1 h. The progress of the reaction was followed by TLC until completion. Ethanol was removed in vacuo and the remaining aqueous solution was extracted with

dichlorome thane. The organic layer was dried over anhydrous Na2S04 and

concentrated under reduced pressure. The crude mixture was further purified by silica gel column chromatography using CH2CI2 as mobile phase yielding compound 2 as a red solid (3.5 g, 85%).

Ή NMR (400 MHz, CDCls) 8(ppm): 1.52 (s, 6H), 2.6 (s, 6H), 3.33 (s, 12H), 3.48-3.66 (m, 56H), 4.45-4.47 (m, 2H), 6.49 (s, 2H), 6.7 (s, 1H).

¹³C NMR (100 MHz, CDCls) 8(ppm): 16.18, 17.11, 59.19, 70.19, 70.59, 70.64, 70.67, 70.77, 72.08, 85.71, 106.45, 108.59, 131.12, 132.98, 136.26, 141.19, 145.41, 160.81. AEI MS-902 (ESI+): Calculated exact mass for C53H85N2O18I2BF2 [M+Na]⁺: 1363.39; found 1363.38.

Elemental analysis: Anal, calculated for C53H85N2O18I2BF2: C, 47.47; H, 6.39; N, 2.09; found: C, 47.51; H, 6.37; N, 2.11. Synthesis of Water-Soluble Monoiodinated BODIPY Substrate

Scheme S2. Synthetic route to the oligoethylene oxide-modified carboxyl- functionalized mono-iodinated BODIPY.

Pd₂(dba)₃ (7.0 mg , 0.008 mmol), P(i-Bu)₃ ( 0.033 mmol, 48 pL), n-Bu₄NI (18 mg, 0.05 mmol) and Cul (5.0 mg, 0.026 mmol) were added to a solution of compound 2 (54.0 mg, 0.04 mmol) in dry 1,4-dioxane (2 mL) and N,N '-cUcy clone xylmethylamine (0.2 mL, 0.94 mmol ) at 24 °C followed by addition of 4-ethynylbenzoic acid (15.0 mg, 0.1 mmol) under argon atmosphere. The reaction mixture was stirred for 2 h at room temperature under continuous argon atmosphere. The progress of the reaction was monitored by TLC. After completion of the reaction, water (10 mL) was added to the reaction mixture. The resulting solution was extracted with CH2CI2 (2 x 50 mL), followed by drying over anhydrous MgS04 and evaporation of the solvent under reduced pressure to obtain the crude product. Silica gel column chromatography using EtOAc/CHCl3/MeOH (10/10/1) as eluent afforded compound 1 as a red solid (12 mg, 25% yield).

¹H NMR (400 MHz, CDCI3) 8(ppm): 1.52 (s, 6H), 2.6 (s, 6H), 3.33 (s, 12H), 3.48-3.66 (m, 56H), 4.46-4.48 (m, 2H), 6.41 (s, 2H), 6.58 (s, 1H), 7.51 (d, 2H, J = 7.42), 8.04 (d, 2H, J = 7.99). ⁱ³C NMR (100 MHz, CDCls) 8(ppm): 16.18, 17.11, 59.19, 70.19, 70.59, 70.64, 70.67, 70.77, 72.08,85.16, 85.54, 95.82, 102.53, 105.95, 115.43, 128.58, 128.75, 130.06, 131.08, 131.79, 135.72, 141.95, 144.75, 145.40, 157.04, 157.96, 161.64, 170.10.

AEI MS-902 (ESI+): Calculated exact mass for C62H90N2O20IBF2 [M+Na]⁺: 1381.52; found: 1381.50.

Elemental analysis: Anal, calculated for C62H90N2O20IBF2: C, 54.79; H, 6.67; N, 2.06; found: C, 54.83; H, 6.69; N, 2.03.

EXAMPLE 2: Palladium catalyzed dehalogenation of precursor dye.

Compounds 1 and 2 were individually subjected to palladium-catalyzed deiodination by dissolution in sodium acetate (NaOAc) buffer (0.5 M, pH = 5.0) in the presence of a water-soluble Pd catalyst (Na2PdCl4 · TPPTS) (see SI 2 - 3 for reaction). The product (3 and 4) UV-Vis absorption and fluorescence spectra obtained after 4 h of shaking at r.t. differed markedly from those of the corresponding precursors. As anticipated, the absorption maxima of the dehalogenated products were strongly blue- shifted (Figures 7). Indeed, the color change of the aqueous solution from red to yellow upon deiodination was clearly visible to the naked eye, which implies the possibility for colorimetric detection (similar to Figure 2b).

The fluorescence emission maxima of 3 and 4 also exhibited a blue shift, with fluorescence intensities increasing 35- and 80-fold, respectively, relative to the profluorescent substrates. The significant difference in fluorescence intensity increase was anticipated in light of the higher background intensity of monoiodo precursor 1, which we attribute to it having fewer heavy atoms than bisiodo precursor 2. Moreover, the fluorescence quantum yields (Off) of fluorophore 3 (Φ113 = 0.68) and 4 (ΦΙ14 = 0.81) were 22 and 40 times greater than those of their precursors (Table 1). At this stage, it is important to note that the fluorogenic reaction does not proceed in the absence of any of three reaction components: phosphine ligands and Pd, which form the catalytic complex, and iodo-BODIPY. Hence, signal generation is dependent on the presence of ligands, catalytic compound and precursor dye.

Scheme S3. Synthetic route to the deiodinated fluorescent products of water-soluble mono- and bisiodo BODIPY reporter dyes.

Table 1. Photophysical properties of the mono- and bisiodinated BODIPY precursor dyes 1, 2 and the dehalogenated fluorescent products 3, 4.

r , Absorbance Excitation coefficient Emission Fluorescence quantum yield ompoun ^ [ M'¹ cm'¹] [nm] (<¾)

1 533 7.8xl0⁴ 577 0.03

533 9.7xl0⁴ 552 0.02

500 9.5xl0⁴ 510

500 10.8xl0⁴ 510 EXAMPLE 3: Probe Synthesis and Characterization

The suitability of this fluorogenic deiodination for DNA detection was tested using a DNA-templated reaction strategy (Figure 2A). This example describes the design, synthesis and characterization of a set of oligonucleotide probes according to the invention, wherein each probe is provided with a terminal triphenylphosphine moiety as ligand.

All oligonucleotides (Table 2) were synthesized in 10 pmol scale on an AKTA oligopilot plus (GE Healthcare) DNA synthesizer using standard 6-cyanoethylphosphoramidite coupling chemistry. Deprotection and cleavage from the PS-support were carried out by incubation in concentrated aqueous ammonium hydroxide solution for 5 h at 55 °C. Following deprotection, the oligonucleotides were purified by using anion exchange chromatography, HiTrap™ Q HP 1 mL or 5 mL column (GE Healthcare) through custom gradients using elution buffers (A: 25 mM Tris, pH = 8.0, B: 25 mM Tris and 1.0 M NaCl). Fractions were further desalted by either desalting column (HiTrap™ desalting, GE Healthcare) or dialysis membrane (MWCO 2000, Spectrum^®

Laboratories). Oligonucleotide concentrations were determined by UV absorbance using extinction coefficients. Finally, the identity of the oligonucleotides (ODN) was confirmed by MALDI-TOF mass spectrometry (Table 2).

Subsequently, triphenylphosphine ligands were individually coupled via amide bonds to the 5'-end of probe L and 3'-end of probe R (Scheme S4).

Scheme S4. Synthetic route for PPli3-labeled ODN probes (L and R). Synthesis of NHS ester of triphenylphosphine (PPhe) ligand

The carboxyl group of triphenylphosphine ligand was activated by reacting compound 9 (0.0306 g, 0.1 mmol) with N-hydroxy succinimide (NHS) (0.0364 g, 0.3 mmol) and N,N '-dicyclohexyl-carbodiimide (0.037 g, 0.32 mmol) in 2 mL of DMF. The reaction was carried out for 24 h under inert atmosphere at room temperature (Scheme S4). Precipitated dicyclohexylurea (DCU) was removed by filtration. The solvent was evaporated under reduced pressure and the crude mixture was purified by column chromatography using hexane/EtOAc (1: 1) as eluent. Activated product 10 was obtained as colorless solid (27 mg, 67%). DNA labeling with PPhs-NHS ester

5 -(C6)-Amino-modified oligonucleotides probe L and R (Table 2) were dissolved in sodium tetraborate buffer (0.1 M, pH = 8.5) in two separate vials at concentrations of 1 nmol/pL. 100 pL of each amino-modified oligonucleotide solution was reacted separately in two different vials, each containing a solution of activated PPI13-NHS ester 10 in dimethylformamide (20 pL, 40 pg/pL). The resulting reaction mixtures were mixed in a shaker for 24 h at ambient temperature (Scheme S4). The reaction mixtures were freeze-dried to remove the DMF-H2O mixture. Purification of the labeled oligonucleotides was carried out by using reverse-phase HPLC on a C 15 RESOURCE RPC™ 1 mL column (GE Healthcare) through custom gradients using elution buffers (A: 100 mM TEAAc and 2.5% acetonitrile, B: 100 mM TEAAc and 65% acetonitrile). The coupling yield of the labeling reaction was estimated to be 60% from the integration of the peaks of the HPLC chromatogram. The purified PPli3-labeled oligonucleotides L and R (band at -20 mL) were analyzed by MALDI-TOF mass spectrometry.

Table 2. Sequences and MALDI-TOF mass spectrometry data of the

triphenylphosphine-modified probe [L], [R] ODNs and target strands T and T- sbm used for templated fluorescence activation studies.

Calculated Found

ODN DNA Sequence (5' to 3') (m/z) (m/z)

L PPh₃- (C6 ) -TAG TAT ATA TCT TGC-3' 502 4 5 026

R 5'-ATC TTT AGT TTA GC- ( C 7 ) PPh₃ 471 7 471 9

5'-GCA AGA TAT ATA CTA GGC TAA ACT AAA GAT-3' 9255 925 8

T-sbm^b 5'-GCA AGA TAT ATA GTA GGC TAA ACT AAA GAT-3' 92 8 9 92 9 1 Fully matched

b Single-base-mismatched (C to G mutation) sequences for T architecture.

EXAMPLE 4: DNA-Templated Deiodination Reaction

After HPLC purification, the phosphine-labeled probes were annealed with the target strand (or template, T) in a hybridization buffer (NaOAc (0.5 M, pH = 5.0) in the presence of 75 mM NaCl). Subsequent addition of Na2PdCL resulted in catalytic complex formation due to the close proximity of the ligands on L and R. Low probe concentrations (< 1 μΜ) were chosen so that Pd complex formation would only occur through hybridization to the template. It should be noted that in initial experiments we investigated different templates T with various nucleotide gaps (0-4 nt) between the two annealing sites of L and R. When the two probes were separated by 0, 1, and 2 nt an active catalyst was formed; at greater distances (3 and 4 nt) dehalogenation did not occur (data not shown). Since the highest activity was achieved with a single nucleotide gap (see Figure 2A), this hybrid catalyst in combination with

profluorescent BODIPYs was used to detect the presence of a target DNA sequence, which we investigated in terms of reaction kinetics and detection limit.

A set of DNA- directed palladium catalyzed deiodination experiments were performed using varied concentration of target strand T (CT = 1 nM to 1 fJVI) in presence of 100 tolOOO fold excess of iodo-BODIPY reporter molecules (1 and 2) under fixed concentration (1 μΜ) of probe and catalyst. DNA strands in NaOAc buffer (0.5 M, pH = 5.0) were mixed for 5 min in the presence of 75 mM NaCl solution and then heated up to 60 °C and cooled down slowly (1 °C / 1 min) to 24 °C using a thermal cycler. Na2PdCl4 solution in water was added to the hybridized DNA solution under argon atmosphere and the reaction mixtures were shaken for additional 10 min followed by the addition of iodo-reporter dyes in water to initiate the catalytic reaction. The reaction mixtures, each with a final volume of 100 pL, were shaken at 24 °C.

After gentle mixing for 4 h, the reaction mixtures exhibited the expected intense fluorescence, due to multiple turnovers of deiodination (Figure 2C). As negative controls, all reactions were also performed without the template or catalyst, or with a single-base mismatched template (T-sbm). When the fully complementary template was used, 90% of the fluorescence maximum was reached after 4 min, and saturation was achieved within 10 min (Figure 3A). This rate of reaction is twice that of a DNA-directed Heck reaction to deiodinate an analogous BODIPY-DNA

conjugate [18a]. A possible explanation for this improvement is that the

dehalogenation reaction entails fewer intermediates than the Heck cross-coupling. [19]

Although the deiodination is generally fast, the use of T-sbm in identical conditions slowed the reaction dramatically, reducing the initial intensity growth 65- fold (data not shown). Comparing this finding to the equivalent figure using the Pd- catalyzed C-C cross-coupling (55-fold slowing), [18a] we see a greater degree of sequence selectivity using the DNA hybrid catalyst and thus a potential enhancement in sensitivity. Kinetics of DNA-Templated Deiodination Reaction

The kinetics of the fluorogenic deiodination reactions were monitored using the following reaction conditions: pH = 5.0, 24 °C, 75 mM NaCl, 1 μΜ Na₂PdCl4, 1 μΜ probe ODNs, 0.1 nM template ODN and 10 nM bisiodo substrate 2. As controls, the kinetics of the same conversion were also monitored without template or catalyst and with a single-base mismatch template. The fluorescence data were recorded on a SpectraMax M2 spectrophotometer (Molecular Devices, USA) using a 1 cm light-path quartz cuvette. The fluorescence signal was monitored every 30 sec at 510 nm

(excitation: 500 nm). Results are shown in Figure 4.

Pd-Catalyzed Dehalogenation Assay in Presence of Crude Extract

and Proteins The DNA-mediated Pd-catalyzed dehalogenation reactions were carried out separately in presence of E. coli cell extract, DNA polymerase and BSA under identical reaction conditions (both probe and palladium concentrations were fixed at 1 μΜ, while the concentration of target and monoiodo substrate 1 were fixed at 100 pM and 1 nM, respectively). In order to obtain bacterial crude cell extract, E.coli ER2738 was grown in LB medium until O. D. 600 = 1. Then the bacterial cells were broken down by the freeze/thaw method. The technique involves freezing a cell suspension in a liquid nitrogen bath and then thawing the cells at 37°C. After this lysis step the insoluble fraction of the suspension was spun down by centrifugation. The

supernatant representing a clear cell extract was used as the medium

for the dehalogenation assay. Both BSA and DNA polymerase were obtained from commercial sources.

The dehalogenation assay in presence of E. coli cell extract was carried out by mixing 50 pL of crude E.coli cell extract with DNA strands (L, R & T) in 50 pL of NaOAc buffer (0.5 M, pH = 5.0) containing 75 mM NaCl solution, 10 μΜ Na2PdC14 and 20 μΜ NaBH4 followed by heating to 60°C and cooling down slowly (1°C / 1 min) to 24°C using a thermal cycler. Dehalogenation assays with DNA polymerase or BSA were carried out by mixing either 20 pL (2U/ pL) of DNA polymerase (40U ~ 500 nM end concentration) or 20 pL of 100 pM BSA (end concentration = 20 pM) with DNA strands (L, R & T) in 80 pL of NaOAc buffer (0.5 M, pH = 5.0) containing 75 mM NaCl solution and 10 μΜ Na₂PdCl₄ and 20 μΜ NaBH₄ followed by heating to 60°C and cooling down slowly (1°C / 1 min) to 24°C using a thermal cycler. Finally, the kinetics of all the dehalogenation reactions were monitored after addition

of 10 pL of monoiodo-reporter dye in water to the reaction mixture (final dye concentration 1 nM). Figure 4 shows the evolution of fluorescence intensity over time for Pd-catalyzed dehalogenation assays in the presence of bacterial cell extracts (curve 1), DNA polymerase (curve 2), and BSA (curve 3). Determination of limit of detection

In terms of sensitivity, we investigated both colorimetric and fluorimetric limits of detection. The detection limit of the DNA-templated fluorogenic conversion was calculated by a reported method [18a] and Baeumner et al., Anal. Chem. 2004, 76, 888). We carried out a number of DNA- directed catalytic deiodination reactions using equimolar concentrations of probe and catalyst (1 μΜ) and a concentration of template ODNs ranging from 10 pM to 1 fJVI in the presence of 1000-fold excess reporter molecules 1 or 2. As a negative control, all reactions were also performed without template. The fluorescence intensity after reaction completion was measured for all reactions using a standard spectrophotometer and the resulting fluorescence intensities were plotted against template concentration. The limit of detection was determined to be the lowest measured concentration for which the mean fluorescence intensity exceeded that of the negative control by at least three standard deviations, oi (10 fM) = 1.8± 0.52; t (10 fM) =8.67 ± 0.85 > oi (10 fM) + 3 x sd. Thus, 10 fJVI was determined to be the detection limit (Figure 3B).

A series of reaction mixtures with differing target concentrations and a fixed concentration of 2 (0.1 μΜ) were incubated for 4 h, and it was possible to observe a clear transition from red to yellow with the naked eye for target concentrations down to 1 nM (Figure 2b). This visual ODN detection limit in our DNA-templated system is somewhat limited compared to other systems, such as plasmonic enhancement of inorganic particles[20] and the DNA-photograph technique [21] because the observation here depends solely upon the absorption shift of the iodinated versus deiodinated BODIPY from Amax = 533 to 500 nm. However, the amplified conversion to reporter dyes in our system and the high extinction coefficient of BODIPY analogs make such simple and immediate visual detection possible without any additional instruments. When fluorescence rather than the relatively small absorption shift is used as the sensing mechanism, the detection limit improves by up to 5 orders of magnitude. This limit of detection was defined as the target concentration at which the fluorescence signal could be distinguished from the corresponding negative control without template, namely CT = 100 fJVI with bisiodo BODIPY 2 and 10 fJVI using monoiodo BODIPY 1 as precursors. The difference in the detection limits for two compounds is a simple consequence of number of iodine atoms in the precursors. Below a target ODN concentration of 100 fJVI, the catalytic complex can still quite efficiently remove single iodine atoms from the abundant profluorescent substrates, yielding a strong signal for the conversion of 1 to 3. However, precursor 2 must be deiodinated twice to produce a significant fluorescence signal, and at such low catalytic complex concentrations the predominant product is monohalogenated 5 (Scheme S3), which has a low FQY (Φ115 = 0.03) and exhibits a much smaller blue shift in fluorescence and absorption. The different conversion capabilities of mono- and bisiodo precursors at low target concentrations were investigated further in terms of the quantitative threshold for complete iluorogenic conversion as a function of CT. Four sets of experiments were performed using mono- and bisiodo precursors to determine the quantitative threshold of complete conversion to fluorescent reporter dye. Either 1 or 2 was added in 30 - 1500-fold excess to a range of target concentrations (1 pM, 500 fJVI, lOOfM and 10 fJVI) with a fixed amount of probe and catalyst. The lowest number of equivalents of precursor which could be completely converted to iluorogenic product at a fixed target concentration was considered the threshold.

The lower conversion efficiency for bisiodo precursor 2 (Figure 3C, black bars) at all probed target concentrations is consistent with a single deiodination event per bisiodo molecule. This result merits further investigation to better understand catalytic conversions at low concentrations. DNA in our catalytic model offers several advantages: facile functionalization, immobilization, and sequence-specific

assemblies. Table 3. Determination of quantitative conversion thresholds for 1 and 2. Each threshold was determined by the complete conversion of the dye added in varied equivalents (from 30 to 1500X) at fixed CT = 10, 100, 500 and 1000 fM.

In summary, we have developed an amplified DNA-detection concept using iodo- BODIPYs and a catalytic DNA-Pd complex allowing multiple signal generation from a single hybridization event. This simple target-directed amplification concept is suitable for rapid colorimetric or fluorimetric detection not only for DNA, but for a wide variety of (biological or chemical) targets. In our novel strategy, ligands for the catalytic complex, rather than dye precursors, are conjugated directly to the probes, yielding an active catalytic complex upon hybridization to the target sequence. As such, each hybridization event can catalyze the fluorogenic conversion of many precursor dyes, producing hundreds of fluorophores even in sub-picomolar target concentration. This is a major improvement over the standard method of conjugating reporter dye precursors to the probes, which is limited to one fluorophore per target hybridization. The result of this strategy is amplified ultrasensitive target detection down to a limit of 10 fM, to the best of our knowledge 2 orders of magnitude better than that reported for any other DNA-templated fluorogenic reaction [18a]. Because our method requires no complex design or external stimuli, it is equally applicable to DNA detection in commercially relevant technologies such as real-time polymerase chain reaction. Likewise, it is advantageously applied in combination with DNA aptamers for sensing of other biomolecules or small molecule analytes. EXAMPLE 5: ATP detection employing phosphine labelled aptameric oligonucleotides.

In this example, DNA-directed transition metal catalysis is employed for sensitive detection of small molecules. As a proof of concept, the detection and quantification of adenosine triphosphate (ATP) is realized by using a set of probes consisting of two split aptamer sequences that are each labelled with a phosphine ligand. Upon addition of the analyte and a palladium salt an active catalyst complex is formed (see Figure 6A). This catalytic centre facilitates the conversion of water soluble non- fluorescent bisiodo boron dipyrromethane (BODIPY) dye 2 into a strongly fluorescent deiodinated product 4.

Triphenylphosphine conjugated aptamer synthesis and characterization.

All oligonucleotides were purchased at Biomer.net at HPLC purification grade and used as received (Table 4)

Table 4. Sequences of aptameric oligonucleotide probes used for the detection of ATP.

Phosphine labelled aptamers were synthesized and characterized as described in Example 3, scheme S4.

Adenosine triphosphate detection assay

For the detection of ATP, samples were prepared in salt buffer (NaOAc, 0.5M, pH = 5.02 and NaCl 75mM) with fixed aptamer concentrations (1 μΜ). Subsequently, samples were heated to 60 °C for 5 minutes and slowly cooled down to 23 °C at a rate of 1 °C per minute. After this, ATP was added to obtain the desired analyte concentration, and all the samples were allowed to shake for 1 hour. Subsequently Na2PdCl4 was added (1 μΜ) and the reaction mixture was shaken for another 10 minutes. Finally, the bisiodo BODIPY was added up to a final concentration of 100 nM. Immediately after addition of the profluorescent reporter, the fluorescence of the mixture was measured (A_ex = 500 510 nm) for 30 minutes, with an interval of 5 seconds (Figure 6B).

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Claims

Claims

1. A method for detecting a target molecule or interacting target molecules in a sample based on catalytic signal amplification, the method comprising the steps of : providing at least a first non-fluorescent and a second non-fluorescent probe capable of binding to distinct binding sites on the target molecule(s), the first probe being provided with a first ligand and the second probe being provided with a second ligand, wherein the first and second ligand can form a catalytic complex in the presence of a catalytic compound and wherein the catalytic complex can catalyze a chromogenic reaction;

contacting a sample known or suspected to contain the target molecule (s) with said at least a first and second non-fluorescent probe under conditions that allow for binding of the probes to the target molecule(s) such that, upon binding of the probes to the target molecule(s), the first and second ligand are brought in close proximity of each other and together with the catalytic compound form a catalytic complex immobilized on the target molecule(s); and

detecting the formation of the catalytic complex based on a non-enzymatic conversion of a water-soluble precursor dye into a chromophoric reporter dye.

2. Method according to claim 1, further comprising the step of quantitating the target molecule(s) in the sample by correlating the amount of chromophoric reporter dye detected with the chromophoric signal intensities obtained using a standard curve.

3. Method according to claim 1 or 2, wherein the sample is known or suspected to contain the target molecule(s) in amount of up to 1 nM, preferably up to 100 pM, more preferably up to 1 pM.

4. A probe set comprising at least a first non-fluorescent and a second non- fluorescent probe, each probe capable of binding to distinct binding sites of a target molecule of interest, the first probe being provided with a first ligand and the second probe being provided with a second ligand, wherein the first and second ligand can form a catalytic complex in the presence of a catalytic compound and wherein the catalytic complex can catalyze a chromogenic reaction, preferably transition-metal catalyzed dehalogenation.

5. Method or probe set according to any one of the preceding claims, wherein the target molecule is a biological molecule, preferably a nucleic acid molecule or a proteinaceous substance.

6. Method or probe set according to any one of the preceding claims, wherein at least one probe is a nucleic acid molecule, preferably between 6 and 50 nucleotides in length.

7. Method or probe set according to any one of the preceding claims, wherein the probes are at least two oligonucleotide probes designed to hybridize to adjacent regions on a target nucleic acid sequence, each probe labeled with a ligand.

8. Method or probe set according to any one of the preceding claims, wherein said chromogenic reaction is a transition metal catalyzed dehalogenation reaction.

9. Method or probe set according to any one of the preceding claims, wherein said catalytic compound is a transition metal salt.

10. Method or probe set according to claim 8, wherein the catalytic compound comprises Pd²⁺, Rh³⁺, Ir³⁺, Ru³⁺, Pt²⁺ or Cu²⁺.

11. Method or probe set according to claim 9, wherein the catalytic compound is selected from the group consisting of Na2PdCLi, PdC , (NPU^PdCU, PdBr2,

Palladium(II) acetate, Tris(dibenzylideneacetone)dipalladium(0), RI1CI3, IrC , RuC , K₂PtCl₄ and CuS0₄.

12. Method or probe set according to any one of the preceding claims, wherein the ligand is capable of forming a catalytic complex with a transition metal catalyst, preferably with Pd.

13. Method or probe set according to claim 12, wherein the ligand is a phosphine4>ased ligand, preferably selected from the group consisting of

triphenylphosphine l, l'-Bis(diphenylphosphino)ferrocene, tricyclohexylphosphine and (l, l'4)iphenyl-2-yl)dicyclohexylphosphine.

14. Method according to any one of the preceding claims, wherein the precursor dye is a halogenated molecule.

15. Method according to any one of the preceding claims, wherein the precursor dye is a boron dipyrromethane (BODIPY) derivative, preferably an iodinated BODIPY derivative.

16. An oligoethylene glycol-modified monoiodonated boron dipyrromethane (BODIPY) derivative, preferably wherein the BODIPY derivative is modified with at least two, preferably at least three, more preferably at least four polyethylene glycol chains.

17. BODIPY derivative having the formula

18. Kit of parts comprising a set of ligand-conjugated non-fluorescent probes according to any one of claims 4 to 13, and an appropriate catalytic compound.

19. Kit according to claim 18, further comprising a water-soluble precursor dye, preferably wherein the dye is a halogenated molecule, more preferably wherein the reporter dye is a halogenated BODIPY derivative.

20. Use of method, set of probes, derivative and/or kit according to any one of claims 1-19 in a real-time polymerase chain reaction (RT-PCR) procedure.