A UNIVERSAL LABELING SYSTEM

FIELD OF THE INVENTION

[0001] This invention relates to a novel method for labeling of target nucleotide strands, such as DNA and RNA.

BACKGROUND OF THE INVENTION

[0002] The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

[0003] Oligonucleotides can be labeled either chemically or enzy- matically. The chemical approach includes often preparation of modified building blocks, and their subsequent insertion to synthetic oligonucleotides during oligonucleotide synthesis [Beaucage, S. L., Iyer, P.P., 1993, Tetrahedron, 49, 1859, US 6,949,639, US 09/985,454]. Alternatively, the oligonucleotide or natural DNA or RNA can be transformed by bisulfite-catalyzed transamination of cytosine residues [Hayatsu, H., 1976, Biochemistry, 15, 2677, Draper, D. E., Gold, L., 1980, Biochemistry, 19, 1774, Molander, J., Hurskainen, P., Hovinen, J., Lahti, M., Lδnnberg, H., 1993, Bioconjugate Chem., 4, 362, Adarichev, V.A., Kalachnikov, S. M., Kiseliova, A.V., Dymshits, G. M., 1998, Bioconjugate Chem., 9, 651], by electrophilic substitution of C8 of guanine residues [Tchen, P., Fuchs, R.P.P., Sage, E., Leng, M., 1984, PNAS, 81 , 3466] or by using label molecules tethered to platinum, which coordinates at N7 of guanine residues [US 5985566]. The enzymatic approach consists of preparation of nucleoside triphosphates derivatized with appropriate labels, and their incorporation into DNA by a polymerase reaction [US 2004214221].

[0004] All the above mentioned methods have their drawbacks. Attachment of linkers or labels to critical positions of DNA weaken hybridization properties of the oligomer. Although platinum predominantly coordinates at N7 of guanine residues, the reactivity of the platinated label is dependent on the base sequence of the target molecule as well as on the label attached. Furthermore, not only do N7 of guanine residues react but also other bases such

Lippert B., 1989, Progr. Inorg. Chem., 37, 1 , Lippert, B., Cisplatin, Chemistry and Biochemistry of a Leading Anticancer Drug/; Ed. B. Lippert, Wiley-VCH, 1999., Perspectives on Bioinorganic Chemistry, Vol. 4, Eds R. W. Hay, J. R. Dilworth and K. B. Nolan, JAI Press, 1999]. Although coordination of the platinum label at N7 of guanine allows formation of natural Watson-Crick base pairs, the bulky substituents definitely change the three dimensional structure of the oligomer, especially in the cases where the label attached is an organic chromophore with intercalating properties. Furthermore, it has been shown that in certain cases platinated oligonucleotides are extremely labile as soon as the platinated oligonucleotide is paired with its complementary strand [Anin, M. -F., Gaugheron, F; Leng, M, 1992, Nucleic Acids Res., 20, 4825]. This, in turn, results in the formation of abasic sites and loss of genetic information and the label.

[0005] The labeled 2 ^' -deoxyribonucleotide 5 ^' -triphosphates (dNTPs) ought to be almost as good substrates to DNA polymerases as normal dNTPs. Since DNA polymerases are very sensitive to structural changes of their substrates, the selection of methods to attach non-radioactive markers into dNTPs are rather limited. Furthermore, the enzymatic approach based on DNA polymerases requires the use of a primer and a template.

[0006] Organic dyes as label molecules suffer from commonly known drawbacks such as Raman scattering, concentration quenching and low water solubility. This is a major problem when these types of labels are statistically introduced in the nucleophilic sites of large bioactive molecules. In the worst case the labeled biomolecule has only a limited water solubility, totally unnatural three dimensional structure and no fluorescence properties derived from the label. Fortunately, several of these problems can be avoided by using lanthanide(lll) chelates as the labels. These molecules have several unique properties such as strong long-decay time luminescence, large Stokes shift and very sharp emission lines. Indeed, time resolved fluorimetric assays based on lanthanide chelates have found increasing applications in diagnostics, research and high throughput screening. The heterogenous DELFIA technique (EP 0139675 B1 ; US 4,808,541 ; EP 0298939 B1 ; US 6,127,529; US

4,565,790; WO 03/076939; Hemmila I., Dakubu, S., Mukkala, V.-M., Siitari, H., Lδvgren, T., 1984, Anal. Biochem. 137, 335; Fl Pat. Appl. 20065030) is applied in assays requiring exceptional sensitivity, robustness and multi-label approach. The development highly stable and luminescent lanthanide(lll) chelates (Hemmila, I.; Mukkala, V.-M. 2001 , Crit. Rev. Clin. Lab. Sci. 38, 441 ) has enabled the use of homogenous assay technologies based on time resolution. The different photochemical properties of europium, terbium, dysprosium and samarium chelates enable development even multiparametric homogenous assays. Accordingly, a number of attempts have been made to develop new highly luminescent chelate labels suitable for time-resolved fluorometric applications. These include e.g. stabile chelates composed of derivatives of pyridines [US 4,920,195; US 4,801 ,722; US 4,761 ,481 ; PCT/FI91 /00373; US 4,459,186; EP A-0770610; Remuinan et al, J. Chem. Soc. Perkin Trans 2, 1993, 1099], bipyridines [US 5,216,134], terpyridines [US 4,859,777; US 5,202,423; US 5,324,825] or various phenolic compounds [US 4,670,572; US 4,794,191 ; ltal Pat. 42508 A789] as the energy mediating groups and polycar- boxylic acids as chelating parts. In addition, various dicarboxylate derivatives [US 5,032,677; US 5,055,578; US 4,772,563] macrocyclic cryptates [US 4,927,923; WO 93/5049; EP-A-493745] and macrocyclic Schiff bases [EP-A- 369000] have been disclosed. Recently, development of neutral, highly luminescent stable europium, terbium, samarium and dysprosium chelates based on azamacrocycles has been disclosed [US Pat. Appl.10/928, 143; US Pat. Appl. 11/004,061]. By changing the nature of the chelating metal these type of molecules can be used also in positron emission tomography, single photon emission tomography and magnetic resonance imaging.

[0007] Universal base analogues of nucleosides are designed to form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Numerous such analogues have been synthesized and their applicability as biochemical toos have been investigated [Loakes, D., 2001 , Nucleic Acids Res., 29, 2437]. Universal bases must exhibit the ability to replace any of the five normal nucleobases without significantly affecting either melting behaviour of duplexes or the normal activities of the modified oligon u-

cleotide. 3-Nitropyrrole [Nichols, R., Andrews, P. C, Zhang, P., Bergstom, D. E., 1994, Nature, 369, 492, Bergstrom, D. E., Zhang, P., Toma, P. H., Andrews, P.C., Nichols, R., 1995, J. Am. Chem. Soc, 117, 1201] or 5-nitroindole [Loakes, D., Brown, D. M, 1994, Nucleic Acids Res., 22, 4039] as well as the naturally occurring base, hypoxantine [Ohtsuka, E., Matsuki, S., Ikehara, M., Takahashi, Y., Matsubara, K., 1985, J. Biol. Chem., 260, 2605, Takahashi, Y., Kato, K., Hayashizaki, Y., Wakabayashi, T., Ohtsuka, E., Matsuki, S., Ikehara, M., Matsubara, K., 1985, PNAS, 82, 1931], are among the most commonly used. Phosphoramidites derived from these and several other universal bases are currently commercially available.

[0008] Oligonucleotide ligation assays, useful for determining of mutations, for example, have been described in the art (US 6,506,594; WO 02/22883; Chackravarty et al., Thromb Haemost 1997; 78:1234-6). Here two nucleotide probes, one immobilized and the other one labeled, where the probes are supposed to be complementary to a target oligonucleotide strand to be studied, are contacted with said target oligonucleotide. Ligation between the two probes occurs if the target oligonucleotide is complementary to the probes and thus able capable of binding the probes.

[0009] However, use a template oligonucleotide, bearing a number of natural bases and a number of universal bases, to enable introduction of a label into any target oligonucleotide strand, has not been described so far.

SUMMARY OF THE INVENTION

[0010] The main object of the present invention is to provide a universal method for the labeling of nucleotide strands, such as DNA and RNA. Accordingly the method according to the invention comprises the steps of bringing into a contact with each other

[0011] a) a synthetic labeled oligonucleotide having one or more lables at its 5 ^' -terminus and a pretetermined number of natural nucleobases, sufficient for targetting to a template at its 3 ^' -terminus;

[0012] b) a synthetic oligonucleotide template having a sufficient number of natural nucleobases at its 5 ^' -terminus complementary to the natural

bases of the said synthetic labeled oligonucleotide, and a number of universal bases at its 3 ^' -terminus required for hybridization with the target nucleotide strand;

[0013] c) the target nucleotide strand; and

[0014] d) an enzyme capable of connecting the target nucleotide to the synthetic labeled oligonucleotide, positioned in relation to the target nucleotide strand with the aid of the template.

[0015] Furthermore, the invention concerns a composition useful in a method for labeling of a target nucleotide strand, comprising the components

[0016] a) a synthetic labeled oligonucleotide having one or more lables at its 5 ^' -terminus and a pretetermined number of natural nucleobases, sufficient for targetting to a template at its 3 ^' -terminus, and

[0017] b) a synthetic oligonucleotide template having a sufficient number of natural nucleobases at its 5 ^' -terminus complementary to the natural bases of the said synthetic labeled oligonucleotide, and a number of universal bases at its 3 ^' -terminus required for hybridization with the target nucleotide strand, and optionally

[0018] c) an enzyme capable of connecting the target nucleotide to the synthetic labeled oligonucleotide, positioned in relation to the target nucleotide strand with the aid of the template.

[0019] Still further, the invention concerns a capture oligonucleotide, useful for the removal of the template as defined above from the reaction mixture after completed ligation, said capture oligonucleotide having a sequence complementary to a target sequence in the template.

[0020] Major advantages of the invention:

[0021] The new approach according to the present invention has the following advantages:

[0022] (i) The target molecule remains intact, and hence its hydridi- zation properties are not changed upon labeling.

[0023] (ii) The oligonucleotide label can be used in the labeling of all target molecules.

[0024] (iii)The oligonucletide label can be synthesized in large scale using standard phosphoramidite chemistry. The labeling can be performed either in solution or on solid phase using commonly employed well documented procedures.

[0025] (iv)lf required, the stability of the oligonucleotide label can be enhanced by modifying the phosphodiester backbone or by using non- nucleosidic labeling reactants.

[0026] (v) The nature of the label can be tailored according to the application.

[0027] (vi)Since the template is constructed from natural bases complementary to the 3 ^' -terminus of the oligonucleotide label and universal bases for hybridization with the target molecule, synthesis of only a single template oligonucleotide is required.

DETAILED DESCRIPTION OF THE INVENTION [0028] Definitions:

[0029] The term "target nucleotide strand" shall mean an oligo- or polynucleotide to be labeled. The term shall be understood to cover natural as well as synthetic nucleotides. As non-restricting examples can be mentioned DNA and RNA nucleotides, ribozymes, siRNA:s and antisense strands.

[0030] The term "label" refers especially to a detectable label, but the term shall also be understood to cover any other useful group, such as groups useful for immobilizing the target nucleotide strand to a solid support.

[0031] "Natural nucleobase" shall be understood to mean adenine, thymine, uracil, cytosine or guanine.

[0032] According to a preferable embodiment, the label is linked to a nucleobase via a linker which is formed from one to ten moieties, each moiety being selected from the group consisting of phenyl, alkyl containing 1 -12 carbon atoms, ethynediyl (-C≡C-), ethylenediyl (-C=C-); ether (-O-), thioether (- S-), amide (-CO-NH- and -NH-CO- and -CO-NR ² and -NR ² -CO-), carbonyl (- CO-), ester (-COO- and -OOC-), disulfide (-SS-), diaza (-N=N-) or a tertiary

amine (-NR -), where R represents an alkyl containing less than 5 carbon atoms.

[0033] According to another preferable embodiment, the label is an organic dye, a spin label, biotin, or a luminescent or non-luminescent lantha- nide chelate or a chelate suitable for positron emission tomography or single photon emission computed tomography.

[0034] According to a further preferable embodiment, the metal is gallium-67, gallium-68, indium-111 , technetium-98m, europium, terbium, samarium, gadolinium or dysprosium.

[0035] According to a further preferable embodiment, the linker is covalently attached to N3 of uracil or N3 of thymidine, C5 of cytosine or C5 of uracil, N6 of adenine, C8 of adenine, 06 of guanine, C7 of deazaadenine, C7- of deazaguanine or C6 of adenine.

[0036] According to a further preferable embodiment, the labeled oligonucleotide is constructed from natural phosphodiester linkages, phos- phoramidates, phosphoromonothioates or -dithioates.

[0037] Preferably, the labeled oligonucleotide has natural DNA bases at its 3 ^' -terminus complementary to the 5 ^' -terminus of the synthetic oligonucleotide template.

[0038] The synthetic oligonucleotide template is preferably constructed from natural DNA bases at its 5 ^' -terminus complementary to the natural DNA bases at the 3 ^' -terminus of the labeled oligonucleotide, and universal DNA bases for hybridization with the target molecule.

[0039] According to a preferable embodiment, the universal bases are selected from hypoxantine, 3-nitropyrrole or 5-nitroindole.

[0040] According to a preferable embodiment, the number of labels, number of natural bases in the labeled oligonucleotide and template, and the number of universal bases in the template is judged according the demands of the application.

[0041] Preferably, the number of natural nucleobases in the synthetic labeled oligonucleotide is in the range of 10-20, and the and the number

of natural bases in the template is at least the same as the number of natural nucleobases in the synthetic labeled oligonucleotide.

[0042] The template oligonucleotide is preferably removed from the reaction mixture after completed ligation by a capture oligonucleotide having a sequence complementary to a target sequence in the template. Such a target sequence is, for example, the strand of natural bases in the template.

[0043] According to a preferable embodiment, the capture oligonucleotide is immobilized to a solid and is added to the reaction mixture after completion of the ligation reaction.

SCHEMATIC PRESENTATION OF THE LABELING PROCEDURE

[0044] The scheme below illustrates the principle of the present labeling strategy.

2 REMOVAL OF THE TEMPLATE G-G-G-G-G-OH (excess)

O

N- A

X J

[0045] The labeling procedure is the following

[0046] 1. The labeled oligonucleotide is allowed to hybridize with the template oligonucleotide in the presence of the target molecule and the enzyme (e.g. ligase). The order of addition of these reagents is not critical.

[0047] 2. When the ligation is completed, the template oligonucleotide is removed by an excess of a capture oligonucleotide has a sequence complementary to a sequence in the template oligonucleotide. The capture oli-

gonucleotide can be bound to a solid matrix. If desired, the template oligonucleotide can be released by increasing the temperature and reused.

[0048] The number of the natural bases at the 3 ^' -terminus of the labeled oligonucleotide and at the 5 ^' -terminus of the template oligonucleotide depends on the structure of nucleobases. Accordingly, in order to form a stabile duplex at room temperature, ca 12 CG pairs or 15 AT pairs is needed.