WO2003052132A2 - Analogues d'oligonucleotides lineaires et en epingle a cheveux comprenant des pseudonucleotides intercalants - Google Patents

Analogues d'oligonucleotides lineaires et en epingle a cheveux comprenant des pseudonucleotides intercalants Download PDF

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WO2003052132A2
WO2003052132A2 PCT/DK2002/000874 DK0200874W WO03052132A2 WO 2003052132 A2 WO2003052132 A2 WO 2003052132A2 DK 0200874 W DK0200874 W DK 0200874W WO 03052132 A2 WO03052132 A2 WO 03052132A2
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oligonucleotide
sequence
intercalator
dna
nucleic acid
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WO2003052132A3 (fr
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Ulf Bech Christensen
Erik Bjerregaard Pedersen
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Human Genetic Signatures Pty Ltd
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6832—Enhancement of hybridisation reaction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
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    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3511—Conjugate intercalating or cleaving agent
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    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag

Definitions

  • the present invention relates to the field of pseudonucleotides comprising interca- lators.
  • the invention relates to the field of linear and hairpin oligonucleotide analogues comprising said intercalator pseudonucleotides
  • the invention relates to such oligonucleotide analogues for use in extension and amplification of nucleic acids and nucleic acid analogues.
  • the invention relates to the field of detecting hybridisation of said oligonucleotide analogues as well as to incorporation of said oligonucleotide analogues into nucleic acids or nucleic acid analogues and to detection of said interca- lator pseudonucleotides in extension products.
  • Nucleic acids such as DNA, RNA as well as a number of nucleic acid analogues such as PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Xylo-LNA, ⁇ -D-Ribo-LNA, [3.2.1]-LNA, Bicyclo- DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, -Bicyclo-DNA, Tricyclo-DNA, Bi- cyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, ⁇ -D-Ribopyranosyl- NA, ⁇ -L-Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR
  • RNA expression studies Detection of the presence and amount of specific nucleic acid sequences is used extensively in particularly RNA expression studies. RT-PCR based techniques have become widespread and are now considered an indispensable method for assaying gene expression.
  • the well-known previous generation detection formats based on end-point quantification of gel-separated e.g. ethidium bromide stained PCR-product or radioactively labeled PCR-product is now often being replaced by fluorescence based detection with new fluorescent molecules. This kind of detection is typically carried out during the PCR process (often in real-time), saving time and providing data of higher quality than using older methods.
  • fluorescence based detection systems for specific nucleic acid sequences are available and all are based on one or more primers/probes detecting the presence of the specific nucleic acid sequence.
  • PCR remains an indispensable tool when validated gene expression studies need to be performed, but the multiplexing and throughput capabilities of PCR still limit its uses in some applications. For genome wide assessment of gene expression, DNA micro array approaches are therefore preferred.
  • Certain synthetic nucleic acids have an increased affinity for nucleic acids in general. High affinity towards target nucleic acids may greatly facilitate hybridization based_assays and high affinity may also be useful for a number of other reactions involving nucleic acids for example amplification reactions.
  • nucleic acids as well as most synthetic nucleic acid analogues do not discriminate rigidly between different kinds of nucleic acid, i.e. they bind roughly equally well to complementary DNA and complementary RNA.
  • Pyrene is an excimer-forming molecule, which has been incorporated into oligode- oxynucleotides (ODNs) by several groups.
  • ODNs oligode- oxynucleotides
  • Ebata et al. incorporated a pyrene- modified nucleotide in the 5' end of one ODN and a pyrene-modified nucleotide into the 3' end of another.
  • an excimer band at 490 nm was generated. Paris et al.
  • US 5,446,578 describes synthetic nucleotide like molecules comprising fluorescent molecules, which show a change in spectra with concentration, for example pyrene.
  • the document describes nucleic acids derivatised with such fluorescent molecule on the phosphate of a nucleic acid backbone or nucleic acids comprising an acyclic backbone monomer unit consisting of 5 atoms between two phosphates of the nucleic acid backbone, coupled to such a fluorescent molecule.
  • the document states that the fluorescent molecules should be positioned at the exterior of a nucleic acid helix so that they are not capable of intercalating with nucleobases of a nucleic acid.
  • the fluorescence of the fluorescent molecule increases upon hybridization and that a cationic surfactant must be present to achieve this effect.
  • Yamana et al., 1999 describes an oligonucleotide containing a 2 ' -O-(1- pyrenylmethyl)uridine at the center position. Said oligonucleotide has higher affinity for DNA and lower affinity for RNA compared to an unmodified oligonucleotide. Upon hybridization monomer and exciplex fluorescence is enhanced.
  • Yamana et al., 1997 describes a phosphoramidit coupled to pyrene, which may be incorporated into a nucleic acid at any desired position.
  • said phospho- ramidit may be incorporated into a nucleic acid, as an acyclic backbone monomer consisting of 5 atoms between two phosphates of the nucleic acid backbone.
  • excimer fluorescence is greatly enhanced and nucleic acids into which said phosphoramidites have been incorporated retain normal binding affinity for DNA.
  • Korshun et al., 1999 describes a phosphoramidit coupled to a pyrene, which may be incorporated into a nucleic acid at any desired position.
  • said phosphoramidit may be incorporated into a nucleic acid, as an acyclic backbone mono- mer consisting of 5 atoms between two phosphates of the nucleic acid backbone.
  • oligonucleotides into which said phospho- ramidits have been incorporated and it is described that the oligonucleotides have higher affinity for DNA, than an unmodified oligonucleotide. It is mentioned that close coplanar mutual approach of two pyrene residues located in the neighboring positions of a modified oligonucleotide chain is strongly inhibited because of the small length of the linker. Excimer fluorescence increases upon hybridization, however oligonucleotides comprising 5 such pyrene pseudonucleotides at the end exhibit high excimer fluorescence when unhybridised as well.
  • pseudonucleotides which may comprise an intercalator such as an acridine or anthraquinone.
  • the pseudonucleotide comprises an achiral or a single enantiomer organic backbone, such as diethanolamine.
  • the pseudonucleotides may be incorporated at any desired position within an oligonucleotide.
  • Such oligonucleotides in general have higher affinity for complemenntary nucleo- tides, in particular when the pseudonucleotides are inserted at the end.
  • the document does not describe fluorescence data.
  • US 6,031,091 describes pseudonucleotides which may be incorporated at any position in an oligonucleotide.
  • the document describes acyclic phosphor containing backbones and it is mentioned that the pseudonucleotides may comprise an intercalator.
  • Specific pseudonucleotides described in the document comprise very long linkers connecting polyaromates to the nucleic acid backbone.
  • EP 0 916 737 A2 describes polynucleotides derivatised with for example intercalat- ing compounds.
  • the intercalating compounds should preferably be positioned with approx. 10 nucleotides in between.
  • the polynucleotide may be derivatised on the phosphate, the sugar or the nucleobase moiety. In particular, they may be derivatised on the nucleobase by a 7 or a 11 atoms long linker coupled to a polyaromate in a manner that does not interfere with Watson-Crick base pairing. It is stated that fluorescence intensity is enhanced by intercalation.
  • WO 97/43298 describes nucleoside analogues comprising a polyaromatic hydrocarbon for example pyrene attached to the 1 ' position of ribose or deoxyribose as well as phosphoramidite derivatives of said polyaromatic hydrocarbons.
  • nucleotide derivatives comprising nucleobases fused to planar polycyclic aromatic compounds. Oligonucleotide comprising said nucleotide derivatives have increased affinity for DNA and fluorescence is decreased by hybridization.
  • Ebata et al., 1995 describes incorporation of a pyrene-modified nucleotide in the 5' end of a DNA oligonucleotide and a pyrene-modified nucleotide into the 3' end of another.
  • an excimer band at 490 nm was generated.
  • Hairpin-forming oligonucleotide hybridization probes with interactive label pairs particularly fluorescent label pairs and fluorescence-quencher label pairs
  • Tyagi et al. PCT application No. WO95/13399
  • Tyagi et al. PCT application No. WO97/39008
  • Tyagi and Kramer (1996) Nature Biotechnology 14:303.
  • These probes, labeled with a fluorophore and a quencher are usually "dark", that is, have relatively little or no fluorescence, when free in solution but fluoresce when hybridized to their nucleic acid targets.
  • oligonucleotides or oligonucleotide analogues comprising at least one intercalator pseudonuclotide of the general structure
  • X is a backbone monomer unit capable of being incorporated into the phosphate backbone of a nucleic acid
  • Q is an intercalator comprising at least one essentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid;
  • Y is a linker moiety linking said backbone monomer unit and said intercalator
  • oligonucleotide comprises a first sequence consisting of n nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides and a second nucleotide sequence consisting of m nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides
  • oligonucleotides or oligonucleotide analogues wherein said second sequence is capable of hybridizing to a homologously complementary target sequence comprised within a nucleic acid or nucleic acid analogue and is capable of priming a template directed extension reaction; and wherein said first sequence will not hybridise with said second sequencelt is also an object of the present invention to provide hairpin oligonucleotide analogues comprising at least one intercalator pseudonucleotide of the general structure
  • X is a backbone monomer unit capable of being incorporated into the phosphate backbone of a nucleic acid
  • Q is an intercalator comprising at least one essentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid;
  • Y is a linker moiety linking said backbone monomer unit and said intercalator
  • the oligonucleotide comprises a first sequence consisting of n nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides and a second nucleotide sequence consisting of m nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides, wherein said first sequence is capable of hybridising to said second sequence.
  • hairpin oligonucleotide analogues wherein said first sequence and said second sequence are separated by a third sequence consisting of p nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides, wherein said first sequence is capable of hybridizing to said second sequence; and wherein the second and third sequence is capable of hybridizing to a homologously complementary target sequence comprised within a nucleic acid or nucleic acid analogue and is capable of priming a template directed extension reaction.
  • a hairpin oligonucleotide analogue comprising an intercalator pseudonucleotide as described herein above and a template comprising a nucleic acid sequence capable of hybridising to the second sequence of said hairpin oligonucleotide analogue comprising the steps of
  • Figure 1 illustrates the synthesis of an intercalator pseudonucleotide, a phospho- ramidite as depited in 5.
  • the pyrene moiety is co-axial stacked with the underlying base pair.
  • the pyrene makes co- axial stacking with both the upper and lower neighboring nucleobases of the opposite strand.
  • the pyrene moiety is able to interact with both the upper and lower neighboring nucleobases of the opposite strand. The distance between the nucleobases and the pyrene moiety is shown to the right.
  • Figure 5 illustrates fluorescent measurements of a 13-mer, mono pyrene inserted ssDNA (*); its duplex with complementary, 12-mer RNA ( ) and its duplex with complementary, 12-mer DNA ( ⁇ ).
  • the sequences are the same as those shown in Table 3.
  • Figure 6 illustrates fluorescent measurements of a 14-mer ssDNA with two pyrene insertions separated by one nucleotide (*); its duplex with complementary, 12-mer RNA ( ) and its duplex with complementary, 12-mer DNA ( ⁇ ). The sequences are the same as those shown in Table 3.
  • Figure 7 illustrates a procedure to prepare a sample for RT-PCR
  • Figure 8 illustrates a procedure to prepare a sample for RT-PCR
  • Figure 9 illustrates a procedure to prepare a sample for RT-PCR
  • Figure 10 illustrates a procedure to prepare sequence specfic DNA
  • Figure 11 illustrates a procedure to prepare a sequence specfic DNA
  • Figure 12 illustrates a method to detect sequence specific DNA using a chip
  • Figure 13 illustrates different kinds of oligonucleotides that may be useful as probes on a chip
  • Figure 14 illustrates PCR quantification.
  • Figure 15 illustrates transcription blockage using a pair of oligonucleotides according to the invention indicated as A and B, respectively.
  • Figure 16 Nuclease resistance of two oligonucleotides whereof one comprises intercalating pseudonucleotides (INA oligo) and the duplex of said two oligonucleotides.
  • INA oligo intercalating pseudonucleotides
  • Figure 17 Secondary structure of the hairpin forming probe I. In this conformation the monomer and excimer fluorescence is quenched.
  • Figure 18 Secondary structure of probe I when hybridised to at target sequence. When hybridized to a target sequence, the excimer complex is free to be formed and hence excimer fluorescence can be observed. The monomer fluorescence is also increased.
  • Figure 19 SYBR green II stained INA oligos, visualized on an ArrayWorx scanner.
  • Figure 20 illustrates a test of oligo binding on Asper SAL slides.
  • Figure 25 Sequence of the employed double-stranded target oligo, the attacking lOs and the complimentary pairing lOs. Y denote intercalating units.
  • Figure 26 lOs spontaneously bind target DNA. Reactions where carried out in 20 ⁇ l volumes containing 126 nM lOs with or without
  • Reactions were carried out in 15 ⁇ l volumes containing the indicated concentrations of lOs with or without 20nM target DNA (single or double stranded), for 2 h at 37 °C. Binding was assayed by electrophoresis in a 10 % polyacrylamide /%xTBE gel and visualized by phosphorimaging.
  • Figure 28 IO pairing in spontaneous target binding.
  • Figure 29 Pairing does not affect the efficiency of spontaneous binding. Reactions were carried out in 15 ⁇ l volumes containing 20nM target DNA and increasing amounts of lOs (40-80-160 nM) as indicated for 4 h at 37 °C. Binding was assayed by electrophoresis in a 10 % polyacrylamide /VixTBE gel and visualized by phosphorimaging. Band intensities are relative numbers representing intensities of the band areas.
  • Figure 30 IO-DNA complex formation in nuclear extracts Reactions were carried out in 15 ⁇ l volumes containing pre-annealed 180 nM lOs and 20 nM target DNA where indicated, nuclear extracts (NE) were added to the reactions as indicated. Reactions were incubated at 37°C for 10 min, and then another 60 min upon addition of 1.125 ⁇ l 10% SDS and 37.5 ⁇ g Proteinase K. Binding was assayed by electrophoresis in a 7 % polyacrylamide IVzxJBE gel and visualized by phosphorimaging.
  • Figure 31 Nuclear factors favour IO-DNA complex formation by IO pairs Reactions were carried out in 15 ⁇ l volumes containing 180 nM lOs and 20 nM target DNA. 10 ⁇ g HeLa nuclear extract were added to the reactions. Reactions were incubated at 37°C for 10 min, and then another 60 min upon addition of 1.125 ⁇ l 10% SDS and 37.5 ⁇ g Proteinase K. Binding was assayed by electrophoresis in a 10 % polyacrylamide /%xTBE gel and visualized by phosphorimaging
  • Figure 32 Chemical structures of LNA and INA P nucleotide monomers.
  • B nucleobase.
  • Figure 33 Melting temperature data of INAs with different insertion patterns when hybridised to the complementary structure and LNA targets.
  • P INA monomer P.
  • T L and Me C L are locked nucleotides of thymine and 5-methylcytosine, respectively.
  • T m Transition temperatures, T m (°C) for hairpin probes with ssDNA targets.
  • T and Me C L are locked nucleotides of thymine and 5-methylcytosine, respectively.
  • Figure 35 A) transition curves of the non-intercalating pseudonucleotide comprising probes B) Two LNA probes comprising one intercalating pseudonucleotide together with the unmodified reference duplex. C) LNA probes comprising one or two intercalating pseudonucleotide together with the unmodified reference duplex. D) A non- intercalating pseudonucleotide comprising LNA probes and two probes comprising one intercalating pseudonucleotide together with homologously complementary
  • DNA probe all hybridized to a target sequence comprising one intercalating pseudonucleotide.
  • Figure 36 Scheme 1. Schematic presentation of the conformations formed by T 4 - LNA oligonucleotides at transition temperature.
  • Figure 38 Sequences and hybridisation data of synthesized ODNs in DNA DNA(RNA) duplexes
  • Figure 40 illustrates a beacon primer Figure 41: - illustrates a PCR quantification strategy using beacon primers
  • Figure 42 illustrates complete complementarity and mismatch/excimer formation
  • nucleic acid covers the naturally occurring nucleic acids, DNA and RNA, including naturally occurring derivatives of DNA and RNA such as but not limited to methylated DNA, DNA containing adducts and RNA covalently bound to proteins.
  • nucleic acid analogues covers synthetic derivatives and analogues of the naturally occurring nucleic acids, DNA and RNA. Synthetic analogues comprise one or more nucleotide analogues.
  • nucleotide analogue comprises all nucleotide analogues capable of being incorporated into a nucleic acid backbone and capable of specific base-pairing (see herein below), essentially like naturally occurring nucleotides.
  • single strands of nucleic acids or nucleic acid analogues according to the present invention are capable of hybridising with a substantially complementary single stranded nucleic acid and/or nucleic acid analogue to form a double stranded nucleic acid or nucleic acid analogue. More preferably such a double stranded analogue is capable of forming a double helix.
  • the double helix is formed due to hydrogen bonding, more preferably, the double helix is a double helix se- lected from the group consisting of double helices of A form, B form, Z form and intermediates thereof.
  • nucleic acids and nucleic acid analogues according to the present invention includes, but is not limited to the kind of nucleid acids and/or nucleic acid analogues selected from DNA, RNA, PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2'-NH)- TNA, (3'-NH)-TNA, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Xylo-LNA, ⁇ -D-Ribo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, ⁇ -Bicyclo- DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide- DNA, ⁇ -D-Ribopyranosyl-NA, ⁇ -L
  • non-phosphorous containing compounds may be used for linking to nucleotides such as but not limited to methyliminomethyl, formacetate, thiof ormacetate and linking groups comprising amides.
  • nucleic acids and nucleic acid analogues may comprise one or more intercalator pseudonucleotides according to the present invention.
  • mixture is meant to cover a nucleic acid or nucleic acid analogue strand comprising different kinds of nucleotides or nucleotide analogues.
  • hybrid is meant to cover nucleic acids or nucleic acid analogues comprising one strand which comprises nucleotides or nucleotide analogues with one or more kinds of backbone and another strand which comprises nucleotides or nucleotide analogues with different kinds of backbone.
  • duplex is meant the hybridisation product of two strands of nucleic acids and/or nucleic acid analogues, wherein the strands preferably are of the same kind of nucleic acids and/or nucleic acid analogues.
  • HNA nucleic acids as for example described by Van Aetschot et al., 1995.
  • MNA nucleic acids as described by Hossain et al, 1998.
  • ANA refers to nucleic acids described by Allert et al, 1999.
  • LNA may be any LNA molecule as described in WO 99/14226 (Exiqon), preferably, LNA is selected from the molecules depicted in the abstract of WO 99/14226. More preferably LNA is a nu- cleic acid as described in Singh et al, 1998, Koshkin et al, 1998 or Obika et al., 1997.
  • PNA refers to peptide nucleic acids as for example described by Nielsen et al., 1991.
  • nucleotide designates the building blocks of nucleic acids or nucleic acid analogues and the term nucleotide covers naturally occurring nucleotides and derivatives thereof as well as nucleotides capable of performing essentially the same functions as naturally occurring nucleotides and derivatives thereof.
  • Naturally occurring nucleotides comprise deoxyribonucleotides comprising one of the four nucleobases adenine (A), thymine (T), guanine (G) or cytosine (C), and ribonucleotides comprising on of the four nucleobases adenine (A), uracil (U), guanine (G) or cytosine (C).
  • Nucleotide analogues may be any nucleotide like molecule that is capable of being incorporated into a nucleic acid backbone and capable of specific base-pairing.
  • Non-naturally occurring nucleotides includes, but is not limited to the nucleotides selected from PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Xylo- LNA, ⁇ -D-Ribo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi- Bicyclo-DNA, ⁇ -Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA,
  • nucleotides and nucleotide analogues according to the present in- vention is to be able to interact specifically with complementary nucleotides via hydrogen bonding of the nucleobases of said complementary nucleotides as well as to be able to be incorporated into a nucleic acid or nucleic acid analogue.
  • Naturally occuring nucleotides, as well as some nucleotide analogues are capable of being enzymatically incorporated into a nucleic acid or nucleic acid analogue, for example by RNA or DNA polymerases, however nucleotides or nucleotide analogues may also be chemically incorporated into a nucleic acid or nucleic acid analogue.
  • nucleic acids or nucleic acid analogues may be prepared by coupling two smaller nucleic acids or nucleic acid analogues to another, for example this may be done enzymatically by ligases or it may be done chemically.
  • Nucleotides or nucleotide analogues comprise a backbone monomer unit and a nucleobase.
  • the nucleobase may be a naturally occuring nucleobase or a derivative thereof or an analogue thereof capable of performing essentially the same function.
  • the function of a nucleobase is to be capable of associating specifically with one or more other nucleobases via hydrogen bonds.
  • nucleobase it is an important feature of a nucleobase that it can only form stable hydrogen bonds with one or a few other nucleobases, but that it can not form stable hydrogen bonds with most other nucleobases usually including itself.
  • base-pairing The specific interaction of one nucleobase with an- other nucleobase is generally termed "base-pairing".
  • Base pairing results in a specific hybridisation between predetermined and complementary nucleotides.
  • Complementary nucleotides according to the present invention are nucleotides that comprise nucleobases that are capable of base-pairing.
  • nucleobases adenine (A) pairs with thymine (T) or uracil (U); and guanine (G) pairs with cytosine (C).
  • A adenine
  • T thymine
  • U uracil
  • G guanine
  • C cytosine
  • a nucleotide comprising A is complementary to a nucleotide comprising either T or U
  • a nucleotide comprising G is complementary to a nucleotide comprising C.
  • Nucleotides according to the present invention may further be derivatised to comprise an appended molecular entity.
  • the nucleotides can be derivatised on the nucleobases or on the backbone monomer unit. Preferred sites of derivatisation on the bases include the 8-position of adenine, the 5-position of uracil, the 5- or 6- position of cytosine, and the 7-position of guanine.
  • the heterocyclic modifications can be grouped into three structural classes: Enhanced base stacking, additional hydrogen bonding and the combination of these.
  • Modifications that enhance base stacking by expanding the ⁇ -electron cloud of planar systems are represented by conjugated, lipophilic modifications in the 5-position of pyrimidines and the 7- position of 7-deaza-purines.
  • Substitutions in the 5-position of pyrimidines modifications include propynes, hexynes, thiazoles and simply a methyl group; and substituents in the 7-position af 7-deaza purines include iodo, propynyl, and cyano groups.
  • a second type of heterocycle modification is represented by the 2-amino-adenine where the additional amino group provides another hydrogen bond in the A-T base pair, analogous to the three hydrogen bonds in a G-C base pair.
  • Heterocycle modifications providing a combination of effects are represented by 2-amino-7-deaza-7-modified andenine and the tricyclic cytosine analog having an ethoxyamino functional group of heteroduplexes.
  • N2-modified 2-amino adenine modified oligonucleotides are among commonly modifications.
  • Preferred sites of derivatisation on ribose or deoxyribose moieties are modifications of nonconnecting carbon positions C-2' and C-4', modifications of connecting carbons C-1', C-3' and C-5', replacement of sugar oxygen, O-4', Anhydro sugar modifications (conformational restricted), cyclosugar modifications
  • connection sites - sugar to sugar (C-3' to C-57 C-2' to C-5'), hetero-atom ring - modified sugars and combinations of above modifications.
  • other sites may be derivatised, as long as the overall base pairing specificity of a nucleic acid or nucleic acid analogue is not disrupted.
  • the backbone monomer unit comprises a phosohate group
  • the phosphates of some backbone monomer units may be derivatised.
  • Oligonucleotide or oligonucleotide analogue as used herein are molecules essentially consisting of a sequence of nucleotides and/or nucleotide analogues and/or intercalator pseudo-nucleotides.
  • oligonucleotide or oligonucleotide analogue comprises 3-200, 5-100, 10-50 individual nucleotides and/or nucleotide analogues and/or intercalator pseudo-nucleotides, as defined above.
  • a target nucleic acid or target nucleic acid analogue sequence refers to a nucleotide or nucleotide analogue sequence which comprise one or more sites/sequences for hybridisation of one or more oligonucleotide(s) and/or oligonucleotide analogue(s), for example primers or probes.
  • Target sequences may be found in any nucleic acid or nucleic acid analogue including, but not limited too, other probes, RNA, genomic
  • DNA, plasmid DNA, cDNA can for example comprise a wild-type or mutant gene sequence or a regulatory sequence thereof or an amplified nucleic acid sequence, for example as when amplified by PCR.
  • a target sequence may be of any length.
  • the site addressed may or may not be one contiguous sequence.
  • said site may be composed of two or more contigous subsequences separated by any number of nucleotides and/or nucleotide analogues.
  • the total length of the site addressed, composed by all subsequences on that particular target nucleic acid or target nucleic acid analogue, by said oligonucleotide and/or oligonucleotide analogue typically is less than 100 nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides.
  • Nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotide analogues are said to be homologously complementary, when they are capable of hybridising.
  • homologously complementary nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotide analogues are capable of hybridising under low stringency conditions, more preferably homologously complementary nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotide analogues are capable of hybridising under medium stringency conditions, more preferably homologously complementary nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotide analogues are capable of hybridising under high stringency conditions.
  • High stringency conditions shall denote stringency as in comparison to, or at least as stringent as, what is normally applied in connection with Southern blotting and hybridisation as described e.g. by Southern E. M., 1975, J. Mol. Biol. 98:503-517. For such purposes it is routine practise to include steps of prehybridiza- tion and hybridization. Such steps are normally performed using solutions containing 6x SSPE, 5% Denhardt's, 0.5% SDS, 50% formamide, 100 ⁇ g/ml denaturated salmon testis DNA (incubation for 18 hrs at 42°C), followed by washings with 2x
  • Medium stringency conditions shall denote hybridisation in a buffer containing 1 mM EDTA, 10mM Na 2 HPO 4 H 2 0, 140 mM NaCl, at pH 7.0, or a buffer similar to this having approximately the same impact on hybridization stringency.
  • a buffer similar to this Preferably, around 1,5 ⁇ M of each nucleic acid or nucleic acid analogue strand is provided.
  • medium stringency may denote hybridisation in a buffer containing 50 mM KCI, 10 mM TRIS-HCI (pH 9,0), 0.1 % Triton X-100, 2 mM MgCI2.
  • Low stringency conditions denote hybridisation in a buffer constituting 1 M NaCl, 10 mM Na 3 PO 4 at pH 7,0, or a buffer similar to this having approximately the same impact on hybridization stringency.
  • homologously complementary nucleic acids, nucleic acid analogues, oligonucleotides or oligonucleotide analogues are nucleic acids, nucleic acid ana- logues, oligonucleotides or oligonucleotide analogues substantially complementary to each other over a given sequence, such as more than 70% complementary, for example more than 75% complementary, such as more than 80% complementary, for example more than 85% complementary, such as more than 90% complementary, for example more than 92% complementary, such as more than 94% comple- mentary, for example more than 95% complementary, such as more than 96% complementary, for example more than 97% complementary.
  • said given sequence is at least 4 nucleotides long, for example at least 10 nucleotides, such as at least 15 nucleotides, for example at least 20 nucleotides, such as at least 25 nucleotides, for example at least 30 nucleotides, such as between 10 and 500 nucleotides, for example between 4 and 100 nucleotides long, such as between 10 and 50 nucleotides long.
  • More preferably homologously complementary oligonucleotides or oligonucleotide analogues are substantially homologously complementary over their entire length.
  • the specificity of hybridisation of nucleic acids and/or nucleic acid analogues and/or oligonucleotides and/or oligonucleotide analogues refers to the ability of which said hybridisation event distinguishes between homologously complementary hybridisation partners according to their sequence differencies under given stringency conditions. Often it is the intention to target only one particular sequence (the target sequence) in a mixture of nucleic acids and/or nucleic acid analogues and/or oligonucleotides and/or oligonucleotide analogues and to avoid hybridization to other se- quences even though they have strong similarity to said target sequence. Some- times only one or few nucleotides differ among target and non-target sequences in the sequence-region used for hybridization.
  • High specificity in hybridisation denotes hybridisation under high stringency conditions at which an oligonucleotide or oligonucleotide analogue will hybridise with a homologous target sequence significantly better than to a nearly identical sequence differing only from said target sequence by one or few base- substitutions.
  • Discrimination refers to the ability of oligonucleotides and/or oligonucleotide analogues, in a sequence-independent manner, to hybridise preferentially with either RNA or DNA. Accordingly, the melting temperature of a hybrid consisting of oligonu- cleotide and/or oligonucleotide analogue and a homologously complementary RNA (RNA hybrid) is either significantly higher or lower than the melting temperature of a hybrid between said oligonucleotide and/or oligonucleotide analogue and a homologously complementary DNA (DNA hybrid).
  • RNA hybrid homologously complementary RNA
  • RNA-like refers to nucleic acid analogues or oligonucleotide analogues behaving like RNA with respect to hybridisation to homologously complementary oligonucleotides and/or oligonucleotide analogues comprising at least one internal pseudonu- cleotide. Accordingly, RNA-like nucleic acid analogues or oligonucleotide analogues can be functionally categorized on the basis of their ability to hybridise with oligonucleotides and/or oligonucleotide analogues able to discriminate between RNA and DNA.
  • said oligonucleotide analogues able to discriminate between RNA and DNA comprises one or more internally positioned pseudonucleotide inter- calators and consequently, said oligonucleotide analogue comprising pseudonucleotide intercalators will preferentially not hybridise to said RNA-like nucleic acid analogues or oligonucleotide analogues.
  • RNA-like molecules are RNA, 2'-O-methyl RNA, LNA, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Xylo-LNA, ⁇ -D-Ribo-LNA, [3.2.1]-LNA, 2'-R-RNA, 2'-OR-RNA, and mixtures thereof.
  • DNA-like refers to nucleic acid analogues or oligonucleotide analogues behaving like DNA with respect to hybridisation to homologously complementary nucleic acids and/or nucleic acid analogues. Accordingly, DNA-like nucleic acids or nucleic acid analogues can be functionally categorized on the basis of their ability to hybridise with oligonucleotides or oligonucleotide analogues able to discriminate between RNA and DNA.
  • said oligonucleotides or oligonucleotide analogues able to discriminate between RNA and DNA comprises one or more internally positioned pseudonucleotide intercalators, and consequently, said oligonucleotide analogue comprising pseudonucleotide intercalators will preferentially hybridise to said DNA-like nucleic acid analogues or oligonucleotide analogues.
  • DNA-like molecules is DNA and INA (Christensen, 2002. Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol: stabilisation of dsDNA and discrimination of DNA over RNA. Nucl. Acids. Res. 2002 30: 4918- 4925).
  • cross-hybridisation covers unattended hybridisation between at least two nucleic acids and/or nucleic acid analogues, i.e. cross-hybridisation may also be denoted intermolecular hybridisation.
  • cross-hybridization may be used to describe the hybridisation of for example a nucleic acid probe or nucleic acid analogue probe sequence to other nucleic acid sequences and/or nucleic acid analogue sequences than its intended target sequence.
  • cross-hybridization occurs between a probe and one or more homologously complementary non-target sequences, even though these have a lower degree of complementarity than the probe and its complementary target sequence. This un- wanted effect could be due to a large excess of probe over target and/or fast an- nealing kinetics.
  • Cross-hybridization also occurs by hydrogen bonding between few nucleobase pairs, e.g. between primers in a PCR reaction, resulting in primer dimer formation and/ or formation of unspecific PCR products.
  • nucleic acids comprising one or more nucleotide analogues with high affinity for nucleotide analogues of the same type tend to form dimer or higher order complexes based on base pairing.
  • probes comprising nucleotide analogues such as, but not limited to, DNA, RNA, 2'-O-methyl RNA, PNA, HNA, MNA, ANA, LNA, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Xylo-LNA, ⁇ -D-Ribo-LNA, [3.2.1]- LNA, 2'-R-RNA, 2'-OR-RNA, and mixtures thereof generally have a high affinity for hybridising to other oligonucleotide analogues comprising backbone monomer units of the same type. Hence even though individual probe molecules only have a low degree of complementarity, they tend to hybridise.
  • self-hybridisation covers the process wherein a nucleic acid or nucleic acid analogue molecule anneals to itself by folding back on itself, generating a secondary structure like for example a hairpin structure, i.e. self-hybridisation may also be defined as intramolecular hybridisation. In most applications it is of importance to avoid self-hybridization.
  • the generation of said secondary structures may inhibit hybridisation with desired nucleic acid target sequences. This is undesired in most assays for example when the nucleic acid or nucleic acid analogue is used as primer in PCR reactions or as fluorophore/ quencher labeled probe for exonuclease assays. In both assays self-hybridisation will inhibit hybridization to the target nucleic acid and additionally the degree of fluorophore quenching in the exonuclease assay is lowered.
  • nucleic acids comprising one or more nucleotide analogues with high affinity for nucleotide analogues of the same type tend to self-hybridise.
  • probes comprising nucleotide analogues such as, but not limited to, DNA, RNA, 2'-
  • Melting of nucleic acids refer to thermal separation of the two strands of a double- stranded nucleic acid molecule.
  • T m The melting temperature denotes the temperature in degrees centigrade at which 50% helical (hybridised) versus coil (unhybridised) forms are present.
  • a high melting temperature is indicative of a stable complex and accordingly of a high affinity between the individual strands.
  • a low melting temperature is indicative of a relatively low affinity between the individual strands. Accordingly, usually strong hydrogen bonding between the two strands results in a high melting temperature.
  • intercalation of an intercalator between nucleobases of a double stranded nucleic acid may also stabilise double stranded nucleic acids and accordingly result in a higher melting temperature.
  • the melting temperature is dependent on the physical/chemical state of the surroundings.
  • the melting temperature is dependent on salt concentration and pH.
  • the melting temperature may be determined by a number of assays, for example it may be determined by using the UV spectrum to determine the formation and breakdown (melting) of hybridisation.
  • the backbone monomer unit of a nucleotide or a nucleotide analogue or an intercalator pseudonucleotide according to the present invention is the part of the nucleo- tide, which is involved in incorporation of the nucleotide or nucleotide analogue or intercalator pseudonucleotide into the backbone of a nucleic acid or a nucleic acid analogue.
  • Any suitable backbone monomer unit may be employed with the present invention.
  • the backbone monomer unit of intercalator pseudonucleotides according to the present invention may be selected from the backbone monomer units mentioned herein below.
  • the backbone monomer unit comprises the part of a nucleotide or nucleotide analogue or intercalator pseudonucleotide that may be incorporated into the backbone of an oligonucleotide or an oligonucleotide analogue.
  • the backbone monomer unit may comprise one or more leaving groups, protecting groups and/or reactive groups, which may be removed or changed in any way during synthesis or subsequent to synthesis of an oligonucleotide or oligonucleotide analogue comprising said backbone monomer unit.
  • backbone monomer unit only includes the backbone monomer unit per se and it does not include for example a linker connecting a backbone monomer unit to an intercalator. Hence, the intercalator as well as the linker is not part of the backbone monomer unit.
  • backbone monomer units only include atoms, wherein the monomer is incorporated into a sequence, are selected from the group consisting of
  • atoms which are capable of forming a linkage to the backbone monomer unit of a neighboring nucleotide or b) atoms which at least at two sites are connected to other atoms of the backbone monomer unit; or c) atoms which at one site is connected to the backbone monomer unit and otherwise is not connected with other atoms
  • backbone monomer unit atoms are thus defined as the atoms involved in the direct linkage (shortest path) between the backbone Phosphor-atoms of neighbouring nucleotides, when the monomer is incorporated into a sequence, wherein the neighbouring nucleotides are naturally occurring nucleotides,.
  • the backbone monomer unit may be any suitable backbone monomer unit.
  • the backbone monomer unit may for example be selected from the group consisting of the backbone monomer units of DNA, RNA, PNA, HNA, MNA, ANA, LNA, CNA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, ⁇ -L- Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Xylo-LNA, ⁇ -D-Ribo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, ⁇ -Bicyclo-DNA, Tricyclo-DNA, Bicy- clo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, ⁇ -D-Ribopyranosyl- NA, ⁇ -
  • oligomers of DNA RNA & PNA
  • Cyclohexenyl-NA (CeNA) .
  • Ref Yamana, K. et al., Tetrahedron Lett., 1991, 63 7-6350. Ref: Sayer, J. et al., J. Org. Chem., 1991, 56, 20-29.
  • the backbone monomer unit of LNA (locked nucleic acid) is a sterically restricted DNA backbone monomer unit, which comprises an intramolecular bridge that restricts the usual conformational freedom of a DNA backbone monomer unit.
  • LNA may be any LNA molecule as described in WO 99/14226 (Exiqon), preferably, LNA is selected from the molecules depicted in the abstract of WO 99/14226. Preferred
  • LNA according to the present invention comprises a methyl linker connecting the 2'- O position to the 4'-C position, however other LNA's such as LNA's wherein the 2' oxy atom is replaced by either nitrogen or sulphur are also comprised within the present invention.
  • the backbone monomer unit of intercalator pseudonucleotides according to present invention preferably have the general structure before being incorporated into an oligonucleotide and/or nucleotide analogue:
  • R T is a trivalent or pentavalent substituted phosphoratom, preferably R ⁇ is
  • R 2 may individually be selected from an atom capable of forming at least two bonds, said atom optionally being individually substituted, preferably R 2 is individually selected from O, S, N, C, P, optionally individually substituted.
  • R 2 can represent one, two or more different groups in the same molecule.
  • the bonds between two R 2 may be saturated or unsaturated or a part of a ring system or a combination thereof
  • Each R 2 may individually be substituted with any suitable substituent, such as a substituent selected from H, lower alkyl, C2-6 alkenyl, C6-10 aryl, C7-11 arylmethyl,
  • alkyl group refers to an optionally substituted saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups.
  • the alkyl group has 1 to 25 carbons and contains no more than 20 heteroatoms. More preferably, it is a lower alkyl of from 1 to 12 carbons, more preferably 1 to 6 carbons, more preferably 1 to 4 carbons. Heteroatoms are preferably selected from the group consisting of nitrogen, sulfur, phosphorus, and oxygen.
  • alkenyl group refers to an optionally substituted hydrocarbon containing at least one double bond, including straight-chain, branched-chain, and cyclic alkenyl groups, all of which may be optionally substituted.
  • the alkenyl group has 2 to 25 carbons and contains no more than 20 heteroatoms. More preferably, it is a lower alkenyl of from 2 to 12 carbons, more preferably 2 to 4 carbons. Heteroatoms are preferably selected from the group consisting of nitrogen, sulfur, phosphorus, and oxygen.
  • alkynyl refers to an optionally substituted unsaturated hydrocarbon containing at least one triple bond, including straight-chain, branched-chain, and cyclic alkynyl groups, all of which may be optionally substituted.
  • the alkynyl group has 2 to 25 carbons and contains no more than 20 heteroatoms. More preferably, it is a lower alkynyl of from 2 to 12 carbons, more preferably 2 to 4 carbons. Heteroatoms are preferably selected from the group consisting of nitrogen, sulfur, phosphorus, and oxygen.
  • aryl refers to an optionally substituted aromatic group having at least one ring with a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl, biaryl, and triaryl groups.
  • aryl substitution substituents include alkyl, alkenyl, alkynyl, aryl, amino, substituted amino, carboxy, hydroxy, alkoxy, nitro, sul- fonyl, halogen, thiol and aryloxy.
  • a “carbocyclic aryl” refers to an aryl where all the atoms on the aromatic ring are carbon atoms. The carbon atoms are optionally substituted as described above for an aryl. Preferably, the carbocyclic aryl is an optionally substituted phenyl.
  • heterocyclic aryl refers to an aryl having 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. Examples of heterocyclic aryls in- elude furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, and imidazolyl. The heterocyclic aryl is optionally substituted as described above for an aryl.
  • the substituents on two or more R 2 may alternatively join to form a ring system, such as any of the ring systems as defined above.
  • R 2 is substituted with an atom or a group selected from H, methyl, P , hydroxyl, halogen, and amino, more preferably R 2 is substituted with an atom or a group selected from H, methyl, R 4 .
  • R 2 is individually selected from O, S, NH, N(Me), N(R 4 ), C(R 4 ) 2 , CH(R 4 ) or CH 2 , wherein R 4 is as defined below,
  • R 3 methyl, beta-cyanoethyl, p-nitrophenetyl, o-chlorophenyl, or p-chlorophenyl.
  • R 4 lower alkyl, preferably lower alkyl such as methyl, ethyl, or isopropyl, or heterocyclic, such as morpholino, pyrrolidino, or 2,2,6,6-tetramethylpyrrolidino, wherein lower alkyl is defined as Ci - C 6 , such as C T - C 4 .
  • R 6 is a protecting group, selected from any suitable protecting groups.
  • R 6 is selected from the group consisting of trityl, monomethoxytrityl, 2-chlorotrityl, 1 ,1 ,1 ,2-tetrachloro-2,2-bis(p-methoxyphenyl)-ethan (DATE), 9-phenylxanthine-9-yl (pixyl) and 9-(p-methoxyphenyl) xanthine-9-yl (MOX) or other protecting groups mentioned in "Current Protocols In Nucleic Acid Chemistry" volume 1 , Beaucage et al. Wiley.
  • the protecting group may be selected from the group consisting of monomethoxytrityl and dimethoxytrityl.
  • the protecting group may be 4, 4'-dimethoxytrityl (DMT).
  • R 9 is selcted from O, S, N optionally substituted, preferably R 9 is selected from O, S, NH, N(Me).
  • R 10 is selected from O, S, N, C, optionally substituted.
  • X 2 Cl, Br, I, N(R 4 ) 2 , or O "
  • the backbone monomer unit can be acyclic or part of a ring system.
  • the backbone monomer unit of an intercalator pseudonucleotide is selected from the group consisting of acyclic backbone monomer units.
  • Acyclic is meant to cover any backbone monomer unit, which does not comprise a ringstructure, for example the backbone monomer unit preferably does not comprise a ribose or a deoxyribose group.
  • the backbone monomer unit of an intercalator pseudonucleotide is an acyclic backbone monomer unit, which is capable of stabilising a bulge insertion (see herein below).
  • the backbone monomer unit of an intercalator pseudonucleotide according to the present invention may be selected from the group consisting of backbone monomer units comprising at least one chemical group selected from the group consisting of trivalent and pentavalent phosphorous atom such as a pentavalent phosphorous atom. More preferably the phosphate atom of the backbone monomer unit of an intercalator pseudonucleotide according to the present invention may be selected from the group consisting of backbone monomer units comprising at least one chemical group selected from the group consisting of, phosphoester, phosphodiester, phosphoramidate and phosphoramidit groups.
  • the backbone monomer unit of an intercalator pseudonucleotide according to the present invention is selected from the group consisting of acyclic backbone monomer units comprising at least one chemical group selected from the group consisting of phosphate, phosphoester, phosphodiester, phosphoramidate and phosphoramidit groups.
  • Preferred backbone monomer units comprising at least one chemical group selected from the group consisting of phosphate, phosphoester, phosphodiester, phosphoramidate and phosphoramidit groups are backbone monomer units, wherein the distance from at least one phosphor atom to at least one phosphor atom of a neighbouring nucleotide, not including the phosphor atoms, is at the most 6 atoms long, for example 2, such as 3, for example 4, such as 5, for example 6 atoms long, when the backbone monomer unit is incorporated into a nucleic acid backbone.
  • the distance is measured as the direct linkage (i.e. the shortest path) as discussed above.
  • the backbone monomer unit is capable of being incorporated into a phosphate backbone of a nucleic acid or nucleic acid analogue in a manner so that at the most 5 atoms are separating the phosphor atom of the intercalator pseudonucleotide backbone monomer unit and the nearest neighbouring phosphor atom, more preferably 5 atoms are separating the phosphor atom of the intercalator pseudonucleotide backbone monomer unit and the nearest neighbouring phosphor atom, in both cases not including the phosphor atoms themselves.
  • the backbone monomer unit is capable of being incorporated into a phosphate backbone of a nucleic acid or nucleic acid analogue in a manner so that at the most 4 atoms are separating the phosphor atom of the intercalator pseudonucleotide backbone monomer unit and the nearest neighbouring phosphor atom, more preferably 4 atoms are separating the phosphor atom of the intercalator pseudonucleotide backbone monomer unit and the nearest neighbouring phosphor atom, in both cases not including the phosphor atoms themselves.
  • the intercalator pseudonucleotide comprises a backbone monomer unit that comprises a phosphoramidit and more preferably the backbone monomer unit comprises a trivalent phosphoramidit.
  • Suitable trivalent phosphoramidits are trivalent phosphoramidits that may be incorporated into the backbone of a nucleic acid and/or a nucleic acid analogue.
  • the amidit group per se may not be incorporated into the backbone of a nucleic acid, but rather the amidit group or part of the amidit group may serve as a leaving group and/ or protecting group.
  • the backbone monomer unit comprises a phosphoramidit group, because such a group may facilitate the incorporation of the backbone monomer unit into a nucleic acid backbone.
  • acyclic backbone monomers may be selected from one of the general structures depicted below:
  • R R 2 and R 6 are as defined above.
  • acyclic backbone monomer unit may be selected from the group depicted below:
  • backbone monomer units numbered I) to wherein Ri and R 6 are as defined above, and R 8 may be R or H, optionally substituted.
  • the backbone monomer unit including optional protecting groups may be selected from the group consisting of the structures I) to XLIV) as indicated herein below:
  • N-Rg R N.
  • N-Rg R N, N-Rg R ⁇ -N N / N-R 6
  • backbone monomer units selected from the group consisting of:
  • the acyclic backbone monomer unit may be selected from the group consisting of the structures a) to g) as indicated below:
  • the backbone monomer unit of an intercalator pseudonucleotide which is inserted into an oligonucleotide or oligonucleotide analogue, according to the present inven- tion may comprise a phosphodiester bond. Additionally, the backbone monomer unit of an intercalator pseudonucleotide according to the present invention may comprise a pentavalent phosphoramidate. Preferably, the backbone monomer unit of an intercalator pseudonucleotide according to the present invention is an acyclic backbone monomer unit that may comprise a pentavalent phosphoramidate.
  • the backbone monomer unit according to the present invention may comprise one or more leaving groups.
  • Leaving groups are chemical groups, which are part of the backbone monomer unit when the intercalator pseudonucleotide or the nucleotide is a monomer, but which are no longer present in the molecule once the intercalator pseudonucleotide or the nucleotide has been incorporated into an oligonucleotide or oligonucleotide analogue.
  • the nature of a leaving group depends of the backbone monomer unit. For example, when the backbone monomer unit is a phosphor amidit, the leaving group, may for example be an diisopropylamine group.
  • the backbone monomer unit is a phosphor amidit
  • a leaving group is attached to the phosphor atom for example in the form of diisopropylamine and said leaving group is removed upon coupling of the phosphor atom to a nucleophilic group, whereas the rest of the phosphate group, may become part of the nucleic acid or nucleic acid analogue backbone.
  • the backbone monomer units according to the present invention may furthermore comprise a reactive group which is capable of performing a chemical reaction with another nucleotide or oligonucleotide or nucleic acid or nucleic acid analogue to form a nucleic acid or nucleic acid analogue, which is one nucleotide longer than before the reaction.
  • nucleotides when they are in their free form, i.e. not incorporated into a nucleic acid, they may comprise a reactive group capable of reacting with another nucleotide or a nucleic acid or nucleic acid analogue.
  • said reactive group may be protected by a protecting group. Prior to said chemical reaction, said protection group may be removed. The protection group will thus not be a part of the newly formed nucleic acid or nucleid acid analogue.
  • reactive groups are nucleophiles such as the 5'-hydroxy group of DNA or RNA backbone monomer units.
  • the backbone monomer unit according to the present invention may also comprise a protecting group, which can be removed, and wherein removal of the protecting group allows for a chemical reaction between the intercalator pseudonucleotide and a nucleotide or nucleotide analogue or another intercalator pseudonucleotide.
  • a nucleotide monomer or nucleotide analogue monomer or intercalator pseudonucleotide monomer may comprise a protecting group, which is no longer present in the molecule once the nucleotide or nucleotide analogue or intercalator pseudonucleotide has been incorporated into a nucleic acid or nucleic acid analogue.
  • backbone monomer units may comprise protecting groups which may be present in the oligonucleotide or oligonucleotide analogue subsequent to incorporation of the nucleotide or nucleotide analogue or intercalator pseudonucleotide, but which may no longer be present after introduction of an additional nucleotide or nucleotide analogue to the oligonucleotide or oligonucleotide analogue or which may be removed after the synthesis of the entire oligonucleotide or oligonucleotide analogue.
  • the protecting group may be removed by a number of suitable techniques known to the person skilled in the art, however preferably, the protecting group may be removed by a treatment selected from the group consisting of acid treatment, thiophenol treatment and alkali treatment.
  • Preferred protecting groups according to the present invention which may be used to protect the 5' end or the 5' end analogue of a backbone monomer unit may be selected from the group consisting of trityl, monomethoxytrityl, 2-chlorotrityl, 1 ,1 ,1 ,2- tetrachloro-2,2-bis(p-methoxyphenyl)-ethan (DATE), 9-phenylxanthine-9-yl (pixyl) and 9-(p-methoxyphenyl) xanthine-9-yl (MOX) or other protecting groups mentioned in "Current Protocols In Nucleic Acid Chemistry" volume 1 , Beaucage et al. Wiley. More preferably the protecting group may be selected from the group consisting of monomethoxytrityl and dimethoxytrityl. Most preferably, the protecting group may be 4, 4'-dimethoxytrityl(DMT).
  • 4, 4'-dimethoxytrityl(DMT) groups may be removed by acid treatment, for example by brief incubation (30 to 60 seconds sufficient) in 3% trichloroacetic acid or in 3% dichlororacetic acid in CH 2 CI 2 .
  • Preferred protecting groups which may protect a phosphate or phosphoramidit group of a backbone monomer unit may for example be selected from the group consisting of methyl and 2-cyanoethyl.
  • Methyl protecting groups may for example be removed by treatment with thiophenol or disodium 2-carbamoyl 2-cyanoethylene- 1 ,1-dithiolate.
  • 2-cyanoethyl-groups may be removed by alkali treatment, for example treatment with concentrated aqueous ammonia, a 1 :1 mixture of aqauos methylamine and concentrated aqueous ammonia or with ammonia gas.
  • intercalator covers any molecular moiety comprising at least one essentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid.
  • an intercalator according to the present invention essentially consists of at least one essentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid or nucleic acid analogue.
  • the intercalator comprises a chemical group selected from the group consisting of polyaromates and heteropolyaromates an even more preferably the intercalator essentially consists of a polyaromate or a heteropolyaromate. Most preferably the intercalator is selected from the group consisting of polyaromates and heteropolyaromates.
  • Polyaromates or heteropolyaromates according to the present invention may consist of any suitable number of rings, such as 1 , for example 2, such as 3, for example 4, such as 5, for example 6, such as 7, for example 8, such as more than 8.
  • polyaromates or heteropolyaromates may be substituted with one or more selected from the group consisting of hydroxyl, bromo, fluoro, chloro, iodo, mer- capto, thio, cyano, alkylthio, heterocycle, aryl, heteroaryl, carboxyl, carboalkoyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl and amido.
  • the intercalator may be selected from the group consisting of polyaromates and heteropolyaromates that are capable of fluorescing. In another more preferred embodiment of the present invention the intercalator may be selected from the group consisting of polyaromates and heteropolyaromates that are capable of forming excimers, exciplexes, fluorescence resonance energy transfer (FRET) or charged transfer complexes.
  • FRET fluorescence resonance energy transfer
  • the intercalator may preferably be selected from the group consisting of phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, napthene, phenanthrene, picene, chrysene, naphtacene, acridones, benzanthra- cenes, stilbenes, oxalo-pyridocarbazoles, azidobenzenes, porphyrins, psoralens and any of the aforementioned intercalators substituted with one or more selected from the group consisting of hydroxyl, bromo, fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, heterocycle, aryl, heteroaryl, carboxyl, carboalkoyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl and/or amido
  • the intercalator is selected from the group consisting of phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, napthene, phenanthrene, picene, chrysene, naphtacene, acridones, benzanthracenes, stilbenes, oxalo-pyridocarbazoles, azidobenzenes, porphyrins and psoralens.
  • intercalator may be selected from the group of intercalators comprising one of the structures as indicated herein below:
  • the intercalator may be selected from the group of intercalators comprising one of the intercalator structures above numbered V, XII, XIV, XV, XVII, XXIII, XXVI, XXVIII, XLVII, Ll and LH as well as derivatives thereof.
  • interacalator is selected from the group of intercalator structures above numbered XII, XIV, XVII, XXIII, Ll.
  • intercalator moiety of the intercalator pseudonucleotide is linked to the backbone unit by the linker.
  • the linker and intercalator connection is defined as the bond be- tween a linker atom and the first atom being part of a conjugated system that is able to co-stack with nucleobases of a strand of a oligonucleotide or oligonucleotide analogue when said oligonucleotide or oligonucleotide analogue is hybridised to an oligonucleotide analogue comprising said intercalator pseudonucleotide.
  • the linker may comprise a conjugated system and the intercalator may comprise another conjugated system.
  • the linker conjugated system is not capable of costacking with nucleobases of said opposite oligonucleotide or oligonucleotide analogue strand.
  • the linker of a intercalator pseudonucleotide according to the present invention is a moiety connecting the intercalator and the backbone monomer of said intercalator pseudonucleotide.
  • the linker may comprise one or more atom(s) or bond(s) between atoms.
  • the linker is the shortest path linking the backbone and the intercalator. If the intercalator is linked directly to the backbone, the linker is a bond.
  • the linker usually consists of a chain of atoms or a branched chain of atoms. Chains can be saturated as well as unsaturated.
  • the linker may also be a ring structure with or without conjugated bonds.
  • the linker may comprise a chain of m atoms selected from the group consisting of C, O, S, N. P, Se, Si, Ge, Sn and Pb, wherein one end of the chain is connected to the intercalator and the other end of the chain is connected to the backbone monomer unit.
  • the total length of the linker and the intercalator of the intercalator pseudonucleotides according to the present invention preferably is between 8 and 13 A (see herein below). Accordingly, m should be selected dependent on the size of the intercalator of the specific intercalator pseudonucleotide.
  • m should be relevatively large, when the intercalator is small and m should be relatively small when the intercalator is large.
  • m will be an integer from 1 to 7, such as from 1-6, such as from 1-5, such as from 1-4.
  • the linker may be an unsaturated chain or another system involving conjugated bonds.
  • the linker may comprise cyclic conjugated structures.
  • m is from 1 to 4 when the linker is an saturated chain.
  • m is preferably an integer from 1 to 7, such as from 1-6, such as from 1-5, such as from 1-4, more preferably from 1 to 4, even more preferably from 1 to 3, most preferably m is 2 or 3.
  • the intercalator has the m is preferably from 2 to 6, more preferably 2.
  • the chain of the linker may be substituted with one or more atoms selected from the group consisting of C, H, O, S, N, P, Se, Si, Ge, Sn and Pb.
  • the linker is an azaalkyl, oxaalkyl, thiaalkyl or alkyl chain.
  • the linker may be an alkyl chain substituted with one or more selected from the group consisting C, H, O, S, N, P, Se, Si, Ge, Sn and Pb.
  • the linker consists of an unbranched alkyl chain, wherein one end of the chain is connected to the intercalator and the other end of the chain is connected to the backbone monomer unit and wherein each C is substituted with 2 H.
  • said unbranched alkyl chain is from 1 to 5 atoms long, such as from 1 to 4 atoms long, such as from 1 to 3 atoms long, such as from 2 to 3 atoms long.
  • the linker is a ring structure comprising atoms selected from the group consisting of C, O, S, N, P, Se, Si, Ge, Sn and Pb.
  • the linker may be such a ring structure substituted with one or more selected from thegroup consisting of C, H, O, S, N, P, Se, Si, Ge, Sn and Pb.
  • the linker consists of from 1-6 C atoms, from 0-3 of each of the following atoms O, S, N. More preferably the linker consists of from 1-6 C atoms and from 0-1 of each of the atoms O, S, N.
  • the linker consists of a chain of C, O, S and N atoms, optionally substituted.
  • said chain should consist of at the most 3 atoms, thus comprising from 0 to 3 atoms selected individually from C, O, S, N, optionally substituted.
  • the linker consists of a chain of C, N, S and O atoms, wherein one end of the chain is connected to the intercalator and the other end of the chain is connected to the backbone monomer unit.
  • such a chain comprise one of the linkers shown below, most preferably the linker consist of one of the molecule shown below:
  • the chain comprise one of the linkers shown below, more preferably the linker consist of one of the molecule shown below:
  • the chain comprise one of th linkers shown below, more preferably the linker consist of one of the molecule shown below:
  • the linker constitutes Y in the formula for the intercalator pseudonucleotide X-Y-Q, as defined above, and hence X and Q are not part of the linker.
  • Intercalator pseudonucleotides according to the present invention preferably have the general structure
  • X is a backbone monomer unit capable of being incorporated into the backbone of a nucleic acid or nucleic acid analogue
  • Q is an intercalator comprising at least one essentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid ;
  • Y is a linker moiety linking said backbone monomer unit and said intercalator
  • the intercalator pseudonucleotide comprises a backbone monomer unit, wherein said backbone monomer unit is capable of being incorporated into the phosphate backbone of a nucleic acid or nucleic acid analogue in a manner so that at the most 4 atoms are separating the two phosphor atoms of the backbone that are closest to the intercalator.
  • intercalator pseudonucleotides preferably do not comprise a nucleobase capable of forming Watson-Crick hydrogen bonding. Hence intercalator pseudonucleotides according to the invention are preferably not capable of Watson-Crick base pairing.
  • the total length of Q and Y is in the range from 7 A to 20 A, more preferably, from 8 A to 15 A, even more preferably from 8 A to 13 A, even more preferably from 8.4 A to 12 A, most preferably from 8.59 A to 10 A or from 8.4 A to 10.5 A.
  • the total length of Q and Y is preferably in the range of 8 A to 13 A, such as from 9 A to 13 A, more preferably from 9.05 A to 11 A, such as from 9.0 A to 11 A, even more preferably from 9.05 to 10 A, such as from 9,0 to
  • the total length of the linker (Y) and the intercalator (Q) should be determined by determining the distance from the center of the non-hydrogen atom of the linker which is furthest away from the intercalator to the center of the non-hydrogen atom of the essentially flat, conjugated system of the intercalator that is furthest away from the backbone monomer unit.
  • the distance should be the maximal distance in which bonding angles and normal chemical laws are not broken or distorted in any way.
  • the distance should preferably be determined by calculating the structure of the free intercalating pseudonucleotide with the lowest conformational energy level, and then determining the maximum distance that is possible from the center of the non- hydrogen atom of the linker which is furthest away from the intercalator to the center of the non-hydrogen atom of the essentially flat, conjugated system of the intercalator that is furthest away from the backbone monomer unit without bending, stretching or otherwise distorting the structure more than simple rotation of bonds that are free to rotate (e.g. not double bonds or bonds participating in a ring structure).
  • the energetically favorable structure is found by ab initio or forcefields calculations.
  • the distance should be determined by a method consisting of the following steps:
  • the structure of the intercalator pseudonucleotide of interest is drawn by computer using the programme ChemWindow® 6.0 (BioRad); and b) the structure is transferred to the computer programme SymAppsTM (BioRad); and c) the 3-dimensional structure comprising calculated lengths of bonds and bonding angles of the intercalator pseudonucleotide is calculated using the computer programme SymAppsTM (BioRad); and d) the 3 dimensional structure is transferred to the computer programme RasWin Molecular Graphics Ver. 2.6-ucb; and e) the bonds are rotated using RasWin Molecular Graphics Ver. 2.6-ucb to obtain the maximal distance (the distance as defined herein above); and f) the distance is determined.
  • intercalator pseudonucleotide has the following structure:
  • the total length of Q and Y is determined by measuring the linear distance from the center of the atom at A to the center of the atom at B, which in the above example is 9,79 A.
  • intercalator pseudonucleotide has the following structure:
  • the total length of Q and Y, which is measured in a straight line from the center of the atom at A to the center of the atom at B is 8.71 A.
  • Intercalator pseudonucleotides according to the present invention may be any combination of the above mentioned backbone monomer units, linkers and intercalators.
  • the intercalator pseudonucleotide is selected from the group consisting of intercalator pseudonucleotides with the structures 1) to 9 as indicated herein below:
  • the intercalator pseudonucleotide is selected from the group consisting of phosphoramidits of 1- (4,4'-dimethoxytriphenylmethyloxy)-3-pyrenemethyloxy-2-propanol. Even more preferably, the intercalator pseudonucleotide is selected from the group consisting of the phosphoramidit of (S)-1-(4,4'-dimethoxytriphenylmethyloxy)-3-pyrenemethyloxy- 2-propanol and the phosphoramidit of (R)-1-(4,4'-dimethoxytriphenylmethyloxy)-3- pyrenemethyloxy-2-propanol .
  • intercalator pseudonucleotides according to the present invention may be synthesised by any suitable method. However preferably the method may comprise the steps of
  • a1) providing a compound containing an intercalator comprising at least one es- sentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid and optionally a linker part coupled to a reactive group;
  • linker precursor molecule comprising at least two reactive groups, said two reactive groups may optionally be individually protected;
  • d1) providing a backbone monomer precursor unit comprising at least two reactive groups, said two reactive groups may optionally be individually protected and/or masked) and optionally comprising a linker part;
  • linker precursor molecule comprising at least two reactive groups, said two reactive groups may optionally be individually protected;
  • d2) providing a compound containing an intercalator comprising at least one es- sentially flat conjugated system, which is capable of co-stacking with nucleo- bases of a nucleic acid and optionally a linker part coupled to a reactive group;
  • a3 providing a compound containing an intercalator comprising at least one es- sentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid and a linker part coupled to a reactive group;
  • b3) providing a backbone monomer precursor unit comprising at least two reactive groups, said two reactive groups may optionally be individually protected and/or masked), and a linker part;
  • the intercalator reactive group is selected so that it may react with the linker reactive group.
  • the linker reactive group is a nucleophil
  • the intercalator reactive group is an electrophile, more preferably an electrophile selected from the group consisting of halo alkyl, mesyloxy alkyl and tosyloxy alkyl. More preferably the intercalator reactive group is chloromethyl.
  • the intercalator reactive group may be a nucleophile group for example a nucleophile group comprising hydroxy, thiol, selam, amine or mixture thereof.
  • the cyclic or non cyclic alkane may be a polysubstituted alkane or alkoxy comprising at least three linker reactive groups. More preferably the polysubstituted alkane may comprise three nucleophilic groups such as, but not limited to, an alkane thole, an aminoalkan diol or mercaptoalkane diol. Preferably the polysubstituted alkane contain one nucleophilic group that is more reactive than the others, alternatively two of the nucleophilic groups may be protected by a protecting group.
  • the cyclic or non cyclic alkane is 2,2-dimethyl-4-methylhydroxy-1 ,3- dioxalan, even more preferably the alkane is D- ⁇ , ⁇ -isopropylidene glycerol .
  • the linker reactive groups should be able to react with the intercalator reactive groups, for example the linker reactivegroups may be a nucleophile group for example selected from the group consisting of hydroxy, thiol, selam and amine, preferably a hyhroxy group.
  • the linker reactive group may be an electrophile group, for example selected from the group consisting of halogen, triflates, mesylates and tosylates.
  • at least 2 linker reactive groups may be protected by a protecting group.
  • the method may furthemore comprise a step of attaching a protecting group to one or more reactive groups of the intercalator-precursor monomer.
  • a DMT group may be added by providing a DMT coupled to a halogen, such as Cl, and reacting the DMT-CI with at least one linker reactive group. Accordingly, preferably at least one linker reactive group will be available and one protected. If this step is done prior to reaction with the phosphor comprising agent, then the phosphor comprising agent may only interact with one linker reactive group.
  • the phoshphor comprising agent may for example be a phosphoramidit, for example NC(CH 2 ) 2 OP(Npr i 2 ) 2 or NC(CH 2 ) 2 OP(Npr i 2 )CI.
  • the phosphor comprising agent may be reacted with the intercalator-precursor in the presence of a base, such as N(et) 3 , N('pr) 2 Et and CH 2 CI 2 .
  • a base such as N(et) 3 , N('pr) 2 Et and CH 2 CI 2 .
  • oligonucleotide or oligonucleotide analogue are preferably chemically synthesised using commercially available methods and equipment:
  • the solid phase phosphoramidite method can be used to produce short oligonucleotide or oligonucleotide analogue comprising intercalator pseudonucleotides.
  • oligonucleotides or oligonucleotide analogues may be synthesised by any of the methods described in "Current Protocols in Nucleic acid Chemistry” Volume 1 , Beaucage et al., Wiley.
  • an intercalator pseudonucleotide according to the invention comprising a reactive group, which may be protected by an acid la- bile protection group into contact with a growing chain of a support- bound oligonucleotide or oligonucleotide analogue; and b. reacting said intercalator pseudonucleotide with said support-bound oligonucleotide or oligonucleotide analogue; and c. washing away excess reactants from product on the support; and d. optionally capping unreacted said support-bound oligonucleotide; and e. oxidizing the phosphite product to phosphate product; and f.
  • step c) bringing an intercalator pseudonucleotide according to the present invention into contact with an universal support; and b) reacting said intercalator pseudonucleotide with the universal support; followed by step c) to j) as described in the method herein above.
  • the last acid labile protection group may be removed prior to cleavage of the support-bound oligonucleotide analogue. Subsequent purification of the oligonucleotide analogue is optional.
  • the method comprises the synthesis an oligonucleotide or oligonucleotide analogue comprising at least one internally positioned intercalator pseudonucleotide, wherein synthesis may comprise the steps of
  • nucleotide or nucleotide analogue protected with an acid labile protection group into contact with a growing chain of a support-bound nucleotide, oligonucleotide, nucleotide analogue or oligonucleotide analogue; and b) reacting the protected nucleotide analogue with the growing chain of said support-bound nucleotide, oligonucleotide, nucleotide analogue or oligonucleotide analogue; and c) washing away excess reactants from product on support; and d) optionally capping unreacted said support-bound nucleotide; and e) oxidizing the phosphite product to phosphate product; and f) washing away excess reactants from product on support; and g) optionally capping unreacted said support-bound nucleotide; and h) removing acid labile protecting group; and i) washing away excess reactants from product
  • the last acid labile protection group may be removed prior to cleavage of the support-bound oligonucleotide analogue. Purification of the oligonucleotide analogue is optional.
  • template refers to a nucleic acid or nucleic acid analogue.
  • a template comprises a nucleotide sequence that corresponds to a specific oligonucleotide or oligonucleotide analogue. If said sequence comprised within said template is internally positioned, hybridization between said template and said oligonucleotide or oligonucleotide analogue will create a 3' free end of said oligonucleotide or oligonucleotide analogue.
  • This 3' free end is capable of being extended by a for example a DNA polymerase in a template directed manner and said oligonucleotide or oligonucleotide analogue is thus said to prime extension.
  • the term "primer” therefore covers a oligonucleotide or oligonucleotide analogue that is able to prime template directed extension by DNA and RNA polymerases.
  • Linear oligonucleotides comprising intercalator pseudonucleotides
  • the present invention relates to an oligonucleotide and/or oligonucleotide analogue comprising at least one intercalator pseudonucleotide of the general structure
  • X is a backbone monomer unit capable of being incorporated into the phosphate backbone of a nucleic acid
  • Q is an intercalator comprising at least one essentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid;
  • Y is a linker moiety linking said backbone monomer unit and said intercalator
  • the oligonucleotide comprises a first sequence consisting of n nucleotides and/or nucleotide analogues and/ or intercalator pseudonucleotides and a second nucleotide sequence consisting of m nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides; and wherein the second sequence is capable of hybridizing to a homologously complementary target sequence comprised within a nucleic acid or nucleic acid analogue and is capable of priming a template directed extension reaction.
  • the at least one intercalator pseudonucleotide may be inserted at any desired position within the first and/ or second sequence. If the oligonucleotide analogue comprises more than one intercalator pseudonucleotides, preferably every two intercalator pseudonucleotides are separated by at least one nucleotide or nucleotide analogue.
  • the intercalator pseudonucleotide may be any of the intercalator pseudonucleotides described herein above.
  • the first sequence is attached as a tail sequence to the second sequence and meant to be rather inactive in sequence specific hybridisation.
  • the second sequence alone is preferentially capable of hybridizing to a target nucleic acid or nucleic acid analogue, and the first sequence preferentially contains neighboring intercalator pseudonucleotides according to the present invention.
  • the first sequence preferably consists of n nucleotides and/or nucleotide analogues and intercalator pseudonucleotides, wherein n may be any suitable integer.
  • n is an integer in the range from 1 to 150, such as from 1 to 50, such as from 5 to 50, such as from to 20, for example n is an integer in the range from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as from 20 to 30, for example from 30 to 50.
  • the second sequence preferably consists of m nucleotides and/or nucleotide analogues and/ or intercalator pseudonucleotides, wherein m may be any suitable integer.
  • m is an integer in the range from 1 to 150, such as from 1 to 50, such as from 5 to 50, such as from to 20, for example n is an integer in the range from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as from 20 to 30, for example from 30 to 50.
  • the oligonucleotide analogue comprises more than one intercalator pseudonucleotides, preferably every two intercalator pseudonucleotides are separated by at least one nucleotide or nucleotide analogue.
  • n is smaller than m. However it is also contained within the present invention that m is equal to n.
  • the first and second sequence of the oligonucleotide analogues according to the present invention may comprise any desirable number of intercalator pseudonucleotides.
  • the oligonucleotide or oligonucleotide analogue may comprise from 2 to 20, such as from 2 to 10, such as from 2 to 5, such as between 5 and 10, such as between 10 and 15, for example between 15 and 20 intercalator pseudonucleotides.
  • said first sequence is not merely a tail attached to said second sequence.
  • the intercalator pseudonucleotides may be placed in any desirable position within the first and the second sequence.
  • an intercalator pseudonucleotide may be placed at either end of the first and second sequence or an intercalator pseudonucleotide may be placed in an internal position within the first and second sequence.
  • the first sequence comprises at least two closely positioned intercalator pseudonucleotides.
  • the oligonucleotide analogue comprises more than one intercalator pseudonucleotide
  • said pseudonucleotides may be placed in relation to each other in any desirable manner. For example, they may be placed so that 0, such as 1 , for example 2, such as 3, for example 4, such as 5, for example from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as more than 20 nucleotides and/ or nucleotide analogues are separating the intercalator pseudonucleotides.
  • the oligonucleotide analogue comprises at least one pair of intercalator pseudonucleotides capable of forming an intramolecular excimer, intramolecular exciplex, a FRET complex and/or a charge-transfer complex.
  • the first sequence is positioned upstream of said second sequence. It is furthermore preferred that said first sequence comprises at least one pair of intercalator pseudonucleotides capable of forming an intramolecular excimer, intramolecular exciplex, a FRET complex and/or a charge-transfer complex.
  • an oligonucleotide analogue comprises said first and second sequence, where said second sequence comprises only one intercalator pseudonucleotide positioned in the 5' end and where said second sequence is able to hybridize to a homologously complementary target sequence and prime template directed extension.
  • the first and second sequence of the oligonucleotide analogue may comprise more than one intercalator pseudonucleotide, wherein said intercalator pseudonucleotides may be similar or may be different. If the oligonucleotide analogue comprises more than one intercalator pseudonucleotides, preferably every two intercalator pseudonucleotides are separated by at least one nucleotide or nucleotide analogue.
  • the oligonucleotide analogues may comprise nucleotides comprised within DNA, RNA, LNA, PNA, ANA, MNA, 2'-O-methyl RNA and HNA. Accordingly, the oligonucleotide analogue may comprise one or more selected from the group consisting of subunits of DNA, RNA, LNA, PNA, ANA, MNA, 2'-O-methyl RNA and HNA, i.e. the oligonucleotide analogue may be selected from the group of DNA, RNA, LNA, PNA, ANA, MNA, 2'-O-methyl RNA, HNA and mixtures thereof.
  • the second sequence of an oligonucleotide analogue comprising a first and a second sequence according to the present invention is capable of hybridizing to a homologously complementary target sequence under high-stringency conditions.
  • said second sequence of said oligonucleotide analogue is only capable of hybridizing to a complementary target sequence under high-stringency conditions.
  • Hairpin oligonucleotides comprising intercalator pseudonucleotides
  • the present invention relates to an oligonucleotide analogue comprising at least one intercalator pseudonucleotide of the general structure X-Y-Q
  • X is a backbone monomer unit capable of being incorporated into the phosphate backbone of a nucleic acid
  • Q is an intercalator comprising at least one essentially flat conjugated system, which is capable of co-stacking with nucleobases of a nucleic acid ;
  • Y is a linker moiety linking said backbone monomer unit and said intercalator
  • the oligonucleotide comprises a first sequence consisting of n nucleotides and/or nucleotide analogues and intercalator pseudonucleotides and a second nucleotide sequence consisting of m nucleotides and/or nucleotide analogues and/ or intercalator pseudonucleotides, wherein said first sequence is capable of hybridizing to said second sequence.
  • the first sequence constitutes a sequence part of the oligonucleotide analogue and the second sequence constitutes another sequence part of the oligonucleotide analogue.
  • the first and the second sequence may be separated by a third consisting of p nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides.
  • the second and third sequence are capable of hybridizing to a homologously complementary target sequence comprised within a nucleic acid or nucleic acid analogue and are capable of priming a template directed extension reaction.
  • the at least one intercalator pseudonucleotide may be inserted at any desired position within the first, second and/or third sequence. Preferably, if more than one intercalator pseudonucleotides are present, every two intercalator pseudonucleotides are separated by at least one nucleotide or nucleotide analogue.
  • the intercalator pseudonucleotide may be any of the intercalator pseudonucleotides described herein above.
  • the first sequence preferably consists of n nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides, wherein n may be any suitable integer.
  • n is an integer in the range from 5 to 50, for example n is an integer in the range from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as from 20 to 30, for example from 30 to 50.
  • the second sequence preferably consists of m nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides, wherein m may be any suitable integer.
  • m is an integer in the range from 5 to 50, for example n is an integer in the range from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as from 20 to 30, for example from 30 to 50.
  • the first sequence may hybridize with the second sequence essentially over the entire length of the first sequence and the second sequence, even more preferably nucleotides and nucleotide analogues in the duplex are essentially complementary.
  • oligonucleotide analogues according to the present invention should as a minimum be as long as the first sequence and the second sequence together, however the oligonucleotide analogues may be longer than the first sequence and the second sequence together, and accordingly the oligonucleotide analogues may comprise other parts than the first sequence and the second sequence.
  • the oligonucleotide analogue preferably comprises between 5 and 100, such as between 5 and 10, such as between 10 and 15, for example between 15 and 20, such as between 20 and 30, for example between 30 and 40, such as between 40 and 50, for example between 50 and 60, such as between 60 and 80, for example between 60 and 100 nucleotides and/or nucleotide analogues and/or intercalator pseudonucleotides. More preferably the oligonucleotide analogue consists of in the range from 15 to 50 nucleotides.
  • an oligonucleotide analogue according to the invention may comprise a first sequence and a second sequence, wherein said first sequence and said second sequence are separated by a third sequence consisting of p nucleotides and/or nucleotide analogues.
  • p may be any desirable integer, for example p may be an integer between 1 and 5, for example between 5 and 10, such as between 10 and 15, for example between 15 and 20, such as between 20 and 30, for example between 30 and 50.
  • the oligonucleotide analogues according to the present invention may comprise any desirable number of intercalator pseudonucleotides.
  • the oligonucleotide analogue may comprise between 2 and 5, such as between 5 and 10, such as between 10 and 15, for example between 15 and 20 intercalator pseudonucleotides.
  • the intercalator pseudonucleotides may be positioned at any position within_the first sequence of the oligonucleotide analogue.
  • the present invention relates to oligonucleotide analogues according to the present invention, wherein the first sequence comprises at least one, for example between 2 and 5, such as between 5 and 10, such as between 10 and 15, for example between 15 and 20 intercalator pseudonucleotides.
  • the invention relates to oligonucleotide and/or oligonucleotide analogues according to the present invention, wherein the second sequence comprises no intercalator pseudonucleotides.
  • the intercalator pseudonucleotides may be placed in any desirable position within the first sequence of the oligonucleotide analogues.
  • an intercalator pseudonucleotide may be placed at either end of the first sequence of the oligonucleotide analogues or an intercalator pseudonucleotide may be placed in an internal position within the oligonucleotide analogues.
  • the oligonucleotide analogue comprises more than one intercalator pseudonucleotides
  • they may be placed in relation to each other in any desirable manner. For example, they may be placed so that 1 , for example 2, such as 3, for example 4, such as 5, for example from 5 to 10, such as from 10 to 15, for example from 15 to 20, such as more than 20 nucleotides are separating the intercalator pseudonucleotides.
  • At least two intercalator pseudonucleotides are placed so the intercalators of said intercalator pseudonucleotides are capable of forming an intramolecular excimer, an intramolecular exciplex, a intramolecular FRET complex and/or a intramolecular charge-transfer complex.
  • the oligonucleotide analogue may comprise more than one intercalator pseudonucleotide, wherein said intercalator pseudonucleotides may be similar or may be different.
  • the oligonucleotide analogues may comprise any nucleotides or nucleotide ana- logues. Nucleotides and nucleotide analogues can for example be chosen from the group consisting of DNA, RNA, LNA, PNA, ANA, MNA, 2'-O-methyl RNA and HNA. Accordingly, the oligonucleotide analogue may comprise one or more selected from the group consisting of subunits of DNA, RNA, LNA, PNA, ANA, MNA, 2'-O-methyl RNA and HNA, i.e. the oligonucleotide analogue may be selected from the group of DNA, RNA, LNA, PNA, ANA, MNA, 2'-O-methyl RNA, HNA and mixtures thereof.
  • an oligonucleotide analogue comprises a first and a second sequence, where said first sequence is positioned upstream of said second sequence and where said second sequence is able to hybridize to a homologously complementary target sequence and prime template directed extension.
  • Said second and third sequence may either hybridise to said target sequence as one consecutive sequence, thus said second sequence is positioned downstream of said third sequence.
  • said third sequence may fold back and hybridise to the elongated second sequence and thus hybridise downstream of said second sequence. This last alternative is referred to as a Scorpion mechanism (Whitcombe et al. 1999, Nature Biotechnology 17: 804-807).
  • the melting temperature of a hybrid of the first sequence and the second sequence is significantly lower than the melting temperature of a hybrid between the second sequence and a homologously complementary, preferably complementary, target nucleic acid or nucleic acid analogue.
  • an oligonucleotide analogue comprises a first, a second and a third sequence, where said first sequence is positioned upstream of said second and third sequence and where said second and third sequence are able to hybridize to a homologously complementary target sequence and prime template directed extension.
  • the melting temperature of a hybrid of the first sequence and the second sequence is significantly lower than the melting temperature of a hybrid between the second sequence and a homologously complementary target nucleic acid or nucleic acid analogue.
  • the melting temperature of a hybrid of the first and second sequence is significantly higher than the melting temperature of a hybrid between the first sequence and a non-corresponding nucleic acid or nucleic acid analogue.
  • the melting temperature of a hybrid of the first and second sequence is significantly higher than the melting temperature of a hybrid between the second sequence and a non-corresponding nucleic acid or nucleic acid analogue.
  • the melting temperature of a hybrid of the first sequence and the second sequence is significantly lower than the melting temperature of a hybrid between the second and third sequence and a homologously complementary nucleic acid or nucleic acid analogue.
  • the melting temperature of a hybrid of the first sequence and the second sequence may be significantly lower than the melting temperature of a hybrid between the second and a homologously complementary nucleic acid or nucleic acid analogue.
  • the melting temperature of a hybrid of the first and second sequence is significantly higher than the melting temperature of a hybrid between the second and third sequence and a non- complementary nucleic acid or nucleic acid analogue.
  • the homologously complementary target nucleic acid or nucleic acid analogue is DNA or a nucleic acid analogue derived from a DNA template by extending oligonucleotides according to the present invention.
  • the melting temperature of a hybrid of the first sequence and the second sequence is significantly lower than the melting temperature of a hybrid between the second and optionally third sequence and a homologously complementary nucleic acid or nucleic acid analogue, for example DNA, that results in the advantageous effect that in a mixture comprising the oligonucleotide analogue comprising the first sequence and the second sequence and said nucleic acid or nucleic acid analogue homologously complementary to the second and optionally third sequence, the second and optionally third sequence will preferably hybridize with said homologously complementary nucleic acid or nucleic acid analogue, rather than with the first sequence.
  • a mixture comprising an oligonucleotide analogue according to the present invention, if the first sequence and the second sequence are hybridized, that is indicative of that only a limiting amount or for example no homologously complementary target nucleic acids and/ or nucleic acid analogues_are available.
  • a mixture comprising the oligonucleotide and/or oligonucleotide analogues, if the first sequence and the second sequence are not hybridized, that is indicative of that the mixture furthermore comprises homologously complementary nucleic acids and/ or nucleic acid analogues.
  • an amount of said oligonucleotide analogues corresponding to the amount of said homologously complementary targets will be hybridized to said homologously complementary targets and the remaining amount of said oligonucleotide analogue first sequences will be hybridized to said second sequences.
  • an oligonucleotide or oligonucleotide analogue comprising a first, a second and optionally a third sequence according to the present invention is affixed to a solid surface.
  • an array of oligonucleotides or oligonucleotide analogues comprising a first, a second and optionally a third sequence according to the present invention is affixed to a solid surface.
  • An excimer is a dimer of compounds, which is associated in an electronic excited state, and which is dissociative in its ground state. When an isolated compoundjs excited it may loose its excitation or it may associate with another compound of the same kind_(which is not excited), whereby an excimer is formed. An excimer emits fluorescence at a wavelength different from monomer fluorescence emission. When the excimer looses its excitation the association is no longer favourable and the two species will dissociate.
  • An exciplex is an excimer like dimer, wherein the two compounds_are different.
  • Intramolecular excimers are formed by two moieties comprised within one molecule, for example 2 polyaromatic groups within the same molecule. Similar intramolecular exciplexes are formed by two moieties comprised within one molecule, for example by 2 different polyaromatic groups.
  • Fluorescence resonance energy transfer is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.
  • FRET Fluorescence resonance energy transfer
  • the donor and the acceptor must be in close proximity (typically between 10 to 100 A) for FRET to occur.
  • the absorption spectrum of the acceptor must overlap with the fluorescence emission spectrum of the donor. It is further preferred that the donor and the acceptor transition dipole orientations must be approximately parallel.
  • a charge transfer complex is a chemical complex in which there is weak coordination involving the transfer of charge between two intermolecular or intramolecular moieties, called an electron donor and an electron acceptor. These two moieties exhibit an observable charge-transfer absorption band during charge-transfer transi- tion._An example is phenoquinone, in which the phenol and quinone molecules are not held together by formal chemical bonds but are associated by transfer of charge between the compounds' aromatic ring systems.
  • Spectral properties such as fluorescence
  • fluorescent labels compatible with association to nucleic acids and nucleic acid analogues are needed.
  • intercalator pseudonucleotides comprise fluorescence properties that may be used for detection of hybridisa- tion and/or extension.
  • monomer fluorescence of intercalator pseudonucleotides may be used to detect hybridisation or to detect the presence of extension products or to follow the real-time formation of extension products.
  • the oligonucleotide analogue comprises a first sequence, which comprises at least two intercalator pseudonucleotides and said intercalator pseudonucleotides are capable of forming an excimer and/ or an exciplex and/ or a charge-transfer and/ or a FRET complex.
  • said two intercalators of an oligonucleotide analogue is placed in a distance from one another within the oligonucleotide analogue so they can interact and hence form an intramolecular excimer, an intramolecular exciplex, a FRET complex or a charge transfer complex.
  • the intercalators of at least two intercalator pseudonucleotides within said first sequence are capable of forming an intramolecular excimer, an intramolecular exciplex, an intramolecular FRET complex and/ or an intramolecular charge transfer complex, when at least one of the k nucleotides at either side of any of said intercalator pseudonucleotides are unhybridised.
  • the intercalators of at least two intercalator pseudonucleotides within said first sequence are capable of forming an intramolecular excimer and/ or an intramolecular exciplex and/ or an intramolecular FRET complex and/ or an intramolecular charge transfer complex, when at least one of the basepairs comprised within the k nucleotides at either side of any of said intercalator pseudonucleotides is unhybridised and said intercalators are not capable of forming an intramolecular excimer, an intramolecular exciplex, an intramolecular FRET complex or an intramolecular charge transfer complex, when all the k nucleotides at either side of any of said intercalator pseudonucleotides are hybridized.
  • k is an integer in the range of 1 to 3.
  • the intercalators of said first sequence are capable of forming an intermolecular excimer, an intermolecular exciplex, an intermolecular FRET complex and/ or an intermolecular charge transfer complex, when the second sequence of the oligonucleotide analogue comprising interalator pseudonucleotides is not hybridized to the first sequence.
  • Intercalators are capable of co-stacking with nucleobases.
  • intercalators When oligonucleotide analogues comprising said intercalators hybridize with homologously complementary DNA, said intercalators will preferably co-stack with the nucleobases of the hybrid. If all k nucleobases, k being an integer in the range of 1 to 3, around each of the at least two intercalators form matched base-pairs, this will preferably result in a steric hindrance of the intercalator moieties, so that said intercalators will not be able to interact and accordingly not be able to form an intramolecular excimer, an intramolecular exciplex, FRET complex or a charge transfer complex.
  • preferred oligonucleotide and/or oligonucleotide analogue according to the present invention are oligonucleotide and/or oligonucleotide analogues, wherein the first sequence comprises at least two intercalator pseudonucleotides, wherein the intercalators of said intercalator pseudonucleotides are capable of forming an intramolecular excimer, an intramolecular exciplex or a FRET complex or a charge- transfer complex, when the first sequence is not hybridized with the second sequence or another nucleic acid.
  • Such intercalators employed according to the present invention for example include, but is not limited to, pyrene.
  • the oligonucleotide analogue according to the present invention may in one embodiment comprise one or more directly or indirectly detectable labels in addition to intercalator pseudonucleotides.
  • the oligonucleotide analogue according to the present invention may comprise fluorescent groups in addition to intercalator pseudonucleotides.
  • the fluorescent groups may be useful for detecting the oligonucleotide analogue.
  • the fluorescent group may be any group capable of fluorescing, in particular any group capable of fluorescing when it is associated with said first or second sequence of the oligonucleotide analogue according to the present invention and/or when it is incorporated into a nucleic acid or nucleic acid analogue.
  • the fluorescent group may be selected from the group consisting of fluorescein, FITC, rhodamine, lissamine rhodamine, rhodamine 123, coumarin, CY-2, CY-3, CY 3.5, CY-5, CY 5.5, FAM, GFP, YFP, BFP, YO-YO, HEX, JOE, Nano Orange, Nile Red, OliGreen, Oregon Green, Pico green, Radiant Red, Ribo Green, ROX, R- phycoerythrin, SYPRO Orange, SYPRO Red, SYPRO Ruby, TAMRA, Texas Red, XRITC, Propidium iodide, Acridine Orange, ethidium bromide, SYBR Gold, SYBR
  • the oligonucleotide analogue may comprise at least one quencher molecule.
  • a quencher molecule according to the present invention is any molecule that is capable of quenching the fluorescence of a fluorescent group in its vicinity. The quencher may function by absorbing energy from the fluorescent group and dissipating the energy as heat or radiative decay. Hence, the signal from the fluorescent group will be reduced or absent. Accordingly, if a fluorescent group and a suitable quencher molecule are placed close to each other, the fluorescence of the fluorescent group will be quenched.
  • quencher molecules include, but are not limited to, DABCYL, DABSYL TAMRA, Methyl red, Black Hole-1 , Black Hole-2, ElleQuencher and QSY-7. However, the quencher molecule must generally be selected according to the fluorescent group.
  • Preferred pairs of fluorescent group-quencher molecules according to the present invention includes, but is not limited to:
  • the oligonucleotide and/or oligonucleotide analogue according to the present invention may comprise both a fluorescent group and a quencher molecule.
  • the fluorescent group may be placed as dangling end in the 5' end or in the 3' end or both ends. It is also preferred that at least one quencher molecule is attached as a dangling end, more preferably all non-intercalator pseudonucleotide quencher molecules are attached as dangling ends.
  • the quencher molecule may be placed as a dangling end in the 5' end or in the 3' end or both ends.
  • one the fluorescent group or one quencher molecule are placed as a dangling end, in the 5' end of the oligonucleotide and/or oligonucleotide analogue.
  • one fluorescent group is placed as a dangling end in the 5' end, preferably a quencher is positioned internally.
  • one quencher molecule is placed as a dangling end in the 5' end of the oligonucleotide and/or oligonucleotide analogue, one fluorescent group is positioned internally.
  • the fluorescent group and the quencher molecule are positioned in relation to each other so that the quencher molecule is capable of quenching the fluorescence or part of the fluorescence of the fluorescent group when the first sequence is hybridized to the second sequence, but is not capable of quenching the fluorescence of the fluorescent group when the first sequence is not hybridized to the second sequence.
  • an oligonucleotide and/or oligonucleotide analogue comprise a first sequence and a homologously complementary second sequence that are separated by a third sequence (a hairpin probe), where at least one of the sequences comprise at least one intercalator pseudonucleotide and the oligonucleotide and/or oligonucleotide analogue has an additional fluorescent group placed as dangling end in the 5' end, and quencher molecule internally positioned.
  • one quencher molecule is placed as a dangling end in the 5' end of the oligonucleotide and/or oligonucleotide analogue, one fluorescent group is positioned internally.
  • the quencher molecule is capable of quenching the fluorescence or part of the fluorescence of the fluorescent group when the first sequence is hybridized to the second sequence, but is not capable of quenching the fluorescence of the fluorescent group when the first sequence is not hybridized to the second sequence. In the most preferred embodiment, this is obtained when said quencher molecule and fluorescent group come into close proximity when the first and second sequences of said oligonucleotide and/or oligonucleotide analogue hybridize.
  • the label is a complex of at least two intercalator pseudonucleotides capable of forming an intramolecular excimer, exciplex, FRET or charge-transfer complex (see herein below) with the proviso that every two intercalator pseudonucleotides are separated by at least one nucleotide or nucleotide analogue
  • quenching of signal could be obtained by hybridization of the nucleotides in a region of at least one nucleotide to either side of any intercalator pseudonucleotide in the complex to a complementary sequence.
  • an oligonucleotide and/or oligonucleotide analogue comprising at least two intercalator pseudonucleotides where the spectral properties are changed upon hybridization to a target nucleic acid or as a consequence of amplification of a target nucleic acid.
  • the spectral signal is low when there is no or small amounts of a target nucleic acids, and high when there is larger amounts of target nucleic acid present.
  • the spectral signal increases in correspondence to the increase of said target nucleic acid sequence.
  • an oligonucleotide and/or oligonucleotide analogue that comprise a first sequence and a complementary second sequence that are separated by a third sequence (a hairpin probe), where second or third sequence comprise at least one intercalator pseudonucleotide and where first sequence comprise an additional complex of intercalator pseudonucleotides according to the present invention, where the spectral signal is low when said first sequence is hybridized to second sequence and high when they are not hybridized.
  • the first sequence may comprise a donor for FRET and the second sequence may comprise an acceptor for a FRET or vice versa.
  • said donor and said acceptor are positioned so that FRET may occur when the first sequence is hybridized to the second sequence and accordingly FRET fluorescence may only be detected when the first sequence is hybridized to the second sequence.
  • FRET donor and acceptor pairs for example includes:
  • nucleotide or nucleotide analogue extension products constituting double stranded nucleic acids and/ or nucleic acid analogues may also be detected using a label not associated directly with the oligonucleotide and/or oligonucleotide analogues and optionally oligonucleotides used for priming the extension.
  • the oligonucleotide and/or oligonucleotide analogues of the present invention are used to prime template directed extension, where after a label specific for double stranded nucleic acids and/ or nucleic acid derivatives is used to detect extension products.
  • such a label may be selected from the group consisting of, but not limited to, Propidium iodide, Acridine Orange, ethidium bromide, SYBR Gold, SYBR Green I, and SYBR Green II.
  • the fluorescence properties of a fluorescent group may be modulated according to buffer conditions and physical factors. For instance temperature, ionic strength and pH are parameters that can affect the strength of the fluorescent signal obtained from a specific fluorophore.
  • An example of a different chemical group of substances that are also known to affect fluores- cence of pyrene excimers is the lipophillic surfactant molecules, as disclosed in US patent 5,466,578.
  • the cationic subgroup of surfactants comprising a quarternary ammonium salt with carbon chains attached, like e.g. hexade- cyltrimethyl ammonium bromide, can be used to significantly increase the signal from oligonucleotides end-labeled with pyrene excimers.
  • oligonucleotides, oligonucleotide analogues, nucleic acids or nucleic acid analogues may be labeled by other means. This may be a preferred option if the fluorescent properties of the oligonucleotide and/or oligonucleotide analogues according to the present invention or other additional fluorescent lables are not used for detection.
  • An example is when exploiting only the abilities of said oligonucleotide and/or oligonucleotide analogues to reduce the formation of unwanted extension products.
  • oligonucleotide and/or oligonucleotide analogues with directly associated fluorescent lables are the use of ligands that bind to labeled antibodies, the use of radioactive lables and the use of chemiluminescent agents.
  • the choice label depends on the sensitivity, ease of conjugation with the oligonu- cleotide and/or oligonucleotide analogue, stability requirements, and available instrumentation.
  • Non-radioactive probes are often labeled by indirect means.
  • Labels include antibodies, which can serve as specific binding pair members for a labeled ligand.
  • one or more ligand molecule(s) is/are covalently bound to the probe, in this case the oligonucleotide and/or oligonucleotide analogue.
  • the ligand(s) then binds to an anti-ligand molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • Ligands and anti-ligands may be varied widely. Where a ligand has a natural anti- ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
  • a natural anti- ligand for example, biotin, thyroxine, and cortisol
  • any haptenic or antigenic compound can be used in combination with an antibody.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phospha- tases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include lucif- erin, AMPPD ([3-(2'-spiroamantane )-4-methoxy-4-(3'- phosphoryloxy)-phenyl-1 ,2- dioxetane]) and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Oligonucleotide analgoues may be labeled also be labeled with 3 H, 125 l, 35 S, 14 C, 33 P or 32 P and subsequently detected by autoradiography.
  • a normal PCR including hot dNTPs is carried out, whereby only the extension product will be radioactively labeled.
  • the above-mentioned methods will typically require some sort of separation of extended and non-extended oligonucleotide and/or oligonucleotide analogues after extension of oligonucleotide and/or oligonucleotide analogues have been carried out, but prior to detection.
  • the method of separation chosen will typically depend on the labeling method used and the availability of procedures and instruments. Examples of separation methods are described herein below.
  • the high affinity of oligonucleotide and/or_oligonucleotide analogues according to the present invention toward nucleic acids or nucleic acid analogues and the spectral properties are not the only special features possessed by this oligonucleotide and/or ligonucleotide analogue.
  • several other features added by the incorporation of intercalator pseudonucleotide moieties may be exploited.
  • the first strategy relates to the presence of intercalator pseudonucleotides, which can decrease hybridization strength of homologously complementary sequences, both comprising intercalator pseudonucleotides, thereby preventing cross-hybridization.
  • the second strategy relates to the sterical hindrance posed by the intercalator pseudonucleotide, being a non-natural nucleotide, which might also inhibit a DNA or RNA polymerase of proceeding beyond an intercalator pseudonucleotide present in the template strand, enabling precise control of extension-termination.
  • the third strategy relates to exploiting the properties of a hairpin structure to inhibit unwanted priming.
  • the present invention provides methods of reducing cross-hybridization between regions of individual oligonucleotide and/or oligonucleotide analogues comprising intercalator pseudonucleotides.
  • Primer-dimers are unwanted PCR extension products arising from cross- hybridization among oligonucleotides or oligonucleotide analogues used for primers in the PCR reaction.
  • extension products arising from cross-hybridization between other nucleic acids or nucleic acid analogues in the PCR reaction, e.g. hybridization probes also lead to unwanted extension products.
  • the formation of unwanted extension products is a phenomenon general to PCR methods, and in particular, it is a problem when performing PCR with many cycles.
  • a template extension product essentially free of DNA contaminants is often required. Therefore, the presence of unwanted extension products may impose the need for additional and time-consuming purification steps.
  • Another example where the presence of unspecific products, especially primer- dimers, is a major concern is in quantitative PCR when using unspecific stains for detection of double stranded nucleic acids or nucleic acid analogues.
  • intercalator pseudonucleotides may provide different degrees of sterical hindrance to a polymerase, different intercalator pseudonucleotides may be chosen according to their properties in hindering the polymerase of proceeding beyond an intercalating pseudonucleotide present in the template strand and chosen according to a particular interest.
  • the use of intercalator pseudonucleotides as PCR terminators does not exclude the use of other PCR terminators, also reffered to as "PCR stoppers", known in the art.
  • oligonucleotide and/or oligonucleotide analogues according to the present invention inhibit the formation of primer-dimers by inhibiting cross-hybridization among said oligonucleotide and/or oligonucleotide analogues when used as primers for template directed extension.
  • oligonucleotide and/or oligonucleotide analogues may also reduce the formation of primer-dimers even if intercalator pseudonucleotides are not capable of reducing cross-hybridization between primers.
  • This second strategy of reducing unwanted extension products exploits that intercalator pseudonucleotides pose a sterical hindrance to polymerases as outlined below.
  • the first sequence of oligonucleotide and/or oligonucleotide analogues comprising intercalating pseudonucleotides as described herein above are positioned in the upstream region of the oligonucleotide and/or oligonucleotide analogue.
  • Another complementary, homologously complementary or non- corresponding oligonucleotide and/or oligonucleotide analogue designed the same way may be capable of hybridizing to the first mentioned oligonucleotide and/or oligonucleotide analogue.
  • intercalator pseudonucleotides present in the oligonucleotide and/or oligonucleotide analogue serving as template for another oligonucleotide and/or oligonucleotide analogue priming extension, will be positioned downstream of the extension site. Since nucleic acids are extended only in the 5' to 3' direction, intercalator pseudonucleotides of said oligonucleotide and/or oligonucleotide analogue template will inhibit a DNA or RNA polymerase in proceeding beyond said intercalator pseudonucleotides. Therefore, only a limited number of nucleotides or nucleotide analogues will be added.
  • oligonucleotide and/or oligonucleotide analogues comprising intercalator pseudonucleotides reduces the formation of unwanted extension products in template directed extension of nucleic acids or nucleic acid analogues by posing sterical hindrances to polymerases were extension is unwanted.
  • oligonucleotide and/or oligonucleotide analogues comprising intercalator pseudonucleotides according to the present invention reduce the formation of unwanted extension products arising from cross-hybridization between oligonucleotides and oligonucleotide analogues in template directed extension of nucleic acids or nucleic acid analogues.
  • said unwanted extension products refer to products arising from extension of said oligonucleotide and/or oligonucleotide analogues where non-target nucleic acids or nucleic acid analogues (including oligonucleotide and/or oligonucleotide analogues) serve as templates.
  • extension products should be interpreted as the situation compared to extension where all conditions for extension are identical except that said oligonucleotide and/or oligonucleotide analogues do not comprise intercalator pseudonucleotides according to the present invention.
  • oligonucleotide and/or oligonucleotide analogues according to the present invention comprise a first sequence comprising intercalator pseudonucleotides, a second and a third sequence, wherein said first sequence is capable of hybridizing to said second sequence.
  • first and second sequences are homologously complementary, they will preferably form a hairpin structure with a duplex without overhang where the 5' and 3' ends of the oligonucleotide and/or oligonucleotide analogue meet.
  • the melting temperature of a hybrid of said second and third sequence and a homologously complementary target sequence is above the melting temperature of a hybrid between the first sequence and the second sequence.
  • the oligonucleotide and/or oligonucleotide analogue will preferably hybridize with said target and prime an extension reaction. If said target is not present, said first and second sequence will hybridize forming a hairpin thus protecting from cross- hybridization and the formation of unspecific products.
  • an oligonucleotide and/or oligonucleotide analogue comprising intercalator pseudonucleotides according to the present invention the duplex formed between the first and second sequence is without overhang.
  • an oligonucleotide or oligonucleotide analogue comprising intercalator pseudonucleotides according to the present invention the duplex formed between the first and second sequence has a 3'-overhang.
  • homologously complementary first and second sequences form duplexes, preventing unwanted extension events when the oligonucleotide and/or oligonucleotide analogue is not hybridized to a target sequence in a template or when oligonucleotide and/or oligonucleotide analogue sequences is not present or present in limiting amounts.
  • Intramolecular duplex formation will happen fast due to the superior kinetics of the uni-molecular hybridization event thus obtained during intramolecular duplex-formation when said template is not present.
  • intercalator pseudonucleotides capable of forming intramolecular excimers, intramolecular exciplexes, FRET complexes or charge-transfer complexes can be positioned in the first sequence of said oligonucleotide and/or oligonucleotide analogue.
  • the fluorescence signal from said intercalator pseudonucleotides is quenched upon the formation of a duplex comprising said first and second sequence.
  • the fast kinetics of uni-molecular hairpin formation provides means of switching between fluorescence on-off states fast. For example fast kinetics are preferred to minimize PCR cycling time.
  • the nm separation between excitation- and emission-peaks are referred to as "the Stokes shift” and typical examples of Stokes shifts found for conventional fluorophores used for quantitative PCR are in the range of 10-25 nm.
  • the Stokes shift is approximately 140 nm. It is preferred to have large Stokes shifts to facilitate separation of excitation light from detected light to avoid false signal and to avoid cross-talk between fluorophores where one fluorophore excites another independently from amplified nucleic acid or nucleic acid analogue.
  • oligonucleotides or oligonucleotide analogues comprising intercalator pseudinucleotides according to the present invention capable of forming intramolecular excimer, and/or intramolecular exciplex, and/or FRET and/or charge transfer complex fluorescence with Stokes shifts significantly larger than the non- intercalating pseudonucleotide fluorophores mentioned herein above.
  • an oligonucleotide or oligonucleotide analogue comprises modifications in backbone moieties not being derived from nucleotides, nucleotide analogues and/or intercalating pseudonucleotides according to the present invention.
  • modifications are capable of being build into an oligonucleotide or an oligonucleotide analogue backbone but are not capable of taking part in base-pairing events or in any autonomous way contribute to increased melting hybridization strength of a nucleic acid and/or nucleic acid analogue
  • said backbone modifications are small chemical moieties capable of adjoining two nucleobases in a nucleic acid or nucleic acid analogue sequence without essentially disturbing the base-pairing properties of said sequence nucleic acid or nucleic acid analogue.
  • said backbone modifications By inserting said backbone modifications into the backbone of a nucleic acid or nucleic acid analogue, small backbone bulges are thus obtained between nucleobases. This provides the backbone of the nucleic acid or nucleic acid analogue with increased local conformational flexibility. Examples of backbone modifications are described herein above, since said backbone modifications largely equals the backbone moieties of intercalating pseudonucleotides.
  • Said flexible backbone units can be inserted at any internal position within a nucleic acid or nucleic acid analogue or a pair of nucleic acids and/or nucleic acid analogues in any distance from intercalating pseudonucleotides if present.
  • said flexible backbone modifications are placed such that they are positioned opposite of intercalator pseudonucleotides when both intercalator pseudonucleotides and said backbone modifications are present within a duplex or hybrid structure.
  • said flexible backbone moieties when placed directly opposite of an intercalator pseudonucleotide in a duplex structure, are capable of accommodating said intercalator pseudonucleotide with better fit than when said backbone moiety is not present.
  • a better fit means that the increased flexibility of the opposite backbone in a duplex will allow the intercalator to adopt a stable conformation with a more favorable energy minimum than when the opposite strand does not comprise said backbone moiety.
  • At least two intercalating pseudonucleotides capable of forming an excimer, exciplex.FRET complex or charge-transfer complex are positioned as bulge insertions in a oligonucleotide or oligonucleotide analogue sequence.
  • each of said intercalator pseudonucleotides are positioned opposite of a flexible backbone moiety.
  • intercalator pseudonucleotides are accommodated with better fit, and in the most preferred embodiment, the increased flexibility in accommodation results in a significant decrease in excimer, exciplex, FRET or charge-transfer fluorescence of intercalator pseudonucleotides present in the duplex structure.
  • Said complementary nucleic acid analogue sequence comprising flexible backbone moieties need not be a separate sequence, but can be covalently linked to said first sequence, according to the hairpin oligonucleotide and oligonucleotide analogue designs described above.
  • said hairpin may both function as an hybridization probe and/or a primer for an elongation process, such as PCR.
  • the present invention also relates to methods of detecting hybridisation between a nucleic acid or nucleic acid analogue comprising an oligonucleotide or oligonucleotide analogue and a template comprising a nucleic acid sequence capable of hybridising to the second sequence comprising the steps of
  • the nucleic acid or nucleic acid analogue comprising an oligonucleotide or oligonucleotide analogue may in one embodiment be equal to said oligonucleotide or oligonucleotide analogue. However, it is also possible, that the nucleic acid or nucleic acid analogue comprising an oligonucleotide or oligonucleotide analogue may be longer than the oligonucleotide or oligonucleotide analogue.
  • nucleic acid or nucleic acid analogue may be an elongated oligonucleotide or oligonucleotide analogue, wherein nucleotides have been added to the 3' end of the oligonucleotide or oligonucleotide analogue.
  • An elongated oligonucleotide or oligonucleotide analogue may be the product of a reaction catalysed by a DNA polymerase.
  • a DNA polymerase may elongate an oligonucleotide or oligonucleotide analogue, when said oligonucleotide or oligonucleotide analogue is hybridised to a longer homologously complementary nucleic acid in a template dependent manner.
  • said homologously complementary nucleic acid may be DNA or RNA and more prerably said homologously complementary nucleic acid might be the template.
  • said nucleic acid or nucleic acid analogue may be a product of a PCR reaction comprising said oligonucleotide or oligonucleotide analogue.
  • Hybridisation may be determined by a number of different techniques taking advantage of the properties of the oligonucleotide or oligonucleotide analogue.
  • detection of hybridisation comprises determining spectral properties of the oligonucleotide or oligonucleotide analogue. Determination of spectral properties is described in more detail herein above.
  • hybridisation may be determined by determining the melting temperature. This may be done when the melting temperature of a hybrid between the first sequence and the second sequence is different to the melting temperature of a hybrid consisting of the second sequence and a homologously complementary nucleic acid or nucleic acid analogue for example the template.
  • nucleic acid or nucleic acid analogue comprising said oligonucleotide or oligonucleotide analogue not hybridised to said template may be separated from said hybridised nucleic acid or nucleic acid analogue prior to detection by any method known to the person skilled in the art.
  • the method of detecting hybridisation may be used for a number of different purposes.
  • the method may be employed for detecting hybridisation in an assay dependent on specific hybridisation, for example Southern Blotting, Northern blotting, FISH or other kinds of in situ hybridisation.
  • the method is employed for quantification of a polymerase chain reaction (PCR).
  • nucleic acid analogue comprising said oligonucleotide or oligonucleotide analogue or said template may be affixed to a solid support.
  • the present invention also relates to methods of detecting template directed extension primed by oligonucleotide and/or oligonucleotide analogues.
  • the template may be an extended/elongated.oligonucleotide or oligonucleotide analogue, wherein nucleotides have been added to the 3' end of an oligonucleotide or oligonucleotide analogue.
  • An elongated oligonucleotide or oligonucleotide analogue may be the product of a reaction catalysed by a DNA polymerase or a ligase.
  • a DNA polymerase may elongate an oligonucleotide analogue, when said oligonucleotide analogue is hybridized to a longer nucleic acid or nucleic acid analogue in a template dependent manner.
  • said nucleic acid or nucleic acid analogue may be DNA or RNA and more preferably said nucleic acid might be the template.
  • detection of extension comprises determining spectral properties of the oligonucleotide analogue.
  • One advantage of such a detection type is that it can be done real-time during a reaction, so that the amount of extended oligonucleotide analogue can be determined at any specific time during a reaction.
  • the reaction may for example be a PCR.
  • a spectral signal above or below a predetermined limit may be indicative of extension, preferably however, a spectral signal above a predetermined limit is indicative of extension.
  • the spectral properties may for example be fluorescent properties.
  • the spectral properties are monomer fluorescence and/or excimer fluorescence and/or exciplex fluorescence and/or FRET fluorescence and/or charge- transfer absorbance.
  • oligonucleotide analogues comprising at least 2 intercalators capable of forming an intramolecular excimer and/or intramolecular exciplex and/or a FRET complex and/or charge-transfer complex as described herein above.
  • said oligonucleotide analogues comprising a first sequence comprising at least 2 intercalators capable of forming an intramolecular excimer and/or intramolecular exciplex and/or FRET and/or charge-transfer complex as described herein above are employed.
  • said oligonucleotide or oligonucleotide analogue is affixed to a solid support.
  • said oligonucleotide or oligonucleotide analogue is affixed to a solid support and capable of priming an extension reaction when said oligonucleotide or oligonucleotide is hybridised to a corresponding, preferentially a complementary, target nucleic acid or nucleic acid analogue.
  • the oligonucleotide analogue according to the present invention preferably comprises at least one intercalator pseudonucleotide capable of fluorescing, more preferably the oligonucleotide analogue comprises only intercalator pseudonucleotide capable of fluorescing.
  • monomer fluorescence of intercalator pseudonucleotides is used for detection of extension of oligonucleotide analogues according to the present invention.
  • the monomer fluorescence of an intercalator pseudonucleotide positioned at the 5' end of the second sequence, where the second sequence is positioned downstream of the first sequence is used to detect extension of oligonucleotide analogue.
  • oligonucleotide analogues comprise at least 2 intercalator pseudonucleotides capable of forming an intramolecular excimer and/or intramolecular exciplex and/or FRET and/or charge-transfer complex.
  • extension of the second downstream sequence may be determined by determining the excimer, exciplex, FRET or charge-transfer fluorescence.
  • high excimer, exciplex, FRET or charge-transfer fluorescence is indicative of template directed extension and low excimer, exciplex, FRET or charge-transfer fluorescence is indicative of no template directed extension.
  • High fluorescence is fluorescence above a predetermined limit and low fluorescence is fluorescence below a predetermined limit.
  • said at least 2 intercalator pseudonucleotides of the first sequence are capable of co-stacking with the end of the hybrid comprised of an extended oligonucleotide analogue and a homologously complementary template.
  • said at least 2 intercalator pseudonucleotides of the first sequence are capable of co-stacking with the end of the hybrid comprised of an extended oligonucleotide analogue and a complementary template.
  • non-extended oligonucleotide analogue is differentiated from extended oligonucleotide analogue by fluorescence polarization or fluorescence correlation spectroscopy.
  • non-extended oligonucleotide analogue is differentiated from extended oligonucleotide analogue by the use of one or more fluorescent groups that give rise to a signal specific to extended oligonucleotide analogues only.
  • fluorescent dyes that are attached to the oligonucleotide and/or oligonucleotide analogues and are specific for double stranded nucleic acids or nucleic acid analogues may be used.
  • the temperature is sufficiently high allowing only homologously complementary hybrids of oligonucleotide analogue comprising nucleic acid analogues to hybridize, such signal may be used for detection.
  • Signal derived from long unspecific extension products may give problems, but that may be removed by using methods for reducing unspecific hybridization and extension events as described above.
  • Fluorescent dyes specific for double stranded nucleic acid or nucleic acid analogue that are not covalently attached to said oligonucleotide or oligonucleotide analogue may also be used. As described herein above, such dyes will simply stain all double stranded nucleic acid or nucleic acid analogue formed during the amplification reaction being present on double stranded form at the chosen measuring temperature.
  • non-extended oligonucleotide or oligonucleotide analogue is separated from extended oligonucleotide or oligonucleotide analogue prior to detection.
  • Separation of non-extended from extended oligonucleotide or oligonucleotide analogues is only carried out when the signal from labels associated with non- extended and extended oligonucleotide or oligonucleotide analogue cannot otherwise be differentiated. Separation may for example be carried out by gel electrophoresis, chromatography, centrifugation, precipitation or any other method known to the person skilled in the art.
  • the present section concerns oligonucleotide and/or oligonucleotide analogues comprising a first, a second and possibly a third sequence, wherein said first sequence is capable of hybridizing to said second sequence, and wherein the first sequence may comprise at least 2 intercalators capable of forming an intramolecular excimer and/or intramolecular exciplex and/or FRET and/or charge-transfer complex as described herein above.
  • hybridisation of the second seqeunce and/or it is possible to detect hybridization by determining fluorescence of non- intercalator pseudonucleotides attached to the oligonucleotide and/or oligonucleotide analogue.
  • oligonucleotide and/or oligonucleotide analogues comprise a fluorescent group and a quencher molecule as described herein above.
  • the oligonucleotide and/or oligonucleotide analogue may comprise a fluorescent group and a quencher molecule positioned in relation to each other so that the quencher molecule is capable of quenching the fluorescence of the fluorescent group when the first sequence is hybridized to the second sequence, but is not capable of quenching the fluorescence of the fluorescent group when the first sequence is not hybridized to the second sequence.
  • fluorescence of the fluorescent group is detectable over a predetermined limit, that is indicative of no hybridization (i.e. no hybridization between first and second sequence) due to extension directed from the template, whereas when fluorescence of the fluorescent group is lower than a predetermined limit, that is indicative of no extension allowing hybridization (i.e. hybridization between first and second sequence).
  • High fluorescence is fluorescence above a predetermined limit and low fluorescence is fluorescence below a predetermined limit.
  • said oligonucleotide or oligonucleotide analogue is affixed to a solid support.
  • the presence of extension product and/or hybridisation may be determined by determining the melting temperature. This may be done when the melting temperature of a hybrid between the first sequence and the second sequence of an oligonucleotide analogue is lower than the melting temperature of an extension product of said oligonucleotide analogue and a homologously complementary or complementary nucleic acid or nucleic acid analogue, for example the template.
  • low melting temperature is indicative of intramolecular hybridization between first and second sequence and essentially no extension
  • high melting temperature is indicative of the presence of extension products.
  • a melting temperature above a predetermined limit may be indicative of extension.
  • the oligonucleotide analogue is designed in a manner so that only part of the oligonucleotide analogue is capable of hybridizing with the preferred template.
  • the template does not comprise a nucleic acid sequence capable of hybridizing with the first sequence.
  • the template does not comprise a nucleic acid sequence capable of hybridizing with for example 1 to 15, such as 1 to 10, for example 1 to 7, such as 1 to 5, for example 1 to 3 nucleotides flanking the intercalator pseudonucleotide(s).
  • the template does not comprise nucleic acid sequences that may hybridize with the part of the first sequence, which is capable of hybridizing to the second sequence.
  • the template comprises a nucleic acid sequence capable of hybridizing with the second sequence and the third sequence, more preferably the template may comprise a nucleic acid sequence complementary to the second sequence and the third sequence.
  • the template may comprise a nucleic acid sequence capable of hybridizing with the part of the first sequence that is not capable of hybridizing to the second sequence.
  • the template does not comprise a nucleic acid sequence capable of hybridizing with the first sequence and the template comprises a nucleic acid sequence capable of hybridizing with the second sequence and the third sequence.
  • the first sequence when the oligonucleotide or oligonucleotide analogue is hybridized to the template, the first sequence is not hybridized. Accordingly, when the first sequence comprises intercalators capable of forming an intramolecular excimer and/or intramolecular exciplex and/or intramolecular FRET and/or intramolecular charge-transfer complex, then excimer fluoresence and/or exciplex fluorerence and/or FRET and/or charge-transfer absorbance may be detected, when the oligonucleotide or oligonucleotide analogue is hybridized to the template.
  • the second sequence preferentially hybridizes with a homologously complementary template
  • the first and the second sequence will only be hybridized, when no homologously complementary template is present. Accordingly, when a homologously complementary template is available the second sequence will preferably be hybridized with said homologously complementary template and hence the first sequence will not be hybridized and excimer fluoresence and/or exciplex fluorerence and/or FRET and/or charge-transfer complex fluorescence above a predetermined limit may be detected.
  • the method is employed for quantification of a polymerase chain reaction (PCR). Because the method allows for real-time detection of hybridization by determining fluorescence properties, the method allows to closely follow a PCR reaction as it is proceeding.
  • PCR polymerase chain reaction
  • ODN Oligodeoxynucleotide
  • INA Intercalating nucleic acid corresponding to intercalator pseudonucleotide
  • 1-Pyrenemethanol is commercially available, but it is also easily prepared from pyrene by Vilsmeier-Haack formylation followed by reduction with sodium borohydride and subsequent conversion of the alcohol with thionyl chloride affords 1- (chloromethyl)pyrene in 98% yield.
  • the acyclic amidite 5 (fig. 1) was prepared from (S)-(+)-2,2-dimethyl-1 ,3-dioxalane-
  • N-formyl-N-methylaniline (68.0 g; 41.4 mL; 503 mmol) and o- dichlorobenzene (75 mL) is cooled on an ice bath and added phosphoroxychloride (68g; 440 mmol) over 2 hours so that the temperature do not exceed 25 °C.
  • Pulverized Pyrene 50 g; 247 mmol is added in small portions over 30 min. and the reaction mixture is equipped with a condenser and heated at 90-95°C for 2 hours. After cooling to room temperature the dark red compound is filtered off and washed with ' ) benzene (50 mL.). Then it is transferred to water (250 mL) and stirred over night.
  • the yellow aldehyde is filtered of and washed with water (3x50 mL). Recrystallized from 75% ethanol 3 times. Yeild: 30.0 g (52.7%).
  • 1-Pyrenylmethanol (6.40 g; 27.6 mmol) is dissolved in a mixture of pyridine (3.3 mL; 41.3 mmol) and CH 2 CI 2 (100 mL) and the mixture is cooled to 0°C. SOCI 2 (3.0 mL; 41.3 mmol) is added slowly over 15 min. and the temperature is allowed to rise slowly to r.t. Stir over night. The mixture is poured into stirring water (200 mL) and added CH 2 CI 2 (100 mL). The mixture is stirred for 30 min.
  • 9-anthracenemethanol (0.81 g; 3.89 mmol; I) was dissolved in dry pyridine (467 ⁇ L; 5.83 mmol) and dry CH 2 CI 2 . Under stirring and at 0°C SOCI 2 (423 ⁇ L; 5.83 mmol) was added dropwise, and the mixture was stirred for 24h during which the tem- perature is allowed to rise to r.t. within 2h. The reaction is poured onto stirring H 2 O
  • 9-anthracenemethylchlorid (628 mg; 277 mmol) was dissolved in dry toluene (25 mL over Na) and 2-[(S)-2',2'-dimethyl-1 ',3'-dioxalan-4'-yl]-ethanol (506 mg; 3.5 mmol) and 3 small spoons of KOH was added. The mixture was connected to a Dean-Stark apparatus and stirred under reflux conditions over night. The reaction mixture was slowly cooled to r.t. and washed with H 2 O (4 ⁇ 25 mL). Dried over Na 2 SO 4 and concentrated in vacuo.
  • the DMT protected anthracene compound was dissolved in dry CH 2 CI 2 (7 mL) and diisopropylammonium tetrazolide (252 mg; 1.5 mmol) and 2-Cyanoethyl N,N,N',N'- tetraisopropyl Phosphane was added. The reaction mixture was stirred for 20h at r.t.
  • nucleoside analogue 5 (fig. 1) it was incor- porated into the 5' end of two different self-complementary strands (5'-XCGCGCG and 5'-XTCGCGCGA).
  • the ODN synthesis is carried out on a Pharmacia LKB Gene Assembler Special using Gene Assembler Special software version 1.53.
  • the pyrene amidite is dis- solved in dry acetonitrile, making a 0.1M solution and inserted in the growing oligonucleotides chain using same conditions as for normal nucleotide couplings (2 min. coupling).
  • the coupling efficiency of the modified nucleotides is greater than 99%.
  • the ODNs are synthesized with DMT on and purified on a Waters Delta Prep 3000 HPLC with a Waters 600E controller and a Waters 484 detector on a Hamilton PRP- 1 column. Buffer A: 950 ml.
  • ODNs are measured in a 150 mM NaCl, 10 mM, Na «Phosphate, 1 mM EDTA pH 7.0, 1.5 ⁇ M of each strand. All melting temperatures giving are with an uncertainty on ⁇ 0.5 °C.
  • the Amber forcefield calculations were done in MacroModel 6.0 and 7.0 with water as solvent and minimization is done by Conjugant Gradient method.
  • the starting oligonucleotide sequences for calculation with the inserted pyrenes is taken from Brookhavens Protein Databank, and modified in MacroModel before minimazation is started.
  • Lam and Au-Yeung solved a structure of a self-complementary sequence, equal to the one used in this work, by NMR. Their structure is prolonged with the pyrene amidite at the 5'-end of each strand and used for the structural calculations.
  • the other sequence is a 13-mer highly conserved HIV-1 long terminal repeat region.
  • G-7 is replaced by the pyrene amidite and calculations are made with and without an across lying C-nucleotide.
  • the pyrene is placed in the interior of the duplex from the beginning. All bonds are free to move and to rotate.
  • the melting temperature of modified and unmodified, self-complementary DNA are shown in Figure 33.
  • Incorporation of the pyrene amidite in the 5' end as a dangling end stabilises the DNA duplex with 19.2 °C - 21.8 °C (8.6 °C - 10.9 °C per modification) depending on the underlying base pair.
  • the stabilizations of the duplexes due to incorporation of 5 at the 5' termini of the nucleic acid strands are similar to those found by Guckian et al. who inserted a pyrene nucleoside at the 5' termini of self complementary ODNs (oligo deoxynucleic acids).
  • the stabilisation can be explained by calculations using "MacroModel" which predict a structure were the pyrene moiety interacts with both nucleosides in the underlying basepair (figure 2).
  • Probe III 3'-CGA ACT CY Ref: 3'-CGA ACT C Target: 5'-GCT TGAG
  • the target strands and probes were annealed by mixing them in the above mentioned buffer at 95°C for 3 min. after which they are slowly cooled to room temperature.
  • the melting temperatures of the hybridised probe-target hybrids were found by slowly heating the solution in a quartz cuvette, while simultaneously determining the absorbance. All melting temperatures presented in this example are with an uncertainty of + 1.0°C as determined by repetitive experiments.
  • probe I and II The difference in melting temperature between probe I and II is due to the short linker of probe I. Hence it is important that the combined length of linker and inter- calator is optimal, to obtain a large increase in affinity between intercalating pseudonucleotide modified oligonucleotides and their taget DNA sequences. Probes II and III have nearly the same affinity for their target sequences, even though the intercalating moieties in the two probes are very different. This shows that the intercalating pseudonucleotides are a class of compounds that, dependent on the wanted feature it should introduce into an oligonucleotide or oligonucleotide analogue, it should be designed by more or less strict rules.
  • oligonucleotides or oligonucleotide analogues comprise intercalating pseudonucleotides at either or both ends.
  • oligonucleotides or oligonucleotide analogues with intercalating pseudonucleotides in the 3'-end can be synthesised using Universal supports.
  • selfcomplementary oligonucleotides compris- ing intercalating pseudonucleotides positioned in the 3'-end form very thermal stable hybrids.
  • Design A was synthesized using a universal support while B was synthesized using standard nucleotide coupled columns and procedures:
  • oligo nucleotide analogues were treated with 2% LiCI in a 32% NH 4 OH solution in order to remove protection groups from the heterocyclic amines and to cleave the oligonucleotide from the universal support. Oligonucleotides comprising intercalating pseudonucleotides were tested on MALDI-TOF and found at the expected values.
  • the target strands and probes were annealed by mixing them in the above mentioned buffer at 95°C for 3 min. after which they are slowly cooled to room temperature.
  • the melting temperatures of the hybridised probe-target hybrids were found by slowly heating the solution in a quartz cuvette, while simultaneously determining the absorbance. All melting temperatures presented in this example are with an uncertainty of ⁇ 1.0°C as determined by repetitive experiments.
  • intercalating pseudonu- cleotides in either end of an oligonucleotide increases the affinity for a complementary target nucleic acid. It is also shown that intercalating pseudonucleotides can be inserted into the 3' end of an oligonucleotide or oligonucleotide analogue by using standard universal base chemistry.
  • Phosphoramidite 5 (fig. 1 ) is prepared as described in example 1.
  • the stabilization of the duplex by co-axial stacking of the pyrene moiety is not large enough to compensate for the loss in binding affinity due to the reduced number of hydrogen bonds by substitution of G with the pyrene moiety, the modified duplex being less stable than the unmodified fully complementary by 8.6 °C.
  • the same trend is found for DNA/RNA duplexes although these have lower melting temperatures in general than the corresponding DNA/DNA duplexes.
  • the stabilization of the pyrene moiety is only_8.2 °C for the DNA/RNA duplex when compared with ethylene glycol whereas the stabilization is 16.4 for the DNA/DNA duplex.
  • the pyrene insertion results in an improved discrimination between ssDNA and ssRNA with 9.0 °C difference in the melting temperatures of their corresponding duplexes.
  • the difference in the melting temperature between the pyrene modified DNA/DNA duplex and the pyrene modified DNA/RNA duplex is increased to 12.6 °C when inserting one pyrene modification as a bulge.
  • This difference is 7.4 °C larger than in the unmodified duplexes and much larger than the differences between the du- plexes containing natural nucleoside or flexible ethylene glycol bulges.
  • the pyrene moiety is selective and only able to stabilize DNA/DNA duplexes and not the DNA/RNA duplex.
  • the duplex have the same melting temperature with the glycerol linker than with the pyrene moiety, indicating that the pyrene does not intercalate into the strands.
  • DNA/DNA stabilization and DNA/RNA discrimination when compared with the duplex with only one insertion.
  • intercalating pseudonucleotides inserted in the middle of a DNA oligonucleotide. When hybridised to target said intercalating pseudonucleotides act as bulge insertions. All intercalating pseudonucleotide modified oligonucleotides shown here have an increased affinity for the complementary DNA target compared to the unmodified oligonucleotide:
  • intercalating pseudonucleotides are a broad group of compounds that obeys some simple rules regarding the combined length of the in- tercalator and linker.
  • the melting temperature of the hybrid increases by introduction of intercalating pseudonucleotides into the probe - regardless if the probe is DNA or RNA (see Table 4 below). Additional the affinity for a RNA target is reduced regardless if the probe is DNA or RNA.
  • intercalator pseudonucleotides can be introduced to oligonucleotides or oligonucleotide analogues giving the oligonucleotide or oligonucleotide analogue increased affinity for DNA and reduced affinity for RNA and RNA- like compounds like LNA, 2'-O-METHYL RNA .
  • Type Xi X 2 Type X 3 X 4
  • Table 4 Three different situations. At the top: DNA duplex affinity is increased or unaltered by the presence of intercalator pseudonucleotides in the hybrid compared to the duplex where none of the strands comprise intercalator pseudonucleotides. In the middle: The RNA duplex is destabilized by the presence of intercalator pseudonucleotides in the hybrid compared to the duplex where none of the strands comprise intercalator pseudonucleotides. At the bottom: Here it is shown how the hybrid between a DNA and a RNA strand is stabilized by intercalator pseudonucleotides if these are comprised by the RNA strand. Furthermore it is shown than when incorporated into the DNA strand the affinity for RNA is decreased.
  • DNA duplex is stabilized by the presence of intercalator pseudonucleotides in the hybrid compared to the duplex where none of the strands comprise intercalator pseudonucleotides. If intercalator pseudonucleotides are positioned in relation to each other, so that they are in close vicinity of each other when the oligonucleotides or oligonucleotide analogues are hybridized the melting temperature is decreased compared to when only one strand comprises intercalator pseudonucleotides.
  • RNA duplex is destabilized by the presence of intercalator pseudonucleotides in the hybrid compared to the duplex where none of the strand comprise the intercalator pseudonucleotides.
  • the hybrid between a DNA and a RNA strand is stabilized by intercalator pseudonucleotides if these are comprised in the RNA strand.
  • intercalator pseudonucleotides are positioned in relation to each other, so that they are in close vicinity of each other when the oligonucleotides or oligonucleotide analogues are hybridized the melting temperature is decreased compared to when only the RNA strand comprises intercalator pseudonucleotides.
  • 5 amidite 5 from example 1 incorporated into the strand according to the procedure described herein above.
  • Double pyrene inserted oligonucleotides gives the same result for all of the different ODNs. This effect is more pronounced when the modified DNA is hybridized with ssDNA than when hybridized with ssRNA ( Figures 5 and 6), indicating less intercalation of pyrene into the DNA/RNA Duplexes.
  • Two pyrene moieties separated by only one nucleotide generates a third peak at 480 nm, due to excimer formation of the pyrene residues. However this band is almost extinguished, when this type of DNA with two insertions with pyrene hybridizes to a complementary DNA strand. This indicates intercalation around an intact base- pair preventing the two pyrene moieties to get into the physical distance of approximately 3.4 nm needed for excimer formation. When a double inserted DNA hybridizes to a complementary RNA the two pyrene moieties are still able to interact since a substantial excimer band is found.
  • Example 14 3-Exonuclease stability of oligonucleotides or oligonucleotide analogues comprising intercalating pseudonucleotides
  • DNA oligo 3'-TGT CGA GGG CGT CGA INA oligo ⁇ 5'- YAC AGC YTC CCY GCA GCY T
  • INA Intercalating Pseudonucleotide comprised Nucleic Acid
  • SVPDE Snake Venom phosphordiesterase
  • Hairpin shape oligonucletides comprising intercalating pseudonucleotides for the detection of nucleic acid
  • hairpin shaped oligonucleotides comprising intercalating pseudonucleotides (probe I) can be used for the detection of nucleic acids. It is further more shown that using this principle it is possible to detect as low as a 5 nM solution (1 pmol in 200 ⁇ L) of target nucleic acid. It is also shown that the addition of Hexadecyl trimethyl ammoniumbromide (HTMAB) can enhance the signal sensitivity in a concentration dependent matter.
  • HTMAB Hexadecyl trimethyl ammoniumbromide
  • sequence of the detection probe comprising intercalating pseudonucleotides.
  • the nucleotides which is involved in the hairpin formation is underlined and the nucleotides that are involved in the binding to target is in shown in bold letters:
  • Probe I 5 - CAT CCG YAY AAG CTT CAA TCG GAT GGT TCT TCG
  • FIGURE 17 is shown the secondary structure of the hairpin.
  • the hydrogen bonds of the basepairs in the stem is shown as dots.
  • the surfactant used in the experiments was HTMAB:
  • FIGURE 18 is shown a figure that illustrates when the probe binds to its target sequence. It is shown that when the probe is hybridised to the Target, the two pyrene moieties from the intercalating pseudonucleotides are no longer separated by an intact base pair. This makes it possible for them to interact more freely, giving rise to higher excimer fluorescence:
  • Measurement time 0.1s, 4.0 mm from the bottom of the plate.
  • the addition of surfactants on the fluorescence level is also shown.
  • the addition of the HTMAB surfactant increases the fluorescence in some cases more than 100 times (column 6), and hence increases the sensitivity of the detection up to a 100 times.
  • probe I can be used as a primer in template directed extension reactions makes oligonucleotides or oligonucleotide ana- logues a very useful tool in e.g. the detection of nucleic acids, for labelling nucleic acids, for the use in extension reactions like ligation and PCR and in real-time quantitative PCR.
  • Oligos, 50 ⁇ M are spotted dissolved in 400 mM Sodium carbonate buffer, pH 9.
  • the chips are centrifuged, 600 rpm for 5 min, to remove excess water from the surface. • The chip is scanned, or stored refrigerated at 4° C.
  • variable background that is shown with different sections of the same chip can be caused by inadequate wash, calibration of scanner, or variation in SAL coating. • Observed tendency: Generally the quality of spots, that is shape and signal- homogeneity, seem to be better, when the oligos contain INA modifications (compare 1 and 7, bottom right.)
  • the target strands and probes were annealed by mixing them in the above mentioned buffer at 95°C for 3 min. after which they are slowly cooled to room temperature.
  • the melting temperatures of the hybridised probe-target hybrids were found by slowly heating the solution in a quartz cuvette, while simultaneously determining the absorbance. All melting temperatures presented in this example are with an uncertainty of ⁇ 1.0°C as determined by repetitive experiments.
  • pH was adjusted with a solution of 25% NH 4 OH and glacial acetic acid.
  • the method of preparing a sample for RT-PCR of a target sequence is depicted in figure 7.
  • the method has the advantage that false positive signals from DNA are largely reduced.
  • a cell sample is provided and the cell walls of the cell are destroyed, thereby releasing DNA and RNA from the cells (figure 7A).
  • an oligonucleotide comprising an intercalator pseudonucleotide, which can hybridise to the target sequence is incubated with the DNA/RNA sample under conditions allowing hybridisation between the oligo and DNA (figure 7B).
  • the sample is then ready to be up- scaled by any standard RT-PCR procedure (figure 7C). Because target DNA present in the sample is blocked by hybridisation to the oligonucleotide, then only RNA may be amplified.
  • RNA may be purified by any standard method for example by extraction and precipitation (figure 7D).
  • the purified RNA may be purified by any standard method for example by extraction and precipitation (figure 7D).
  • the purified RNA may be purified by any standard method for example by extraction and precipitation (figure 7D).
  • RNA will comprise small amounts of DNA contamination.
  • an oligonucleotide comprising an intercalator pseudonucleotide, which can hybridise to the target sequence is incubated with the RNA sample under conditions allowing hybridisation between the oligo and DNA (figure 7E).
  • the sample is then ready to be upscaled by any standard RT-PCR procedure (figure 7F). Because target DNA contamination present in the sample is blocked by hybridisation to the oligonucleotide, then only RNA may be amplified.
  • RNA may be purified by any standard method from the sample (figure 8B), however it is also possible to perform the subsequent steps on the DNA/RNA sample.
  • the sample is incubated with beads linked to an oligonucleotide comprising an intercalator pseudonucleotide (Figure 8C), which can hybridise to the target sequence under conditions allowing hybridisation between the oligo and DNA. After hybridisation the sample is filtered to remove the beads together with bound target DNA from the sample (figure 8D).
  • Figure 8C an oligonucleotide comprising an intercalator pseudonucleotide
  • the sample is ready for RT-PCR (figure 8E). Because the sequence specific target DNA has been removed from the sample, the risk of false positives of the RT-PCR due to DNA contamination is largely reduced.
  • the sample is incubated with a solid support linked to an oligonucleotide comprising an intercalator pseudonucleotide ( Figure 9B), which can hybridise to the target sequence under conditions allowing hybridisation between the oligo and DNA.
  • Figure 9B the solid support is removed from the sample together with bound target DNA.
  • the sample may once again be incubated with a solid support linked to an oligonucleotide comprising an intercalator pseudonucleotide to remove traces of sequence specific DNA still left in the sample.
  • the solid support is removed from the sample after hybridisation to sequence specific DNA (figure 9C).
  • the sample is then ready for RT-PCR.
  • a cell sample is treated with GnSCN thereby releasing nucleic acids.
  • the sample is incubated with beads linked to an oligonucleotide comprising an intercalator pseudonucleotide (Figure 10A), which can hybridise to the target sequence under conditions allowing hybridisation between the oligo and DNA.
  • the sample is filtrated and washed to remove non-bound nucleic acids (figure 10B).
  • the beads are subjected to heating and filtration, releasing pure, sequence specific DNA largely free of se- quence specific RNA (figure 10C).
  • the nucleic acid sample is incubated with a solid support linked to an oligonucleotide comprising an intercalator pseudonucleotide (Figure 11B), which can hybridise to the target sequence under conditions allowing hybridisation between the oligo and DNA.
  • the solid support is separted from the rest of the sample and subjected to heating, which releases the sequence specific DNA (figure 11C).
  • the sequence specific DNA will be largely free of sequence specific RNA and is ready for diagnosis, PCR or other purposes.
  • Oligonucleotides comprising pyrene pseudonucleotides are linked to a chip.
  • the oligonucleotides are designed so that a part of it may hybridise to a specific target DNA and so that the oligonucleotide may also self-hybridise.
  • 3 pairs of pyrene pseudonucleotides are facing each other, and accordingly the melting temperature of a DNA/oligo hybrid is higher than the melting temperature of the selfhybrid.
  • the oligonucleotide com- prises two pyrenes capable of forming an excimer, only when the probe is not hybridised to itself (figure 12 and figure 13A).
  • Different oligonucleotides recognising different target DNAs may be added to various defined regions of the chip. In the present example 2 different oligonucleotides are linked to spot 1 and spot 2, respectively.
  • a crude mixture of DNA fragments containing the target DNA is added to the chip at a temperature where the oligonucleotide can not selfanneal.
  • the oligonucleotide may be designed so that it comprises a fluorophore and a quencher, wherein the fluorophore signal may only be quenched by the quencher, when the oligonucleotide is self-hybridised (figure 13B).
  • oligonucleotides which each comprises 3 pyrenes pseudonucleotides that are facing each other when the oligonucleotides are hybridised.
  • the oligonucleotides also contains a fluorophore and a quencher each, posi- tioned so that the fluorophore signals may only be quenched by the quencher when the oligonucleotides are hybridised (figure 13C).

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Abstract

L'invention porte sur des oligonucléotides analogues d'oligonucléotides linéaires et en épingle à cheveux comprenant au moins un pseudonucléotide intercalant. Les pseudonucléotides intercalants, qui peuvent s'incorporer au squelette de phosphate d'un acide nucléique, comprennent un intercalant comportant au moins un système conjugué pouvant se co-empiler avec les nucléobases d'un acide nucléique. L'invention porte également sur des procédés de détection d'hybridations entre ces (parties de) pseudonucléotides ou leurs analogues, et une matrice. Elle porte en outre sur des procédés de détection d'expansions en direction de la matrice d'un analogue d'oligonucléotide comprenant au moins un pseudonucléotide intercalant. Dans l'exécution préférée, l'analogue d'oligonucléotide est en épingle à cheveux.
PCT/DK2002/000874 2001-12-18 2002-12-18 Analogues d'oligonucleotides lineaires et en epingle a cheveux comprenant des pseudonucleotides intercalants WO2003052132A2 (fr)

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WO2004065625A1 (fr) * 2003-01-24 2004-08-05 Human Genetic Signatures Pty Ltd Essai pour detecter des modifications de methylation dans des acides nucleiques au moyen d'un acide nucleique intercalant
WO2006026828A1 (fr) 2004-09-10 2006-03-16 Human Genetic Signatures Pty Ltd Bloqueur d'amplification comprenant des acides nucleiques intercalants (tna) contenant des pseudonucleotides intercalants (ipn)
WO2006032487A2 (fr) * 2004-09-24 2006-03-30 Universität Bern Balises moleculaires
WO2006125267A1 (fr) 2005-05-26 2006-11-30 Human Genetic Signatures Pty Ltd Amplification par deplacement de brin isotherme utilisant des amorces contenant une base non reguliere
US7799525B2 (en) 2003-06-17 2010-09-21 Human Genetic Signatures Pty Ltd. Methods for genome amplification
US7833942B2 (en) 2004-12-03 2010-11-16 Human Genetic Signatures Pty. Ltd. Methods for simplifying microbial nucleic acids by chemical modification of cytosines
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US9879222B2 (en) 2007-12-14 2018-01-30 Mofa Group Llc Gender-specific separation of sperm cells and embryos
US8518908B2 (en) 2009-09-10 2013-08-27 University Of Idaho Nucleobase-functionalized conformationally restricted nucleotides and oligonucleotides for targeting of nucleic acids
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WO2003052133A2 (fr) 2003-06-26
WO2003052133A3 (fr) 2003-10-02
AU2002358464B2 (en) 2006-04-27
WO2003052132A3 (fr) 2003-10-09
CN1653079B (zh) 2010-06-16
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WO2003051901A2 (fr) 2003-06-26
CN1653079A (zh) 2005-08-10
AU2002358463A1 (en) 2003-06-30
AU2002358464A1 (en) 2003-06-30
WO2003051901A3 (fr) 2003-11-27
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