WO2005083084A1

WO2005083084A1 - Intercalating triplex forming oligonucleotide derivatives and process for the preparation thereof

Info

Publication number: WO2005083084A1
Application number: PCT/IB2005/000472
Authority: WO
Inventors: Erik Bjerregaard Pedersen; Carsten Jessen
Original assignee: Syddansk Universitet
Priority date: 2004-02-25
Filing date: 2005-02-24
Publication date: 2005-09-09

Abstract

The present invention relates to novel intercalating oligonucleotide derivatives, which are useful for stabilizing natural or modified DNA and RNA triplexes and hybrids thereof. A linker for alternate strand triplex formation is described. The linker is designed so that it is able to stabilize the triplex by intercalation. The alternate strand triplex shows higher stability than the one of a homotriplex of the same length. The specificity of the alternate strand triplex is in the same range as for a traditional triplex in the case of a single mismatch.

Description

INTERCALATING TRIPLEX FORMING OLIGONUCLEOTIDE DERIVATIVES AND PROCESS FOR THE PREPARATION THEREOF

FIELD OF THE INVENTION

The present invention relates to the field of oligonucleotides and oligonucleotide analogues comprising intercalating compounds. In particular the present invention relates to novel intercalating oligonucleotide derivatives, which are useful for stabilizing natural or modified DNA and RNA triplexes and hybrids thereof. More specifically one part of the invention is directed to a novel linker for alternate strand triplex formation, which is designed so that it is able to stabilize the triplex by intercalation.

BACKGROUND OF THE INVENTION

In recent years there has been a growing interest in exploiting the increasing knowledge of the humane genome in the design of new therapeutics. Attempts to target either DNA or mRNA with modified oligonucleotides have been made and the efforts have lead to several drug candidates, which all are antisense drugs, targeting mRNA.

The rational design of sequence-specific ligands of nucleic acid is an active field of research with several goals i) to provide molecular biologists with new tools to investigate the function of specific genes and the role of specific sequences in the control of gene expression, ii) to develop sequence-specific artificial nucleases that could cleave long DNA fragments at selected sites for, e.g., mapping genes on chromosomes, iii) to provide a rational basis for the development of new therapeutic agents based on selective modulation of gene expression.

Oligonucleotides and analogues have received considerable attention during the past few years, as a very versatile class of reagents for sequence-specific recognition and modification of nucleic acids. In the "antisense" strategy an oligonucleotide is targeted to a specific messenger RNA (mRNA). It recognizes its target by hydrogen bonding interactions, forming Watson-Crick base pairs with its complementary sequence. Translation of the mRNA is inhibited according to several mechanisms. The oligonucleotide should have a minimum length to recognize a single mRNA species within a living cell.

So far there have been no antigene drugs targeting DNA that made it into clinical trials. In spite of lacking success, the antigene strategy is still the most logical approach to generate new therapeutics because each cell only contains two targets per diploid cell.

In the "antigene" strategy the oligonucleotide is targeted to double-helical DNA. It recognizes Watson-Crick base pairs by hydrogen bonding interactions within the major groove and forms a local triple-helical structure. Triple helix formation may inhibit transcription. Covalent modifications have been introduced into triple helixforming oligonucleotides in order i) to stabilize triple-helical structures, ii) to induce irreversible modifications of the target sequence, iii) to create artificial sequence-specific nucleases.

The basic principles and recent developments in this area of research are presented below.

In the antigene strategy an oligodeoxynucleotide (ODN) binds to the major groove of double helical DNA strand, forming a triple helical structure (triplex). The triplex forming oligodeoxynucleotide (TFO) consists of either pyrimidine nucleotides or purine nucleotides. When the TFO consists of pyrimidine nucleotides, the TFO binds to the target DNA strand through Hoogsteen Py PuPy base triplets (T AT and C⁺ GC) in a parallel orientation compared to the purine strand of the DNA duplex. When the TFO consists of purine nucleotides, the TFO is able to bind to the target DNA strand through reverse Hoogsteen Pu PuPy base triplets (A AT and G GC) in an antiparallel orientation. In both cases the target sequence needs to contain a purine tract of a considerable length both to ensure specificity of the TFO and to obtain a stable triplex. This limits the number of targets available to the antigene strategy considerably. One method to increase the number of targets is the alternate strand triplex formation. When the target DNA sequence consists of two adjacent purine tracts on opposite strands it is possible to design a TFO with inverted polarity and a suitable linker, which is capable of binding to both purine tracts. In this approach two different types of linkers are needed to meet the requirements of the parallel Hoogsteen triplexes, whereas another set of linkers has to be constructed for the reverse Hoogsteen triplexes. DNA target sequences are mostly restricted to oligopurine-oligopyrimidine tracts even though more complex heterogeneous sequences can be recognized by oligomers containing modified bases or intercalators that bind base pair inversions or by switching from one strand of DNA to the other when oligopurine sequences alternate on DNA strands. In most cases, the binding of triplexforming oligonucleotides to target DNA sequences is not strong enough to expect the development of antigene oligonucleotides as therapeutically useful drugs. The binding strength can be increased by tethering intercalating agents to the ends of triplex-forming oligonucleotides or by inserting intercalating agents at internal sites to recognize base pair inversions in oligopurine- oligopyrimidine sequences.

There have been several successful designs of 3'-3' linkers for the alternate strand Hoogsteen triplex, but until now no satisfactory linker for the 5'-5' linkage has been developed. The lack of a good 5'-5' linker has limited the number of possible targets. A suitable 5'-5' linker would substantially increase the number of possible targets. When combined with a 3'-3' linker, it will be possible to design a TFO, which is able to switch back and forth between several adjacent purine tracts on alternate strands.

SUMMARY OF THE INVENTION

The problem that the present invention solves is the provision of triplex-forming oligonucleotide derivatives with an improved binding to target DNA sequences to enable the development of antigene oligonucleotides as therapeutically useful drugs. In particular the problem of the present invention is the provision of a suitable 5'-5' linker in order to increase the number of possible targets.

The present inventors have surprisingly found that novel intercalating oligonucleotide derivatives with three benzene rings interconnected with triple bonds that allow the aromatic rings to intercalate with the duplex and with both the TFO's. Only an additional short flexible linker at both ends is then required to make the correct length and to make the formation of a 5'-5' linked alternate strand triplex possible.

The invention provides an intercalating oligonucleotide for stabilizing natural or modified DNA and RNA triplexes and hybrids thereof wherein the intercalating oligonucleotide is a compound of the general structure:

wherein

• Ri and R₂ are independently of each other a mono-cyclic or polycyclic aromatic ring system, • Oligo-1 and Oligo-2 are independently selected from the group consisting of hydrogen, DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)-TNA, (3'- NH)-TNA, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Ribo-LNA, β-D-Xylo-LNA, [3.2.1J-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo- DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D- Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR-RNA, α-L-RNA, β-D- RNA, and modifications thereof,

• Linker-1 and Linker-2 do independently from each other comprise 1-60 atoms and may contain non aromatic cyclic regions, wherein Oligo-1 is connected via the linkage Linker-1 to the aromatic ring system R^ which in turn is connected via a Conjugator defining a conjugated system, such as a mono-cyclic or polycyclic aromatic ring system, to the aromatic ring system R₂ which in turn is connected by the linkage Linker-2 to the Oligo-2, where Linker-1 and Linker-2 are independently of each other attached via a phosphate moiety, or a sugar moiety, or a nucleobase, or a modified oligo backbone, to Oligo-1 and Oligo-2.

Waybright et al (J. Am. Chem. Soc. 2001, 723, 1828-1833) disclose oligonucleotide modified organics (OMOs) in nano-architectural systems utilizing DNA to self-assemble specific modules of interest. Although a few of these compounds fall under the definition of intercalating oligonucleotides according to the present invention Waybright et al do not envisage the use of these particular compounds as intercalating nucleotides. In order to render the present invention novel over Waybright et al. following compounds are excluded from protection:

wherein R is H, DMT or an oligonucletide. The Conjugator of the intercalating oligonucleotide derivatives of the present invention is aryl, R₃, linked to R via x single bonds, n double bonds and/or m triple bonds and linked to R₂ via y single bonds, k double bonds and/or I triple bonds where k, I, m, n, x and y independently from each other are integers of 0-5. Thus the 1-60 atoms of comprised in Linker-1 and Linker-2 may constitute alkyl, alkenyl and/or alkynyl groups.

More specifically the invention provides intercalating oligonucleotides of the formula:

Oligo-2

wherein the three benzene rings independently from each other can be ortho, meta or para substituted, r and s independently of each other are integers of 0-10, and Oligo-1 and Oligo-2 are independently selected from the group consisting of hydrogen, DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)- TNA, α-L-Ribo-LNA, ot-L-Xylo-LNA, β-D-Ribo-LNA, β-D-Xylo-LNA, [3.2.1J-LNA, Bicyclo- DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D-Ribopyranosyl- NA, α-L-Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR-RNA, α-L-RNA, β-D-RNA, and modifications thereof.

A preferred intercalating oligonucleotide of the present invention has the formula:

Ol

wherein

Oligo-1 and Oligo-2 are independently selected from the group consisting of hydrogen, DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)- TNA, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Ribo-LNA, β-D-Xylo-LNA, [3.2.1J-LNA, Bicyclo- DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D-Ribopyranosyl- NA, α-L-Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR-RNA, α-L-RNA, β-D-RNA, and modifications thereof.

The intercalating triplex forming oligonucleotides of the present invention are capable of forming triplexes with a target DNA duplex, or RNA duplex, or hybrids thereof. Moreover the Oligo-1 part of the intercalating triplex forming oligonucleotide is capable of forming Hoogsteen triplex or reverse Hoogsteen triplex with one of the duplex strands whereas the Oligo-2 part is capable of forming a Hoogsteen triplex or a reverse Hoogsteen triplex with the other strand of the duplex, the duplex being a DNA duplex, RNA duplex or hybrids thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows two possibilities for parallel alternate strand Hoogsteen triplex formation.

Figure 2 shows a duplex structure with two different TFO's bound to purine tracts on opposite strands. The circles mark the two 5'-ends to be connected by a linker.

Figure 3 shows a structure obtained from the conformational search of the alternate strand triplex with the intercalating linker (L). As appears from the figure the structure of the designed 5'-5' linker is flexible enough to allow stacking interaction with the surrounding nucleobases and stiff enough to ensure a favourable entropy effect for the alternate strand triplex formation.

Figure 4 shows the synthesis of the intercalating 5'-5' linker. The synthesis starts from commercially available 4-iodobenzyl bromide (1). Reaction with ethylene glycol and sodium hydride yielded the alcohol 2 in excellent yield. The subsequent Sonogashira coupling of 2 with the commercially available 1 ,3-diethynylbenzene afforded the core structure 3 of the intercalating linker. The intercalating linker was mono protected with dimethoxytrityl chloride in 41% overall yield from 2. The low yield is not a result of low reactivity of the starting diol 3, but rather a result of very poor selectivity between mono- and di-protected products, which, however, were easy to separate by chromatography. Attempts to optimize the reaction by varying the temperature and/or the amount dimethoxytrityl chloride were unsuccessful. The phosphoramidite 5 was synthesized in 87% yield by reaction with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite. Conditions: (i) Ethylene glycol, NaH, 5min, reflux, (ii) 1 ,3-Ethynylbenzene, Pd(PPh₃)₂CI₂, Cul, DIPEA, THF, 23h, rt. (iii) DMT-CI, NEt₃, CH₂CI₂, 15h, rt. (iv) NC(CH₂)₂OP(NPrⁱ ₂)₂, diisopropylammonium tetrazolide, NEt₃, MeCN, 3h, rt.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions apply to the present invention:

An "alkyl" group refers to an optionally substituted saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 20 carbons and contains no more than 10 heteroatoms. More preferably, it is a lower alkyl of from 1 to 12 carbons, more preferably 1 to 6 carbons, and more preferably 1 to 4 carbons. Heteroatoms are preferably selected from the group consisting of nitrogen, sulfur, phosphorus, and oxygen.

An "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. Preferably, 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.

An "alkynyl" group 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. Preferably, 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. An "aryl" refers to an optionally substituted aromatic group having at least one ring, i.e. monocyclic or polycyclic, with a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl, biaryl, and triaryl groups. Examples of aryl substitution substituents include alkyl, alkenyl, alkynyl, aryl, amino, substituted amino, carboxy, hydroxy, alkoxy, nitro, sulfonyl, 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.

A "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 include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, and imidazolyl. The heterocyclic aryl is optionally substituted as described above for an aryl.

Oligonucleotides according to the present invention include, but are not limited to, the kind of nucleic acids and/or nucleic acid analogues selected from DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, α-L-Ribo-LNA, α-L- Xylo-LNA, β-D-Ribo-LNA, β-D-Xylo-LNA, [3.2.1RNA, Bicyclo-DNA, 6-Amino-Bicyclo- DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D-Ribopyranosyl-NA, α-L-

Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR-RNA, α-L-RNA, β-D-RNA and mixtures thereof and hybrids thereof, as well as phosphorous atom modifications thereof, such as but not limited to, phosphorothioates, methyl phospholates, phosphoramidiates, phosphorodithiates, phosphoroselenoates, phosphotriesters and phosphoboranoates. In addition nonphosphorous containing compounds may be used for linking to nucleotides, such as but not limited to, methyliminomethyl, formacetate, thioformacetate and linking groups comprising amides. In particular nucleic acids and nucleic acid analogues may comprise one or more intercalator pseudonucleotides according to the present invention.

By HNA is meant nucleic acids as for example described by Van Aetschot et al., 1995. By

MNA is meant 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 nucleic 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.

The term 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 one of the four nucleobases adenine (A), uracil (U), guanine (G) or cytosine (C).

The function of nucleotides and nucleotide analogues according to the present invention 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 occurring 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.

Furthermore 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 occurring 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. Thus 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. The specific interaction of one nucleobase with another 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.

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 n-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. It is also possible to modify the 5-position of cytosine from propynes to fivemembered heterocycles and to tricyclic fused systems, which emanate from the 4- and 5-position (cytosine clamps). 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 deaza modified andenine and the tricyclic cytosine analogue having an ethoxyamino functional group of heteroduplexes. Furthermore, N2-modified 2-amino adenine modified oligonucleotides are among common 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 (conformational restricted), ribofuranosyl ring size change, connection sites - sugar to sugar, (C-3' to C-5' / C-2' to C-5'), hetero-atom ring - modified sugars and combinations of above modifications.. However, other sites may be derivatised, as long as the overall base pairing specificity of a nucleic acid or nucleic acid analogue is not disrupted. Finally, when the backbone monomer unit comprises a phosphate 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. Preferably 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 refers to a nucleotide or nucleotide analogue sequence which comprises 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 and 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. For example said site may be composed of two or more contiguous subsequences separated by any number of nucleotides and/or nucleotide analogues. Preferentially 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. Preferably 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.

Melting of nucleic acids refer to thermal separation of the two strands of a doublestranded nucleic acid molecule. The melting temperature (Tm) 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. Vice versa 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. Furthermore, as disclosed by the present invention, 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.

In addition the melting temperature is dependent on the physical/chemical state of the surroundings. For example 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 intercalating oligonucleotides of the present invention have at least following applications:

i) A method for fluorescence in situ hybridization FISH which method uses intercalating oligonucleotides according to the present invention; these intercalating oligonucleotides optionally being labelled with fluorescence markers, as is generally known in the art.

ii) The intercalating oligonucleotides according to the present inventions conjugated to DNA reactive agents. DNA reactive agents are mutagenic agents capable of directing mutagenesis, or are photoinducable crosslinkers, or are radioactive agents, or are alkylating groups, or are molecules that can recruit DNA-damaging cellular enzymes.

iii) A pharmaceutical composition suitable for use in antisense therapy and antigene therapy comprising one or more intercalating oligonucleotides according to the present invention.

iv) A method to treat diseases or conditions mediated by the presence of unwanted duplex polynucleotides, which method comprises administering to a subject in need of such treatment an effective amount of an intercalating oligonucleotide according to the present invention or a pharmaceutical composition thereof. Effect of the intercalating linker on triplex stability

The intercalating effect of the intercalating linker into a duplex, it was attached as a dangling end to a TFO was demonstrated. The thermal stability was measured of the so formed TFO I against a 26-mer duplex containing the target sequence of I (Table 1). Comparison of the stability of the triplex formed with I with the one formed with the original TFO without the intercalating linker, showed that the intercalating linker resulted in an increase in melting temperatures of 18°C and >20°C at pH5 and pH6, respectively. In the latter case, the unmodified 8-mer TFO did not form a triplex above 5°C. The very large stabilization showed that the intercalating linker was indeed able to stabilize the triplex structure.

Target DNA Entry TFO 3'-TATGTATGGGGAAAGAAATTCTTCTT-5' 5'-ATACATACCCCTTTCTTTAAGAAGAA-3' pH5 pH6 T_m (°C) T_m (°C) 1 3'-CTTTCTTT-5' + 5'-TTCTTCTT-3' 19 <5 2 3'-CTTTCTTT-5'-L (I) 37 25 3 3'-CTTTCTTT-5'-L-5'-TTCTTCTT-3' (II) >60 49

3'-TATGTATGGGGAAAGAAAAAGAAGAA-5' 5^,-ATACATACCCCTTTCTTTTTCTTCTT-3^■ pH5 pH6 T_m (°C) Tm (°C) 3'-CTTTCTTTTTCTTCTT-5^, >60 44

Table 1 : Thermal stability studies of triplexes comparing an unmodified TFO, with a TFO with the intercalating linker L attached to the 5'-end and with a 5'-5' linked (via L) TFO forming an alternate strand triplex. Bold faces mark the binding region of the TFO's.

Alternate strand triplex formation via the intercalating 5'-5' linker

The TFO II (Table 1) was synthesized with two regions of opposing orientations connected at the 5'-ends to the intercalating linker. The two regions matched two sequences on the alternating purine strands of the 26-mer target duplex. Thermal stability measurements of the triplex between II and the target duplex showed additional increases in melting temperatures of >23°C and 24°C at pH5 and pH6, respectively when compared to the melting temperatures obtained from I (Table 1). In this case the full effect could not be estimated at pH5 because of melting of the target duplex. The large additional gain in stability is best explained as a result of an alternate strand triplex formation. To investigate the stability of the alternate strand triplex compared to a traditional triplex, the melting temperature was measured of a triplex between a 16-mer TFO consisting of the same T/C ratio as II and a 26-mer target duplex. The melting temperature of the homotriplex was measured to 44°C at pH6, which are 5°C lower than the one observed for the alternate strand triplex. One should expect lower stability of an alternate triplex, because of interruption of the intra strand stacking. However, with the 5'-5' linkage, the loss in stability due to intra strand interruption is more than compensated by stacking onto the TFO regions and by intercalation into the duplex.

Mismatch investigations of 5'-5' linked alternate strand triplex

It is known that the thermal stability of a homotriplex decreases dramatically (>10°C) in case of a mismatch between the TFO and the target duplex. Therefore it was of interest to investigate whether this specificity is maintained for the 5'-5' linked alternate strand triplex using the TFO II. The thermal stability was measured of alternate strand triplexes with a mismatch between the duplex and the TFO at the position next to the 5'-5' linkage. The mismatch studies showed that the alternate strand triplex is very sensitive to a mismatch with a decrease in the melting temperature in the range of 13°C to 19°C at pH 6 (Table 2). Finally the stability was measured of an alternate strand triplex between II and a duplex where the two target purine tracts where separated by an additional nucleotide. In this case, the intercalating linker is not able to intercalate as efficiently as in the previous cases although the nucleobases of the TFO is fully matched with the target duplex. The combination resulted in a decrease in melting temperature of 7°C at pH6, which is smaller than the one observed for a simple mismatch next to the linker.

TFO Entry Target DNA 3'-CTTTCTTT-5'-L-5'-TTCTTCTT-3' (ll) pH6 T_m (°C) ΔT_m (°C) S'-TATGTATGGGGAAAGAAATTCTTCTT-S' 49 5'-ATACATACCCCTTTCTTTAAGAAGAA-3' 3'-TATGTATGGGGAAAGAAGTTCTTCTT-5' 32 -17 5'-ATACATACCCCTTTCTTCAAGAAGAA-3' 3'-TATGTATGGGGAAAGAACTTCTTCTT-5' 30 -19 5'-ATACATACCCCTTTCTTGAAGAAGAA-3' 3'-TATGTATGGGGAAAGAAITTCTTCTT-5' 36 -13 5'-ATACATACCCCTTTCTTAAAGAAGAA-3' 3'-TATGTATGGGGAAAGAAACTTCTTCTT-5' 42 5'-ATACATACCCCTTTCTTTGAAGAAGAA-3'

Table 2: Thermal stability studies of mismatched alternate strand triple helices. Bold faces mark the binding regions of the TFO's and underlined basepairs mark mismatches of the TFO.

EXAMPLES

Example 1 Preparation of 2-(4-iodobenzyloxy)ethanol (compound no. 2 in Scheme 1).

The reaction is carried out under nitrogen. NaH (60% in mineral oil, 2.61 g, 0.065 mol) is added in portions to ethylene glycol (25mL) at 0°C. After addition the mixture is allowed to come to RT, while stirring for 30min. 4-lodobenzyl bromide (3.88g, 0.013mol) is added and the reaction is heated with a heatgun until a clear solution is obtained. The mixture is poured into water (300mL) and extracted with ether (3x100mL). The combined organic phases are washed with brine (100mL) and dried over magnesium sulphate. After evaporation of the solvent, column chromatography (EtOAc/PE 1 :1 v/v) affords 2 (3.46g, 95%) as a clear oil. ¹H-NMR (CDCI₃): δ 2.30 (1H, br s, OH), 3.57 (2H, t, J = 4.5Hz, OCH₂CH₂OH), 3.74 (2H, m, CH₂OH), 4.49 (2H, s, PhCH₂O), 7.08 (2H, d, J = 8.1 , Ph), 7.67 (2H, d, J = 8.1 , Ph). ¹³C-NMR (CDCI₃): δ 61.7 (CH₂OH), 71.5 (OCH₂CH₂OH), 72.5 (PhCH₂O), 93.2, 129.5, 137.4, 137.6 (Ph). Example 2

Preparation of 1 ,3-bis[4-(2-hydroxyethoxymethyl)phenylethynyl]benzene (compound no. 3 in Scheme 1).

1 ,3-Diethynylbenzene (0.69g, 5.5mmol), compound 2 (3.06g, 11mmol), Pd(PPh₃)₂CI₂ (86mg, 0.12mmol) and copper(l) iodide (86mg, 0.45mmol) are suspended in 40mL dry tetrahydrofuran under nitrogen. N-Ethyldiisopropylamine (3.25mL) is added and the reaction is stirred at RT for 23h. Water (50mL) is added and the mixture is extracted with ethyl acetate (3x25mL). The combined organic phases are washed with brine (2x25mL) and dried over magnesium sulphate. After removal of the solvent, column chromatography (EtOAc/PE 3:1 v/v) affords 3 (1.80g, 77%) as a white solid, mp 125°-127°C. ¹H-NMR (CDCI₃): δ 2.15 (2H, m, 2xOH), 3.61 (4H, t, J = 4.5Hz, 2xOCH₂CH₂OH), 3.77 (4H, m, 2xCH₂OH), 4.57 (4H, s, 2xPhCH₂). 7.30-7.71 (12H, m, Ph). ¹³C-NMR (CDCI₃): δ 61.9 (CH₂OH), 71.5 (CH₂CH₂OH), 72.8 (PhCH₂O), 88.6, 89.8 (C≡C), 122.4, 123.6, 127.6, 128.5, 131.3, 131.7, 134.6, 138.4 (Ph). MALDI HRMS, found m/z 449.1732; calculated for C₂₈H₂₆O₄ (M + Na)⁺ 449.1723.

Example 3

Preparation of 2-{4-[3-(4-{2-[Bis(4-methoxyphenyl)phenylmethoxy]ethoxy-methyl}phenyl- ethynyl)phenylethynyl]benzyloxy}ethanol (compound no. 4 in Scheme 1).

The diol 3 (1.16g, 2.71 mmol) and dry triethylamine (454μL, 3.26mmol) is dissolved in dry methylene chloride (40mL) under nitrogen and dimethoxytrityl chloride (1.103g, 3.26mmol) in methylene chloride (15mL) is slowly added during 15min. The mixture is stirred at RT for 15h. The solvent is removed in vacuo and the residue is purified by column chromatography (EtOAc/PE/TEA 50:50:1 v/v/v) yielding 4 (0.81g, 41%) as a clear oil. ¹H- NMR (CDCI₃): δ 2.08 (1H, br s, OH), 3.28 (2H, t, J = 4.9Hz, CH₂ODMT), 3.60 (2H, t, J = 4.5Hz, CH₂CH₂OH), 3.68 (2H, t, J = 4.9Hz, CH₂CH₂ODMT), 3.77 (8H, br s, 2xOCH₃, CH₂OH), 4.57 (2H, s, PhCH₂O), 4.61 (2H, s, PhCH₂O), 6.81 (4H, d, J = 8.8Hz, DMT), 7.17-7.72 (21 H, m, ArH). ¹³C-NMR (CDCI₃): δ 55.2 (2xCH₃O), 61.9 (CH₂OH), 63.2 (CH₂ODMT), 69.9 (CH₂CH₂ODMT), 71.5 (CH₂CH₂OH), 72.6 (PhCH₂O), 72.8 (PhCH₂O), 85.9 (OCPh₃), 88.4, 88.7, 89.7, 90.0 (2xC≡C), 113.0, 126.6, 127.3, 128.2, 130.0, 136.3, 145.0, 158.4 (DMT), 122.0, 122.4, 123.5, 123.6, 127.6, 127.7, 128.4, 131.19, 131.24, 131.6, 131.7, 134.6, 138.4, 139.2 (Ar). MALDI HRMS, found m/z 751.3033; calculated for

Example 4

Preparation of Phosphoramidite of 2-{4-[3-(4-{2-[Bis(4methoxy-phenyl)phenylmethoxy]- ethoxymethyl}phenylethynyl)phenylethynyl]ben-zyloxy}ethanol (compound no. 5 in Scheme 1).

The alcohol 4 (0.33g, 0.45mmol) is dissolved under nitrogen in dry acetonitrile (6mL). Triethylamine (124μL, 0.90mmol), 2-cyanoethyl N,N,N',N'-tetraisopropylphosphordiamidite (283μL, 0.90mmol) and diisopropylammonium tetrazolide (0.145g, 0.85mmol) are added and the mixture is stirred at RT for 3h. The solution is filtered through silica gel (EtOAc/PE/TEA 25:75:1 v/v/v) leading to 5 (0.36g, 87%) as a yellow oil. ¹³C-NMR (CDCI₃): δ 20.25, 20.34 (CH₂CN), 24.5, 24.57, 24.59, 24.7 (4xCH₃), 43.0, 43.1 (2xCH(CH₃)₂), 55.2 (2xOCH₃), 58.3, 58.5 (CH₂OP), 62.6, 62.8 (CH₂CH₂OP), 63.2 (CH₂ODMT), 69.9 (CH₂CH₂ODMT), 70.3, 70.4 (PhCH₂O), 72.6 (PhCH₂O), 85.9 (OCPh₃), 88.4, 88.5, 89.8, 89.9 (2xC-≡C), 113.0, 126.6, 127.3, 128.2, 130.0, 136.3, 145.0, 158.4 (DMT), 122.0, 122.1 , 123.58, 123.61 , 127.5, 127.7, 128.4, 131.2, 131.6, 134.5, 138.8, 139.2 (Ar). ³¹P-NMR (CDCI₃): δ 149.5

Example 5 Oligodeoxynucleotide synthesis, purification and thermal stability studies.

The phosphoramidite 5 was dissolved in dry acetonitrile as a 0.1 M solution and used for oligo-synthesis under standard conditions for nucleotide couplings (2 min. coupling). The coupling efficiency of the intercalating linker was -90%. In case of II the change in polarity around the linker was accomplished by using 5'-phosphoramidites instead of the usual 3'- phosphoramidites. The TFO's were synthesized with dimethoxytrityl on and purified by HPLC Waters Xterra™ MS Cι₈ column. Buffer A, 950mL of 0.1 M NH₄HCO₃ and 50mL of acetonitrile, pH 9.0; buffer B, 250mL of 0.1 M NH₄HCO₃ and 750mL of acetonitrile, pH 9.0. Gradient 5 min. 100% A, linear gradient to 70% B in 30 min., 2 min. 70% B, linear gradient to 100% B in 8 min. and then 100% A in 15 min. Retention time 46min and 38min for I and II, respectively. Dimethoxytrityl was removed by treatment with 20μL of water and 80μL of acetic acid. Both modified TFO's were confirmed by MALDI-TOF analysis on a Voyager Elite Biospectrometry Research Station from PerSeptive Biosystems. The sequences synthesized are shown in Table 1. Example 6

The thermal stability studies.

The melting temperatures were measured on a 1.5μM scale in 1mL of buffer consisting of 0.1 M NaCI, 0.01 M MgCI₂ and 0.02M sodium cacodylate where the desired pH was obtained by addition of cone. HCI. The samples were initially heated to 80°C followed by cooling to 5°C before the melting temperatures were measured by increasing the temperature 2°C/min from 5°C to 85°C. The thermal melting data are shown in Tables 1 and 2.

Claims

1. An intercalating oligonucleotide for stabilizing natural or modified DNA and RNA triplexes and hybrids thereof having the general structure

wherein

Ri and R₂ are independently of each other a mono-cyclic or polycyclic aromatic ring system, Oligo-1 and Oligo-2 are independently of each other an oligonucleotide selected from the group consisting of hydrogen, DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Ribo-LNA, β-D-Xylo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D-Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR-RNA, α-L-RNA, β-D-RNA, and modifications thereof, Linker-1 and Linker-2 do independently from each other comprise 1-60 atoms and may contain non aromatic cyclic regions, wherein Oligo-1 is connected via the linkage Linker-1 to the aromatic ring system R^ which in turn is connected via the Conjugator defining a conjugated system, such as a mono-cyclic or polycyclic aromatic ring system, to the aromatic ring system R₂ which in turn is connected by the linkage Linker-2 to the Oligo-2, where Linker-1 and Linker-2 are independently of each other attached via a phosphate moiety, a sugar moiety, a nucleobase, or a modified oligo backbone, to Oligo-1 and Oligo-2, and with the proviso that following compounds are excluded from protection:

wherein R is H, DMT or an oligonucletide

2. An intercalating oligonucleotide according to claim 1 , wherein the Conjugator is an aryl, R₃, linked to R via x single bonds, n double bonds and/or m triple bonds and linked to R₂ via y single bonds, k double bonds and/or I triple bonds where k, I, m, n, x and y independently from each other are integers of 0-5.

3. An intercalating oligonucleotide according to claim 2, wherein the linkage between R₃ and Ri is composed of alkyl, alkenyl and/or alkynyl groups.

4. An intercalating oligonucleotide according to claim 2 or 3, wherein the linkage between R₃ and R₂ is composed of alkyl, alkenyl and/or alkynyl groups.

5. An intercalating oligonucleotide according to any one of claims 2 to 4, wherein the linkage between R₃ and R or R₃ and R₂ is independently selected from the group consisting of alkyl of from 1 to 12 carbons, alkenyl of from 2 to 12 carbons, alkynyl of from 2 to 25 carbons and combinations thereof.

6. An intercalating oligonucleotide according to claim 5, wherein the alkynyl group has 2 to 4 carbons.

7. An intercalating oligonucleotide according to claim 6, wherein the alkynyl group is acetylene.

8. An intercalating oligonucleotide according to any one of the preceding claims, wherein Linker-1 and Linker-2 are independently selected from the group consisting of straight- chain, branched-chain, and cyclic alkyl groups.

9. An intercalating oligonucleotide according to claim 9, wherein the alkyl group has heteroatoms selected from nitrogen, sulfur, phosphorus, and oxygen.

10. Intercalating oligonucleotides of the formula:

wherein the three benzene rings independently from each other can be ortho, meta or para substituted, r and s independently of each other are integers of 0-10, and Oligo-1 and Oligo-2 are independently of each other an oligonucleotide selected from the group consisting of hydrogen, DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)-TNA, (3'-NH)-TNA, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Ribo-LNA, β-D-Xylo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D-Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR-RNA, α-L-RNA, β-D-RNA, and modifications thereof, and with the proviso that following compounds are excluded from protection:

wherein R is H, DMT or an oligonucletide.

11. Intercalating oligonucleotide of the formula:

Ol

igo-2

wherein

• Oligo-1 and Oligo-2 are independently selected from the group consisting of hydrogen, DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2'-NH)-TNA, (3'- NH)-TNA, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Ribo-LNA, β-D-Xylo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo- DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D- Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA, 2'-R-RNA, 2'-OR-RNA, α-L-RNA, β-D- RNA, and modifications thereof.

12. Intercalating oligonucleotide according to claim 11 , wherein Oligo-1 and Oligo-2 are single-stranded pyrimidin-rich oligodeoxyribonucleotides.