EP2126083A1 - Hoogsteen-type triplex formation of pyrene labelled probes for nucleic acid detection in fluorescence assay - Google Patents

Abstract

The present invention relates to fluorescent oligonucleotides and methods of providing and using fluorescent oligonucleotides. In particular, the present invention relates to fluorescent oligonucleotides that are capable of triplex formation, said triplex formation resulting in changed fluorescent properties such as to allow detection of triplex formation.

Description

HOOGSTEEN-TYPE TRIPLEX FORMATION OF PYRENE LABELLED PROBES FOR NUCLEIC ACID DETECTION IN FLUORESCENCE ASSAY

Background of the invention

During the last decade, the sequencing of the human genome has lead to a high need for the development of probes, able to detect specific sequences of nucleic acids in cytogenetic research and clinical diagnosis. The demand for such assays is caused by a need of clinics to reduce time in the detection of diseases. One of the ways in solving this problem is the development of assays, where nucleic acid species are recognized directly in the cell, without any amplification steps, like PCR.

In 2003 Hausmann et al.³ introduced the combinatorial oligonucleotide fluorescence in situ hybridisation (COMBO-FISH) that uses oligonucleotides (ONs) that form triple helices with intact duplexes to label chromosomes in a cell nucleus under non- denaturing conditions. Triplexes are formed when triplex forming oligonucleotides (TFO's) bind to a homopurine/homopyrimidine DNA duplex in the major groove via the formation of Hoogsteen base-pairs with the homopurine strand. As this triplex formation is highly specific, it can be used for sequence-specific recognition of dsDNA, without prior denaturation of it.⁵ However, one of the disadvantages is the use of nucleic acid probes possessing a fluorophore, which is not sensitive enough to changes in the microenvironment, especially after hybridization of the probe to duplex/triplex. This leads to a high background signal and the requirement of washing out the excess of the probes which makes labelling very difficult. To overcome these problems, novel fluorescent modified nucleic acids are needed.

Previous research in which a pyrene unit was attached to the 2'-position via different linkers studied the influence upon binding to ssDNA or ssRNA. These modified oligomers showed an increase in the fluorescence signal upon binding to ssRNA.^7"11 Thus far, no similar effects have been described for triplex formation.

Obika et al.¹² demonstrated that several 2'-5'-linkages in a 3'-5' TFO strand led to stabilisation of triplexes. Summary of the invention

The present invention provides oligonucleotides with new fluorescent properties that are useful for detection of nucleic acids. The oligonucleotides of the invention are particular useful for detection of doublestranded nucleic acids. Another aspect of the invention is a monomer unit for synthesis of the oligonucleotide of the invention. Still other aspects are a method of forming a triplex complex and a method of detecting a nucleic acid.

Brief description of the figures

Figure 1. Structures of the incorporated triplex-stabilisators.

Figure 2. Steady-state fluorescence emission spectra of 0N2 and the corresponding complexes with Dl, 0N15 and 0N16 upon excitation at 350 nm. Spectra of 1.0 μM solutions were recorded in thermal denaturation buffer at 10°C at pH 5.0, emission slit 0.0 nm (figure 2a) and pH 6.0, emission slit 2.5 nm (figure 2b).

Figure 3. Steady-state fluorescence of ONlO and the corresponding complexes with Dl, ON15 and ON16 upon excitation at 350 nm. Spectra of 1.0 μM ON solutions were recorded in thermal denaturation buffer at 10⁰C at pH 5.0 (figure 3a) and pH 6.0 (figure 3b).

Figure 4. Steady-state fluorescence of ssONIO, corresponding triplexes with Dl and parallel and antiparallel duplexes with ON15 and ON16, respectively. Spectra were recorded in thermal denaturation buffer at pH 7.2 at 10⁰C, using an excitation wavelength of 350 nm.

Figure 5. Steady-state fluorescence emission spectra of ON12 and the corresponding duplexes with complementary ssDNA/RNA. Spectra were recorded in the thermal denaturation buffer at 10⁰C using an excitation wavelength of 350 nm and an excitation slit of 4.0 nm and emission slit of 2.5 nm.

Figure 6. Structures obtained by molecular modeling studies of the triplex formed by ON2 with dsDNA. The upper pictures show minimisation result with pyrene inside the duplex, the lower pictures with pyrene outside the duplex. Pictures to the left show the side-view of the triplex. Pictures to the right show the top-view. Figure 7. Structures obtained by molecular modeling studies of the duplex formed by ON 12 with complementary DNA. The modified building block is shown in green. Picture to the left shows the side-view of the duplex. Picture to the right shows the top-view.

Detailed description of the invention:

Here we present the synthesis of a novel pyrene containing nucleoside monomer which after incorporation into the middle of 14-mer homopyrimidine ONs exhibited strong increase of fluorescence quantum yield (Φ_F) from 0.004 for ssON to 0.061 for a corresponding triplex DNA upon excitation at 350 nm, at pH 6.0. Under similar conditions Φ_F for fully matched antiparallel duplex was 0.036. Substitution of five native nucleobases by α-L-locked nucleic acids (α-L-LNA) monomers in modified TFO led to further increase of fluorescence quantum yield to 0.158 for Hoogsteen-type triplex and to 0.081 for Watson-Crick duplex. The fluorescent nucleoside for ON synthesis was prepared by click chemistry between 1-ethynylpyrene and 3'-azidomethyl-3'- deoxyribothymidine and incorporated into several ONs. Thermal stabilities and fluorescence spectra of the different probes, containing also other nucleic acid analogues, e.g. twisted intercalating nucleic acids (TINA) and α-L-LNA and their corresponding duplexes and triplexes with complementary ssDNA/RNA and dsDNA's were examined. Modelling was used as a tool to explain the results of thermal stability studies and the fluorescence properties observed.

Thus, in a first aspect, the present invention provides an oligonucleotide with the general structure

• which can be (or is) incorporated into the backbone of an oligonucleotide or an oligonucleotide analogue, or PNA, or PNA analogues, wherein B is a nucleobase or modified nucleobase which can form hydrogen bonding to an natural nucleobase or a modified nucleobase, Z is a linker comprising 0-60 atoms, X and Y do independently of each other comprise 0-20 atoms and X and Y can independently of each other form a linkage to an oligonucleotide backbone, or to a nucleobase, or to an oligonucleotide, W comprise 10-90 atoms and comprises 2-14 condensed aromatic rings,

• N₁ and N₂ 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.I]-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 combinations and modifications thereof,

• with proviso that N₁ and N₂ can not both be hydrogen, and with proviso that following compounds is excluded from protection:

The structure depicted above as part of an oligonucleotide above, confers surprising fluorescent properties on the oligonucleotide as will be discussed further below and demonstrated in the examples section. The structure is herein termed "fluorescent triplex forming oligonucleotide monomer" and the oligonucleotide comprising the monomer is also herein termed a "fluorescent triplex forming oligonucleotide".

In a preferred embodiment, the linker Z comprises an aromatic ring, and/or heteroaromatic ring, and/or an alkene, and/or an alkyne and comprises an atom which forms the connection to the 3'-position of the nucleoside.

In yet another preferred embodiment, the oligonucleotide is according to general structure

• wherein L is an atom which may be substituted with hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylcarbonylalkyl, heteroarylcarbonylalkyl, alkylthio, arylthio, heteroarylthio, aralkyl, hydroxyl, mercapto, heteroarylcarbonyloxy, formyloxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxy, azido, cyano, amino, alkoxycarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, N- alkylcarbamoyloxy, N-arylcarbamoyloxy, N-heteroarylcarbamoyloxy, N- alkylthiocarbamoyloxy, N-arylthiocarbamoyloxy, N-heteroarylthiocarbamoyloxy, alkoxy, aralkyloxy, aralkylthio, aryl, polyaryl, heteroaryl, heteropolyaryl, benzocycloalkyl,

• Ar is an aromatic ring, or a heteroaromatic ring, or an alkene or an alkyne,

• Ar may be substituted with hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylcarbonylalkyl, heteroarylcarbonylalkyl, alkylthio, arylthio, heteroarylthio, aralkyl, hydroxyl, mercapto, heteroarylcarbonyloxy, formyloxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxy, azido, cyano, amino, alkoxycarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, N-alkylcarbamoyloxy, N- arylcarbamoyloxy, N-heteroarylcarbamoyloxy, N-alkylthiocarbamoyloxy, N- arylthiocarbamoyloxy, N-heteroarylthiocarbamoyloxy, alkoxy, aralkyloxy, aralkylthio, aryl, polyaryl, heteroaryl, heteropolyaryl, benzocycloalkyl,

• W comprises 2-14 condensed aromatic rings which may be substituted with hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylcarbonylalkyl, heteroarylcarbonylalkyl, alkylthio, arylthio, heteroarylthio, aralkyl, hydroxyl, mercapto, heteroarylcarbonyloxy, formyloxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxy, azido, cyano, amino, alkoxycarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, N-alkylcarbamoyloxy, N-arylcarbamoyloxy, N- heteroarylcarbamoyloxy, N-alkylthiocarbamoyloxy, N-arylthiocarbamoyloxy, N- heteroarylthiocarbamoyloxy, alkoxy, aralkyloxy, aralkylthio, aryl, polyaryl, heteroaryl, heteropolyaryl, benzocycloalkyl,

Preferably, W comprises 2-6 condensed aromatic rings and/or heteroaromatic rings.

More preferably, W comprises derivatives of fluorescent molecules, i.e. derivatives of naphthalene, anthracene, acridine, acridone, phenanthrene, phenanthroline, pyrene, perylene.

Most preferably, W comprises pyrene.

Preferably, Y and X independently of each other are equal to O, or S, or NH.

Preferably, Ar comprises a five membered heterocyclic aromatic ring, wherein W comprises 2-6 condensed aromatic rings and/or heteroaromatic rings, wherein Y and X independently of each other are equal to O, or S, or NH

Oligonucleotide comprising triazole linker

In an even more preferred embodiment, the oligonucleotide of the invention comprises a triazole linker, i.e. L is triazole and W comprises 2-14 condensed aromatic rings which may be substituted with hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylcarbonylalkyl, heteroarylcarbonylalkyl, alkylthio, arylthio, heteroarylthio, aralkyl, hydroxyl, mercapto, heteroarylcarbonyloxy, formyloxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxy, azido, cyano, amino, alkoxycarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, N-alkylcarbamoyloxy, N-arylcarbamoyloxy, N- heteroarylcarbamoyloxy, N-alkylthiocarbamoyloxy, N-arylthiocarbamoyloxy, N- heteroarylthiocarbamoyloxy, alkoxy, aralkyloxy, aralkylthio, aryl, polyaryl, heteroaryl, heteropolyaryl, benzocycloalkyl,

Preferably, W comprises 2-6 condensed aromatic rings and/or heteroaromatic rings.

In a preferred embodiment, W comprises derivatives of fluorescent molecules, i.e. derivatives of naphthalene, anthracene, acridine, acridone, phenanthrene, phenanthroline, pyrene, perylene.

Most preferably, W comprises pyrene.

In an alternative embodiment, the triplex forming oligonucleotide is of the formula:

Oligonucleotide comprising pyrene

As mentioned above, in a preferred embodiment the oligonucleotide of the invention comprises pyrene and is of the formula

wherein B is a nucleobase selected from the group consisting of Thymine, Adenine, Cytosine, Guanine, Uracil, or modifications thereof,

Ni and N₂ are independently of each 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.I]-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 combinations and modifications thereof

As described in the examples section, the oligonucleotide of the invention has very useful fluorescent properties. The oligonucleotide has a very low fluorescence signal as single stranded oligonucleotide and upon hybridisation to a target double stranded nucleic acid, a considerable increase in fluorescent signal is observed.

The discrimination in fluorescence properties upon binding of ONs possessing pyrene residue to dsDNA and ssDNAs of different nature has never been observed hitherto. The strong increase of fluorescence intensity upon binding to dsDNA is a very important feature and an advantage of the described probe, as it opens novel possibilities for the detection of nucleic acids upon triplex formation. Earlier described attempts were devoted to the development of probes based on the principle of molecular beacons, i.e. a fluorophore and a quencher were placed separately at the 3' and 5'-ends of the TFO, which was extended with a number of complementary bases in order to make a clamp and place a fluorophore and a quencher in a close proximity. However, it is known that molecular beacons sometimes open up in a cell media in the absence of complementary targets bringing false positives in the detection assays. Existing nucleic acids dyes, based on small molecules, like TOTO and others, which are used nowadays for labelling of dsDNA have two main disadvantages. First, such staining dyes bind to dsDNA unspecifically or have preferences only to certain regions, which usually are repetitive in the genome. Second, these dyes have a very low affinity to triplexes as a consequence of electrostatic repulsion between nucleic acid triplexes and charged structure of dyes and therefore cannot be used as a combination of TFO/staining dye for sequence selective labelling of dsDNA. Therefore, nucleic acids probes having one or several modified nucleotides with a fluorophore, which give a positive signal upon binding to complementary strands, are of high importance for any detection technique.

The oligonucleotides of the invention enable sequence specific detection of double stranded nucleic acids without washing out excess probe. This is useful for various gene typing methods such as e.g. analysis of single nucleotide polymorphisms and detection of genefusions. The fluorescent signal of the oligonucleotide also increases upon base pairing with a single stranded nucleic acid, wherefore the oligonucleotide can also be used for detection of single stranded nucleic acids. Interestingly, the fluorescent signal increases more by base pairing to ssRNA than to ssDNA. Importantly, the oligonucleotide can form both parallel duplexes and antiparallel duplexes.

Preferably, the oligonucleotide of the invention comprises at least 10 oligonucleotide monomer units. More preferably, the oligonucleotide comprises 14 oligonucleotide monomer units.

The term oligonucleotide monomer unit as used herein refers to a RNA nucleotide, DNA nucleotide, LNA nucleotide or any other monomer units that can be build into the oligonucleotide. Thus, as herein, also the nucleotide substituted at the 3'-position with a pyrene is regarded as a monomer unit.

As the skilled man will understand, the oligonucleotide of the invention will display an increasing melting temperature base paired to single stranded nucleic acid or double stranded nucleic acid with increasing length. Thus, the length of the oligonucleotide may be increased if a stronger complex (duplex or triplex) is desired. On the other hand, specificity may be compromised when using a long oligonucleotide. In certain situations, also bioavailability may be compromised. Therefore, in one embodiment, it is preferred that the oligonucleotide is between 10 and 40 oligonucleotides in length. More preferred is a length between 15 and 30 monomer units.

In another embodiment, the oligonucleotide comprises less than 30 oligonucleotide monomer units.

In yet another embodiment, the oligonucleotide comprises at least 8 DNA monomer units, such as 10, 12, 14, 16, 18 and 20 monomer units.

In yet another embodiment, the oligonucleotide does not comprise any RNA units. In certain embodiments, RNA monomer units may be omitted from the oligonucleotide of the invention, as incorporation of RNA monomer units may decrease biostability of the oligonucleotide.

Preferably, the oligonucleotide of the invention comprises at least 7 contiguous pyrimidine nucleobases, i.e. 7 oligonucleotide monomer units that all comprise a pyrimidine nucleobase. In other embodiments, the oligonucleotide comprises at least 10, 14, 16, 20, and 24 contiguous pyrimidine nucleobases, respectively. Preferably, the oligonucleotide comprises between 10 and 30 contiguous pyrimidine nucleobases and more preferably between 15 and 25 contiguous pyrimidine nucleobases.

In another embodiment, 60% of the monomer units of the oligonucleotide comprise pyrimidine nucleobases. More preferably, 80% of the monomer units comprise pyrimidine nucleobases.

In still another embodiment, the oligonucleotide of the invention comprises exclusively pyrimidine nucleobases.

A pyrimidine rich sequence in the oligonucleotide of the invention will enable triplex complex formation with a purine rich double stranded nucleic acid. As mentioned earlier, triplex forming oligonucleotides (TFOs) are of interest for sequence specific detection of double stranded DNA.

Preferably, the oligonucleotide of the invention comprises at least 7 contiguous purine nucleobases, i.e. 7 oligonucleotide monomer units that all comprise a purine nucleobase. In other embodiments, the oligonucleotide comprises at least 10, 14, 16, 20, and 24 contiguous purine nucleobases, respectively. Preferably, the oligonucleotide comprises between 10 and 30 contiguous purine nucleobases and more preferably between 15 and 25 contiguous purine nucleobases.

In another embodiment, 60% of the monomer units of the oligonucleotide comprise purine nucleobases. More preferably, 80% of the monomer units comprise purine nucleobases.

In still another embodiment, the oligonucleotide of the invention comprises exclusively purine nucleobases.

A purine rich sequence in the oligonucleotide of the invention will enable triplex formation complex with a pyrimidine rich double stranded nucleic acid by formation of an antiparallel triplex complex.

In yet another embodiment, the oligonucleotide comprises a contiguous sequence of G and T bases (GT rich sequence) of at least 10 bases, and more preferably of at least 15 bases.

In a preferred embodiment, the oligonucleotide of the invention further comprises modifications or nucleotide analogues that increase its affinity to a double stranded target sequence.

Thus, in a preferred embodiment, the oligonucleotide comprises a LNA (locked nucleic acid) monomer unit.

Even more preferably, the oligonucleotide comprises at least one LNA monomer unit per 4 oligonucleotide monomer units. Thus, if the oligonucleotide consists of 20 monomer units; the oligonucleotide will comprise at least 4 LNA monomer units. In another embodiment, every fourth monomer unit is LNA.

Preferably, the LNA unit is selected from the group consisting of: LNA, α-L-amino-LNA, α-D-amino-LNA, α-L-thio-LNA, α-D-thio-LNA, β -L-amϊno-LNA, β -D-amino-LNA, β-L- thio-LNA, β-D-thio-LNA, α-L-LNA and β-L-LNA.

Most preferably, the LNA unit is α-L-LNA.

Preferably, Nl and N2 each comprise 5 oligonucleotide monomer units. Thus, the nucleotide monomer unit comprising pyrene is located at least 5 monomer units from either end of the oligonucleotide. In another embodiment, the monomer unit comprising pyrene is located at the central part of the oligonucleotide, wherein the central part comprises half the number of monomer units relatively to the total length of the oligonucleotide. If the oligonucleotide consists of 21 monomer units, the central part comprises 11 units.

Phosphoramidate

A second aspect of the invention is a fluorescent triplex forming oligonucleotide monomer adapted for incorporation into an oligonucleotide synthesis.

In a preferred embodiment, the monomer is a phosphoramidate of the formula

• Where B is a nucleobase or modified nucleobase which can form hydrogen bonding to an natural nucleobase or a modified nucleobase

More preferably, B is a nucleobase selected from the group consisting of Thymine, Adenine, Cytosine, Guanine, Uracil, or modifications thereof,

Method of synthesizing oligonucleotide

A third aspect the invention is a method for the preparation of an oligonucleotide of the invention comprising the steps

a. Providing a fluorescent triplex forming oligonucleotide monomer adapted for incorporation into a oligonucleotide synthesis b. Providing standard reagents for oligonucleotide synthesis c. During standard oligonucleotide synthesis incorporating one or more fluorescent triplex forming oligonucleotide monomer(s) into the oligonucleotide d. Thereby generating a fluorescent triplex forming oligonucleotide comprising one or more fluorescent triplex forming oligonucleotide monomer(s) In a preferred embodiment, the oligonucleotide monomer adapted for incorporation is a phosphoramidate as described in the second aspect of the invention.

Method of forming triplex complex

A fourth aspect of the invention is a method of forming a triplex nucleic acid comprising the steps:

a. Providing an oligonucleotide of the invention b. Providing a double stranded target nucleic acid c. Incubating the oligonucleotide of step a with the double stranded target nucleic acid of step b under conditions of triplex formation d. Thereby forming a triplex nucleic acid structure

Method of detection

A fifth aspect of the invention is a method of detecting a nucleic acid comprising the steps of

a. Providing an oligonucleotide of the invention. b. Providing a test sample c. Incubating the test sample and the oligonucleotide under conditions allowing triplex formation d. Measuring fluorescence of the mixed sample of step c

The test sample may e.g. comprise genomic DNA from an individual that is to be tested for the presence of particular gene sequences, e.g. SNPs, gene fusions or gene translocations. The sample could also comprise bacterial DNA if the aim of the test is determining the presence of a certain bacterial strain in a clinical sample. Either way, the oligonucleotide is designed such as to be able to form a triplex with the target sequence. If the target sequence is present in the sample, an increased fluorescent signal will be observed.

In another embodiment, the sample is a tissue slice, e.g. from a solid tumour. In a preferred embodiment, the fluorescence measurement comprises excitation at a wavelength between 340 nm and 360 nm. Even more preferred is a wavelength between 345 and 355 and most preferred is a wavelength between.

In yet another embodiment, the test sample is a PCR reaction mixture. I.e. the oligonucleotide of the invention is used to determine the presence of a PCR product in a PCR reaction. Preferably, detection of PCR is performed quantitatively and for each PCR round such as to enable quantitative PCR.

Examples

Example 1

Materials and Methods

NMR spectra were recorded on a Varian Gemini 2000 spectrometer at 300 MHz for ¹H using TMS (δ: 0.00) as an internal standard and at 75 mHz for ¹³C using CDCI₃ (δ: 77.0) or DMSO (δ: 39.44) as an internal standard. Accurate ion mass determinations of the synthesised compounds were performed using the 4.7 T Ultima Fourier transform (FT) mass spectrometer (Ion Spec, Irvine, CA). The [M + Na]⁺ ions were peak matched using ions derived from the 2,5-dihydroxybenzoic acid matrix. MALDI-TOF mass spectra of isolated oligodeoxynucleotides (ONs) were determined on a Voyager Elite biospectrometry research station (PerSeptive Biosystems). Thin-layer chromatography (TLC) analyses were carried out with use of TLC-plates 60 F_2S4 purchased from Merck and were visualized in UV light (254 nm). The silica gel (0.040-0.063 mm) used for column chromatography was purchased from Merck. Solvents used for column chromatography were distilled prior to use, while reagents were used as purchased.

Synthesis of l-{3-[(4-(pyren-l-yl)-lH-l,2,3-triazol-l-yl)methylene]-3-deoxy- β-D-ribofuranosyl}thymine (compound 2 in Scheme 1).

To a solution of compound 1 (in Scheme 1) (420 mg, 1.41 mmol) in 8 mL of a DMF/H₂O 19:1 mixture in a microwave tube, freshly prepared IM CuSO₄ in water (283 μM, 0.28 mmol) was added. The solution was bubbled with Argon for 1 minute. Afterwards, a freshly prepared aqueous sodium ascorbate solution (424 μM, 0.42 mmol) and 1-ethynylpyrene (480 mg, 2.12 mmol, dissolved in 8 mL DMF/ H₂O 19:1) were added. The mixture was bubbled again with Argon for 3 minutes. The resulting mixture in a sealed microwave tube was treated with a microwave synthesiser Emrys Creator. The heating temperature was set to 125 °C, with a 30 s premixing time. The reaction mixture was irradiated for 15 min followed by N₂ cooling to 40 ⁰C. Afterwards water was added and the mixture was kept at 6 °C during 5 h for complete precipitation of the triazol formed and unreacted alkyn. The precipitate was filtered off and washed with water to remove all catalysts. After this, the solid on the filter was washed with methanol to dissolve the triazol. The collected methanol was evaporated to dryness and the residue was purified by silica gel column chromatography (CH₂CI₂/Me0H 95:5) to yield the pure triazol 2 (583 mg, 79%). ¹H NMR (300 MHz, DMSO): δ 1.79 (3H, s, 5- CH₃), 2.96 (IH, m, H-3'), 3.51 (IH, d, J = 12.3 Hz, H-5'), 3.81 (IH, J = 12.6 Hz, H- 5"), 4.19 (IH, m, H-4'), 4.32 (IH, m, H-2'), 4.67 (IH, dd, J = 6.0 and 14.1 Hz, H-6'), 4.81 (IH, J = 7.5 and 14.1 Hz, H-6"), 5.33 (IH, br s, 5'-OH), 5.78 (IH, br s, 2'-OH), 6.24 (IH, d, J = 5.1 Hz, H-I'), 8.04-8.41 (9H, m, Pyrene-H and H-6), 8.77 (IH, s, triazol-H), 8.91 (IH, d, J = 6.3 Hz, Pyrene-H), 11.31 (IH, br s, N(6)H); ¹³C NMR (300 MHz, CDCI₃): 5 12.24 (5-CH₃), 41.17 (C-3'), 46.10 (C-6'), 60.12 (C-5'), 74.80 (C-2'), 82.77 (C-4'), 90.87 (C-I'), 108.30 (C-5), 123.86, 124.24, 124.89, 125.04, 125.10, 125.38, 125.46, 126.41, 127.01, 127.27, 127.43, 127.61, 127.92, 130.31, 130.49, 130.87 (Pyrene and triazol C-5), 136.14 (C-6), 146.03 (triazol C-4), 150.39 (C-2), 163.80 (C-4); HRMS (ESI-MS) for C₂₉H₂₅N₅O₅Na [M+Na]⁺ found, 546.1769; calcd, 546.1753.

Synthesis of l-{3-deoxy-5-O-(4_/4'-dimethoxytπphenylmethyl-3-[(4-(pyren-l- yl)-lH-l,2,3-triazol-l-yl)methylene]-β-D-ribofuranosyl>thymine (compound 3 in Scheme 1). Compound 2 (in Scheme 1) (530 mg, 1.01 mmol) was coevaporated three times with pyridine and dissolved in anhydrous pyridine (4 ml_). 4,4'-dimethoxytrityl chloride (461 mg, 1.36 mmol) was added under nitrogen. After 16 h, MeOH (0.5 ml.) and EtOAc (20 mL) were added and the mixture was extracted with NaHCO₃ (20 mL). The water layer was extracted twice with EtOAc (20 mL). The combined organic layers were dried over MgSO₄, evaporated to dryness, and purified by column chromatography (CH₂CI₂/Me0H/Pyridine 97/2.5/0.5). Compound 3 was obtained (760 mg, 91%) as a yellow foam. ¹H NMR (300 MHz, CDCI₃): δ 1.42 (3H, s, 5-CH₃), 3.10 (IH, m, H-3'), 3.29 (2H, m, H-5' and H-5"), 3.67 (6H, s, 2x OCH₃), 4.39 (3H, m, H-2', H-6' and H-6"), 4.71 (IH, m, H-4'), 5.71 (2H, m, H-I' and 2'-OH), 6.79 (4H, d, J = 8.7 Hz, DMT), 7.15-7.43 (1OH, m, DMT and H-6), 7.72 (IH, s, triazol-H), 7.89-8.10 (8H, m, Pyrene-H), 8.61 (IH, d, J = 9.3 Hz, Pyrene-H), 10.91 (IH, br s, N(6)H); ¹³C NMR (300 MHz, CDCI₃): 5 12.06 (5-CH₃), 42.86 (C-3'), 49.69 (C-6'), 55.11 (OCH₃), 72.57 (C-2⁷), 75.95 (C-5⁷), 82.01 (C-4'), 87.53 and 87.08 (C-I' and C(Ar₃)), 110.64 (C-5), 113.35, 124.93- 131.28, 135.40 (Pyrene, DMT and triazol C-5), 136.11 (C-6), 144.18 (triazol C-4), 146.55 (DMT), 150.58 (C-2), 158.70 (DMT), 164.55 (C-4); HRMS (ESI-MS) for C₅₀H₄₃N₅O₇Na [M+Na]⁺ found, 848.3084; calcd, 848.3060.

Synthesis of l-{2-O-[2-cyanoethoxy(diisopropyIamino)phosphino]-3-deoxy-5- O-(4,4'-dime-thoxytriphenylmethyl-3-[(4-(pyren-l-yl)-lH-l,2,3-triazol-l- yl)methylene]-β-D-ribofuranosyl}-thymine (compound 4 in Scheme 1).

Compound 3 (in Scheme 1) (510 mg, 0.62 mmol) was dissolved under nitrogen in anhydrous CH₂CI₂ (10 mL). Λ/,Λ/-Diisopropylammonium tetrazolϊde (159 mg, 0.93 mmol) was added followed by the dropwise addition of 2-cyanoethyl tetraisopropylphosphordiamidite (223 mg, 0.74 mmol) under external cooling with an ice-water bath. After 16h, the reaction was quenched with H₂O (6 mL). The layers were separated and the organic phase was washed with H₂O (6ml_). The water layers were washed with CH₂CI₂ and the resulting organic phases were combined, dried over MgSO₄ and filtered. The solvent was removed under reduced pressure, and the residue was purified using silica gel column chromatography (CHCI₃/MeOH/pyridine 99/0.5/0.5). The purified compound 4 (412 mg, 65%) was obtained as a foam that was used in DNA synthesis. ³¹P NMR (CDCI₃) δ 148.40, 150.34 in a ratio of 2:3; HRMS (ESI-MS) for C₅₉H₆₁N₇O₈P [M + H]⁺ found, 1026.4338; calcd, 1026.4318.

Synthesis and Purification of Modified Oligonucleotides. DMT-on oligo-deoxynucleotides were synthesized in a 0.2 μmol scale on CPG supports using Expedite Nucleic Acid Synthesis System Model 8909 (Applied Biosystems). Standard procedures were used for phosphoramidite 4 with 4,5-dicyanoimidazole as an activator except for extended coupling time (10 min) and an increased deprotection time (100 s), resulting in step-wise coupling yields of 98% for monomer 4 and >99% for unmodified DNA phosphoramidites.

The obtained DMT-on oligonucleotides bound to CPG-supports were treated with 32% aqueous ammonia (1.3 mL) at room temperature for 2 hours and then at 55°C overnight. Purification of the 5'-O-DMT-on ONs was carried out by using a reverse- phase semipreparative HPLC on a Waters Xterra MS C_i8 column. DMT groups were cleaved by treatment with 80% AcOH (100 μL) for 20 minutes, followed by addition of H₂O (100 μl_) and 3 M aq NaOAc (50 μL). The ONs were precipitated from 99% EtOH (600 μL). The precipitate was washed with chilled 70% aqueous ethanol. The purity of the obtained ONs was checked by ion-exchange chromatography on a LaChrom system (Merck Hitachi) using a GenPak-Fax column (Waters) and it was found to be 100% for all ONs.

Molecular Modeling. Molecular modeling experiments were performed with Maestro v7.5 from Schrδdinger. All calculations were conducted with AMBER* force field and the GB/SA water model. The dynamics simulations were performed with stochastic dynamics, a SHAKE algoritm to constrain bonds to hydrogen, time step 1.5 fs and simulation temperature of 300 K. Simulation for 0.5 ns with an equilibration time of 150 ps generated 250 structures, which all were minimized using the PRCG method with convergence threshold of 0.05 kJ/mol. The minimized structures were examined with Xcluster from Schrδdinger, and representative low-energy structures were selected. The starting structures were generated with Insight II v97.2 from MSI, followed by incorporation of the modified nucleoside building block.

Melting Temperature Measurements. Melting profiles were measured on a Perkin- Elmer UV-vis spectrometer Lambda 35 fitted with a PTP-6 temperature programmer. The triplexes were formed by first mixing the two strands of the Watson-Crick duplex, each at a concentration of 1.0 μM, followed by the addition of the TFO at a concentration of 1.5 μM in the corresponding buffer solution. The solution was heated to 80⁰C for 5 min and afterward cooled to 15°C and kept at this temperature for 30 minutes. The duplexes were formed by mixing the two strands, each at a concentration of 1.0 mM in the corresponding buffer solution followed by heating to 80⁰C for 5 minutes and then cooling to room temperature. The absorbance of both triplexes and duplexes was measured at 260 nm from 5 to 70⁰C with a heating rate of 1.0 °C/min. The melting temperatures (7^" _m, ⁰C) were determined as the maximum of the first derivative plots of the melting curves. All melting temperatures are within the uncertainty ± 0.5 ⁰C as determined by repetitive experiments.

Synthesis of phosphoramidate and oligonucleotides

The synthesis of phosphoramidite 4 (Scheme 1) was started from compound 1, which was obtained in 11 steps from 1,2-O-isopropylidine-α-D-xylofuranose with an overall yield of 17%.¹³ The formation of the 1,4-triazol was performed in the microwave cavity during 15 minutes at 125 ⁰C with Cu (I) as a catalyst in 79% yield. 5'-O- Dimethoxytrityl-protection followed by phosphytilation at the 2'-position were performed under standard conditions in 95% yield over two steps.

DNA-synthesis of ON's containing compound 4 was performed on a 0.2 μmol scale under standard conditions except for an increased coupling time (10 min) and an extended deprotection step (100 sec), using 4,5-dicyanoimidazole as an activator, which resulted in a coupling efficiency of 98%. The obtained ON's were purified by reverse-phase HPLC, their composition was verified by MALDI-TOF (Table Sl) and the purity was found to be over 82% by ion-exchange HPLC.

Thermal denaturation studies

The thermal stability of triplexes and duplexes (DNA/DNA and DNA/RNA) using synthesized oligonucleotides was determined by thermal denaturation studies. The melting temperatures (7^" _m, ⁰C) determined as the first derivatives of melting curves at 260 nm are listed in Tables 1, 3 and 4.

For homopyrimidine sequences, pH-dependent Hoόgsteen-type base-pairing was studied, by determining the thermal stability both of parallel triplexes toward duplex Dl and of parallel duplexes toward ON15 (Table 1). Here T_m values of Watson-Crick type antiparallel duplexes formed by ONl-IO and ON16 are also shown. Since the latter type of duplexes can also be studied by mixed pyrimidine/purine sequences, mixed 9- mer sequences were used for hybridization with ssDNA and ssRNA (Table 4).

1

O

Scheme 1. Reagents and conditions: a) 1-ethynylpyrene, CuSO₄, ascorbic acid, DMF/H₂O 19 : 1, 15 min, 125⁰C, microwave; b) DMT-CI, Pyridine, 16h; c) NC(CH₂)₂OP(NPri₂)2, diisopropyl-ammonium tetrazolide, CH₂CI₂, 16h.

Tables with data from melting temperature measurements and quantum yields

Table 1. T_m [⁰C] Data for Duplex Melting of ONl-IO and Dl, ON15 and ON16, Taken from UV Melting Curves (λ= 260 nm)^a

. ^a C= 1.5 μM of ONl-IO and 1.0 μM of each strand of dsDNA (Dl) in 20 imM sodium cacodylate, 100 mM NaCI, 10 mM MgCI₂, pH 5.0, 6.0 and 7.2; duplex 7^~ _m = 56,5 ⁰C (pH 5.0), 58.5 (pH 6.0) and 57.0 (pH 7.2); ^b C= 1.0 μM of each strand in 20 mM sodium cacodylate 100 mM NaCI, 10 mM MgCI₂, pH 5.0, 6.0 and 7.2; ^c Third strand and duplex melting overlaid. 7^" _m was determined at 350 nm; ^d Third strand and duplex melting overlaid; ^e n.d. not determined; p = TINA, T^L = thymin-1-yl α-L- LNA monomer, ^MeC^L = 5-methylcytosin-l-yl α-L-LNA monomer. Structure of monomer X is shown in Scheme 1 as the 5'-DMT- 2'-phosphoramidite-derivative 4, structures of p and α-L-LNA are shown in Figure 1.

Triplex³

3' CTGCCCCTTTC I I I I I I Parallel duplex⁶ Antiparallel duplex⁶

5' GACGGGGAAAGAAAAAA 5' GACGGGGAAAGAAAAAA 3' GGGGAAAGAAAAAA

(Dl) (ON15) (ON16)

pH = pH = pH = pH = pH = pH = pH= 5.0 pH= 6.0 pH= 7.2

5.0 6.0 7.2 5.0 6.0 7.2

ONl 3' TTT CTT TCC CC 55.0° 28.0 <5.0 29.5 19.5 n.d. 47.5 48.5 47.0

ON2 3' TTT CTX TCC CC 55.0' 25.0 <5.0 20.0 <5.0 n.d. 45.0 47.5 45.5

ON3 3' TTT CXX TCC CC 33.0 19.5 n.d.^e 32.0 21.5 n.d. 36.0 38.0 n.d.

ON4 3' TTTTTTCXXXCCCC 32.0 < 5.0 n.d. 31.5 24.0 n.d. 36.5 38.5 n.d.

ON5 3' TTT TXT CTT TCC CC 55.5^C 16.0 n.d. 23.5 <5.0 n.d. 39.5 43.0 n.d.

ON6 3' TTTTTTCXTXCC CC 25.5 <5.0 n.d. 21.5 <5.0 n.d. 31.5 37.5 n.d.

ON7 3' TTTTXTCTXTCCCC 27.0 <5.0 n.d. 22.5 <5.0 n.d. 34.5 39.5 n.d.

ON8 3¹ TTTTTTCTPXTCC CC 54.0 ^C 20.5 n.d. 27.5 16.5 n.d. 43.0 47.5 n.d.

ON9 3' TTT TTT CTXp TCC CC 53.0 ^c 22.5 n.d. 29.0 20.0 n.d. 44.0 46.5 n.d.

ONlO 3' TT^LT TT^LT ^MeC^LTX T^MeC^LC ^MeC^LC 65.5 ^d 49.5^C 35.5^C 53.0 43.5 38.5 58.5 59.0 58.5

Table 2. Quantum Yields at λ= 350 nm for Single-Stranded Probes ON2, ON5-ON7 and ONlO, and the Corresponding Parallel Triplexes, Parallel and Antiparallel Duplexes in Aerated Thermal Denaturation Buffer at 10°C. ^a C = 1.0 μM of each strand in 20 mM sodium cacodylate 100 mM NaCI, 10 mM MgCI₂, pH 5.0, 6.0 and 7.2; ^b n.d. not determined because of low thermal stability of triplexes; ^c exciplex band at 460 nm was observed.

SS strand Triplex*

3' CTGCCCCTTTC I I I I I I

5' GACGGGGAAAGAAAAAA

(Dl)

PH pH = pH = pH = pH = pH =

10 6.0 7.2 5.0 6.0 7.2

5.0

ON 2 3¹ TTT TTT CTX TCC CC 0.0 0.004 n.d.¹ 0.179 0.061 n.d. 01

ON 5 3' TTT TXT CTT TCC CC 0.0 0.003 n.d. 0.138 0.023 n.d.

01

ON 6 3¹ TTT TTT CXT XCC CC 0.0 0.003 n.d. 0.036 n.d. n.d.

01

15

ON 7 3' TTT TXT CTX TCC CC 0.0 0.003 n.d. 0.037 n.d. n.d.

02

ON lO 3' TT^LT TT^LT C^LTX TC^LC C^LC 0.0 0.003 0.05 0.179 0.158 0.073^c

01

Table 2 continuous on next side

Table 2 continued from previous side

Parallel duplex^a Antiparallel duplex^a

5' 3' GGGGAAAGAAAAAA GACGGGGAAAGAAAAA (ON 16)

A (ON 15)

PH pH = pH = pH = pH = pH =

10 6.0 7.2 5.0 6.0 7.2 5.0 SJ

ON 2 3' TTT TTT CTX TCC CC 0.0 n.d. n.d. 0.032 0.036 n.d. 01

ON 5 3' T TT TXT CTT TCC CC 0.0 n. d. n.d. 0.035 0.039 n.d.

05

ON 6 3¹ T TT TTT CXT XCC CC 0.0 n. d. n.d. 0.005 0.008 n.d.

09

15 ON 7 3' TTT TXT CTX TCC CC 0.0 n.d. n.d. 0.037 0.028 n.d.

09

ON lO 3' 7T^LT TT^LT C^LTX TC^LC C^LC 0.0 0.032 0.081^c 0.046 0.081 0.038^c

08

Table 3. 7^" _m [⁰C] Data for Mismatched Parallel Triplex of ON2, Taken from UV Melting Curves (λ= 260 nm)^a and Fluorescence

Quantum Yields at 350 nm (in brackets). For 7^~ _m measurement: C = 1.5 μM of ON2 and 1.0 μM of each strand of mismatche dsDNA in 20 mM sodium cacodylate, 100 mM NaCI, 10 mM MgCI₂, pH 5.0 and 6.0; for quantum yield measurement: C = 1.0 μM o each strand in the same buffer at pH 5.0.

3' TTT TTT CTX TCC CC (ON2) pH = 5.0 pH = 6.0

3' AAA AAA GAC AGG GGC AG 28.0 <5.0 N> (JI 5' TTT TTT CTG TCC CCG TC (0.001)

3' AAA AAA GAG AGG GGC AG 21.0 <5.0

5' TTT TTT CTC TCC CCG TC (0.001)

3' AAA AAA GAT AGG GGC AG 29.5 <5.0

5' TTT TTT CTA TCC CCG TC (0.001)

Table 4. T_m [⁰C] Data for Duplex Melting of ON11-14 and DNA/RNA Complements, Taken from UV Melting Curves (λ= 260 nm)^a and Fluorescence Quantum Yields for ON12 Determined (in brackets). ^aThermal denaturation temperatures measured as the maximum of the first derivative of the melting curve (A₂₆o vs temperature) recorded in high salt buffer (1400 mM NaCI, 20 mM sodium phosphate, pH 7.0), using 1.0 μM concentrations of each complementary strands; ^b Fluorescence quantum yields were measured in the aerated thermal denaturation buffer at 10⁰C, using 1.0 μM concentrations of each complementary strands at excitation wavelength of 350 nm.

5' GTG AAA TGC 5" GUG AAA UGC DNA (ON17) RNA (ON18)

ONIl 3¹ CAC TTT ACG 36.0 34.0

ON12 3' CAC TXT ACG 20.0 (0.04) 19.0 (0.07) (0.02)^b

ON13 3¹ CAC TXX ACG < 5.0 13.5

ON14 3' CAC XXX ACG < 5.0 < 5.0

Results & Discussion

Obika et al.¹² described the stabilising effect of the replacement of 3',5'- phosphodiester linkages in triplex forming oligonucleotides (TFO's) by 2',5'- linkages. As we can see from Table 1 for ON2 and ON5 toward Dl at pH 6.0, insertion of the fluorophore positioned at 3'-carbon via the triazole linker led to destabilization of parallel triplexes with Δ7^" _m values of 3.0 and 12.0 ⁰C as compared to unmodified triplex ON1/D1, respectively. Introduction of the second 2'-5' thymidine with pyrene residue led to further destabilization of the parallel triplexes (ON3, ON6, ON7 toward Dl). Destabilisation was found to be higher for triplexes formed with ON6 and ON7 having one or three nucleosides between two X's than in the case for ON3, with two adjacent pyrene containing thymidines. Moreover it is interesting to mention that at pH 5.0 T_m value for the triplex with ON4 possessing three Xs in the row was higher than those for ON6 and ON7, however all these values were lower than T_m for the unmodified triplex. Such behaviour can be explained by non-interrupted neighbouring 2'-5'-linkages in ON4 compared to the separate 2'-5'-linkages in ON6 and ON7. This can also be a reason for stabilization of parallel duplexes surprisingly observed for ON3 and ON4 in comparison with the wild-type triplex ON1/ON15 at pH 5.0 and 6.0 (Table 1). This is an interesting observation because both parallel triplexes and parallel duplexes are formed according to Hoogsteen base-pairing. However, a duplex as a more flexible structure than a triplex can easier accommodate repetitive insertions of 2'-5' thymidine linkages with pyrene in the middle of one of the strands. This unfortunately was not a case with Watson-Crick type duplexes, because a drop in thermal stability was detected for ON2-ON7 toward ON16 as compared to ON1/ON16 at pH 5.0 and 6.0. The presence of the pyrene residue in ONs resulted in the formation of an additional band in UV spectra with maxima at 350 nm. The single stranded probes containing a single incorporation of the monomer X exhibited a fluorescence with λ_max « 386 (band I) and 402 nm (band III) upon excitation at 350 nm. This emission appears at a higher wavelength than the typical pyrene monomer emission with λ_max « 378 (band I) and 391 nm (band III), which can be explained by the conjugated triazole ring in the monomer X. To our surprise a very low intensity of fluorescence was observed for single-stranded ONs, which resulted in a huge increase of intensity upon formation of parallel triplexes. For more detailed study of fluorescence properties we decided to determine fluorescence quantum yield (Φ_F) for ssONs and corresponding complexes with complementary strands. Φ_F is defined as the ratio of the number of photons emitted by fluorescence at a certain wavelength to the number of photons absorbed at this wavelength. In our study Φ_F was determined in the thermal denaturation buffer at 10⁰C using equimolar quantities of each strand (1.0 μM) upon excitation at 350 nm relative to anthracene (Φ_F = 0.36) and 9,10- diphenylanthracene (Φ_F =0.95) in cyclohexane¹⁴ following standard procedures (Table 2 ).¹⁵'¹⁶

The fluorescence emitted by single stranded ON2 and ON5-ON7 was strongly quenched with Φ_F in a range from 0.001 to 0.004, which was most probably a result of communication with neighbouring bases including electron transfer.¹⁷ Upon binding of these TFOs to the dsDNA (Dl), an increase in fluorescence intensity was detected at pH 5.0. As parallel triplexes are pH sensitive and more thermally stable complexes are formed at acidic conditions due to required protonation of cytosine, values of fluorescence quantum yield were higher at pH 5.0 than at pH 6.0. For example, Φ_F for ON2/D1 was almost three times higher in more acidic buffer (0.179 and 0.061 at pH 5.0 and 6.0, respectively). A correlation between thermal stability of parallel triplexes and fluorescence quantum yields can be also illustrated by comparison of ON2 and ON5. Thus T_m for ON5/D1 at pH 6.0 was 9.0 ⁰C lower than T_m for ON2/D1, which resulted in lower Φ_F value observed, 0.023 versus 0.061, respectively. Surprisingly a large difference was observed in fluorescence quantum yields between parallel triplexes, parallel and antiparallel duplexes formed by the same ON. Almost complete quenching of fluorescence was detected for all parallel duplexes, even at pH 5.0, when complexes were most stable. Despite of higher thermal stability, lower Φ_F values for antiparallel duplex ON2/ON16 was observed in comparison with the triplex at pH 6.0. As an outcome of low triplex thermal stability, this was not a true for ON5 at pH 6.0, however for ON2, ON5 and ON6 fluorescence intensities of triplexes were much higher than those for antiparallel duplexes at pH 5.0. All measurements of Φ_F were performed at 10 ⁰C and lower Φ_F values for parallel triplexes were detected at 20 ⁰C (results are not shown), because of partial melting of triplexes. To ensure that these effects are not a consequence of interactions of the pyrene residue in the structure X with metal ions presented in a cacodylate buffer solution we performed measurements in sodium phosphate buffer at pH 5.0 and got the same tendency described above in fluorescence spectra upon binding of ON2 to Dl, ON15 and ON16 (results are not shown).

The melting temperatures and fluorescence intensities of the triplexes formed by ON2 with a mismatching duplex were determined and are shown in Table 3. The mismatched triplexes showed much lower thermal stability compared to matched triplexes and no changes in fluorescent spectra of ssON2 was observed upon its hybridization to mis-matched dsDNAs. Important to mention is that mis-matched nucleic bases were positioned opposite insertion of X, and the presence of pyrene in the molecule did not reduce sensitivity of TFO to mismatches. Therefore, binding of ON2 to its corresponding dsDNA via formation of parallel duplex can be detected by fluorescence selectively.

The main drawback of the monomer X is its destabilizing effect upon triplex formation. For this reason attempts were made to develop a TFO, which can form stable triplexes at realistic cell pH's 6.0 and 7.2, and at the same time shows the same favourable fluorescence intensity increase that we observed for the above described ON's.

In ON8 and ON9, we used bulged insertions of TINA monomer, which stabilizes parallel triplexes with Δ7^~ _m up to 19.0 ⁰C upon single insertion.¹⁸ A disadvantage of TINA monomer in this particular case is that excitation wavelengths for X and p are very close to each other, 350 and 373 nm, respectively, meaning that irradiation of only one of these monomers is hardly achieved. We assumed that by placing of p as left or right bulged neighbour to modification X in ON2, we could transfer strong monomer fluorescence coming from TINA in a single- stranded state to an excimer band appearing at longer wavelengths by interaction of two pyrenes. In contrast to these considerations, fαrttFer destabilization σf^~ parallel triplexes was observed (Table 1, ON8 and ON9 toward Dl in comparison with ON2/D1 at pH 6.0). Moreover, high monomer fluorescence was detected for ssONδ and ssON9 with only very small excimer band at 480 nm upon excitation at 350 nm, which hampered the detection of binding these probes to dsDNA by fluorescence. However, these results do not exclude a use of TINA monomers in a combination with the monomer X in future. For this purpose fluorescently orthogonal intercalators to pyrene, like naphthalene derivative,¹⁸ should be used in TINA structure and it could be placed three or four bases away from insertion of X in the TFO.

An alternative to intercalating nucleic acids is sugar modified nucleotides, which are fluorescently silent and give thermal stabilization upon insertion into TFOs. We decided to use α-L-LNA, which stabilizes triplexes up to 5 ⁰C per insertion.¹⁹ As a general rule,¹⁹ every third or fourth nucleotide in the TFO should be substituted by α-L-LNA monomer to achieve high T_m values at neutral pH. A use of 5-methyl-cytosine derivative of α-L-LNA is also an advantage for parallel triplex formation, because 5-methyl-cytosine is easier protonated than cytosine. In the sequence ONlO, five α-L-LNA nucleotides were incorporated leading to increased thermal stability in all cases (Table 1). Thus, at pH 6.0 a triplex stabilisation of 5 °C per insertion of α-L-LNA was observed and at pH 7.2 triplex ON10/D1 melted at 35.5 ⁰C. Concerning the fluorescence properties of ONlO, a similar increase in fluorescence intensity was observed upon triplex formation at pH 5.0 and 6.0 caused by monomer X (Figure 3). Interesting to note, there was the same value of fluorescence quantum yield (0.179) for triplexes formed by ON2 and ONlO at pH 5.0 (Table 2). A considerably lower decrease of Φ_F was detected for ON10/D1 than for ON2/D1 upon changing pH from 5.0 to 6.0, which is clearly a consequence of higher triplex thermal stability for ONlO. Important, the fluorescence intensity for antiparallel duplex ON10/ON16 was lower than that for the corresponding triplex at pH 5.0 and 6.0, meaning that fluorescent discrimination caused by X in ON2 between triplex and antiparallel duplex remained after substitutions of some natural bases in TFO by α-L-LNA.

At pH = 7.2, parallel duplexes and triplexes with ONlO were less stable due to the deprotonation of cytosine (Table 1). Under these conditions ONlO formed a triplex with T_m of 35.5 ⁰C. However, deprotonation of cytosine had a large influence on the fluorescence properties of ONlO and its complexes at pH 7.2. In the fluorescence spectra of ssONIO we observed a change in the ratio of band III/I (Figure 3), which was reversed in comparison with ssON2 (Figure 1). It is known that the ratio of band III/I is affected by local environmental polarity.²⁰ Upon binding of ONlO to complementary dsDNA and ssDNAs a new broad emission appeared at longer wavelength (460 nm, Figure 3). However, it differed from an excimer emission band (480-500 nm), which usually is caused by interactions of at least two pyrene units positioned in a closed proximity. Similar results have been observed by Yamana et al.²¹ upon duplex formation of pyrene labeled oligonucleotides. The increased intensity ratio of band III/I was associated with a decrease in local environmental polarity. In our case the pyrene residue was most probably transferred into the more hydrophobic base-pair pocket, discussed later on in molecular modeling section, which leads to a new broad emission band around 460 nm. This emission can be ascribed to the exciplex of the pyrene and an adjacent base. This effect was observed upon triplex formation, but was even more obvious in the fluorescence spectrum for the parallel duplex at pH 7.2 (Figure 4). The reason why the exciplex was only observed at pH = 7.2 upon hybridization, could be explained by higher flexibility of ONlO in the complexes with complementary strands because of partially deprotonation of cytosine. This led to interaction of pyrene with one of the surrounding nucleobases and was responsible for transferring energy into the exciplex band instead of an increase in the monomer fluorescence.

Although the formation of the exciplex band was unexpected at pH 7.2 for complexes of ONlO, it opens new possibilities for the use of this probe for diagnostic properties. Fluorescence intensity upon hybridization to dsDNA at pH 7.2 was much lower (ca. 100 arb. units, Figure 3) than that at pH 6.0 (750 arb. units, Figure 2). Such pH-depending fluorescence signal can be useful for detection in cells at lower pH. It is known that tumor cells have a natural tendency to overproduce acids, resulting in more acidic pH values in the tumor microenvironment, which can be down to 6.4 for human cancers, and down to 6.12 for mouse cancers, depending on the tumor type.²² Therefore, the described probe is an ideal platform to develop sequence-specific detectors for tumor cells. Moreover, as COMBO-FISH experiments are so far performed under conditions with pH around 6.0,³ ONlO forms a suitable probe for such experiments. To study the influence of the monomer X on Watson-Crick base-pairing, a mixed sequence was synthesised with one, two or three neighbouring modified nucleotides incorporated. Table 4 shows the thermal stabilities of ON11-14 upon hybridization to ssDNA/ssRNA and the fluorescence quantum yields for ON12 and its complexes determined at 350 nm. Earlier reports for 2',5'-linked oligoribonucleotides showed a decrease of -1.3 ⁰C per each 2',5'-linkage in ON in their thermal stability toward ssRNA and a much higher decrease toward ssDNA.²³ As no transition in melting curves was observed in a medium salt phosphate buffer for duplexes with ON12-14 we used a high salt concentration for this study. When adding a pyrene unit to the 3'-position of 2',5'-linkages (structure X), a considerable decrease in antiparallel duplex thermal stability, more than 15 ⁰C per modification, was observed in comparison with the wild-type duplex (ON11/ON17). This indicates that pyrene could not stabilise duplexes by intercalation, but most probably was directed towards the outside of the duplex. This can also be seen in the steady-state fluorescence emission spectra of ON12 and the corresponding duplexes with ssDNA and ssRNA (Figure 5). Interesting to mention is that pyrene fluorescence was not quenched in ssON12 as much as we have already seen for single-stranded homopyrimidine sequences in Table 2. This can be a result of the presence of purines in the same sequence for ON12. Upon hybridisation of ON12 with ssDNA a two-fold increase in fluorescence intensity was observed. Binding to complementary ssRNA resulted in an approximately 3.5-fold increase in fluorescence intensity. Similar results have previously been described for 2'-pyrene-modified oligoribonucleotides. The 2'-N-position of 2'-amino-DNA⁷'⁸ and -LNA⁹ or 2'-O-position of RNA¹⁰'¹¹ have been substituted with pyrene via a short tether. In these cases the increase of fluorescence upon hybridisation to RNA is very strong, and much stronger than is the case for binding to DNA.

This short study about the influence of X on Watson-Crick base pairing shows that a much larger decrease in stability is obtained, while a smaller increase in fluorescence signal upon binding can be observed. Duplexes with RNA show a higher increase compared to duplexes with DNA, which is similar to previous reported analogue studies.

Molecular modeling studies were performed for ON2 with dsDNA. For ON2, its corresponding parallel triplex was minimized with pyrene located inside or outside of the duplex (Figure 6). Comparing the resulting triplexes, a larger distortion of the triplex structure was observed when the pyrene was intercalating with bases of the triplex. This promotes the pyrene ring to be oriented outside of the triplex structure, which is in agreement with our observation of very strong fluorescence signal for ON2/D1. An extra stabilizing factor was observed in molecular modelling. Thus when the pyrene moiety was positioned outside the triplex, a hydrogen bond (distance: 2,584 A) can be formed between the hydrogen of the triazole ring and the phosphate backbone (Figure 6, phosphate B). Despite of such a large distortion of the structure upon intercalation of pyrene, this was most probably the case for triplex ON10/D1 at pH 7.2 when exciplex band was observed in fluorescence spectra.

Modeling studies for the duplex formed between ON12 and complementary ssDNA showed serious distortions of the phosphate backbone and the Watson-Crick base-pairs while the pyrene moiety was placed between bases of dsDNA. Moreover this also led to reduced stacking interactions formed with upper- and underlying nucleobases. This forces the pyrene to be directed towards the outside of the triplex and results in an increase of the fluorescence signal, which is also in agreement with our thermal stability and fluorescence data.

A novel 3'- substituted ribothymidine possessing pyrene via a triazole linker, compound X, was synthesized and incorporated as a 2',5'-linker into different ONs. Thermal stability studies showed that single incorporation of X in the middle of ONs instead of thymidine resulted in lowering T_m values for either Watson-Crick or Hoogsteen type complexes. While the single stranded homopyrimidine probes were fluorescently silent with the quantum yield (Φ_F) of 0.001-0.004, formation of parallel triplexes with the complementary dsDNA led to the very strong increase of the fluorescence signal with the value of Φ_F in a range from 0.023 to 0.179 depending on the site of insertion and thermal stability. Lowering the pH resulted in higher thermal stability of Hoogsteen-type triplexes and higher fluorescence quantum yields. Antiparallel duplexes of the same probes had lower values of Φ_F than parallel triplexes under the same conditions. Molecular modelling of triplexes showed that the pyrene residue at the 3'-position of 2',5'-ribothymidine prefers to be positioned outside of the triplex as less distortion of the complex was observed in this case than when it was intercalating between nucleic bases. To compensate lowering of triplex thermal stability caused by X, known triplex stabilizators were additionally incorporated into ONs. In a first attempt phenylethynylpyrene glycerol (TINA) was incorporated as a neighbouring bulge to the modification X. However, neither stabilisation nor the previously observed fluorescence enhancement were observed in triplexes formed by these oligo's. For this reason α-L-LNA, a non-fluorescent nucleotide monomer was used to stabilize triplexes. As a result of five substitutions of native nucleic bases by α-L- LNA in the homopyrimidine sequence possessing X, enhancement in thermal stability and fluorescent quantum yield was observed at pH 6.0 and 5.0 upon triplex formation while an oligomer had very low fluorescence signal as single stranded probe. Upon binding to dsDNA at pH 6.0, a very specific 50-fold increase of the fluorescence signal was observed and it was stronger than for antiparallel duplex formed by the same probe. However, at pH 7.2 a lower increase in fluorescence in a combination with a shift to a longer wavelength, known as exciplex, was observed. This feature allows using that kind of probe to detect species of dsDNA in an environment of tumor cells, which are known to overproduce acids and have lower pH values than normal cells. For the first time, a pyrene labelled oligonucleotide probe was developed that has discrimination in increase of fluorescence signal upon binding to dsDNA over ssDNAs. This is a promising platform for further development of fluorescent probes, which can bind sequence selectively to dsDNA without denaturation of the target and gives a positive signal upon hybridization. A perspective for such probes is their use in fluorescence in vivo hybridization under vital conditions and in living cells.

References

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Claims

Claims

1 . A fluorescent triplex forming oligonucleotide with the general structure

• which can be incorporated into the backbone of an oligonucleotide or an oligonucleotide analogue, or PNA, or PNA analogues, wherein B is a nucleobase or modified nucleobase which can form hydrogen bonding to an natural nucleobase or a modified nucleobase, Z is a linker comprising 0-60 atoms, X and Y do independently of each other comprise 0-20 atoms and X and Y can independently of each other form a linkage to an oligonucleotide backbone, or to a nucleobase, or to an oligonucleotide, W comprise 10-90 atoms and comprises 2-14 condensed aromatic rings, • Ni and N₂ 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.I]-LNA, Bicyclo-DNA, 6-Amϊno- 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 combinations and modifications thereof,

• with proviso that N₁ and N₂ can not both be hydrogen, and with proviso that following compounds is excluded from protection:

2. A fluorescent triplex forming oligonucleotide according to claim 1, wherein linker Z comprises an aromatic ring, and/or heteroaromatic ring, and/or an alkene, and/or an alkyne and comprises an atom which form the connection to the 3'-position of the nucleoside.

3. A fluorescent triplex forming oligonucleotide according to claim 2 according to general structure

L is an atom which may be substituted with hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylcarbonylalkyl, heteroarylcarbonylalkyl, alkylthio, arylthϊo, heteroarylthio, aralkyl, hydroxyl, mercapto, heteroarylcarbonyloxy, formyloxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxy, azido, cyano, amino, alkoxycarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, N-alkylcarbamoyloxy, N-arylcarbamoyloxy, N-heteroarylcarbamoyloxy, N-alkylthiocarbamoyloxy, N-arylthiocarbamoyloxy, N-heteroarylthiocarbamoyloxy, alkoxy, aralkyloxy, aralkylthio, aryl, polyaryl, heteroaryl, heteropolyaryl, benzocycloalkyl,

• Ar is an aromatic ring, or a heteroaromatic ring, or an alkene or an alkyne,

• Ar may be substituted with hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylcarbonylalkyl, heteroarylcarbonylalkyl, alkylthio, arylthio, heteroarylthio, aralkyl, hydroxyl, mercapto, heteroarylcarbonyloxy, formyloxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxy, azido, cyano, amino, alkoxycarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, N-alkylcarbamoyloxy, N-arylcarbamoyloxy, N- heteroarylcarbamoyloxy, N-alkylthiocarbamoyloxy, N- arylthiocarbamoyloxy, N-heteroarylthiocarbamoyloxy, alkoxy, aralkyloxy, aralkylthio, aryl, polyaryl, heteroaryl, heteropolyaryl, benzocycloalkyl,

• W comprises 2-14 condensed aromatic rings which may be substituted with hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, arylcarbonylalkyl, heteroarylcarbonylalkyl, alkylthio, arylthio, heteroarylthio, aralkyl, hydroxyl, mercapto, heteroarylcarbonyloxy, formyloxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxy, azido, cyano, amino, alkoxycarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, N- alkylcarbamoyloxy, N-arylcarbamoyloxy, N-heteroarylcarbamoyloxy, N- alkylthiocarbamoyloxy, N-arylthiocarbamoyloxy, N- heteroarylthiocarbamoyloxy, alkoxy, aralkyloxy, aralkylthio, aryl, polyaryl, heteroaryl, heteropolyaryl, benzocycloalkyl.

4. A fluorescent triplex forming oligonucleotide according to claims 1-3 wherein W comprises 2-6 condensed aromatic rings and/or heteroaromatic rings.

5. A fluorescent triplex forming oligonucleotide according to claims 1-4 wherein Y and X independently of each other are equal to O, or S, or NH.

6. A fluorescent triplex forming oligonucleotide according to claim 3 wherein Ar comprises a five membered heterocyclic aromatic ring, wherein W comprises 2-6 condensed aromatic rings and/or heteroaromatic rings, wherein Y and X independently of each other are equal to O, or S, or NH.

7. A fluorescent triplex forming oligonucleotide according to any of claims 1-6 of the formula

wherein B is preferentially is a Thymine, or an Adenine, or a Cytosine, or a Guanine, or a Uracil, or modifications thereof,

N₁ and N₂ 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- Ri bo -LNA, α-L-Xylo- LNA, β-D-Ribo-LNA, β-D-Xylo-LNA, [3.2.I]-LNA, Bicyclo-DNA, 6-Amino- Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.O]-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 combinations and modifications thereof.

8. The oligonucleotide of any of the preceding claims, comprising 10 oligonucleotide monomer units.

9. The oligonucleotide of any of the preceding claims, comprising less than 30 oligonucleotide monomer units.

10. The oligonucleotide of any of the preceding claims comprising at least 8 DNA monomer units.

11. The oligonucleotide of any of the preceding claims comprising exclusively pyrimidine nucleobases

12. The oligonucleotide of any of the preceding claims comprising one LNA monomer unit per 4 oligonucleotide monomer units.

13. The oligonucleotide of any of the preceding claims, wherein Nl and N2 I each comprises 5 oligonucleotide monomer units.

14. A phosphoramidate of the formula

15. A method for preparation of a fluorescent triplex forming oligonucleotide according to claims 1-13 comprising the steps

a. Providing a fluorescent triplex forming oligonucleotide monomer adapted for incorporation into a oligonucleotide synthesis b. Providing standard reagents for oligonucleotide synthesis c. During standard oligonucleotide synthesis incorporating one or more fluorescent triplex forming oligonucleotide monomer(s) into the oligonucleotide d. Thereby generating a fluorescent triplex forming oligonucleotide comprising one or more fluorescent triplex forming oligonucleotide monomer(s)

16. A method of forming triplex acids comprising the steps:

a. Providing a fluorescent triplex forming oligonucleotide according to claims 1-13 b. Providing a double stranded target nucleic acid c. Incubating the oligonucleotide of step a with the double stranded target nucleic acid of step b under conditions of triplex formation d. Thereby forming a triplex nucleic acid structure.

17. A method of detecting a nucleic acid comprising the steps:

a. Providing a oligonucleotide according to any of claims 1-13 b. Providing a test sample c. Incubating the test sample and the oligonucleotide under conditions allowing triplex formation d. Measuring fluorescence of the mixed sample of step c

18. The method of claim 17, wherein the test sample comprises genomic DNA.

19. The method of any of claims 17 and 18, wherein the fluorescence measurement comprises excitation at a wavelength between 340 nm and 360 nm.