WO2008067825A1 - Formation de triplex de type hoogsteen de sondes marquées au pyrène pour une détection d'acide nucléique dans un dosage par fluorescence - Google Patents

Formation de triplex de type hoogsteen de sondes marquées au pyrène pour une détection d'acide nucléique dans un dosage par fluorescence Download PDF

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WO2008067825A1
WO2008067825A1 PCT/DK2007/050182 DK2007050182W WO2008067825A1 WO 2008067825 A1 WO2008067825 A1 WO 2008067825A1 DK 2007050182 W DK2007050182 W DK 2007050182W WO 2008067825 A1 WO2008067825 A1 WO 2008067825A1
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oligonucleotide
dna
lna
bicyclo
rna
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PCT/DK2007/050182
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English (en)
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Erik Bjerregaard Pedersen
Ineke Van Daele
Vyachelsav V Filichev
Niels Bomholt
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Erik Bjerregaard Pedersen
Ineke Van Daele
Vyachelsav V Filichev
Niels Bomholt
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Priority to EP07846444A priority Critical patent/EP2126083A1/fr
Publication of WO2008067825A1 publication Critical patent/WO2008067825A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs

Definitions

  • 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.
  • TFO's triplex forming oligonucleotides
  • this triplex formation is highly specific, it can be used for sequence-specific recognition of dsDNA, without prior denaturation of it.
  • 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.
  • novel fluorescent modified nucleic acids are needed.
  • 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.
  • 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.
  • 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 ⁇ -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.
  • the present invention provides an oligonucleotide with the general structure
  • 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
  • N 1 and N 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, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Ribo- LNA, ⁇ -D-Xylo-LNA, [3.2.I]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi- Bicyclo-DNA, ⁇ -Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]- DNA, Bicyclo[4.3.0]amide-DNA, ⁇ -D-Ribopyranosyl-NA, ⁇ -L-Lyxopyranosyl-NA, 2'-
  • N 1 and N 2 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”.
  • 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.
  • the oligonucleotide is 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, arylthio, heteroarylthio, aralkyl, hydroxyl, mercapto, heteroarylcarbonyloxy, formyloxy, alkylcarbonyloxy, cycloalkylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxy, azido, cyano, amino, alkoxycarbonyloxy, alkoxycarbonyl, aryloxycarbonyl, N- alkylcarbamoyloxy, N-arylcarbamoyloxy, N-heteroarylcarbamoyloxy, N- alkylthiocar
  • 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
  • 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-alkylthiocar
  • W comprises 2-6 condensed aromatic rings and/or heteroaromatic rings.
  • W comprises derivatives of fluorescent molecules, i.e. derivatives of naphthalene, anthracene, acridine, acridone, phenanthrene, phenanthroline, pyrene, perylene.
  • W comprises pyrene.
  • Y and X independently of each other are equal to O, or S, or NH.
  • 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
  • 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-alkylcarbam
  • W comprises 2-6 condensed aromatic rings and/or heteroaromatic rings.
  • W comprises derivatives of fluorescent molecules, i.e. derivatives of naphthalene, anthracene, acridine, acridone, phenanthrene, phenanthroline, pyrene, perylene.
  • W comprises pyrene.
  • the triplex forming oligonucleotide is of the formula:
  • Oligonucleotide comprising pyrene
  • the oligonucleotide of the invention comprises pyrene and is of the formula
  • B is a nucleobase selected from the group consisting of Thymine, Adenine, Cytosine, Guanine, Uracil, or modifications thereof,
  • Ni and N 2 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, ⁇ -L-Ribo-LNA, ⁇ -L-Xylo-LNA, ⁇ -D-Ribo-LNA, ⁇ -D-Xylo- LNA, [3.2.I]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, ⁇ - Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, ⁇ -D-Ribopyranosyl-NA, ⁇ -L-Lyxopyranosyl-NA, 2'-R- RNA, 2'-OR-
  • 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.
  • 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.
  • the fluorescent signal increases more by base pairing to ssRNA than to ssDNA.
  • the oligonucleotide can form both parallel duplexes and antiparallel duplexes.
  • the oligonucleotide of the invention comprises at least 10 oligonucleotide monomer units. More preferably, the oligonucleotide comprises 14 oligonucleotide monomer units.
  • oligonucleotide monomer unit refers to a RNA nucleotide, DNA nucleotide, LNA nucleotide or any other monomer units that can be build into the oligonucleotide.
  • nucleotide substituted at the 3'-position with a pyrene is regarded as a monomer unit.
  • 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.
  • the length of the oligonucleotide may be increased if a stronger complex (duplex or triplex) is desired.
  • 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.
  • the oligonucleotide comprises less than 30 oligonucleotide monomer units.
  • the oligonucleotide comprises at least 8 DNA monomer units, such as 10, 12, 14, 16, 18 and 20 monomer units.
  • the oligonucleotide does not comprise any RNA units.
  • RNA monomer units may be omitted from the oligonucleotide of the invention, as incorporation of RNA monomer units may decrease biostability of the oligonucleotide.
  • the oligonucleotide of the invention comprises at least 7 contiguous pyrimidine nucleobases, i.e. 7 oligonucleotide monomer units that all comprise a pyrimidine nucleobase.
  • the oligonucleotide comprises at least 10, 14, 16, 20, and 24 contiguous pyrimidine nucleobases, respectively.
  • the oligonucleotide comprises between 10 and 30 contiguous pyrimidine nucleobases and more preferably between 15 and 25 contiguous pyrimidine nucleobases.
  • 60% of the monomer units of the oligonucleotide comprise pyrimidine nucleobases. More preferably, 80% of the monomer units comprise pyrimidine nucleobases.
  • 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.
  • triplex forming oligonucleotides are of interest for sequence specific detection of double stranded DNA.
  • the oligonucleotide of the invention comprises at least 7 contiguous purine nucleobases, i.e. 7 oligonucleotide monomer units that all comprise a purine nucleobase.
  • the oligonucleotide comprises at least 10, 14, 16, 20, and 24 contiguous purine nucleobases, respectively.
  • the oligonucleotide comprises between 10 and 30 contiguous purine nucleobases and more preferably between 15 and 25 contiguous purine nucleobases.
  • 60% of the monomer units of the oligonucleotide comprise purine nucleobases. More preferably, 80% of the monomer units comprise purine nucleobases.
  • 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.
  • 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.
  • the oligonucleotide of the invention further comprises modifications or nucleotide analogues that increase its affinity to a double stranded target sequence.
  • the oligonucleotide comprises a LNA (locked nucleic acid) monomer unit.
  • the oligonucleotide comprises at least one LNA monomer unit per 4 oligonucleotide monomer units.
  • the oligonucleotide consists of 20 monomer units; the oligonucleotide will comprise at least 4 LNA monomer units.
  • every fourth monomer unit is LNA.
  • the LNA unit is selected from the group consisting of: LNA, ⁇ -L-amino-LNA, ⁇ -D-amino-LNA, ⁇ -L-thio-LNA, ⁇ -D-thio-LNA, ⁇ -L-am ⁇ no-LNA, ⁇ -D-amino-LNA, ⁇ -L- thio-LNA, ⁇ -D-thio-LNA, ⁇ -L-LNA and ⁇ -L-LNA.
  • the LNA unit is ⁇ -L-LNA.
  • Nl and N2 each comprise 5 oligonucleotide monomer units.
  • the nucleotide monomer unit comprising pyrene is located at least 5 monomer units from either end of the oligonucleotide.
  • 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.
  • a second aspect of the invention is a fluorescent triplex forming oligonucleotide monomer adapted for incorporation into an oligonucleotide synthesis.
  • the monomer is a phosphoramidate of the formula
  • B is a nucleobase or modified nucleobase which can form hydrogen bonding to an natural nucleobase or a modified nucleobase
  • B is a nucleobase selected from the group consisting of Thymine, Adenine, Cytosine, Guanine, Uracil, or modifications thereof,
  • 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.
  • the oligonucleotide monomer adapted for incorporation is a phosphoramidate as described in the second aspect of the invention.
  • 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
  • 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.
  • the sample is a tissue slice, e.g. from a solid tumour.
  • 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.
  • the test sample is a PCR reaction mixture.
  • the oligonucleotide of the invention is used to determine the presence of a PCR product in a PCR reaction.
  • detection of PCR is performed quantitatively and for each PCR round such as to enable quantitative PCR.
  • NMR spectra were recorded on a Varian Gemini 2000 spectrometer at 300 MHz for 1 H using TMS ( ⁇ : 0.00) as an internal standard and at 75 mHz for 13 C using CDCI 3 ( ⁇ : 77.0) or DMSO ( ⁇ : 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).
  • FT Fourier transform
  • MALDI-TOF mass spectra of isolated oligodeoxynucleotides 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.
  • the heating temperature was set to 125 °C, with a 30 s premixing time.
  • the reaction mixture was irradiated for 15 min followed by N 2 cooling to 40 0 C. 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 (CH 2 CI 2 /Me0H 95:5) to yield the pure triazol 2 (583 mg, 79%).
  • 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 ⁇ L) for 20 minutes, followed by addition of H 2 O (100 ⁇ l_) and 3 M aq NaOAc (50 ⁇ L). The ONs were precipitated from 99% EtOH (600 ⁇ L). 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 Schr ⁇ dinger. 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 Schr ⁇ dinger, 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 ⁇ M, followed by the addition of the TFO at a concentration of 1.5 ⁇ M in the corresponding buffer solution. The solution was heated to 80 0 C 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 80 0 C for 5 minutes and then cooling to room temperature.
  • the absorbance of both triplexes and duplexes was measured at 260 nm from 5 to 70 0 C with a heating rate of 1.0 °C/min.
  • the melting temperatures (7 " m , 0 C) were determined as the maximum of the first derivative plots of the melting curves. All melting temperatures are within the uncertainty ⁇ 0.5 0 C as determined by repetitive experiments.
  • phosphoramidite 4 (Scheme 1) was started from compound 1, which was obtained in 11 steps from 1,2-O-isopropylidine- ⁇ -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 0 C 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.
  • ON's containing compound 4 DNA-synthesis of ON's containing compound 4 was performed on a 0.2 ⁇ mol 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.
  • 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 , 0 C) determined as the first derivatives of melting curves at 260 nm are listed in Tables 1, 3 and 4.
  • 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.
  • TFO's triplex forming oligonucleotides
  • insertion of the fluorophore positioned at 3'-carbon via the triazole linker led to destabilization of parallel triplexes with ⁇ 7 " m values of 3.0 and 12.0 0 C 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).
  • 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 ⁇ 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 ⁇ max « 378 (band I) and 391 nm (band III), which can be explained by the conjugated triazole ring in the monomer X.
  • ⁇ F fluorescence quantum yield
  • 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.
  • 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.
  • TINA monomer In ON8 and ON9, we used bulged insertions of TINA monomer, which stabilizes parallel triplexes with ⁇ 7 ⁇ m up to 19.0 0 C 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.
  • 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.
  • triplex stabilizators were additionally incorporated into ONs.
  • phenylethynylpyrene glycerol (TINA) was incorporated as a neighbouring bulge to the modification X.
  • TAA phenylethynylpyrene glycerol
  • ⁇ -L-LNA a non-fluorescent nucleotide monomer was used to stabilize triplexes.
  • ⁇ -L-LNA a non-fluorescent nucleotide monomer was used to stabilize triplexes.

Abstract

La présente invention porte sur des oligonucléotides fluorescents et sur des procédés destinés à fournir et utiliser des oligonucléotides fluorescents. En particulier, l'invention porte sur des oligonucléotides fluorescents qui sont capables d'une formation de triplex, ladite formation de triplex conduisant à des propriétés fluorescentes modifiées de façon à permettre la détection d'une formation de triplex.
PCT/DK2007/050182 2006-12-06 2007-12-06 Formation de triplex de type hoogsteen de sondes marquées au pyrène pour une détection d'acide nucléique dans un dosage par fluorescence WO2008067825A1 (fr)

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EP07846444A EP2126083A1 (fr) 2006-12-06 2007-12-06 Formation de triplex de type hoogsteen de sondes marquées au pyrène pour une détection d'acide nucléique dans un dosage par fluorescence

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015021432A1 (fr) * 2013-08-08 2015-02-12 The Scripps Research Institute Procédé d'étiquetage enzymatique spécifique de site d'acides nucléiques in vitro par incorporation de nucléotides non naturels
US10626138B2 (en) 2013-08-08 2020-04-21 The Scripps Research Institute National Institutes Of Health (Nih), U.S. Dept Of Health And Human Services (Dhhs) Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides
EA038576B1 (ru) * 2013-08-08 2021-09-16 Дзе Скриппс Рисёч Инститьют Способ сайт-специфического ферментативного мечения нуклеиновых кислот in vitro введением не встречающихся в природе нуклеотидов
US11634451B2 (en) 2013-08-08 2023-04-25 The Scripps Research Institute Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides
US11834689B2 (en) 2017-07-11 2023-12-05 The Scripps Research Institute Incorporation of unnatural nucleotides and methods thereof
US10610571B2 (en) 2017-08-03 2020-04-07 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
US11622993B2 (en) 2017-08-03 2023-04-11 Synthorx, Inc. Cytokine conjugates for the treatment of autoimmune diseases
US11701407B2 (en) 2017-08-03 2023-07-18 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
US11077195B2 (en) 2019-02-06 2021-08-03 Synthorx, Inc. IL-2 conjugates and methods of use thereof

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