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WO2015162130A1 - Analogues de nucléosides à base modifiée pour la détection de la 06-alkylguanine - Google Patents

Analogues de nucléosides à base modifiée pour la détection de la 06-alkylguanine Download PDF

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WO2015162130A1
WO2015162130A1 PCT/EP2015/058600 EP2015058600W WO2015162130A1 WO 2015162130 A1 WO2015162130 A1 WO 2015162130A1 EP 2015058600 W EP2015058600 W EP 2015058600W WO 2015162130 A1 WO2015162130 A1 WO 2015162130A1
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oligonucleotide
target
nucleic acid
dna
duplex
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Shana STURLA
Ioannis TRANTAKIS
Heidi DAHLMANN
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Eidgenoessische Technische Hochschule Zurich ETHZ
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Eidgenoessische Technische Hochschule Zurich ETHZ
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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
    • C07H19/052—Imidazole radicals

Definitions

  • the present disclosure provides novel compounds and methods for detecting the presence of 0 6 -alkyl guanine in a nucleic acid and for isolating 0 6 -alkyl guanine comprising nucleic acid.
  • the disclosure is based on base-modified-nucleoside analogs that form stable base pairs with 0 6 -alkyl guanine.
  • DNA adducts In our daily life we are constantly exposed to genotoxic chemicals that may interact with cellular DNA, become covalently bound, and generate products called DNA adducts. If DNA adducts persist and evade repair they can lead to mutations in cell- cycle regulatory genes that may cause uncontrolled cell division leading to cancer. Therefore DNA adduct formation is considered one of the key events in tumor initiation during chemical carcinogenesis 1 3 . Levels of DNA adducts represent the amount of carcinogen absorbed by the body, escaped detoxification, encountered a critical cellular macromolecule (DNA) and has not been repaired. Therefore, DNA adducts serve as chemical-specific biomarkers of biologically significant exposure to genotoxins and individual susceptibility to carcinogenesis 4 ' 5 .
  • DNA adducts that trigger cell death in rapidly dividing target cancer cells
  • DNA adducts serve in this situation as chemical biomarkers of drug sensitivity.
  • these chemical biomarkers are measured in cancer epidemiology population studies and in
  • a current difficulty in detecting DNA adducts is that they are formed at exceedingly low levels.
  • the ability of DNA lesions to induce toxicity depends on their chemical structure, abundance of formation and propensity to be recognized by specialized DNA repair proteins, and their positions within the genome 8 ' 9 .
  • DNA sequence context influences the rate of DNA adduct formation, repair and mispairing potency 10 .
  • the location of a DNA lesion within the gene will determine whether it results in mutations influencing the structure and function of the corresponding protein.
  • a first aspect of the disclosure provides an oligonucleotide comprising at least one base-modified-nucleotide analog wherein the base-modified-nucleotide comprises the following structure:
  • Rl is selected from OH or H; and R2 is selected from a deoxyribose moiety of a polynucleotide, a ribose moiety of a polynucleotide, and a nucleic acid backbone mimic moiety.
  • Rl is OH and R2 is selected from a ribose moiety, a deoxyribose moiety, and a nucleic acid backbone mimic moiety
  • Rl is H and R2 is selected from a deoxyribose moiety and a nucleic acid backbone mimic moiety.
  • Rl is selected from OH or H and R2 is a deoxyribose moiety of a
  • Rl is OH.
  • said oligonucleotide is immobilized to a solid surface, more preferably a nanoparticle, more preferably a gold nanoparticle.
  • a further aspect of the disclosure provides a kit of parts comprising the oligonucleotides and a solid substrate, preferably particles, more preferably gold nanoparticles.
  • a further aspect provides the use of said oligonucleotides for detecting the presence of 0 6 -alkyl guanine, in particular the presence of 0 6 -alkyl guanine in a target polynucleotide.
  • a further aspect of the disclosure provides a base-modified-nucleoside analog having the following structure:
  • Rl is OH and R2 is selected from a ribose moiety, a deoxyribose moiety, and a nucleic acid backbone mimic moiety or Rl is H and R2 is selected from a deoxyribose moiety and a nucleic acid backbone mimic moiety.
  • Rl is OH.
  • the base modified nucleoside analog has the following structure:
  • R3 is selected from H, phosphorothioate monophosphate, (PO3), (POs)2, (POaXs, and a protective group for a hydroxyl group for synthesis of nucleic acid, preferably R3 is H and
  • R4 is selected from H, a protective group for a hydroxyl group for synthesis of nucleic acid, a phosphate group, a phosphate group protected with a protective group for synthesis of nucleic acid, or -P(R7)R8 [where R7 and R8 are identical or different, and each represent a hydroxyl group, a hydroxyl group protected with a protective group for synthesis of nucleic acid, a mercapto group, a mercapto group protected with a protective group for synthesis of nucleic acid, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a primary or secondary amine substituted by an alkyl group having 1 to 5 carbon atoms, preferably R4 is H, and R5 is selected from H, OH, 0-alkyl(Cl-C6), F, CI, NH2, CN
  • said method comprising a) contacting the target polynucleotide (or a sample comprising said target polynucleotide) with a first oligonucleotide that is
  • said oligonucleotide comprises at least one base-modified-nucleotide analog comprising the following structure:
  • Rl is selected from OH or H, preferably OH; and R2 is selected from a deoxyribose moiety of a polynucleotide, a ribose moiety of a polynucleotide, and a nucleic acid backbone mimic moiety and b) measuring the formation of said duplex.
  • said oligonucleotide is immobilized to a solid surface, more preferably a nanoparticle, more preferably a gold nanoparticle.
  • the method further comprises contacting the target polynucleotide with a second oligonucleotide that is complementary to said polynucleotide such that said polynucleotide can simultaneously hybridize to both the first and second
  • oligonucleotides forming a duplex and b) measuring the formation of said duplex.
  • the method alternatively comprises contacting said target polynucleotide with a second oligonucleotide that is complementary to said first oligonucleotide such that said second oligonucleotide competes with the target polynucleotide for binding to the first oligonucleotide (comprising the base-modified nucleotide analog).
  • a further aspect of the disclosure provides a method for isolating an 0 6 -alkyl guanine containing target polynucleotide, said method comprising a) contacting a nucleic acid containing sample with an oligonucleotide that is complementary to said target polynucleotide such that said target polynucleotide and said oligonucleotide form a target duplex, said oligonucleotide comprises at least one base-modified-nucleotide analog comprising the following structure:
  • Rl is selected from OH or H, preferably OH; and R2 is selected from a deoxyribose moiety of a polynucleotide, a ribose moiety of a polynucleotide, and a nucleic acid backbone mimic moiety and b) isolating said target duplex.
  • said oligonucleotide is coupled to a purification label or a solid substrate and the target duplex is isolated via the purification label or solid substrate.
  • 0 6 -alkyl guanine is 0 6 -methyl guanine.
  • Figure 1 A) 0 6 -methyl-G and 0 6 -benzyl-G. B) Hydrophophobic nucleoside analogs designed to pair opposite 0 6 -alkyl-G lesions.
  • dR deoxyribose.
  • Figure 2 Statistical analysis of AGCF° of duplex melting (Table 2). Error bars indicate 95% confidence intervals. Analog:target pairs that do not differ significantly (i.e, pairs in which p > 0.05) according to the Games-Howell test are denoted (-).
  • Figure 3 Fluorescence emission spectra of 5 ⁇ dsDNA in PBS buffer containing nucleoside analogs ExBIM and ExBenzi excited at 250 nm.
  • Figure 7 A) Dependence of absorbance ratios on temperature of aggregates formed from target mixtures (20 nM of each G and irrelevant target) supplemented with either 0 6 -meG- (20 nM final concentration) or G-containing targets (40 nM final concentration). An aggregate solution formed from the initial mixture (blank) served as a control. Data points indicate mean ⁇ SD from three independent experiments.
  • Figure 8 A) Linear plot of absorbance ratios (700/530 nm) versus relative 0 6 -meG target concentration ([0 6 -meG]/[total target]). B) Linear plot of absorbance ratios (700/530 nm) versus relative 0 6 -meG target concentration ([0 6 -meG]/[G]). Values are mean ⁇ SD from three independent experiments.
  • N 7 -methylguanine N 7 -meG
  • 0 6 -methylguanine 0 6 -meG
  • Radiolabelling, immunohistochemistry, or HPLC coupled with MS or electrochemical detection are useful for detecting low-abundance 0 6 -alkyl-G DNA lesions in biological samples; however, these methods involve time- and labor-intensive sample preparation and analysis(de Groot et al., 1994; Dennehy and Loeppky, 2005; Georgiadis et al., 2011; Huh et al., 1989; Kang et al., 1992; Yang et al., 2002).
  • Sequence-specific detection of DNA is typically carried out by hybridizing
  • oligonucleotide probes with complementary target DNA This method is used for example in microarrays or DNA chips, which have become ubiquitous in biological and biomedical research.
  • Hybridization probes containing nucleoside analogs that selectively interact with specific DNA lesions as opposed to non- damaged DNA have been developed to monitor biochemical transformations such as abasic site formation, DNA repair, and translesion and postlesion DNA synthesis on damaged DNA substrates.
  • nucleoside analogs utilized in these hybridization probes often contain fluorescent conjugated ring systems that facilitate spectroscopic detection; such nucleoside analogs have been incorporated into hybridization probes for 8-oxo-G and abasic sites.
  • An alternative type of hybridization probe for site-specific detection of 06-Me-G contains hydrophobic nucleoside analogs (Benzi, BIM, Peri, and Per, Figure IB) that form more stable base pairs opposite 0 6 -alkyl-G lesions than opposite non-damaged G, A, T, and C.(Gahlon and Sturla, 2013b; Gong and Sturla, 2007)
  • the present disclosure provides nucleoside analogs Exbenzi and ExBIM for the detection of 0 6 -alkyl guanine ( Figure IB).
  • Figure IB The thermal stabilities of duplexes containing ExBenzi and ExBIM paired opposite G, 0 6 -Me-G, O e -Bn-G, and a model abasic site (THF) as a control were evaluated according to their free energies of duplex melting, which were derived from UV-absorbance melting curves.
  • the oligonucleotide probe containing Exbenzi was more selective for 0 6 -alkyl-G vs G strands than the previously described Per nucleoside.
  • the disclosure provides base-modified-nucleoside analogs and
  • oligonucleotides comprising said nucleoside analogs for detecting the presence of O 6 - alkyl guanine or for isolating a nucleic acid containing 0 6 -alkyl guanine.
  • O"- alkyl guanine is O l! - methyl guanine.
  • a base-modified-nucleoside analog as disclosed herein has the following
  • Rl is OH or H and R2 is selected from a ribose moiety, a deoxyribose moiety, and a nucleic acid backbone mimic moiety.
  • Rl is OH and R2 is selected from a ribose moiety, a deoxyribose moiety, and a nucleic acid backbone mimic moiety or Rl is H and R2 is selected from a deoxyribose moiety and a nucleic acid backbone mimic moiety.
  • Rl is OH.
  • the sugar moiety of the nucleosides may contain additional natural and/or non- natural modifications and/or substitutions, including stabilizing
  • the base-modified- nucleosides also include mono-, di-, and tri-phosphate nucleosides, i.e., "nucleotides”. Additional modifications are known to the skilled person.
  • EP0,302,175 describes modification of nucleotides at the 5' or 3' end, as well as modification of the phosphate moiety and US5446137 describes nucleotides that are substituted at the 4' position of the sugar moiety. Locked Nucleic Acid derivatives are also encompassed by the disclosure.
  • LNAs are modified nucleotides or ribonucleotides that contain an extra bridge between the 2' and 4' carbons of the ribose sugar moiety resulting in a "locked" conformation, and/or bicvclic structure.
  • Nucleic acid backbone mimic moieties are well-known in the art and include, e.g. GNAs, PNA, and TNAs.
  • GNA glycol nucleic acid
  • PNA peptide nucleic acid
  • TNA threose nucleic acid
  • GNA glycol nucleic acid
  • PNA peptide nucleic acid
  • TNA threose nucleic acid
  • the nucleosides may also include protective groups, e.g., protective groups for the synthesis of nucleic acid.
  • protective group refers to a chemical group that changes a functional group so as to disguise the chemical reactivity of the functional group and prevent it from undesirably reacting during reactions occurring at other sites on the compound.
  • Suitable protecting groups include phthalyl, carbobenzyloxy, benzyl, benzoyl, trityl, monomethoxytrityl, dimethoxytrityl (DMT), acetyl, trifluoroacetyl, trimethylsilyl, t-butyl(dimethyl)silyl, t-butyl(diphenyl)silyl, carbonate, 2- trimethylsilylethyl, methoxymethyl, 2-methoxyethoxymethyl and dihydropyranyl groups.
  • the base-modified-nucleoside analog has the following
  • Rl is selected from OH or H, preferably Rl is OH;
  • R3 is selected from H, phosphorothioate monophosphate. (PO ;). (POs)2, (POa s and a protective group for a hydroxyl group for synthesis of nucleic acid,
  • R4 is selected from H, a protective group for a hydroxyl group for synthesis of nucleic acid, a phosphate group, a phosphate group protected with a protective group for synthesis of nucleic acid, or -P(R7)R8 [where R7 and R8 are identical or different, and each represent a hydroxyl group, a hydroxyl group protected with a protective group for synthesis of nucleic acid, a mercapto group, a mercapto group protected with a protective group for synthesis of nucleic acid, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or a primary or secondary amine substituted by an alkyl group having 1 to 5 carbon atoms], and
  • R5 is selected from H, OH, 0-alkyl(Cl-C6), F, CI, NH2, CN, SH, and a 2'- 4' bridge locking the sugar in the C3 endo configuration.
  • R5 is not OH when Rl is H.
  • R5 is H.
  • the base-modified-nucleoside analogs disclosed herein and the oligonucleotides comprising base-modified-nucleotide analogs disclosed herein (referred herein collectively as "the compounds of the disclosure") may be used to detect the presence of 0 6 -alkyl guanine in a sample.
  • the compounds of the disclosure may also be used to isolate or enrich for 0 6 -alkyl guanine.
  • the 0 6 -alkyl guanine is present as a unit of a polynucleotide, i.e., the compounds detect the presence of 0 6 -alkyl guanine in a nucleic acid sequence.
  • a polynucleotide comprises at least two nucleotide monomers, preferably at least 3, 4, 5 or more nucleotide monomers.
  • the terms "polynucleotide” and “nucleic acid sequence” are used interchangeably herein.
  • 0 6 -alkyl guanine can be detected in or isolated from any sample that contains or is suspected of containing nucleic acid.
  • Samples include water, food, cells, organisms, tissue, stool, and biological fluids (e.g., plasma, serum, urine, tears, saliva, spinal fluid or lymph fluids).
  • the samples are obtained from individuals at risk or suspected of being at risk of having or developing a hyperproliferative disorder, e.g., tumor, cancer, neoplastic tissue.
  • the individuals have been exposed to or are suspected of being exposed to a carcinogen, e.g., radiation, a carcinogen in cigarettes, etc.
  • the individual is undergoing chemotherapy.
  • the nucleic acid may be RNA or DNA or fragments thereof.
  • the nucleic acid is DNA.
  • the compounds of the disclosure are used to detect the presence of 0 6 -alkyl guanine in a target polynucleotide or to enrich or isolate for a O 6 - alkyl guanine containing target polynucleotide.
  • one preferred object of the disclosure is to provide compounds that target DNA adducts in sequence specific context.
  • oligonucleotides are designed to hybridize with nucleic acid susceptible to damage or to nucleic acid whose damage leads to harmful effects.
  • Preferred target polynucleotides include oncogenes (e.g., K-ras and p53).
  • the target sequence may comprise nucleic acid fragments, e.g., fragments that comprise mutational "hotspots". Fragments having a known sequence can be prepared by, e.g., digesting a nucleic acid sample with one or more restriction enzymes.
  • the disclosed nucleotide analogs are also used as intermediates for production of oligonucleotides. Accordingly, the disclosure provides an oligonucleotide comprising at least one base-modified-nucleotide analog as disclosed herein.
  • an oligonucleotide is provided comprising at least one base-modified-nucleotide analog wherein the base-modified-nucleotide comprises the following structure:
  • Rl is selected from OH or H; and R2 is selected from a deoxyribose moiety of a polynucleotide, a ribose moiety of a polynucleotide, and a nucleic acid backbone mimic moiety.
  • oligonucleotides are from about 6 to about 50 nucleotides in length. In still more preferred embodiments oligonucleotides are from about 12 to about 20 nucleotides in length.
  • the oligonucleotides are complementary to a target polynucleotide such that the oligonucleotide is capable of forming a stable duplex with a target
  • polynucleotide comprising 0 6 -alkyl guanine. It is understood in the art that a sequence need not be 100% complementary to that of its target sequence to form a duplex.
  • complementarity is used herein to refer to a stretch of nucleic acids which can hybridise to another stretch of nucleic acids. It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. Mismatches may to some extent be allowed, if under the circumstances, the stretch of nucleotides is capable of hybridising to the complementary part.
  • a complementary part comprises at least 3, 4, 5, or 6 consecutive nucleotides complementary to the target polynucleotide.
  • the oligonucleotides may comprise additional nucleoside analogs containing natural and/or non-natural modifications and/or substitutions as disclosed herein, including stabilizing modifications/substitutions and purification or detection labels.
  • the additional nucleoside analogs may contain synthetic and natural bases such as 5- methylcytosine (5-Me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases.
  • 5-Me-C 5-methylcytosine
  • 5-hydroxymethyl cytosine 5-hydroxymethyl cytosine
  • xanthine xanthine
  • hypoxanthine 2- aminoadenine
  • 6-methyl and other alkyl derivatives of adenine and guanine 2-propyl and other alkyl derivatives of adenine and guan
  • the oligonucleotide may contain a backbone of ribose moieties, deoxyribose moieties, or nucleic acid backbone mimic moieties.
  • the moieties may be connected by linkers well-known in the art such phosphodiester bonds, peptide bonds, and
  • oligonucleotides are well-known to a skilled person any includes the phosphodiester method disclosed in Brown E. L. et al (1979) Methods Enzyrnol., 68: 109-151, the phosphotriester method described in Narang S. A. et al (1979) Methods Enzyrnol., 68: 90-98, the diethylphosphoramidate method disclosed in Beaucage S. L., Caruthers M. H. (1981) Tetrahedron Lett., 22: 1859-1862, as well as the use of either phosphoramidite chemistry or H-phosphonate chemistry.
  • Other methods include those described in U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,
  • the compounds of the disclosure may be linked directly or indirectly to labels which aid in detection or purification.
  • Suitable labels include, enzymes such as alkaline phosphatase and horseradish peroxidase, fluorescent proteins (e.g., GFP),
  • bioluminescent proteins e.g. aequorin
  • dyes e.g., fluorescers (e.g., FRET probes)
  • fluorescers e.g., FRET probes
  • chemiluminescers radioactive groups e.g., fluorescers, fluorescers, chemiluminescers radioactive groups.
  • the detection of target duplexes may be performed using any number of methods known to a skilled person including fluorescence, chemiluminescence, bioluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman
  • solid substrate refers to any material that provides a solid structure with which an oligonucleotide or nucleoside can be attached. Such materials include silicon, plastic, metal (e.g. gold electrode), glass, polymers, ceramic surfaces, and the like.
  • a solid substrate may be of a rigid or non-rigid nature like gels, rubbers, polymers, etc. and it may be any type of shape including spherical shapes like beads and particles.
  • Compounds of the disclosure are immobilized to a solid substrate directly or indirectly, preferably through a covalent bond using a spacer molecule or linker group.
  • the immobilization of the compounds of the disclosure on a solid substrate can aid in isolating target duplexes and for immobilizing bound target nucleic acid.
  • the solid substrates act aid in the detection of duplex formation (e.g., quantum dots (fluorescence), gold nanoparticles (aggregation), gold electrode (conductivity), cantilever (surface tension)).
  • the solid substrates may also be useful to provide oligonucleotides in an arrayed format.
  • a solid surface e.g., DNA chip/microarray
  • an array of oligonucleotides comprising ExBIM or Exbenzi.
  • the solid substrate is a particle such as latex or carbon particle.
  • the particle is a nanoparticle such as metal (preferably gold or silver), semiconductor, insulator, magnetic, or a quantum dot, more preferably the particle is a gold nanoparticle.
  • the size of the nanoparticles is preferably from about 5 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm. Methods of making such nanoparticles are well-known in the art. Either the nanoparticles, the oligonucleotides or both are functionalized by methods known in the art in order to attach the oligonucleotides to the nanoparticles. In exemplary embodiments, oligonucleotides are terminated with a 5' thionucleoside or a 3' thionucleoside for attaching to solid surfaces.
  • Preferred aspects of the disclosure provide for the use of the compounds of the disclosure for detecting the presence of O'-alkyl guanine, preferably for detecting the presence of 0 6 -alkyl guanine in a target polynucleotide.
  • the disclosure provides for the use of an oligonucleotide comprising at least one base-modified- nucleotide analog as disclosed herein for detecting the presence of 0 6 -alkyl guanine in a target polynucleotide, wherein said oligonucleotide is complementary to said polynucleotide such that said target polynucleotide and said oligonucleotide form a target duplex.
  • the detection of the presence of 0' ! -alkyl guanine can be determined by measuring the binding of the base-modified-nucleoside analogs to 0 6 -alkyl guanine or by measuring the formation of a duplex between the oligonucleotide and the 0' ! -alkyl guanine i a target polynucleotide("target duplex").
  • Binding and duplex formation can be measured by various assays well-known in the art.
  • the stability of duplex formation can be determined by analysing duplex formation under various conditions.
  • the formation of a duplex formed by the target polynucleotide and the oligonucleotide i.e., the target duplex
  • the control nucleic acid sequence preferably has the same nucleic acid sequence as the target sequence but lacks 0 6 -alkyl guanine.
  • An increase in the amount of duplex formation of the target duplex over the control duplex indicates the presence of 0 6 -alkyl guanine in the target polynucleotide.
  • methods for detecting the presence of 0' ! -alkyl guanine in a target polynucleotide comprising a) contacting a target polynucleotide with an oligonucleotide comprising Exbenzi or ExBIM, wherein said oligonucleotide is complementary to said target polynucleotide such that said target sequence and said oligonucleotide form a duplex and b) measuring the formation or stability of said duplex.
  • the method further comprises c) comparing the formation of the target duplex to the formation or stability of a duplex formed by said oligonucleotide with a control nucleic acid sequence not having 0 6 -alkyl guanine, wherein an increased formation or stability of the target duplex over the control duplex indicates the presence of 0 6 -alkyl guanine in said nucleic acid sequence.
  • the methods comprise a) contacting a target polynucleotide with a first oligonucleotide that is complementary to said target polynucleotide and a second oligonucleotide that is complementary to said target polynucleotide such that said target polynucleotide can simultaneously hybridize to both the first and second oligonucleotides forming a duplex; wherein the first oligonucleotide is an
  • oligonucleotide comprising Exbenzi or ExBIM and b) measuring the formation or stability of said duplex.
  • the resulting close proximity of the two oligonucleotides may be used to measure the formation or stability of said duplex.
  • the method further comprises c) comparing the formation of the target duplex to the formation or stability of a duplex formed by said oligonucleotides with a control nucleic acid sequence not having 0 6 -alkyl guanine, wherein increased formation or stability of the target duplex over the control duplex indicates the presence of 0 6 -alkyl guanine in said nucleic acid sequence.
  • the second oligonucleotide is complementary to said first oligonucleotide such that said second oligonucleotide competes with the target polynucleotide for binding to the first oligonucleotide.
  • the first oligonucleotide comprises Exbenzi or ExBIM and the second oligonucleotide does not comprise 0 6 -alkyl guanine.
  • the first oligonucleotide (comprising Exbenzi or ExBIM) will form a more stable duplex with a target polynucleotide which contains 0 6 -alkyl guanine as compared to the second oligonucleotide (not containing 0 6 -alkyl guanine).
  • the second oligonucleotide in these embodiments has either the same sequence as the target polynucleotide or differs only by the presence of guanine instead of 0 6 -alkyl guanine.
  • the contacting of the oligonucleotides with the target polynucleotide takes place under conditions effective for hybridization of the ExBIM/Exbenzi oligonucleotide with the target sequence of the nucleic acid.
  • hybridization conditions are well known in the art and can readily be optimized for the particular system and target polynucleotide employed. It is clear to a skilled person that these conditions can be optimized such that the target duplex is able to form but the conditions are too stringent for control duplex formation. This can be achieved, for example, by increasing temperature and altering salt concentrations. In such an embodiment, control duplex formation does not occur (or occurs at a much reduced rate as compared to target duplex formation).
  • the duplex is heated in solution until the strands of the duplex separate.
  • the stability of a duplex is expressed by the temperature at which one-half the base pairs have dissociated, commonly known as the "melting temperature" or 'I'm . In practice, this is usually measured by monitoring the ultraviolet absorbance of a solution of nucleic acid while the temperature is increased and denoting the Tm as the temperature at half the maximal absorbance at 260 nm.
  • Methods to measure formation and stability can also be performed using labelled nucleoside analogs or oligonucleotides.
  • the first labelled nucleoside analogs or oligonucleotides can also be performed using labelled nucleoside analogs or oligonucleotides.
  • oligonucleotides is provided with a second detectable label such that the close physical proximity of the first and second labels resulting from duplex formation leads to a measurable parameter.
  • the first and second detectable label are the same, in some embodiments they are different.
  • Exemplary labels include hybridization probes such as FRET probes.
  • FRET probe refers to a fluorescent oligonucleotide which is used for detection of a target polynucleotide wherein detection is based on the FRET effect.
  • the FRET probe commonly contains two chromophores, an acceptor chromophore and a reporter fluorophore.
  • the acceptor chromophore is usually a non-fluorescent dye chosen to quench fluorescence of the reporting fluorophore. Formation of target duplexes leads to a change in fluorescent properties, (see Didenko, Biotechniques. Nov 2001; 31(5): 1106 1121 for a review of FRET based hybridization probes).
  • a hairpin oligonucleotide complementary to the target polynucleotide comprises both acceptor and reporter chromophores and Exbenzi or ExBIM. Binding of the oligonucleotide to the target polynucleotide results in a change in fluorescent properties.
  • a first oligonucleotide having an acceptor chromophore is provided together with a second oligonucleotide having a reporter chromophore, such that both oligonucleotides can simultaneously bind the target nucleic acid, wherein at least one of the oligonucleotides comprises Exbenzi or ExBIM. Formation of a target duplex comprising both oligonucleotides and the target polynucleotide results in a change in fluorescent properties.
  • competitive hybridization is performed wherein a first oligonucleotide being complementary to the target polynucleotide and having an acceptor chromophore and comprising Exbenzi or ExBIM is provided together with a second oligonucleotide being complementary to the first oligonucleotide a d having a reporter chromophore, such that the target polynucleotide and the second
  • oligonucleotide compete for binding to the first oligonucleotide. If the target nucleotide contains 0 6 -alkyl guanine it will bind more stably to the first oligonucleotide than the second oligonucleotide not containing 0 6 -alkyl guanine.
  • the oligonucleotides are attached to quantum dots.
  • quantum dot refers to semiconductor or insulator nanoparticles that may comprise a dopant.
  • the wavelength of the light emitted (e.g., color) by the QD can be selected according to the physical properties of the nanoparticles, such as size, material, and the dopant.
  • QD assays are known to a skilled person and are also described in US20130029333.
  • an oligonucleotide comprising ExBIM or Exbenzi is attached to a QD.
  • the first oligonucleotide is attached to a first quantum dot and the second oligonucleotide is attached to a second quantum dot.
  • the quantum dots are selected such that each emits at a different wavelength under the same excitation source.
  • the first oligonucleotide comprising ExBIM or Exbenzi is attached to QD565 and the second oligonucleotide is attached to QD655.
  • the oligonucleotides are contacted with a nucleic acid sample.
  • Target duplexes are detected by measuring fluorescence.
  • oligonucleotides are attached to nanoparticles and the aggregation of the nanoparticles is used as a readout for target duplex formation.
  • said nanoparticles are gold nanoparticles (AuNP). Aggregation of said particles can be detected by color change, fluorescence, radioactivity, quartz crystal microbalance, Raman spectroscopy, light scattering, and electrical signals.
  • AuNPs are easily conjugated with biomolecules and retain the biochemical activity of the tagged biomolecules. Methods for preparing oligonucleotide bound gold particles are disclosed in the examples and are also described in WO 2008042156.
  • the examples describe an exemplary embodiment in which AuNPs are conjugated to oligonucleotides designed to hybridize with DNA bearing an 0 6 -meG adduct. Selective hybridization is enabled by incorporating into the oligonucleotide probe the novel nucleoside ExBIM. AuNP probes were constructed such that ExBIM is positioned to pair opposite the target DNA adduct within a defined DNA sequence.
  • the oligonucleotides comprising ExBIM or Exbenzi are also useful for enriching or isolating a target nucleic acid. Isolating a target polynucleotide refers to increasing the number or concentration of said target or reducing the amount of non-target polynucleotide from a sample. Accordingly, a method is provided for isolating an 0' !
  • oligonucleotide comprising ExBIM or Exbenzi, wherein said oligonucleotide is complementary to said target polynucleotide such that said target sequence and said oligonucleotide form a target duplex and b) isolating said target duplex.
  • the oligonucleotide is coupled to a purification label or a solid substrate and the target duplex is isolated via the label or substrate.
  • Suitable solid substrates have been disclosed herein.
  • the substrate may be a magnetic bead and target duplexes are separated from the sample with a magnetic field.
  • Example 3 provides an exemplary embodiment of a method for isolating a O 6 - alkyl guanine containing target nucleic acid. It is understood that methods for isolating an 0 6 -alkyl guanine containing target polynucleotide can be used in conjunction with methods for detecting the presence of 0 6 -alkyl guanine in a target nucleic acid.
  • a first oligonucleotide comprising ExBIM or Exbenzi is attached to a magnetic bead.
  • the nucleic acid containing sample is contacted with said first oligonucleotide and a magnetic field is applied to separate the target duplexes from the rest of the sample.
  • the target duplex is heated to denature the duplex.
  • the first oligonucleotide is removed by applying a magnetic field.
  • the isolated target polynucleotide can then be detected by the methods disclosed herein, e.g., using FRET hybridization probe or gold particle aggregation. Combining an isolation step with a detection step can increase the sensitivity and/or reduce the background of the methods.
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb "to consist” may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • Phosphoramidite and oligonucleotide synthesis Phosphoramidite and oligonucleotide synthesis.
  • Phosphoramidites of Exbenzi and ExBIM nucleosides were synthesized (Scheme 1; Figure4) for their site- specific incorporation into oligonucleotides.
  • Exbenzi nucleobase 7 was prepared in a solvent - free reaction by heating 2,3-diaminonaphtalene and urea in a microwave, (Kidwai et al., 2005) and ExBIM nucleobase 8 was prepared by heating 2,3-diaminonapthalene in formic acid.(Herbert et al., 1987; Sachs, 1909)
  • the nucleobases were glycosylated by nucleophilic displacement of chloride from l-(a)-chloro-3,5-di-0-p-toluoyl-2- deoxyribose (9) to yield tolouylated nucleosides, which were deprotected by trans- esterification with NaOMe to yield free nucleosides 5 and 6.
  • Oligonucleotides with sequences 5'-CCGXTATACCGACAA-3' were prepared by solid-phase DNA synthesis; this sequence was selected to allow direct comparison with published data examining related interactions. (Gahlon and Sturla, 2013a; Gong and Sturla, 2007)
  • the sequence 5'- XCGCGCG-3' which has previously been used to characterize n- stacking of both natural nucleosides and nucleoside analogs, (Gao et al., 2004; Guckian et al., 2000; Lai and Kool, 2004; Lee and Kool, 2005; Liu et al., 2005; Lu et al., 2004; Rosemeyer and Seela, 2002) was selected.
  • nucleoside analog phosphoramidites were typically half as efficient as that of the natural nucleotide phosphoramidites (as indicated by UV absorbance trityl cation monitoring), and the major side-products were oligonucleotides truncated immediately prior to Exbenzi or ExBIM
  • DNA duplex stability The melting of DNA duplexes containing nucleoside analogs of Per, Exbenzi, and ExBIM paired opposite G, 0 6 -Me-G or 0 6 -Bn-G was monitored by UV spectroscopy.
  • the thermodynamic stabilities of the duplexes i.e., the free energies of melting, AG
  • AG the free energies of melting
  • AAG° AG°(analog:adduct) - AG°(analog:G)
  • the elongated nucleobases of Exbenzi and ExBIM are better than those of Benzi and BIM at filling the gap of the abasic site and n-stacking with neighboring nucleobases.
  • n- stacking To assess the n-stacking of nucleoside analogs, their ability to stabilize the termini of short DNA duplexes was evaluated. Such experiments have been used to systematically explore the impact of dipole moment, polarizability, and hydrophobicity on the ability of natural and non-natural bases to contribute to DNA stabilization apart from hydrogen bonding or other interactions with complementary partners.
  • nucleobase polarizability and surface area (which influences both polarizability and shielding of the duplex from solvent) robustly correlated to dangling nucleoside stabilization.
  • Quantum yields of nucleoside analogs A quantitative measure of the fluorescence of a compound that allows comparison to other fluorophores is the quantum yield Of. Quantum yield is defined by the ratio of amount of photons absorbed to the amount of photons emitted through fluorescence. (Sinkeldam et al., 2010) Fluorescence quantum yields of nucleoside analogs were determined relative to standards tryptophan (water, pH 7) and 2-aminopyridine (0.05 M H2SO4) using the comparative method.
  • nucleosides showed no detectable fluorescence Upon hybridization of the nucleoside analog-containing probe strands to complementary strands containing G, 0 6 -Me-G, or THF, the fluorescence was quenched relative to that of ssDNA (on the order of 20 fold for ExBIM and 10 fold for Exbenzi).
  • Anhydrous solvents were purified through an LC Technology Solutions solvent purification system, in which the solvent is passed through a column of activated alumina before being dispensed under nitrogen, or were purchased from Sigma-Aldrich and used without further drying or purification.
  • Diamines, nitrogen bases, and sodium hydride were purchased from Sigma-Aldrich and used without further purification.
  • l-(a)-Chloro-3,5-di-0-p-toluoyl-2-deoxy-D-ribose (9) was obtained from Berry & Associates, and 2-cyanoethoxy-N,N-diisopropylaminochlorophosphine (12) was obtained from Link Technologies and Sigma-Aldrich.
  • 4,4'-Dimethoxyltrityl tetraflouroborate (DMT BF4) was prepared from 4,4'-dimethoxytrityl chloride
  • ⁇ NMR spectra were acquired at 400 MHz and 13 C NMR at 100 MHz on a Bruker NMR spectrometer. Chemical shifts are reported relative to the non-deuterated solvent signals (3.31 and 49.05 ppm in MeOH-cfe, 2.50 and 39.43 in DMSO-de, and 2.05 and 30.83 in acetone-de for ⁇ and 13 C NMR spectra, respectively). Flash chromatography (silica gel 60A 200-400 mesh) was used for product purification. Normal phase thin-layer chromatography (TLC; plates obtained from Silicycle) was used for reaction monitoring and the spots were visualized under UV light (254 nm).
  • TLC normal phase thin-layer chromatography
  • High-resolution ESI-MS HRMS was performed on a Thermo Scientific Exactive mass spectrometer. UV absorptions were measured at 260 nm using a Cary 100 UV- Vis spectrophotometer equipped with a Peltier thermal programmer.
  • Exbenzi nucleoside was adapted from that previously described for the preparation of the Per nucleoside. (Gong and Sturla, 2007) Exbenzi nucleobase 7 (1.103 g, 5.50 mmol) and anhydrous THF (50 mL) were added to a flame-dried 100 mL flask and flushed with nitrogen for several minutes. To the mixture, NaH (60% in mineral oil, 333 mg, 8.25 mmol) was added portion-wise over 2 minutes.
  • Oligonucleotides The oligonucleotides containing O e -Me-G, THF, and G were purchased from the Midland Certified Reagent Company Inc. The oligonucleotides containing O e -Bn-G and nucleoside analogs were synthesized using 5'-0- dimethoxytrityl protected phosphoramidites (natural nucleotides from Glen Research, Inc, and Link Technologies) on a Mermade 4 DNA synthesizer.
  • Oligonucleotides were purified by reversed phase HPLC (Agilent 1100) on a Luna 5 ⁇ C18 (2) 100 A 250 mm x 4.60 mm column (Phenomenex), and were eluted with acetonitrile and 50 mM triethylammonium acetate buffer using a gradient of 5% to 16% acetonitrile over 21 min. The identity of each oligonucleotide was confirmed by ESI-MS. Concentrations of oligonucleotides solutions were determined by measuring the UV absorbance at 260 nm. The molar extinction coefficients were determined via the nearest-neighbor method using the IDT calculator at
  • heating/cooling rates (0.75 °C/min and 1 °C/min) were tested; no significant differences in transition temperatures due to these different rates were observed.
  • concentrations ranging from 0.8 to 10 ⁇
  • DNA solutions (of single-strand concentrations ranging from 12 to 40 ⁇ ) were prepared in deionized water, then combined with equal volumes of buffer (2 M NaCl, 0.2mM EDTA, 20 mM sodium phosphate, pH 7.0) to generate sample solutions (with duplex concentrations ranging from 3 to 10 uM).
  • the transition temperature (Tt) of each melting curve was determined by the Cary thermal application software (using the software's derivative method), with smoothing of both the melting curve and derivative curve by 15 data points taken at 0.5 minute time increments for the non-complementary duplexes. For purposes of calculating thermodynamic parameters by evaluating the dependence of the transition temperature on duplex concentration, the transition temperatures were assumed to be approximately equal to the duplex melting temperature. Melting Temperature Analysis. The melting temperature (T m ) of each melting curve was found using the hyperchromicity method available in the Cary Thermal application software.
  • the software allows the user to select upper and linear baselines for the melting curve and subsequently graphs the fraction of annealed duplex (i.e., fraction-folded, or a) vs temperature.
  • thermodynamic parameters Calculating thermodynamic parameters by analyzing the shape of the melting curve.
  • nucleoside analog dilutions and oligonucleotides were excited at 250 nm (5 nm band width for excitation, 20 nm band width for emission, 25°C) in black 96-well plates (Nunc).
  • Quantum yields were determined using the comparative method of Williams et al., (Williams, 1983) in which well characterized standards of a known Of are used. Standards should absorb and emit fluorescence at the same wavelength as the samples to be tested (if recorded under identical conditions). The quantum yield can be calculated with the following e
  • the emission curves of nucleoside analogs BIM and Benzi were cut off at the lower wavelength. Therefore the integrals of fluorescence intensity for BIM and Benzi were calculated differently: the right half of the emission peak was integrated and multiplied by two. Integrated fluorescence intensities were then plotted against calculted absorbance values (at least 5 data points) and the slope of the corresponding linear trendlines were used to calculate the quantum yields with the equation above. The quantum yield of nucleoside analogs was calculated twice, using the
  • Dimethoxytrityl and 4-monomethoxytrityl tetrafluoroborate convenient reagents for the protection of primary alcohols including sugars. J. Chem. Soc. Perk. T. 2. 0:803- 805.
  • the nanoparticle-based detection system discussed herein involves two different oligonucleotide-modified AuNP probes that align in a tail-to-tail fashion onto a complementary target oligonucleotide (Fig. 5b).
  • Probe 1 was a 16-mer consisting of an (A)io spacer and a 6-mer recognition sequence.
  • Probe 2 was a 17-mer consisting of an (A)io spacer and a 7-mer recognition sequence and the first base at the 5'-end was ExBIM.
  • the 13-base oligonucleotide target strand was designed to have its 5'-end
  • the target could cross-link the two distinct sets of AuNPs through
  • Dispersed functionalized 20- nm AuNPs have an intense red color due to the localized surface plasmon resonance (SPR) and exhibit a characteristic SPR band at 530 nm (Fig. 6).
  • SPR surface plasmon resonance
  • the close proximity of the cross-linked AuNPs caused coupling of their individual localized plasmon fields, leading to a shift in their absorbance to a longer wavelength, and the color of the solution changed from red to purple. Therefore, upon addition of target the 530-nm SPR peak decreased while the absorbance in the 700-nm region increased (Fig. 6).
  • the absorbance ratio at 700 nm vs. 530 nm was used for characterizing the aggregation state.
  • the specificity of the AuNP probes was assessed by using as target synthetic oligonucleotides that were designed to represent a mutational hotspot codon in exon 2 of the K-ras oncogene.
  • the RAS gene family is among the most studied and best characterized of the known cancer-related genes.
  • the RAS oncogene is well established to influence cell growth and regulation; its protein product affects cell proliferation, apoptosis, migration, fate specification, and differentiation.
  • KRAS mutations in exon 2 (codons 12 and 13), and to a lesser extent in exon 3 (codon 61), are associated with colorectal cancer outcome. Nearly 97% of all KRAS mutations are considered an early event in the adenoma-carcinoma sequence and are localized to codons 12 or 13, with a large majority located in codon 12 28 . Mutations in each of the three codons compromise the ability of GTPase-activating proteins to inactivate hydrolysis of Ras-bound GTP to GDP 29 . Other mutations are uncommon as they result in lower constitutive Ras signaling than mutations in codons 12, 13 and 61, and so are selected against in carcinogenesis 30 .
  • the target oligonucleotides (13-mers) were designed so that the middle base would correspond to the K-ras codon 13 mutational hotspot (Table 5).
  • the target oligomers varied in the identity of the base placed opposite ExBIM. This position was at the centre of the target sequence to achieve highest discrimination between the adduct and the natural base, as it was shown by previous studies concerning mismatch discrimination 45 47 .
  • targets 1) an oligonucleotide containing the alkylated base 0 6 -meG and 2) an oligonucleotide containing the natural base G.
  • we synthesized a non-complementary 13-mer sequence (Table 1, irrelevant target) as a negative control.
  • Tm melting temperatures
  • the Tm of the aggregates formed from the AuNP probes and 20 nM (Fig. 7a) of the 0 6 -meG-containing target was 32.7 °C.
  • the T m values for aggregates formed from the G- containing target was 29.4 °C. Therefore the difference in the melting temperatures (AT m ) between the aggregates formed from the 0 6 -meG and G containing targets was 3.3 °C.
  • Dissociation curves were also obtained for DNA duplexes formed from the different targets and the conventional oligonucleotide Probe 3 whose sequence combines the recognition sequences of Probes 1 and 2. Higher concentrations of oligomers (2.2 ⁇ in each oligonucleotide) were used in order to obtain a measurable signal at 260 nm; the experiment could not be performed at concentrations comparable to those used for the aforementioned nanoparticle probe studies since the conventional oligonucleotides do not exhibit measurable absorbance changes upon hybridization in this
  • the detection limit for this assay was 96 fmol of the 0 6 -meG-containing target (0.96 nM) in the presence of 4 pmol of the G target (40 nM), which corresponds to a relative 0 6 -meG- containing target concentration of 2.4 % (Fig. 8A). Furthermore, when the presence of the irrelevant target is taken into account, the detection limit for this assay was 96 fmol of the 0 6 -meG target (0.96 nM) in the presence of 6 pmol of total DNA targets (60 nM), which corresponds to a relative O 6 - meG-containing target concentration of 1.6 %.(Fig. 8B)
  • DNA is isolated from a biological sample (e.g., stool, blood, or tissue) and is subjected to digestion with a restriction enzyme that digests the Kras gene upstream and downstream of a DNA adduct hotspot.
  • a biological sample e.g., stool, blood, or tissue
  • a restriction enzyme that digests the Kras gene upstream and downstream of a DNA adduct hotspot.
  • Digested DNA is mixed with biotinylated oligonucleotides that are designed to hybridize with the DNA fragments of the mutational hotspot.
  • biotinylated oligos also comprise ExBIM or Exbenzi.
  • Biotinylated hybrids are mixed with Streptavidin coated magnetic beads (e.g.
  • biotinylated hybrids are captured on the magnetic beads through biotin- streptavidin interaction.
  • Magnetic field is applied so that hybrids are separated from unhybridized DNA fragments.
  • Magnetic beads are resuspended in water or appropriate buffer. Dispersion is briefly heated (e.g. 5 min at 70 °C), so that the hybrid duplex is denatured.
  • the target polynucleotide fragments are in solution and can be collected.

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Abstract

La présente invention concerne de nouveaux composés contenant un nucléoside à base modifiée présentant la formule décrite, et des méthodes permettant de détecter la présence de guanine dans un acide nucléique et d'isoler l'acide nucléique comprenant la 06-alkylguanine. L'invention repose sur des analogues de nucléosides à base modifiée qui forment des paires de bases stables avec la 06-alkylguanine.
PCT/EP2015/058600 2014-04-24 2015-04-21 Analogues de nucléosides à base modifiée pour la détection de la 06-alkylguanine Ceased WO2015162130A1 (fr)

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WO2018035410A1 (fr) * 2016-08-19 2018-02-22 Temple University-Of The Commonwealth System Of Higher Education Compositions et procédés de traitement utilisant des analogues d'adn de taille étendue
TWI673273B (zh) * 2018-11-14 2019-10-01 Winsense Biotech Corp. 作為氧化還原嵌入型探針之雙-萘醌衍生物及其製備方法
CN115232073A (zh) * 2022-09-21 2022-10-25 南京邮电大学 基于1H-萘[2,3-d]咪唑的光响应性有机长余辉材料的制备及其应用
WO2024039516A1 (fr) * 2022-08-19 2024-02-22 Illumina, Inc. Détection de la troisième paire de bases de l'adn spécifique de site

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018035410A1 (fr) * 2016-08-19 2018-02-22 Temple University-Of The Commonwealth System Of Higher Education Compositions et procédés de traitement utilisant des analogues d'adn de taille étendue
TWI673273B (zh) * 2018-11-14 2019-10-01 Winsense Biotech Corp. 作為氧化還原嵌入型探針之雙-萘醌衍生物及其製備方法
WO2024039516A1 (fr) * 2022-08-19 2024-02-22 Illumina, Inc. Détection de la troisième paire de bases de l'adn spécifique de site
CN115232073A (zh) * 2022-09-21 2022-10-25 南京邮电大学 基于1H-萘[2,3-d]咪唑的光响应性有机长余辉材料的制备及其应用
CN115232073B (zh) * 2022-09-21 2022-11-22 南京邮电大学 基于1H-萘[2,3-d]咪唑的光响应性有机长余辉材料的制备及其应用

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